Il-15 composition and method for using the same

Abstract

To provide a composition containing activatable proprotein which is fused to a masked part including an antibody Fc region, and includes a first IL-15 or a variant thereof and a second IL-15Rα or a variant thereof, and a method of using the composition for cancer immunotherapy and other therapy.SOLUTION: Activatable proprotein contains a first polypeptide and a second polypeptide, where each of the first and second polypeptides includes a masked part operably connected to IL-15 or a variant thereof via a first linker of C-terminal, IL-15 or a variant thereof is connected to IL-15Rα or a variant thereof via a second linker of C-terminal, and the masked part masks an active part of proprotein.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to U.S. patent application Ser. No. 62 / 779,793, filed Dec. 14, 2018, which is incorporated by reference in its entirety. SEQUENCE LISTING STATEMENT

[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is VIVA_001_01WO_ST25.txt. The text file is approximately 1,733KB, was created on December 14, 2019, and has been submitted electronically via EFS-Web. [Background technology]

[0003] IL-15 is a pleiotropic cytokine that has been demonstrated to induce and regulate a myriad of immune functions. In particular, IL-15 is important for lymphocyte development and peripheral maintenance of innate immune cells and immunological memory of T cells, specifically natural killer (NK) and CD8+ T cell populations. However, IL-15 does not promote the maintenance of CD4+CD25+FOXP3+ regulatory T cells (Tregs), whereas IL-2 has been demonstrated to induce their development, and IL-15 has been shown to protect effector T cells from IL-2-mediated activation-induced cell death (AICD). For these reasons, IL-15 has long been presumed to have high therapeutic potential for long-term antitumor immunity. IL-15 is a four α-helical protein that belongs to a family of cytokines consisting of the interleukins IL-2, IL-4, IL-7, IL-9 and IL-21. IL-15 signals through a receptor complex composed of the IL-2 / IL-15 receptor beta (IL-15Rβ) (CD122) subunit shared with IL-2 and the common gamma chain (γC) (CD132) receptor subunit also utilized by all additional family members. Although it does not have a critical direct role in IL-15 signaling itself, IL-15Rα is a critical component of the IL-15 cytokine-receptor complex. IL-15Rα is a transmembrane protein with extremely high affinity for IL-15, which promotes IL-15 transport from the endoplasmic reticulum (ER) through the cytoplasm and presentation of the IL-15 / IL-15Rα complex at the cell surface. In addition to remaining associated in the cytoplasm and cell surface expression, IL-15 / IL-15Rα can also be cleaved as a complex into the extracellular space.

[0004] When expressed on the cell surface, the IL-15 / IL-15Rα complex can stimulate neighboring or opposing cells in trans via IL-15Rβ / γC. This type of cytokine stimulation requires cell-cell contact and is called trans-presentation. Under steady-state conditions, trans-presentation is thought to be the primary mode of action for IL-15-mediated development and homeostasis of CD8 T cells, NK cells, NKT cells and intraepithelial lymphocytes. Trans-presentation offers tighter regulation than that of secreted cytokines by providing cell-directed delivery of IL-15 to specific cell types. Nevertheless, soluble (s)IL-15 / IL-15Rα complexes are also cleaved from the cell surface in response to various inflammatory signals, e.g., TLR ligation, type I interferon and CD40 ligation. This generation of sIL-15 complexes is generally transient in nature and can provide a short-lived but potent burst of IL-15 activity to support earlier immune responses. In both of these situations, IL-15 binding to IL-15Rα is not only a platform for IL-15 delivery, but may also increase the half-life of IL-15 and increase the affinity of IL-15 for IL-15Rβ / γC. Indeed, sIL-15 complexes have been shown to be superior in their ability to stimulate IL-15 responses compared to unassociated rIL-15. This has brought much attention to IL-15 and led to the generation of multiple formulations of sIL-15 complexes that are currently being investigated for their therapeutic efficacy.

[0005] The immune effects of IL-15 in cancer patients have been investigated and it has been demonstrated to induce the expansion of NK cells, and significantly increased the proliferation of γδ T cells and CD8+ T cells after administration in humans.

[0006] Despite its promising antitumor immunity, IL-15 has been found to exhibit a short half-life and high doses were required to achieve a biological response in vivo, which resulted in clinical toxicity and limited antitumor responses in patients. To increase therapeutic efficacy and facilitate the use of IL-15 in immunotherapy of cancer and chronic infections, several companies are currently developing IL-15 and IL-15 derivatives, but the main drawbacks of the mentioned drug candidates exist: (1) high serum Cmax, which initially causes overactivation of the immune system, (2) short PK due to the small molecular size of IL-15 (13-14 kD) or the large number of IL-15 catabolism by immune cells expressing IL-15 receptor or IL-15Fc fusion protein, (3) insufficient accumulation in target tumors due to short PK, lack of tumor targeting or ineffective tumor targeting, and (4) undesirable accumulation and immune activation in normal tissues.

[0007] The present invention solves all of the above problems by providing activatable proproteins, including interleukin 15 (IL-15), that are activated in cancer tissues or tumors. This platform has the potential to address the shortcomings of IL-15 and its derivatives currently being developed for cancer immunotherapy. Summary of the Invention [Means for solving the problem]

[0008] In one aspect, the present invention relates to an activatable proprotein comprising a first polypeptide and a second polypeptide, wherein the first and second polypeptide each comprise a masking moiety operably linked to IL-15 or a variant thereof via a first linker at the C-terminus, and IL-15 or a variant thereof is linked to IL-15Rα or a variant thereof via a second linker at the C-terminus, wherein the masking moiety masks an active portion of the proprotein.

[0009] In one aspect, the present invention relates to an activatable proprotein comprising a first polypeptide and a second polypeptide, wherein the first and second polypeptide each comprise IL-15 or a variant thereof operably linked to IL-15Rα or a variant thereof via a first linker at the C-terminus, and the IL-15Rα or a variant thereof is linked to a masking moiety present on each of the first and second polypeptide via a second linker at the C-terminus, wherein the masking moiety masks an active portion of the proprotein.

[0010] In a related embodiment, the first linker on the first or second polypeptide, or on both the first and second polypeptide, is a cleavable linker. In another related embodiment, the second linker on the first or second polypeptide, or on both the first and second polypeptide, is a cleavable linker.

[0011] In a related embodiment, the IL-15 or variant thereof and the IL-15Rα or variant thereof in the first polypeptide and the IL-15 or variant thereof and the IL-15Rα or variant thereof in the second polypeptide comprise one or more Cys substitution mutations.

[0012] In another related aspect, IL-15 or a variant thereof in a first polypeptide comprising one or more Cys substitution mutations forms a disulfide bond with IL-15Rα or a variant thereof in a second polypeptide comprising one or more Cys substitution mutations. In another embodiment, IL-15 or a variant thereof in a second polypeptide comprising one or more Cys substitution mutations forms a disulfide bond with IL-15Rα or a variant thereof in a first polypeptide comprising one or more Cys substitution mutations.

[0013] In a related embodiment, the proprotein is a dimeric proprotein. In another related embodiment, the dimeric proprotein comprises a first polypeptide and a second polypeptide, each comprising a masking moiety linked N-terminus to C-terminus via a first linker to IL-15 or a variant thereof, wherein IL-15 or a variant thereof is linked to IL-15Rα or a variant thereof via a second linker, and wherein the masking moiety of the first polypeptide forms a covalent disulfide bond or is non-covalently bonded fused to the masking moiety of the second polypeptide. In another related aspect, the dimeric proprotein comprises a first polypeptide and a second polypeptide, each comprising a masking moiety linked N-terminus to C-terminus via a first linker to IL-15 or a variant thereof, and IL-15 or a variant thereof linked to IL-15Rα or a variant thereof via a second linker, and the masking moiety of the first polypeptide forms one or more disulfide bonds or non-covalent bonds with the masking moiety of the second polypeptide. In one embodiment, the dimeric proprotein comprises a significantly reduced biological activity compared to native IL-15. In another embodiment, the dimeric protein is activatable by protease cleavage to restore the activity of IL-15 or a variant thereof present in the dimeric proprotein.

[0014] In a related aspect, a recombinant nucleic acid molecule is provided that encodes an activatable proprotein as disclosed herein. In another related aspect, a vector is provided that includes a recombinant nucleic acid molecule that encodes an activatable proprotein.

[0015] In a related aspect, a pharmaceutical composition is provided that includes an activatable proprotein disclosed herein and a pharma- ceutically acceptable carrier.

[0016] In another aspect, the present invention relates to a method for treating cancer in a subject, comprising administering to a subject having cancer an activatable proprotein or a pharmaceutical composition comprising an activatable proprotein as disclosed herein, wherein following administration, the activatable proprotein is activated by protease cleavage in the cancerous tissue. In another embodiment, the activatable proprotein is activated following or after protease cleavage in the cancerous tissue.

[0017] In a related aspect, the invention relates to a method comprising administering to a subject in need thereof an activatable proprotein or a pharmaceutical composition comprising an activatable proprotein as disclosed herein, to induce or enhance an anti-tumor immune response in the subject, wherein following administration, the activatable proprotein is activated by protease cleavage in the tumor, hi another embodiment, the activatable proprotein is activated following or after protease cleavage in the tumor.

[0018] In another aspect, the present invention relates to the use of an activatable proprotein or a pharmaceutical composition comprising an activatable proprotein as disclosed herein for treating cancer in a subject, comprising the step of administering the activatable proprotein or a pharmaceutical composition comprising same, wherein following administration, the activatable proprotein is activated by protease cleavage in the cancerous tissue.

[0019] In another aspect, the present invention relates to the use of an activatable proprotein or a pharmaceutical composition comprising an activatable proprotein as disclosed herein for inducing or enhancing an anti-tumor immune response in a subject, comprising the step of administering the activatable proprotein or pharmaceutical composition, wherein following administration, the activatable proprotein is activated by protease cleavage in the tumor.

[0020] In another embodiment of the methods or uses disclosed herein, protease cleavage partially or completely removes a masking moiety in the activatable proprotein such that the IL-15 / IL-IL-15Rα complex in the first and second polypeptides can bind to IL-15Rβ / γC present on the surface of lymphocytes of blood cells in vitro or in vivo.

[0021] Other features and advantages of the present invention will become apparent from the following detailed description, examples, and drawings. It should be understood, however, that the detailed description and specific examples while indicating embodiments of the present invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0022] Various objects and advantages of the present invention and a more complete understanding thereof will become apparent and will be more readily appreciated by reference to the following detailed description and appended claims, when taken in conjunction with the accompanying drawings in which: [Brief description of the drawings]

[0023] [Figure 1-1] FIG. 1A is a diagram showing the protein topology of human interleukin-15 (IL-15) and the human interleukin-15 receptor alpha chain (IL-15Rα).

[0024] [Figure 1-2]FIG. 1B shows human_IL-15_LSP: amino acid sequence of IL-15 with a long signal peptide (1-162), human_IL-15_mature: amino acid sequence of IL-15 without a signal peptide (49-162), human_IL-15Rα_FL: full-length sequence of IL-15Rα with the signal peptide underlined (1-267), human_IL-15Rα-ECD: extracellular domain sequence of IL-15Rα (31-205), human_IL-15Rα-sushi+: extracellular sushi domain sequence and the first 13 amino acids from the linker region (31-98), and human_IL-15Rα-sushi: extracellular sushi domain sequence (31-85).

[0025] [Figure 1-3] FIG. 1C shows the quaternary structure of IL-15 in complex with its receptors IL-15Rα (CD215), IL-15Rβ (CD122) and common gamma chain (CD132) (PDB: 4GS7) and a schematic of the plasma membrane and transmembrane and signaling domains of the receptors.

[0026] FIG. 1D illustrates a schematic diagram of IL-15 and the cell surface IL-15 receptor complex.

[0027] [Figure 2-1] Figure 2A illustrates the IL-15 complex with its receptor IL-15Rα-sushi (PDB:2Z3Q) with an artificial flexible peptide linker between the C-terminus of IL-15 and the N-terminus of IL-15Ra-sushi (to form an ILR). The interaction site of IL-15 in the ILR with its signaling IL-15Rβ / γc receptor is shown at the IL-15Rβ / γc interaction interface.

[0028] Figure 2B illustrates the homodimeric association of the ILR fusion proteins described in Figure 2A. The interaction site of IL-15 in the homodimeric ILR with its signaling IL-15Rβ / γc receptor is shown at the IL-15Rβ / γc interaction interface. The ILR fusion proteins, unlike the RLI fusion proteins, favor homodimer formation.

[0029] [Figure 2-2] Figure 2C illustrates a schematic of the ILR structure described in Figure 2A. IL-15 in the ILR can bind to and signal through the IL-15Rβ / γc receptor.

[0030] Figure 2D illustrates a schematic diagram of the dimeric ILR structure described in Figure 2B. IL-15 in the dimeric ILR can bind to and signal through the IL-15Rβ / γc receptor.

[0031] [Figure 3-1] Figure 3A illustrates the fusion of the C-terminus of the ILR (depicted in Figure 2B) to the N-terminus of the fusion moiety. Similar to the structure depicted in Figure 2B, this fusion also favors dimer formation. The interaction site of IL-15 in the homodimeric ILR with its signaling IL-15Rβ / γc receptor is shown at the IL-15Rβ / γc interaction interface.

[0032] Figure 3B illustrates a schematic of the dimeric fusion structure described in Figure 3A. IL-15 in the dimeric ILR can bind to and signal through the IL-15Rβ / γc receptor. IL-15 in this dimeric fusion can bind to and signal through the IL-15Rβ / γc receptor.

[0033] FIG. 3C illustrates protein sequence motifs and configuration diagrams of the proteins described in FIGS. 3A and 3B.

[0034] [Figure 3-2]Figure 3D illustrates the fusion of the N-terminus of the ILR (as depicted in Figure 2B) to the C-terminus of the fusion moiety (masking moiety). Similar to the structure depicted in Figure 2B, this fusion also favors dimer formation. The interaction site of IL-15 in the homodimeric ILR with its signaling IL-15Rβ / γc receptor is shown at the IL-15Rβ / γc interaction interface.

[0035] Figure 3E illustrates a schematic of the dimeric fusion structure described in Figure 3D. IL-15 in the dimeric ILR can bind to and signal through the IL-15Rβ / γc receptor. IL-15 in this dimeric fusion cannot bind to and signal through the IL-15Rβ / γc receptor due to steric hindrance caused by the masking moiety and the linker between the ILR and the masking moiety.

[0036] FIG. 3F illustrates protein sequence motifs and configuration diagrams of the proteins described in FIGS. 3D and 3E.

[0037] [Figure 3-3] FIG. 3G illustrates a schematic diagram of a dimeric fusion structure with a protein domain at the C-terminus of the first polypeptide, IL-15Rα.

[0038] FIG. 3H illustrates protein sequence motifs and configuration diagrams of the proteins depicted in FIG. 3G.

[0039] [Diagram 3-4] FIG. 3I illustrates a schematic of a dimeric fusion structure with a protein domain at the C-terminus of IL-15Rα of a second polypeptide.

[0040] FIG. 3J illustrates protein sequence motif and configuration diagrams of the proteins depicted in FIG. 3I.

[0041] [Figure 3-5]FIG. 3K illustrates a schematic diagram of a dimeric fusion structure having a protein domain at the C-terminus of IL-15Rα of the first and second polypeptides.

[0042] FIG. 3L illustrates protein sequence motifs and configuration diagrams of the proteins depicted in FIG. 3K.

[0043] [Figure 4-1] Figure 4A illustrates, but is not limited to, protein sequence motif and configuration diagrams similar to those depicted in Figure 3F. Any one or more of the protein sequence motifs in the diagram of a first polypeptide may differ from the corresponding motif in a second polypeptide, as indicated by the double arrow. The linker sequences depicted in the diagram may be mobile and protease cleavable, or mobile and protease non-cleavable, or a combination thereof.

[0044] [Figure 4-2] Figure 4B illustrates examples of masking motifs in a first and a second polypeptide. The masking motif in a first polypeptide may be the same as or different from the masking motif in a second polypeptide. The masking motif from a first polypeptide may form either non-covalent or covalent interactions, or both covalent and non-covalent interactions, with the masking motif in a second polypeptide.

[0045] [Figure 4-3] 4C-4D illustrate examples of Fab-CH2CH3 (4C) and Fab-Cys-CH2CH3 (4D) as masking moieties in polypeptide fusions with ILR.

[0046] [Figure 4-4] 4E-4F are diagrams illustrating examples of CH2CH3 (4E) and Cys-CH2CH3 (4F) as masking moieties in polypeptide fusions with ILR.

[0047] [Figure 4-5] 4G-4H show examples of CH3(4G) and Cys-CH3(4H) as masking moieties in polypeptide fusions with ILR.

[0048] [Figure 4-6] Figures 4I-4J show examples of VH-CH1 and VL-CL (4I) and VH-CH1-Cys and VL-CL-Cys (4J) as masking moieties in polypeptide fusions with ILR.

[0049] [Diagram 4-7] 4K-4L show examples of heterodimer CH2CH3 (4K) and heterodimer Cys-CH2CH3 (4L) as masking moieties in polypeptide fusions with ILR.

[0050] [Figure 4-8] 4M-4N show examples of leucine zippers (coiled coils) (4M) and Cys-leucine zippers (coiled coils) (4N) as masking moieties in polypeptide fusions with ILRs.

[0051] [Figure 4-9] FIG. 4O shows an example of a Cys(4O) containing flexible peptide as a masking moiety in a polypeptide fusion with an ILR.

[0052] [Diagram 5] Figure 5 shows a schematic of the dimeric Cys-CH2-CH3-ILR. The lack of IL15 signaling activity through the IL15Rβ / γc receptor suggests that inter-chain association of IL15 and IL15R is favored over intra-chain association of IL15 and IL15R, which results in masking of the IL15Rβ / γc interaction site of IL15.

[0053] [Figure 6] Figure 6 shows a schematic diagram of activation of dimeric Cys-CH2-CH3-ILR by protease cleavage of substrate linker sequences in a fusion polypeptide. Examples of protease substrate sequences for linker 1 and linker 3 are shown. Digestion of one of the protease substrate sequences in the linker (partial digestion) releases the steric hindrance imposed by the masking moiety, thus allowing IL-15 in the fusion to bind to and signal through the IL15Rβ / γc receptor. Digestion of both cleavable linkers releases the ILR from the Fc fusion, resulting in full activity of the ILR towards the IL15Rβ / γc receptor.

[0054] [Figure 7] Figure 7 shows a schematic diagram of activation of dimeric Cys-CH2-CH3-ILR by protease cleavage of substrate linker sequences in a fusion polypeptide. Examples of protease substrate sequences for linker 2 and linker 4 are shown. Digestion of one of the two protease substrate sequences in the linker does not efficiently release the steric hindrance imposed by the masking moiety. Thus, complete digestion of both protease linkers is required to activate fusion polypeptide signaling through the IL15Rβ / γc receptor.

[0055] [Figure 8] FIG. 8 shows sequence identifiers for exemplary fusion proteins.

[0056] [Figure 9] 9A to 9F show representative SDS-PAGE results of purified proteins. "M" on the figures represents a protein standard marker.

[0057] [Figure 10-1] 10A to 10G show representative examples of HPLC analysis results of purified proteins. [Figure 10-2] 10A to 10G show representative examples of HPLC analysis results of purified proteins.

[0058] [Figure 11] 11A-11B illustrate representative fluorescence assisted cell sorting (FACS) gating and dot plots of pSTAT5 cell signaling activity of the IL-15 fusion protein P1185.

[0059] 11C-11D illustrate the cell signaling activity of IL-15 fusion proteins as measured by pSTAT5 FACS assay.

[0060] [Figure 12] 12A-12C illustrate representative FACS gating of IL-15 stimulated samples in a cell signaling assay using human whole blood.

[0061] 12D-12F illustrate the cell signaling activity of IL-15 fusion proteins using human whole blood, as measured by a pSTAT5 FACS assay.

[0062] [Figure 13-1] 13A-13H illustrate the activity of IL15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 13-2] 13A-13H illustrate the activity of IL15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 13-3] 13A-13H illustrate the activity of IL15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0063] [Figure 14]FIG. 14A illustrates representative gating of lymphocytes on side scatter (SSC) and forward scatter (FSC) of IL-15 stimulated samples.

[0064] 14B-14F illustrate the results of a human PBMC proliferation assay of IL-15 fusion proteins.

[0065] [Figure 15-1] 15A-15I are diagrams illustrating protease (TEV) cleavage of IL-15 fusion proteins. "M" on the diagrams represents the protein standardmaker. [Figure 15-2] 15A-15I are diagrams illustrating protease (TEV) cleavage of IL-15 fusion proteins. "M" on the diagrams represents the protein standardmaker. [Figure 15-3] 15A-15I are diagrams illustrating protease (TEV) cleavage of IL-15 fusion proteins. "M" on the diagrams represents the protein standardmaker.

[0066] [Figure 16-1] 16A-16G are diagrams illustrating the cleavage of IL-15 fusion protein with uPA, matriptase, legumain, MMP-2 and MMP-9. M represents protein standard marker, B represents sample before protease cleavage, uPA represents uPA protease, Mat represents matriptase protease, L represents legumain protease, M-2 represents MMP-2 protease, and M-9 represents MMP-9 protease. Bands marked with red arrows: uncleaved protein; bands marked with pink arrows: Fc / Fc-L15R; bands marked with yellow arrows: Fc dimer; bands marked with green arrows: L15R. [Figure 16-2]16A-16G are diagrams illustrating the cleavage of IL-15 fusion protein with uPA, matriptase, legumain, MMP-2 and MMP-9. M represents protein standard marker, B represents sample before protease cleavage, uPA represents uPA protease, Mat represents matriptase protease, L represents legumain protease, M-2 represents MMP-2 protease, and M-9 represents MMP-9 protease. Bands marked with red arrows: uncleaved protein; bands marked with pink arrows: Fc / Fc-L15R; bands marked with yellow arrows: Fc dimer; bands marked with green arrows: L15R. [Figure 16-3] 16A-16G are diagrams illustrating the cleavage of IL-15 fusion protein with uPA, matriptase, legumain, MMP-2 and MMP-9. M represents protein standard marker, B represents sample before protease cleavage, uPA represents uPA protease, Mat represents matriptase protease, L represents legumain protease, M-2 represents MMP-2 protease, and M-9 represents MMP-9 protease. Bands marked with red arrows: uncleaved protein; bands marked with pink arrows: Fc / Fc-L15R; bands marked with yellow arrows: Fc dimer; bands marked with green arrows: L15R.

[0067] [Figure 17-1] 17A-17B illustrate the cell signaling activity of IL15 fusion proteins as measured by pSTAT5 FACS assay. [Figure 17-2] 17A-17B illustrate the cell signaling activity of IL15 fusion proteins as measured by pSTAT5 FACS assay.

[0068] [Figure 18-1] 18A-18I show the activity of IL15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 18-2] 18A-18I show the activity of IL15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 18-3] 18A-18I show the activity of IL15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0069] [Figure 19] Figures 19A-19D show human PBMC proliferation assay results of IL-15 fusion protein P1256 with and without TEV protease cleavage. P1185 was used as a positive control. Digested P1256 represents P1256 protein digested overnight at 37°C by TEV protease at a TEV to protein ratio of 1:2. P1256+TEV 1-2 represents P1256 mixed with TEV at a TEV to protein ratio of 1:2 for proliferation assay. Cell culture medium was used in the TEV digestion reaction.

[0070] [Figure 20] 20A-20F are diagrams showing protease (TEV) cleavage of IL-15 fusion proteins. "M" represents a protein standard marker, B represents a protein sample before TEV cleavage, and A represents a protein sample after TEV cleavage.

[0071] [Figure 21] FIG. 21 shows the cell signaling activity of IL15 fusion proteins as measured by pSTAT5 FACS assay.

[0072] [Figure 22-1] 22A-22F show the activity of IL15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 22-2]22A-22F show the activity of IL15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0073] [Figure 23-1] 23A to 23F are diagrams showing the results of reducing SDS-PAGE of purified proteins. "M" on the diagrams represents a standard protein marker. [Figure 23-2] 23A to 23F are diagrams showing the results of reducing SDS-PAGE of purified proteins. "M" on the diagrams represents a standard protein marker. [Figure 23-3] 23A to 23F are diagrams showing the results of reducing SDS-PAGE of purified proteins. "M" on the diagrams represents a standard protein marker.

[0074] [Figure 24-1] 24A to 24F are diagrams showing the results of non-reducing SDS-PAGE of purified proteins. "M" on the diagrams represents a protein standard marker. [Figure 24-2] 24A to 24F are diagrams showing the results of non-reducing SDS-PAGE of purified proteins. "M" on the diagrams represents a protein standard marker. [Figure 24-3] 24A to 24F are diagrams showing the results of non-reducing SDS-PAGE of purified proteins. "M" on the diagrams represents a protein standard marker.

[0075] [Figure 25-1] 25A-25X show representative examples of HPLC analysis of purified proteins. [Figure 25-2] 25A-25X show representative examples of HPLC analysis of purified proteins. [Figure 25-3] 25A-25X show representative examples of HPLC analysis of purified proteins. [Figure 25-4] 25A-25X show representative examples of HPLC analysis of purified proteins. [Figure 25-5] 25A-25X show representative examples of HPLC analysis of purified proteins. [Figure 25-6] 25A-25X show representative examples of HPLC analysis of purified proteins.

[0076] [Figure 26-1] 26A-26P are diagrams showing the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the diagram represents a protein standard marker. "B" represents the protein before protease cleavage. "B-4" represents the protein stored at 4 degrees before cleavage. "B-37" represents the protein stored at 37 degrees before cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "M-9" represents MMP-9 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease. [Figure 26-2] 26A-26P are diagrams showing the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the diagram represents a protein standard marker. "B" represents the protein before protease cleavage. "B-4" represents the protein stored at 4 degrees before cleavage. "B-37" represents the protein stored at 37 degrees before cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "M-9" represents MMP-9 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease. [Figure 26-3]26A-26P are diagrams showing the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the diagram represents a protein standard marker. "B" represents the protein before protease cleavage. "B-4" represents the protein stored at 4 degrees before cleavage. "B-37" represents the protein stored at 37 degrees before cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "M-9" represents MMP-9 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease. [Figure 26-4] 26A-26P are diagrams showing the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the diagram represents a protein standard marker. "B" represents the protein before protease cleavage. "B-4" represents the protein stored at 4 degrees before cleavage. "B-37" represents the protein stored at 37 degrees before cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "M-9" represents MMP-9 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease. [Figure 26-5] 26A-26P are diagrams showing the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the diagram represents a protein standard marker. "B" represents the protein before protease cleavage. "B-4" represents the protein stored at 4 degrees before cleavage. "B-37" represents the protein stored at 37 degrees before cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "M-9" represents MMP-9 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease. [Figure 26-6]26A-26P are diagrams showing the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the diagram represents a protein standard marker. "B" represents the protein before protease cleavage. "B-4" represents the protein stored at 4 degrees before cleavage. "B-37" represents the protein stored at 37 degrees before cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "M-9" represents MMP-9 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease. [Figure 26-7] 26A-26P are diagrams showing the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the diagram represents a protein standard marker. "B" represents the protein before protease cleavage. "B-4" represents the protein stored at 4 degrees before cleavage. "B-37" represents the protein stored at 37 degrees before cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "M-9" represents MMP-9 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease. [Figure 26-8] 26A-26P are diagrams showing the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the diagram represents a protein standard marker. "B" represents the protein before protease cleavage. "B-4" represents the protein stored at 4 degrees before cleavage. "B-37" represents the protein stored at 37 degrees before cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "M-9" represents MMP-9 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease.

[0077] [Figure 27-1]27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 27-2] 27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 27-3] 27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 27-4] 27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 28-1] 27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 28-2] 27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 28-3] 27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 28-4] 27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 28-5]27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 28-6] 27A-27N and 28A-28W show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0078] [Figure 29] Figures 29A-29F illustrate representative results of in vitro serum stability. Western blots were probed with biotinylated anti-IL-15 antibody followed by streptavidin-HRP. Figure 29A: In vitro stability of P1482 in human serum. Figure 29B: In vitro stability of P1482 in mouse serum. Figure 29C: In vitro protein stability in human serum at 164 hours. Figure 29D: In vitro protein stability in mouse serum at 96 hours. Figure 29E: In vitro protein stability in rat serum at 96 hours. Figure 29F: In vitro protein stability in cynomolgus monkey (cyno) serum at 96 hours.

[0079] [Figure 30-1] Figures 30A and 30B are diagrams showing the results of SDS-PAGE of purified proteins. Figure 30A shows the results of reduced SDS-PAGE, and 30B shows the results of non-reduced SDS-PAGE. "M" on the figures represents a protein standard marker. [Figure 30-2] Figures 30A and 30B are diagrams showing the results of SDS-PAGE of purified proteins. Figure 30A shows the results of reduced SDS-PAGE, and 30B shows the results of non-reduced SDS-PAGE. "M" on the figures represents a protein standard marker.

[0080] [Figure 31-1]31A-31F show representative examples of HPLC analysis of purified proteins. [Figure 31-2] 31A-31F show representative examples of HPLC analysis of purified proteins.

[0081] [Diagram 32] Figure 32 shows the SDS-PAGE results of cleavage of IL-15 fusion protein. "M" on the figure represents a protein standard marker. "B" represents the protein before protease cleavage. "uPA" represents uPA protease. "Mat" represents matriptase protease. "L" represents legumain protease. "M-2" represents MMP-2 protease. "K5" represents KLK-5 protease. "K7" represents KLK-7 protease.

[0082] [Figure 33-1] 33A-33J show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 33-2] 33A-33J show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 33-3] 33A-33J show the activity of IL-15 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0083] [Diagram 34] Figures 34A-34B illustrate representative results of in vitro serum stability. Western blots were probed using HRP-anti-human IgG antibody. Figure 34A: In vitro stability of P18121450 in mouse serum. Figure 34B: In vitro stability of P18121450 in rat serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] The present invention provides an activatable proprotein comprising a masking portion and an active portion, also interchangeably referred to herein as an "active domain", wherein the active domain comprises a complex of IL-15 / IL-15Rα. The present invention further provides compositions comprising same and methods of administering or using same to prevent or treat tumors or cancer. definition

[0085] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0086] For purposes of interpreting this specification, the following definitions shall apply and, whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0087] The term "active" as used herein refers to a portion, domain, or fragment that has a biological activity or function. In one embodiment, the terms "active domain" or "active portion" are used interchangeably herein in the context of the activatable proteins disclosed herein. In some embodiments, the activity is equivalent to or approximates that of the wild-type protein. Thus, the active domain of an activatable proprotein comprising an IL-15 / IL-15Rα complex as described herein, whether in soluble form or when bound to a cell surface, is expected to have an activity equivalent to or approximate that of the wild-type IL-15 / IL-15Rα complex.

[0088] The term "subject" as used herein includes, but is not limited to, mammals, including, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep or other non-human mammals, non-mammals, including non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammalian invertebrates. In some embodiments, the methods and compositions of the invention are used to treat (both prophylactically and / or therapeutically) non-human animals.

[0089] The term "pharmaceutical composition" as used herein means a composition suitable for pharmaceutical use in a subject, including an animal or a human. A pharmaceutical composition generally comprises an effective amount of an active agent (e.g., an activatable proprotein provided herein) and a pharma- ceutical acceptable carrier (e.g., buffers, adjuvants, etc.).

[0090] The term "effective amount" refers to a dosage or amount sufficient to effect a desired result, which may include an objective or subjective improvement in the recipient of the dosage or amount (e.g., prolonged survival, reduction in the number and / or size of tumors, effective prevention of a disease condition, etc.).

[0091] "Prophylactic treatment" means treatment to prevent or reduce the appearance of signs or symptoms of a disease, condition, or medical disorder. Or a treatment administered to a subject who shows only early signs or symptoms of a disease, condition or disorder, such that the treatment is administered for the purpose of reducing, preventing or lowering the risk of developing a disease, condition or medical disorder. Prophylactic treatment serves as a preventative treatment against a disease or disorder. "Prophylactic activity" is the activity of an agent, such as an activatable proprotein or composition thereof disclosed herein, that, when administered to a subject who does not show signs or symptoms of a condition, disease or disorder (or who shows only early signs or symptoms of a condition, disease or disorder), reduces, prevents or lowers the risk of the subject developing a condition, disease or disorder. A "prophylactically useful" agent or compound (e.g., an activatable proprotein provided herein) refers to an agent or compound that is useful for reducing, preventing, treating or lowering the development of a condition, disease or disorder.

[0092] A "therapeutic treatment" is a treatment administered to a subject exhibiting symptoms or signs of a condition, disease or disorder, the treatment being administered to the subject with the purpose of reducing or eliminating these signs or symptoms of the condition, disease or disorder. A "therapeutic activity" is the activity of an agent, such as an activatable proprotein or composition thereof provided herein, that eliminates or reduces a sign or symptom of a condition, disease or disorder when administered to a subject suffering from such a sign or symptom. A "therapeutically useful" agent or compound (e.g., an activatable proprotein provided herein) indicates that the agent or compound is useful in reducing, treating, or eliminating such signs or symptoms of a condition, disease or disorder.

[0093] The term "treating," as used herein, unless otherwise indicated, means partially or completely reversing, alleviating, or reducing tumor growth, tumor metastasis, or the recurrence of other cancer-causing or neoplastic cells in a patient. The term "treatment," as used herein, unless otherwise indicated, refers to the act of treating a disease, e.g., cancer or tumor.

[0094] The term "preventing," as used herein, unless otherwise indicated, means partially or completely inhibiting the progression of, delaying the onset of, or reducing the occurrence of tumor growth, tumor metastasis, or other cancer-causing or neoplastic cells in a patient. The term "preventing," as used herein, unless otherwise indicated, refers to the act of preventing a disease, e.g., cancer or tumor.

[0095] The term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, are identical or have a certain percentage of identical nucleotides or amino acid residues. To determine percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by an amino acid residue or nucleotide that is identical to the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In some embodiments, the two sequences are of identical length.

[0096] The term "substantially identical" in reference to two nucleic acids or polypeptides means at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identity. Or it refers to two or more sequences or subsequences that have at least 99% identity (eg, as determined using one of the methods set out above).

[0097] As used herein, the terms "bind", "specifically bind to" or "specific for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM.

[0098] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Reference to a "formulation" or "method" includes one or more formulations, methods, and / or steps of the kind described herein and / or that will be apparent to those skilled in the art upon reading this disclosure.

[0099] "Activatable proprotein", "activatable prodrug", "prodrug" or "proprotein" are used interchangeably herein and refer to an activatable proprotein or derivative / variant thereof comprising at least a masking moiety and an active domain as described herein. In one embodiment, the proprotein may also comprise one or more protein domains.

[0100] The term "polypeptide" in one embodiment refers to a polymer of amino acids and their equivalents, and does not refer to a specific length of the product; thus, "peptide" and "protein" are included within the definition of a polypeptide. Also included within the definition of a polypeptide is a "masking moiety" as defined herein. A "polypeptide region" refers to a segment of a polypeptide, which may, for example, contain one or more domains or motifs (e.g., a polypeptide region of an antibody may, for example, contain one or more constant domains). The term "fragment" refers to a portion of a polypeptide, preferably having at least 10 consecutive amino acids of the polypeptide. In another embodiment, the term refers to a portion of a polypeptide, which has at least 20, 30, 40, 50, 60 or 70 consecutive amino acids of the polypeptide.

[0101] Unless otherwise indicated by context, a "derivative" is a polypeptide or fragment thereof having one or more non-conservative or conservative amino acid substitutions relative to another polypeptide (also referred to as a "variant"), or a polypeptide or fragment thereof that has been modified by the covalent attachment of another molecule, e.g., by a heterologous polypeptide attachment, or by glycosylation, acetylation, phosphorylation, etc. For example, polypeptides containing one or more analogs of an amino acid (e.g., unnatural amino acids, etc.), naturally occurring and non-naturally occurring unsubstituted linkages as well as other modifications known in the art are further included within the definition of "derivative."

[0102] An "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. An isolated polypeptide includes an isolated antibody or fragment thereof. Fragments or derivatives thereof.

[0103] The term "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) chain and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0104] Antibodies exist as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies at the disulfide linkage in the hinge region to produce F(ab')2, a dimer of Fab, itself a light chain connected to VH-CH1 by a disulfide bond. F(ab')2 can be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, New York (1999)). Various antibody fragments are derived from intact antibodies. Although defined in terms of digestion of the Fab' fragment, one of skill in the art will appreciate that such Fab' fragments and the like may be synthesized de novo, either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody as used herein also includes antibody fragments produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodology. Antibodies include single chain antibodies, including single chain Fv (sFv or scFv) antibodies in which a variable heavy chain and a variable light chain are linked together (directly or via a peptide linker) to form a continuous polypeptide.

[0105] In one embodiment, the terms "antibody portion" and "antibody domain" are used interchangeably herein and refer to the portion / domain of an antibody that is used to create the masking moieties described herein.

[0106] The term "about" as used herein means quantitatively plus or minus 5%, or in another embodiment, plus or minus 10%, or in another embodiment, plus or minus 15%, or in another embodiment, plus or minus 20%.

[0107] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.In carrying out or testing the present invention, any method and material similar or equivalent to those described herein can be used, but the preferred method and material are described here.All publications described herein are incorporated by reference for the purpose of disclosing and describing the material in which the reference is cited in relation thereto. Activatable Proproteins

[0108] In one embodiment, the activatable proproteins provided herein are In another embodiment, the activatable proprotein is a dimer. In one embodiment, the active domain comprises a dimeric complex of IL-15 / IL-15Rα. It should be understood that the terms "IL-15 / IL-15Rα complex" and "IL-15 / IL-15Rα complex" are used interchangeably herein and refer to a monomer that is connected to another monomeric complex of IL-15 / IL-15Rα via one or more linkers and / or one or more disulfide bonds and / or one or more non-covalent bonds to form a dimer that constitutes the active domain of the activatable proprotein described herein.

[0109] In one embodiment, the activatable proprotein provided herein comprises a first polypeptide and a second polypeptide, each of which comprises a masking moiety operably linked to IL-15 or a variant thereof via a first linker at the C-terminus, and IL-15 or a variant thereof is linked to IL-15Rα or a variant thereof via a second linker at the C-terminus, and the masking moiety masks the active portion of the proprotein. In one embodiment, such an activatable proprotein comprises the format Fc-IL-15-linker-IL-15Rα (Fc-ILR), where "Fc" corresponds to the masking domain. In another embodiment, the activatable proprotein homodimer comprises the amino acid sequence set forth in SEQ ID NO:26.

[0110] Fc-GS-ENLYFQG-GS-IL-15-(GGS)5-IL-15Rα (homodimer) [ka]

[0111] In one embodiment, the activatable proprotein disclosed herein comprises SEQ ID NO: 26. In another embodiment, the activatable proprotein disclosed herein consists of SEQ ID NO: 26. In another embodiment, the activatable proprotein disclosed herein comprises a variant or homologue of SEQ ID NO: 26.

[0112] In one embodiment, a cleavable linker is provided that includes a protease cleavage site. An exemplary cleavable linker is shown in bold and italics in SEQ ID NO:26 above, and includes a protease (TEV) cleavage site represented by SEQ ID NO:344, and the italicized underlined sequence in SEQ ID NO:26 above represents the linker sequence surrounding the TEV cleavage site. It should be understood that any cleavage site derived from any protease disclosed herein or available in the art can be inserted into the cleavable linker linking any of the components of the activatable proprotein, as further described herein. In one embodiment, the sequence of the proprotein without a protease cleavage site comprises P1187 (SEQ ID NO: 18), P1188 (SEQ ID NO: 19), P1250 (SEQ ID NO: 20), P1251 (SEQ ID NO: 21) and P1252 (SEQ ID NO: 22), P1254 (SEQ ID NO: 24), P1255 (SEQ ID NO: 25), P1279 (SEQ ID NO: 41) and P1280 (SEQ ID NO: 42), and is described in Example 1 and the present invention. See the following sequences in the specification:

[0113] In a related embodiment, the activatable proprotein is a dimeric proprotein, and in another embodiment, the dimeric proprotein comprises a first and a second polypeptide, each comprising IL-15 or a variant thereof operably linked to IL-15Rα or a variant thereof via a first linker at the C-terminus, the IL-15Rα or a variant thereof being linked to a masking moiety present on each of the first and second polypeptides via a second linker at the C-terminus, the masking moiety masking the active portion of the proprotein, the masking moiety of the first polypeptide forming one or more covalent disulfide bonds or one or more non-covalent bonds with the masking moiety of the second polypeptide. In one embodiment, such an activatable proprotein comprises the format IL-15-linker-IL-15Rα-Fc (ILR-Fc), where "Fc" corresponds to the masking domain.

[0114] In one embodiment, the Fc-ILR form of an activatable proprotein comprises one or more protein domains attached to the C-terminus of IL-15Rα of the first polypeptide, the second polypeptide, or both the first and second polypeptides of the activatable proprotein (see, e.g., Figures 3G-L).

[0115] In one embodiment, the activatable proproteins provided herein are designed to be preferentially activated inside tumor or cancerous tissue (as opposed to in normal tissue).

[0116] In one embodiment, provided herein is an activatable proprotein comprising a first polypeptide and a second polypeptide, each of the first and second polypeptides comprising IL-15 or a variant thereof operably linked to IL-15Rα or a variant thereof via a first linker at the C-terminus, and IL-15Rα or a variant thereof linked to a masking moiety present on each of the first and second polypeptides via a second linker at the C-terminus, the masking moiety masking the active portion of the proprotein. In another embodiment, such an activatable proprotein comprises SEQ ID NO: 17 or SEQ ID NO: 73 (see also FIG. 3B herein).

[0117] In another embodiment, the activatable proprotein disclosed herein is a dimeric proprotein.In another embodiment, the activatable proprotein disclosed herein is a dimeric fusion protein.In another embodiment, the dimeric proprotein comprises a first polypeptide and a second polypeptide, each comprising IL-15 or a variant thereof linked to IL-15Rα or a variant thereof via a first linker at the C-terminus from N-terminus to C-terminus, wherein IL-15Rα or a variant thereof is linked to a masking moiety via a second linker at the C-terminus, and the masking moiety of the first polypeptide forms one or more covalent disulfide bonds or non-covalent bonds with the masking moiety of the second polypeptide.

[0118] In another embodiment, the activatable proprotein is a heterodimeric protein that comprises a heterodimeric masking portion. In another embodiment, the activatable proprotein is a heterodimeric protein that comprises a heterodimeric active domain portion. In another embodiment, the activatable proprotein provided herein is a heterodimeric protein that comprises a heterodimeric masking portion and an active domain portion (see the figures herein). Activatable proprotein-activation domain

[0119] In one embodiment, the active domain of the activatable proprotein is IL-15 / IL- The present invention includes IL-15Rα complex or variants thereof. In one embodiment, upon protease cleavage and exposure of the active domain in the activatable proprotein disclosed herein, IL-15 of the IL-15 / IL-15Rα complex binds to IL-15Rβ / γC on the cell surface of lymphocytes of blood cells, activating a series of downstream pathways that result in cell growth, reduced apoptosis, enhanced immune cell activation and migration. Indeed, upon ligand binding, IL-2 / 15Rβ and γC subunits stimulate the Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription (STAT)-5 pathways. After phosphorylation, STAT5 homodimerizes and translocates to the nucleus, promoting the transcription of target genes. In addition, IL-15 stimulates both the PI3K-AKT and RAS-MAPK pathways. Overall, IL-15 signaling stimulates numerous downstream pathways that result in responses that have critical roles in the development, function, and survival of CD8 T cells, NK cells, NKT cells, and intestinal intraepithelial lymphocytes.

[0120] In some embodiments, the IL-15 or variant thereof is human IL-15 or an amino acid mutant derived therefrom. In another embodiment, the IL-15Rα or variant thereof comprises human IL-15Rα, a truncated human IL-15Rα or an amino acid mutant derived therefrom. In another embodiment, the IL-15 / IL-15Rα complex or variant thereof disclosed herein as part of an activatable proprotein comprises human IL-15 or an amino acid mutant derived therefrom and human IL-15Rα, a truncated human IL-15Rα or an amino acid mutant derived therefrom.

[0121] In one embodiment, the precursor amino acid sequence of human IL-15 comprises or consists of SEQ ID NO: 1 (see examples below; the underlined sequence corresponds to the signal sequence or signal peptide). In another embodiment, the underlined sequence in SEQ ID NO: 1 corresponds to the signal sequence or signal peptide. In one embodiment, the mature amino acid sequence of human IL-15 with the S162A point mutation comprises or consists of SEQ ID NO: 3.

[0122] In another embodiment, the human IL-15 amino acid sequence comprises a variant or homologue of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0123] In one embodiment, the full-length amino acid sequence of IL-15Rα comprises or consists of SEQ ID NO:4 (see Examples below; underlined sequences correspond to signal sequences or peptides). In another embodiment, the IL-15Rα extracellular domain (ECD) comprises or consists of SEQ ID NO:5. In another embodiment, the IL-15Rα sushi+ domain comprises or consists of SEQ ID NO:6. In another embodiment, the IL-15Rα, sushi domain comprises or consists of SEQ ID NO:7.

[0124] In one embodiment, the IL-15 or variant thereof and IL-15Rα or variant thereof in the first polypeptide and the IL-15 or variant thereof and IL-15Rα or variant thereof in the second polypeptide comprise one or more Cys substitution mutations that facilitate association of the IL-15 / IL-15Rα complex of the first polypeptide with the IL-15 / IL-15Rα complex of the second polypeptide through disulfide bonds.

[0125] In another embodiment, IL-15 or a variant thereof in a first polypeptide comprising one or more Cys substitution mutations forms a disulfide bond with IL-15Rα or a variant thereof in a second polypeptide comprising one or more Cys substitution mutations. The IL-15 or variant thereof forms a disulfide bond with the IL-15Rα or variant thereof comprising one or more Cys substitution mutations in the first polypeptide. Activatable Proprotein-Linker

[0126] In one embodiment, the first linker of the first or second polypeptide of the activatable proprotein disclosed herein, or of both the same first and second polypeptide, is a cleavable linker. In another embodiment, the second linker of the first or second polypeptide of the activatable proprotein disclosed herein, or of both the same first and second polypeptide, is a cleavable linker.

[0127] The term "linker" is art-recognized and refers to a molecule (including but not limited to unmodified or modified nucleic acids or amino acids) or group of molecules (e.g., two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more) that connects two compounds, e.g., two polypeptides. A linker may consist of a single linking molecule or may include the linking molecule and at least one spacer molecule that is intended to separate the linking molecule and the compound by a particular distance.

[0128] In one embodiment, the linker comprises a cleavable or non-cleavable linker. An example of a cleavable linker is a linker that has a cleavage site of a protease, for example, TEV protease. An example of a non-cleavable linker is a linker that is removed or degraded in cells or is not cleaved.

[0129] In one embodiment, the activatable proproteins and pharmaceutical compositions comprising same described herein may be used directly to target cancer cells such that the activatable proprotein is essentially activated once the protease-cleavable linker is cleaved to provide the active payload, or once the non-cleavable linker is degraded or removed intracellularly by the targeted cell type.

[0130] In one embodiment, a non-cleavable linker is used to link any of the components of the activatable proprotein described herein. A non-limiting example of a non-cleavable linker comprises or consists of the amino acid sequence GGSGGSGSGGSGGS (SEQ ID NO: 345) or a homologue or variant thereof. It should be understood that any non-cleavable linker known in the art may be used and is not limited to those disclosed herein.

[0131] In one embodiment, the linker used in the activatable proproteins described herein comprises an immunoglobulin (Ig) / antibody hinge region. In one embodiment, the hinge region is obtained from an IgG1 antibody. In one embodiment, the terms Ig "hinge" and region refer to a polypeptide comprising an amino acid sequence that shares sequence identity or similarity with a portion of a naturally occurring Ig hinge region sequence that includes the cysteine ​​residues where the disulfide bonds link the two heavy chains of the immunoglobulin. The sequence similarity of the hinge region linkers of the invention to a naturally occurring immunoglobulin hinge region amino acid sequence can range from at least 50% to about 75-80%, and typically is greater than about 90%.

[0132] Derivatives and analogs of the hinge region can be obtained by mutation. As referred to herein, a derivative or analog is a wild-type (or naturally occurring) variant, except that it has one or more amino acid sequence differences that may result from deletions, insertions and / or substitutions. A polypeptide that contains an amino acid sequence that shares sequence identity or similarity with the full-length sequence of a protein present in

[0133] The present invention also encompasses the fragment of hinge region for use as linker.Such fragment must be long enough to allow the protein attached by hinge region fragment to achieve biologically active conformation.In one embodiment, the heterodimeric masking portion of the activatable proprotein provided herein is connected to the heterodimeric active domain by fusion using Ig hinge region.In one embodiment, the masking portion and the active domain are homodimers.

[0134] In one embodiment, the cleavable linker comprises or consists of a protease-cleavage site flanked by linker sequences. In one embodiment, a non-limiting example of a cleavable linker comprises or consists of the amino acid sequence of GSENLYFQGGS (SEQ ID NO: 346) or a homologue or variant thereof. It should be understood that any cleavable linker known in the art may be used and is not limited to those disclosed herein. The cleavage sites of the proteases disclosed herein are known in the art and are contemplated for use with any of the proteases disclosed herein or available in the art.

[0135] In one embodiment, the non-cleavable or cleavable linker is inserted in the activatable proprotein (in the order from C-terminus to N-terminus) between the masking portion and IL-15 and between IL-15 and IL-15Rα of the activatable proprotein disclosed herein. In another embodiment, the non-cleavable or cleavable linker is inserted in the activatable proprotein (in the order from C-terminus to N-terminus) between IL-15 and IL-15Rα and between IL-15Rα and the masking portion of the activatable proprotein disclosed herein. In one embodiment, the cleavable linker in the activatable proprotein comprises a cleavage site of a protease. In another embodiment, the first linker and / or the second linker of the activatable proprotein disclosed herein is a cleavable linker comprising a cleavage site of a protease. In another embodiment, the protease is selected from a metalloprotease, a serine protease, a cysteine ​​protease, an aspartic acid protease, or any combination thereof. In another embodiment, the protease cleavage site is cleavable by MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV, matriptase, uPA, FAP, legumain, PSA, kallikrein, cathepsin A or cathepsin B protease or any protease available in the art. In one embodiment, the protease is any protease known in the art and is not limited to those disclosed herein.

[0136] In one embodiment, after intact protease (e.g., TEV protease) cleavage of the homodimer of an activatable proprotein disclosed herein, the activatable proprotein is cleaved into two cleavage products each comprising two chains:

[0137] Cleavage product 1) Fc-GSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 27)

[0138] Strands 1 and 2 (each with the same sequence)

[0139] [ka]

[0140] In one embodiment, SEQ ID NO:27 comprises a masking moiety after protease cleavage of the activatable proprotein.

[0141] Cleavage product 2) GGS-IL-15-(GGS)5-IL-15Rα homodimer (product of complete cleavage, active domain) (SEQ ID NO: 28)

[0142] Strands 1 and 2 (each with the same sequence)

[0143] [ka]

[0144] In one embodiment, SEQ ID NO:28 comprises an activation domain of a proprotein that is activatable following protease cleavage.

[0145] In one embodiment, after partial protease cleavage (e.g., using -TEV protease) of the homodimer of an activatable proprotein disclosed herein, the masking moiety in the activatable proprotein is partially removed, allowing access of the active domain (IL-15 / IL-15Rα complex) for binding by IL-15Rβ / γC (see FIG. 6). In another embodiment, the partially cleaved activatable proprotein comprises SEQ ID NOs: 29, 30 and 31.

[0146] Cleavage product 3) Fc-GS-ENLYFQG-GS-IL-15-(GGS)5-IL-15Rα / Fc-GSENLYFQ / GGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active) (SEQ ID NOs: 29, 30 and 31).

[0147] Lock 1: Fc-GS-ENLYFQG-GS-IL-15-(GGS)5-IL-15Rα

[0148]

Chem.

Chem.

[0149] Lock 2: Fc-GSENLYFQ

[0150]

Chem.

[0151] Lock 3: GGS-IL-15-(GGS)5-IL-15Rα

[0152]

Chem.

[0153] Linkers suitable for use in the compositions described herein are generally those that provide flexibility of the masking moiety or activatable proprotein to facilitate inhibition of the binding active portion of the activatable proprotein, i.e., IL-15 / IL-15Rα complex. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can be of any of a variety of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. In one embodiment, the linker is 26 amino acids in length. In another embodiment, the linker is 5-9, 10-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, or 46-50 amino acids in length.

[0154] Exemplary flexible linkers include glycine polymers (G), glycine-serine polymers (including, for example, (GS), (GSGGS), and (GGGS), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, see, for example, U.S. Patent Application No. 10,059,762 B2, which is incorporated herein by reference in its entirety. Glycine and glycine-serine polymers are relatively unstructured and therefore can act as neutral tethers between components. Glycine has access to significantly more phi-psi space than even alanine, and is much less constrained than residues with long side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One of skill in the art will recognize that the design of an activatable proprotein can include a linker that is fully or partially flexible, such that the linker can include a flexible linker as well as one or more moieties that confer a less flexible structure to provide the desired activatable proprotein structure.

[0155] In one embodiment, protease cleavage of the cleavable linker in the first or second polypeptide results in binding of the IL-15 / IL-15Rα complex in the first or second polypeptide to IL-15Rβ / γC present on the surface of lymphocytes or blood cells in vivo. In one embodiment, protease cleavage of the cleavable linker in the first and second polypeptides completely removes the masking moiety in the activatable proprotein such that the IL-15 / IL-IL-15Rα complex in the first and second polypeptides can bind to IL-15Rβ / γC present on the surface of lymphocytes or blood cells in vivo.

[0156] In one embodiment, a linker may include a spacer element and a cleavable element to make the cleavable element more accessible to the enzyme responsible for cleavage. Activatable proprotein-masking moiety

[0157] In one embodiment, the activatable proprotein of the present invention comprises a masking moiety dimer that masks the active domain. Thus, within the context of the activatable proproteins provided herein, when an active domain comprising an IL-15 / IL-IL-15Rα complex is modified by the addition of a masking moiety (via one or more cleavable linkers), in the presence of a target (IL-15Rβ / γC), the specific binding of the active domain to its target is blocked, reduced, or inhibited, compared to the specific binding of an active domain that is not modified by the addition of a masking moiety. In another embodiment, when an active domain comprising an IL-15 / IL-IL-15Rα complex is modified by the addition of a masking moiety (e.g., via one or more cleavable linkers), in the presence of a target (IL-15Rβ / γC), the specific binding of the active domain to its target is blocked, reduced, or inhibited, compared to the specific binding of an active proprotein in the presence of a protease enzyme or protease enzyme activity sufficient to partially or completely cleave the masking moiety from the activatable proprotein. In some embodiments, protease cleavage of the linker between the masking moiety and the active domain in the activatable proprotein results in activation ("release") of the active domain, which preferably occurs at the tumor site or in cancer tissue.

[0158] In one embodiment, the masking moiety is fused to the active domain to form a fusion protein. In one embodiment, the fusion protein is recombinantly produced. In another embodiment, the fusion protein is chemically produced (i.e., chemically fused).

[0159] In one embodiment, the masking moiety specifically binds to the active domain. In another embodiment, the masking moiety lacks the ability to specifically bind to the active domain. In one embodiment, the masking moiety and the linker between the ILR and the masking moiety sterically hinder the ability of IL-15 in the active domain to bind to and signal through IL-15Rβ / γc.

[0160] In one embodiment, the masking portion of an activatable proprotein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In another embodiment, the masking portion of an activatable proprotein disclosed herein comprises the amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0161] In one embodiment, the masking moiety can sterically inhibit the binding of the active domain of the activatable proprotein to its target, and in another embodiment, the target is IL-15Rβ / γC. In another embodiment, the masking moiety can allosterically inhibit the binding of the activatable proprotein to its target. In these embodiments, when the active domain is modified by coupling to a masking moiety (to form an activatable proprotein as described herein), in the presence of the target, it inhibits the binding of the active domain to its target, as described in the art. In vivo or targeted replacement There is no binding, or substantially no binding, or 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% or less binding of the active domain to a target compared to binding of an active domain not modified with a masking moiety or binding of an active domain not coupled to a masking moiety for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer as measured in an in vitro assay.

[0162] In one embodiment, when the active domain is in a "masked" state, even in the presence of the target, the masking moiety interferes with or inhibits the binding of the active domain to the target, but with the active domain in an unmasked state, the interference of the masking moiety with the target binding of the active domain is reduced, thereby allowing the active domain more access to the target and providing target binding.

[0163] In one embodiment, the active domain in the activatable proprotein can be unmasked upon cleavage in the presence of an enzyme, which in a preferred embodiment is a disease-specific enzyme, for example, a cancer-specific or tumor-specific protease.Thus, the masking moiety provides masking of the active domain from target binding when the active domain in the activatable proprotein is not cleaved, but does not substantially or significantly interfere or compete with the binding of the target to the active domain when the activatable proprotein is cleaved.Thus, the activatable proprotein promotes a switchable / activatable phenotype, the masking moiety reduces target binding when the activatable proprotein is not cleaved, and cleavage of the masking moiety by the protease provides increased target binding by the active domain.

[0164] In one embodiment, the structural characteristics of the masking moiety will vary according to various factors, such as the minimum amino acid sequence required to interfere with active domain binding to the target, the length of the linker between the masking moiety and the active domain, the presence or absence of cysteines within or adjacent to the active domain suitable for providing dissociation of a cysteine-cysteine ​​disulfide bond, etc.

[0165] In one embodiment, the masking moiety in the first polypeptide and / or in the second polypeptide comprises one or more protein domains. In another embodiment, the masking moiety in the first polypeptide and / or in the second polypeptide comprises a CH3 variant domain of an antibody constant (Fc) region. In another embodiment, the CH3 variant domain of an antibody constant (Fc) region comprises a CH1, CH2, CH3, CH2CH3 or CH1CH2CH3 domain. In another embodiment, the masking moiety in the first polypeptide and / or in the second polypeptide comprises a CH1, CH2, CH3, CH2CH3 or CH1CH2CH3 domain of an antibody constant (Fc) region. In another embodiment, the masking moiety in the first polypeptide comprises one or more heterodimers of at least two different CH3 variant domains of an antibody constant (Fc) region. In another embodiment, the masking moiety in the first polypeptide and / or in the second polypeptide comprises a CH3 variant domain of an antibody constant (Fc) region, the CH3 variant domain comprising one or more mutations.

[0166] In one embodiment, the masking moiety in the first polypeptide and / or in the second polypeptide comprises a fusion of an antigen-binding domain with a CH3 variant domain of an antibody constant (Fc) region. The masking moiety in the two polypeptides comprises a fusion of the antigen-binding domain with a CH1, CH2, CH3, CH2CH3 or CH1CH2CH3 domain of an antibody constant (Fc) region.

[0167] In one embodiment, the masking moiety in the first polypeptide and / or in the second polypeptide does not bind to an antigen. In another embodiment, the masking moiety in the first polypeptide and / or in the second polypeptide binds to an antigen. In another embodiment, the masking moiety in the first polypeptide and / or in the second polypeptide comprises an antigen-binding domain. In another embodiment, the antigen-binding domain is a variable heavy chain domain (VH), a variable domain from a heavy chain antibody (VHH) or an antigen-specific peptide. In another embodiment, the antigen-binding domain comprises an antibody light chain (LC) and an antibody heavy chain (HC). In another embodiment, the masking moiety comprises a constant domain of an antibody light chain (CL), and the light chain is a lambda or kappa chain.

[0168] The antibody domain of the fusion proteins disclosed herein optionally comprises all or a portion of an immunoglobulin molecule, and optionally comprises all or a portion of an immunoglobulin constant domain region (Fc region).

[0169] The antibodies used herein optionally include an IgG domain. However, other embodiments include alternative immunoglobulins, such as IgM, IgA, IgD, and IgE. Additionally, all possible isotypes of various immunoglobulins are also encompassed by this embodiment. Thus, IgG1, IgG2, IgG3, etc. are all possible molecules in the antibody domain of the antibody-immunostimulant fusion protein used in the present invention. In addition to the choice of choosing the type of immunoglobulin and isotype, different embodiments of the present invention include various hinge regions (or functional equivalents thereof). Such hinge regions provide flexibility between different domains of the antibody-immunostimulant fusion protein. See, for example, Penichet, et al. 2001 "Antibody-cytokine fusion proteins for the therapy of cancer" J Immunol Methods 248:91-101. In some embodiments, the antibodies described herein may include any of the following: The antibody portions used in the compositions and methods provided herein are from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA, or IgM.

[0170] In one embodiment, the masking moiety may be selected by a screening procedure to determine the optimal combination of masking moiety and cleavable linker that results in optimal inhibition of the active domain within the context of the activatable proprotein.

[0171] In one embodiment, the masking moiety in the first and / or second polypeptide prevents the binding of the IL-15 / IL-15Rα complex in the first and / or second polypeptide to IL-15Rβ / γC present on the surface of lymphocytes or blood cells in vitro or in vivo.

[0172] In one embodiment, cleavage of the masking moiety in either the first and / or second polypeptide in the activatable proprotein results in partial activation of the activatable proprotein. In one embodiment, the masking moiety can or cannot interact with the IL-15 / IL-15Rα complex after partial activation (partial cleavage) of the activatable proprotein. In another embodiment, cleavage of the masking moiety in both the first and second polypeptide in the activatable proprotein results in full activation of the activatable proprotein to the active IL-15 / IL-15Rα complex or a variant thereof.

[0173] In one embodiment, a humanized antibody may be used in the compositions and methods provided herein. In another embodiment, a humanized antibody may be used to generate a masking moiety provided herein. In another embodiment, a humanized antibody may be used to generate a masking moiety comprising an antigen-binding domain provided herein. In another embodiment, a humanized antibody may be used to generate an antigen-binding domain provided herein. In some embodiments, the term "humanized antibody" or "humanized version of an antibody" refers to an antibody in which the framework or "complementarity determining regions" (CDRs) have been modified to include CDRs of an immunoglobulin of different specificity compared to those of the parent immunoglobulin. In other embodiments, the CDRs of VH and VL are grafted into the framework regions of a human antibody to prepare a "humanized antibody". See, for example, Riechmann, L., See, for example, Neuberger, MS, et al, Nature 332 (1988) 323-327 and Neuberger, MS, et al, Nature 314 (1985) 268-270. The heavy and light chain variable framework regions may be derived from the same or different human The human antibody sequence may be derived from a human antibody sequence. The human antibody sequence may be the sequence of a naturally occurring human antibody. Human heavy and light chain variable framework regions are listed, for example, in Lefranc, M.-P., Current Protocols in Immunology (2000) - Appendix IP A.1P.1-A.1P.37, accessible via IMGT, the international ImMunoGeneTics information system (registered trademark) (http: / / imgt.cines.fr) or via http: / / vbase.mrc-cpe.cam.ac.uk. If necessary, the framework regions may be modified by further mutations. The term "humanized antibody" as used herein also includes such antibodies that have been modified in the constant region, for example by "class switching", i.e., by alteration or mutation of the Fc part (e.g., from IgG1 to IgG4 and / or IgG1 / IgG4 mutations), to generate properties according to the invention, in particular with regard to Clq binding and / or FcR binding. The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the state of the art (vanDijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies also represent the full repertoire of antibodies that, upon immunization, are produced in the absence of endogenous immunoglobulin production. Human antibodies can be produced in transgenic animals (e.g., mice) capable of producing a selection of human or human antibodies. Introduction of a human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen exposure (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al, Nature 362 (1993) 255-258; Brueggemann, MD, et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced using phage display libraries. (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole, A., et al. and Boerner, P., et al. can also be utilized for the preparation of human monoclonal antibodies. (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, AL, p. 77 (1985) and Boerner, P., et al, J. Immunol. 147 (1991) 86-95). As already described for the humanized antibodies of the present invention, the term "human antibody" as used herein also includes those antibodies or portions thereof which have been modified in the constant region to generate the properties according to the present invention.

[0174] In a specific embodiment, the mAb comprised by the activatable proprotein provided herein comprises at least one or more domains from the Fc region and / or one or more domains from the variable region.

[0175] In one embodiment, recombinant human antibodies can be a source of masking portions of the disclosed activatable proproteins. The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, produced, or isolated by recombinant means, e.g., produced by host cells that are transgenic for human immunoglobulin genes. The term "human antibody" includes antibodies isolated from cells, such as NS0 or CHO cells, or from animals, such as mice, or expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in rearranged form. The recombinant human antibodies according to the present invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but may not naturally occur in the human antibody germline repertoire in vivo.

[0176] In some embodiments, a masking moiety comprising an antigen-binding domain that binds to an antigen as disclosed herein binds to the antigen with optimal binding affinity. In other embodiments, a masking moiety comprising an antigen-binding domain that binds to an antigen as disclosed herein binds to the antigen with optimal binding affinity at 25° C., with a KD of 1.0×10 -8 mol / l~1.0×10 -13 The antibodies bind to the antigen with a binding affinity in the mol / l range. Binding affinity is measured at 25° C. using standard binding assays, e.g., surface plasmon resonance technology (BIAcore®, GE-Healthcare The temperature is determined using the FT-IR spectrometer (Uppsala, Sweden). In one embodiment, the masking moiety comprised by the activatable proproteins disclosed herein comprises a variable heavy (VH) chain and / or a variable light (VL) chain.

[0177] In one embodiment of the present invention, the antibody constant domain present in the masking portion of the activatable proprotein provided herein is glycosylated.In some embodiments, the glycosylation is N-glycosylation.In other embodiments, the glycosylation is O-glycosylation. Constructs encoding activatable proproteins

[0178] In one embodiment, provided herein is a vector comprising a recombinant nucleic acid molecule or polynucleotide encoding an activatable proprotein disclosed herein.

[0179] In one embodiment, recombinant nucleic acid molecules encoding the activatable proproteins of the present invention are provided herein. In one embodiment, one or more natural or recombinant nucleic acid molecules are used to arrive at any of the amino acid sequences disclosed in the sequence listing, including SEQ ID NO:1 to SEQ ID NO:346.

[0180] The present disclosure further provides vectors and nucleic acid constructs comprising sequences encoding the proproteins disclosed herein.Suitable nucleic acid constructs include, but are not limited to, constructs that can be expressed in prokaryotic or eukaryotic cells.Expression constructs are generally selected to be compatible with the host cell to be used.In certain embodiments, the vector encodes an activatable proprotein (masking moiety, IL-15 / IL-15Rα complex and cleavable linker).

[0181] For example, non-viral and / or viral construct vectors can be prepared and used, including plasmids that provide replication and / or expression in host cells of DNA encoding proproteins. The choice of vector depends on the type of cell desired to grow and the purpose of growth. Certain constructs are useful for amplifying and producing large amounts of desired DNA sequences. Other vectors are suitable for expression in cells in culture. The selection of appropriate vectors is well within the skill of the art. Many such vectors are commercially available. Methods for generating constructs can be achieved using methods well known in the art.

[0182] To achieve expression in a host cell, the polynucleotide encoding the activatable proprotein disclosed herein or its components is operably linked to regulatory sequences as necessary to promote the desired expression characteristics. These regulatory sequences include promoters, enhancers, terminators, operators, repressors and inducers. The expression construct also generally provides, where necessary or desired, transcription and translation initiation regions, which may be inducible or constitutive, with the coding region operably linked under the transcriptional control of a transcription initiation region and a transcription and translation termination region. These control regions may be native to the species from which the nucleic acid is obtained or may be derived from a foreign source. In the context of the activatable proproteins provided herein, the masking moiety may be produced by recombinant means along with additional components of the first and second polypeptides of the activatable proprotein (i.e., IL-15 / IL-15Rα and a linker), with the first and second polypeptides being connected by disulfide bonds and / or non-covalent bonds to form a dimer. Such methods are widely known in the state of the art and include protein expression in prokaryotic and eukaryotic cells and subsequent isolation and purification of the activatable proprotein, usually to a pharma- ceutically acceptable purity. For protein expression, nucleic acids encoding the components of the activatable proprotein (including the light and heavy chains of the masking moiety or fragments thereof) are inserted into expression vectors by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells, such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast or E. coli cells, and the activatable proprotein is recovered from the cells (from the supernatant or after cell lysis).

[0183] A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a nucleic acid presequence or secretory leader is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the nucleic acid sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, are optionally contiguous. Linkage can be accomplished, for example, by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors, linkers or other methods known in the art can be used. In another embodiment, "operably linked" also refers to the functional pairing of separate amino acid sequences, peptides or proteins, such as in the pairing of the masking moieties and IL-15 described herein via linker sequences also described herein.

[0184] Constructs containing expression constructs can also include a selectable marker that is operative in the host, for example, to facilitate growth of host cells containing the construct of interest. Such selectable marker genes can provide a phenotypic trait for selection of transformed host cells, for example, dihydrofolate reductase or neomycin resistance for eukaryotic cell culture.

[0185] In one embodiment, the heavy and light chain constant domains in the masking moiety of the present invention are combined with the sequences of promoter, translation initiation, constant region, 3' untranslated region, polyadenylation and transcription termination to form an expression vector construct.The heavy and light chain expression constructs can be combined into a single vector and co-transfected, sequentially transfected or separately transfected into host cells, which are then fused to form a single host cell expressing both chains.In one embodiment, the heavy and light chains are recombinantly constructed together with other components of the activatable proprotein disclosed herein.

[0186] In some embodiments, the activatable proproteins utilized in the present invention can be obtained or produced by any method known in the art, as needed. For example, nucleic acid sequences encoding the appropriate components are cloned and ligated into an appropriate vector (e.g., an expression vector for prokaryotic or eukaryotic organisms), as needed. Furthermore, nucleic acid sequences encoding the appropriate components are cloned into the same vector, as needed, in the appropriate orientation and position, so that expression from the vector results in the activatable proprotein of the present invention. Some optimal embodiments require post-expression modifications, such as assembly of antibody subunits. The techniques and skills for the above (and similar) manipulations are well known to those skilled in the art. Relevant instructions are found, for example, in Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 and Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented in 1999). In some alternative embodiments, components of the activatable proprotein are assembled after expression, for example, by chemical means.

[0187] In some embodiments, the activatable proproteins provided herein are administered to a patient in a therapeutically effective amount, that is the amount of the compound or combination of interest that elicits the biological or medical response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, physician or other clinician. Activatable Proproteins - Compositions

[0188] In one embodiment, the present invention provides compositions, such as pharmaceutical compositions, comprising the activatable proproteins of the present invention. In another embodiment, provided herein are pharmaceutical compositions comprising the activatable proproteins and a pharma- ceutically acceptable carrier. In some embodiments, provided are pharmaceutical compositions comprising the activatable proproteins disclosed herein formulated together with a pharmaceutical carrier.

[0189] In one embodiment, the activatable proprotein amino acid sequence is homologous to any amino acid sequence encoding an activatable proprotein disclosed herein. The term "homology" may refer to greater than 70% identity to a sequence disclosed herein. In another embodiment, "homology" refers to greater than 72% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 75% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 78% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 80% identity to any of SEQ ID NO: XY. In another embodiment, "homology" refers to greater than 82% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 83% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 85% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 87% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 88% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 90% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 92% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 93% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 95% identity to any sequence disclosed herein. In one embodiment, "homology" refers to greater than 96% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 97% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 98% identity to any sequence disclosed herein. In another embodiment, "homology" refers to greater than 99% identity to any sequence disclosed herein. In another embodiment, "homology" refers to 100% identity to any sequence disclosed herein.

[0190] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the modified version of the algorithm described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. The algorithm is that of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschule tal., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to nucleic acids encoding proteins of interest. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to proteins of interest. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschule tal., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. As another non-limiting example of a mathematical algorithm utilized for the comparison of sequences, Myersand Miller, CABIOS (1989) algorithm. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, including ADVANCE and ADAM, as described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5, and FASTA, as described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and search speed. When ktup=2, aligned residue pairs are examined to find similar regions in the two sequences being compared, and when ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, and 1-6 for DNA sequences. If ktup is not specified, it defaults to 2 for proteins and 6 for DNA. Alternatively, protein sequence alignments can be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0191] In some embodiments, the polynucleotides of the present invention are prepared using PCR techniques using procedures and methods known to those skilled in the art. In some embodiments, the procedures include ligation of two different DNA sequences (see, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).

[0192] In one embodiment, an affinity tag is included in the activatable proprotein disclosed herein. Affinity tags are well known in the art and are attached to a target and used to detect or isolate the target using a molecule that binds to the affinity tag. In principle, Any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Exemplary affinity tags suitable for use include, but are not limited to, monocytic adaptor protein (MONA) binding peptide, T7 binding peptide, streptavidin binding peptide, polyhistidine tracts, protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione S-transferase (Smithand Johnson, Gene 67:31 (1988) ), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (Hoppetal., Biotechnology 6:1204 (1988)) or other antigenic epitopes or binding domains. See generally Ford et al., Protein Expression and Purification 2:95 (1991). DNA molecules encoding affinity tags are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, NJ). In one embodiment, a His6 tag is included in the activatable proprotein disclosed herein.

[0193] In one embodiment, the polynucleotide of the present invention is inserted into an expression vector (i.e., a nucleic acid construct) to allow expression of the polypeptide described herein. In one embodiment, the expression vector of the present invention comprises additional sequences that make the vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention comprises additional sequences that make the vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention comprises a shuttle vector that makes the vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, the cloning vector comprises transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).

[0194] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems for expressing the polypeptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the polypeptide coding sequence, yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence.

[0195] In some embodiments, non-bacterial expression systems are used to express the polypeptides of the invention (e.g., mammalian expression systems, such as CHO cells, etc.). In one embodiment, the expression vector used to express the polynucleotides of the invention in mammalian cells is the pCI-DHFR vector, which contains the CMV promoter and the neomycin resistance gene.

[0196] In some embodiments, in the bacterial system of the present invention, several expression vectors are advantageously selected depending on the intended use for the expressed polypeptide. In one embodiment, large amounts of the polypeptide are desired. In one embodiment, a vector is desired that directs high levels of expression of the protein product, perhaps as a fusion with a hydrophobic signal sequence that directs the expressed product to the bacterial periplasm or culture medium where the protein product is easily purified. In one embodiment, vectors that can be adapted for such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[0197] In one embodiment, a yeast expression system is used. In one embodiment, several vectors containing constitutive or inducible promoters in yeast can be used, as disclosed in U.S. Patent No. 5,932,447. In another embodiment, the foreign DNA sequence can be expressed in the yeast chromosome. A vector that promotes integration of the vector is used.

[0198] In one embodiment, the expression vector of the present invention may further comprise additional polynucleotide sequences allowing translation of several proteins from a single mRNA, such as, for example, an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0199] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV available from Strategene, pTRES and derivatives thereof available from Clontech.

[0200] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p205. Other exemplary vectors include pMSG, pAV009 / A, pAV009 / B, pAV009 / C, pAV009 / D, pAV009 / E, pAV009 / F, pAV009 / G, pAV009 / H, pAV009 / I, pAV009 / I, pAV009 / N, pAV009 / P ... + , pMTO10 / A + , pMAMneo-5, baculovirus pDSVE and any other vector that allows expression of proteins under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter or other promoters shown to be effective for expression in eukaryotic cells.

[0201] In some embodiments, recombinant viral vectors are useful for in vivo expression of the polypeptides of the present invention because they offer advantages such as lateral infection and targeting specificity. In one embodiment, lateral infection is inherent in, for example, the life cycle of retroviruses, a process in which a single infected cell produces many progeny virions that bud and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which were not initially infected by the original viral particle. In one embodiment, a viral vector is generated that cannot spread laterally. In one embodiment, this feature can be useful when the desired objective is to introduce a specific gene into only a localized number of targeted cells.

[0202] In one embodiment, various methods can be used to introduce an expression vector encoding a polypeptide of the present invention into a cell. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York (1992), and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York (1992). Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich.(1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. at. [Biotechniques 4 (6): 504-512,1986], including, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, for positive-negative selection methods, see U.S. Patent Nos. 5,464,764 and 5,487,992.

[0203] In some embodiments, introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, as higher transfection efficiencies can be obtained due to the infectious nature of viruses.

[0204] It will be appreciated that the activatable proproteins of the present invention or the polypeptides contained thereby may also be expressed from a nucleic acid construct administered to an individual using any suitable mode of administration described herein above (i.e., in-vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into a suitable cell, as appropriate, by an appropriate gene delivery vehicle / method (transfection, transduction, homologous recombination, etc.) and expression system, and the modified cells are then expanded in culture and returned to the individual (i.e., ex-vivo gene therapy).

[0205] It will be understood that, in addition to containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding a polypeptide), the expression constructs of the invention may also contain sequences engineered to optimize the stability, production, purification, yield or activity of the expressed polypeptide.

[0206] In some embodiments, the transformed cells are cultured under effective conditions that allow for the expression of large amounts of the activatable proprotein or its constituent polypeptides (i.e., the first and second polypeptides as further described herein). In some embodiments, effective culture conditions include, but are not limited to, efficient media, bioreactors, temperature, pH and oxygen conditions that allow for protein production. In one embodiment, effective media refers to any medium in which cells are cultured to produce the activatable proproteins provided herein. In some embodiments, media typically include aqueous solutions having assimilable carbon, nitrogen and phosphorus sources and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, the cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, the culture is carried out at a temperature, pH and oxygen content appropriate for the recombinant cells. In some embodiments, the culture conditions are within the expertise of one of ordinary skill in the art.

[0207] In some embodiments, depending on the vector and host system used for production, the resulting polypeptide of the activatable proprotein provided herein remains inside the recombinant cell, is secreted into the fermentation medium, is secreted into the space between two cell membranes, e.g., the periplasmic space in E. coli, or is retained on the outer surface of a cell or viral membrane. In another embodiment, after a predetermined time in culture, recovery of the activatable proprotein or its constituent polypeptides (including any precursors) is achieved.

[0208] In one embodiment, the phrase "recovering the activatable proprotein or a polypeptide contained thereby or constituting it" as used herein refers to collecting the whole fermentation medium containing the activatable proprotein / polypeptide and does not necessarily imply a further step of separation or purification.

[0209] In one embodiment, the activatable proprotein of the present invention or its constituent polypeptides are purified using a variety of standard protein purification techniques, including but not limited to affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, isoelectric focusing and differential solubilization.

[0210] In one embodiment, the expressed coding sequence can be engineered to encode an activatable proprotein of the invention for ease of recovery, hi one embodiment, the activatable proprotein can be designed such that it can be easily isolated by affinity chromatography, for example, by immobilization on a column specific for the cleavable moiety.

[0211] In one embodiment, the activatable proproteins of the present invention are recovered in "substantially pure" form.

[0212] In one embodiment, the phrase "substantially pure" refers to a purity that allows for the effective use of the activatable proprotein in the applications described herein.

[0213] In one embodiment, the activatable proproteins provided herein can also be synthesized using an in vitro expression system. In one embodiment, in vitro synthesis methods are well known in the art and the components of the system are commercially available.

[0214] In some embodiments, once the activatable proproteins disclosed herein are synthesized and purified, their therapeutic effectiveness can be assayed in vivo or in vitro.

[0215] In one embodiment, the pharmaceutical compositions provided herein that include the activatable proproteins of the invention are further formulated with a pharmaceutical carrier. As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically acceptable. Activatable Proproteins - Therapeutic Methods

[0216] The activatable proproteins described herein can be selected for use in a method of treatment of a suitable subject in need thereof. The activatable proproteins or pharmaceutical compositions provided herein can be administered by any suitable means, including oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, spinal or epidermal administration (e.g., by injection or infusion), and, if desired, for local injection (e.g., at the site of a solid tumor). Parenteral administration routes include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

[0217] In one embodiment, provided herein is a method comprising administering to a subject having cancer an activatable proprotein or pharmaceutical composition disclosed herein to prevent or treat cancer in the subject, wherein following administration, the activatable proprotein is activated by protease cleavage in the cancerous tissue.

[0218] In another embodiment, provided herein is a method comprising administering to a subject in need thereof an activatable proprotein or pharmaceutical composition disclosed herein to induce or enhance an anti-tumor immune response in the subject, wherein following administration, the activatable proprotein is activated by protease cleavage in the tumor.

[0219] In one embodiment, provided herein is a use of an activatable proprotein or pharmaceutical composition disclosed herein for preventing or treating cancer in a subject, comprising administering the activatable proprotein or pharmaceutical composition, wherein following administration, the activatable proprotein is activated by protease cleavage in the cancerous tissue.

[0220] In another embodiment, provided herein is a use of an activatable proprotein or pharmaceutical composition disclosed herein for inducing or enhancing an anti-tumor immune response in a subject, comprising administering the activatable proprotein or pharmaceutical composition, wherein following administration, the activatable proprotein is activated by protease cleavage in the tumor.

[0221] The term treatment site or disease site is meant to refer to a site where an activatable proprotein is designed to be switchable as described herein, for example, a site where a masking moiety is cleaved from an active domain. Treatment sites include tissues that can be accessed by local administration (e.g., injection, infusion (e.g., by catheter), etc.) or by systemic administration (e.g., administration to a site remote from the treatment site). Treatment sites include those that are relatively biologically confined (e.g., organs, pouches, tumor sites, etc.).

[0222] In one embodiment, the disease disclosed herein is cancer. In another embodiment, the cancer disclosed herein is adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, lymphoma, thyroid cancer, pancreatic cancer, anal cancer, anorectal cancer, anal canal cancer, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, ... cancer), cancer of bone and joints, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, neuroblastoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, gastrointestinal, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancer, leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasms, mycosis fungoides, Seziary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumors, extragonadal germ cell Tumors, extrahepatic bile duct cancer, eye cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, eye cancer, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, renal cancer, kidney cancer, kidney cancer, laryngeal cancer, acute lymphocytic leukemia disease, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstrom's hypergammaglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous cell neck cancer, mouth cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, chronic myeloid leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer), oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian tumor of low malignant potential, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing family of sarcoma tumors, Kaposi's sarcoma, soft tissue sarcoma, epithelioid sarcoma, synovial sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, renal pelvis and ureter and other Selected from, but not limited to, transitional cell carcinoma of the urinary organs, gestational trophoblastic neoplasia, urethral cancer, endometrial uterine cancer, uterine sarcoma, endometrial cancer, vaginal cancer, vulvar cancer or Wilms' tumor or any tumor associated with the above cancer types or any metastasis following, but not limited to, any of the above cancers.

[0223] In one embodiment, the present invention provides a method of treating a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of an activatable proprotein or pharmaceutical composition disclosed herein.

[0224] In one embodiment, the present invention provides a method of treating a cancer or tumor as disclosed herein in a subject in need thereof, comprising administering a therapeutically effective amount of an activatable proprotein or pharmaceutical composition as disclosed herein.

[0225] In one embodiment, the present invention provides a method for preventing, inhibiting, suppressing, or delaying the onset of cancer or tumor in a subject, comprising administering an effective amount of an activatable proprotein or pharmaceutical composition described herein.

[0226] In some embodiments, treating a patient with a pharmaceutical composition comprising an activatable proprotein provided herein can result in an increase in the average survival time of the treated population compared to a population receiving monotherapy with a drug that is not a compound of the present disclosure or its pharma-ceutically acceptable salt, solvate, analog or derivative.After treatment with the strategies, treatment modalities, methods, combinations and compositions provided herein, the average survival time is preferably increased by more than 30 days, more preferably more than 60 days, more preferably more than 90, 120 or 365 days, more preferably more than 365 days.The increase in the average survival time of a population can be measured by any reproducible means.The increase in the average survival time of a population can be measured, for example, by calculating the average length of survival after the start of treatment with an active compound for a population.The increase in the average survival time of a population can also be measured, for example, by calculating the average length of survival after the completion of the first round of treatment with a pharmaceutical composition disclosed herein.

[0227] In some embodiments, treating a patient with a pharmaceutical composition comprising an activatable proprotein provided herein can result in a reduction in the mortality rate of the treated population compared to a population receiving carrier alone. Treating cancer can result in a reduction in the mortality rate of the treated population compared to a population receiving untreated. Treating cancer can result in a reduction in the mortality rate of the treated population compared to a population receiving monotherapy with a drug that is not a compound of the present disclosure or a pharmacologic acceptable salt, solvate, analog or derivative thereof. After treatment with the strategies, treatment modalities, methods, combinations and compositions provided herein, the mortality rate is preferably reduced by more than 2%, more preferably by more than 5%, more preferably by more than 10%, and most preferably by more than 25%. The reduction in the mortality rate of the treated population can be measured by any reproducible means. The reduction in the mortality rate of the population can be measured, for example, by calculating the average number of disease-related deaths per unit time after the start of treatment with the active compound for the population. The reduction in mortality rate of a population can also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with a pharmaceutical composition disclosed herein. Activatable Proproteins - Administration, Dosing

[0228] The activatable proprotein or pharmaceutical composition comprising same may be administered parenterally to a subject in need thereof or by a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or mode of administration will vary depending on the desired result. It depends. In order to administer the compound of the present invention by a certain administration route, it may be necessary to coat the compound with a material that prevents its inactivation, or to administer the compound simultaneously with it.For example, the compound can be administered to a subject in a suitable carrier, such as liposome or diluent.Pharmaceutical acceptable diluents include physiological saline and aqueous buffer solutions.Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutical active substances is known in the art.

[0229] In typical embodiments, preparations for administration to a subject include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Some embodiments include non-aqueous solvents, such as propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), organic esters (e.g., ethyl oleate) and other solvents known to those skilled in the art. In certain embodiments of the present invention, physiologically acceptable carriers (or excipients) are used as needed. Examples of such include, for example, saline, PBS, Ringer's solution, lactated Ringer's solution, and the like. In addition, preservatives and additives are added to the composition as needed to help ensure stability and sterility. For example, in various embodiments of the compositions herein, antibiotics and other bactericides, antioxidants, chelating agents, and the like are all present as needed.

[0230] The phrases "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0231] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention are formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0232] The activatable proprotein or a pharmaceutical composition comprising it is administered in any suitable sterile pharmaceutical carrier to a subject in need of treatment (either therapeutically or prophylactically). Such pharmaceutical carriers act to maintain the solubility and activity of the activatable proprotein at the time. In some embodiments, it may be desirable to administer additional components in conjunction with the activatable proprotein. For example, some treatment regimes include chemotherapeutic agents, antibiotics, activators as provided herein, and the like. Further formulations, including possible proproteins, and one or more standard care agents, etc., are all optionally included within the compositions of the invention.

[0233] As used herein, the terms "combination treatment", "combination therapy" and "co-therapy" are used interchangeably and generally refer to a treatment modality characterized by an activatable proprotein as provided herein or a pharmaceutical composition comprising the same and an additional therapeutic agent. Typically, a combination treatment modality is part of a specific treatment regimen intended to provide a beneficial effect from the simultaneous action of a combination of therapeutic agents. The beneficial effect of the combination can include, but is not limited to, a pharmacokinetic or pharmacodynamic synergy resulting from the combination of therapeutic agents. The administration of these therapeutic agents in combination is usually carried out over a defined period of time (usually minutes, hours, days or weeks depending on the combination selected). In some embodiments, combination treatment includes sequential administration of two or more therapeutic agents, where each therapeutic agent is administered at a different time, as well as substantially simultaneous administration of these therapeutic agents, or at least two of the therapeutic agents. Substantially simultaneous administration can be achieved, for example, by administering separate dosage forms of the therapeutic agents to the subject, in a single dosage form having a fixed ratio of each therapeutic agent or multiple times. Sequential or simultaneous administration of each therapeutic agent can be performed in a single dosage form or multiple times. The substantially simultaneous administration may be accomplished by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes. The therapeutic agents may be administered according to the same or different administration intervals. For example, a first therapeutic agent of a selected combination may be administered by intravenous injection, while other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally, or all therapeutic agents may be administered by intravenous injection.

[0234] In some embodiments, combination therapy also includes the administration of the therapeutic agent as described above in further combination with other biologically active ingredients and non-drug therapy (e.g., surgery or radiation treatment).When combination therapy further includes non-drug treatment, the non-drug treatment can be carried out at any suitable time, as long as beneficial effects are achieved from the combined action of the therapeutic agent and non-drug treatment.For example, in appropriate cases, beneficial effects are still achieved when the non-drug treatment is removed from the administration of the therapeutic agent in time, perhaps by several days or even weeks.

[0235] In some embodiments, the additional therapeutic agent includes a chemotherapeutic agent (also called antineoplastic or anti-proliferative agent), such as an alkylating agent, an antibiotic, an antimetabolite, an antidote, an interferon, a polyclonal or monoclonal antibody, an EGFR inhibitor, an HER2 inhibitor, a histone deacetylase inhibitor, a hormone, a mitosis inhibitor, an MTOR inhibitor, a multikinase inhibitor, a serine / threonine kinase inhibitor, a tyrosine kinase inhibitor, a VEGF / VEGFR inhibitor, a taxane or taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule targeting drug, a topoisomerase poison, an inhibitor of a molecular target or enzyme (e.g., a kinase or protein methyltransferase), a cytidine analog drug, or any chemotherapeutic agent, an immune checkpoint inhibitor, or any antineoplastic or anti-proliferative agent known to one of skill in the art.

[0236] Exemplary alkylating agents suitable for use in accordance with the combination treatment modalities provided herein include, but are not limited to, cyclophosphamide (Cytoxan; Neosar), chlorambucil (Leukeran), melphalan (Alkeran), carmustine (BiCNU), busulfan (Busulfex), lomustine (CeeNU), dacarbazine (DTIC-Dome), oxaliplatin (Eloxatin), carmustine (Gliadel), ifosfamide (Ifex), mechlorethamine (Mustargen), busulfan (Myleran), carboplatin (Paraplatin), cisplatin (CDDP; Platinol), temozolomide (Temodar), thiotepa (Thioplex), bendamustine (Treanda), or streptozocin (Zanosar).

[0237] Exemplary suitable anthracyclines include, but are not limited to, doxorubicin (Adriamycin), doxorubicin liposomal (Doxil), mitoxantrone (Novantrone), bleomycin (Blenoxane), daunorubicin (Cerubidine), daunorubicin liposomal (DaunoXome), dactinomycin (Cosmegen), epirubicin (Ellence), idarubicin (Idamycin), plicamycin (Mithracin), mitomycin (Mutamycin), pentostatin (Nipent), or valrubicin (Valstar).

[0238] Exemplary antimetabolites include, but are not limited to, fluorouracil (Adrucil), capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinethol), pemetrexed (Alimta), fludarabine (Fl These include valproate, nelarabine (Arranon), cladribine (Cladribine Novaplus), clofarabine (Clolar), cytarabine (Cytosar-U), decitabine (Dacogen), cytarabine liposomal (DepoCyt), hydroxyurea (Droxia), pralatrexate (Folotyn), floxuridine (FUDR), gemcitabine (Gemzar), cladribine (Leustatin), fludarabine (Oforta), methotrexate (MTX; Rheumatrex), methotrexate (Trexall), thioguanine (Tabloid), TS-1 or cytarabine (Tarabine PFS).

[0239] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0240] Exemplary interferons include, but are not limited to, interferon alpha-2b (IntronA) or interferon alpha-2a (Roferon-A).

[0241] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin), ofatumumab (Arzerra), bevacizumab (Avastin), rituximab (Rituxan), cetuximab (Erbitux), panitumumab (Vectibix), tositumomab / iodine-131 tositumomab (Bexxar), alemtuzumab (Campath), ibritumomab (Zevalin, In-111, Y-90 Zevalin), gemtuzumab (Mylotarg), eculizumab (Soliris), or denosumab.

[0242] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa), lapatinib (Tykerb), cetuximab (Erbitux), erlotinib (Tarceva), panitumumab (Vectibix), PKI-166, canertinib (CI-1033), matuzumab (EMD 72000) or EKB-569.

[0243] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin), lapatinib (Tykerb) or AC-480.

[0244] Histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

[0245] Exemplary hormones include, but are not limited to, tamoxifen (Soltamox, Nolvadex), raloxifene (Evista), megestrol (Megace), leuprolide (Lupron, Lupron Depot, Eligard, Viadur), fulvestrant (Faslodex), letrozole (Femara), triptorelin (Trelstar LA, Trelstar Depot), exemestane (Aromasin), goserelin (Zoladex), bicalutamide (Casodex), anastrozole (Arimidex), fluoxymesterone (Androxy, Halotestin), medroxyprogesterone (Provera, Depo-Provera), estramustine (Emcyt), flutamide (Eulexin), toremifene (Fareston), degarelix (Firmagon), nilutamide (Nilandron), abarelix (Plenaxis) or testolactone (Teslac).

[0246] Exemplary mitotic inhibitors include, but are not limited to, paclitaxel (Taxol , Onxol, Abraxane), docetaxel (Taxotere), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), etoposide (Toposar, Etopophos, VePesid), teniposide (Vumon), ixabepilone (Ixempra), nocodazole, epothilone, vinorelbine (Navelbine), camptothecin (CPT), irinotecan (Camptosar), topotecan (Hycamtin), amsacrine or lamellarin D (LAM-D).

[0247] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel), rapamune, ridaforolimus, or AP23573.

[0248] Exemplary multikinase inhibitors include, but are not limited to, sorafenib (Nexavar), sunitinib (Sutent), BIBW2992, E7080, Zd6474, PKC-412, motesanib, or AP24534.

[0249] Exemplary serine / threonine kinase inhibitors include, but are not limited to, ruboxistaurin, eril / fasudil hydrochloride, flavopiridol, seliciclib (CYC202, Roscovitine), SNS-032 (BMS-387032), Pkc412, bryostatin, KAI-9803, SF1126, VX-680, Azd1152, Arry-142886 (AZD-6244), SCIO-469, GW681323, CC-401, CEP-1347, or PD332991.

[0250] Exemplary tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Sutent), trastuzumab (Herceptin), bevacizumab (Avastin), rituximab (Rituxan), lapatinib (Tykerb), cetuximab (Erbitux), panitumumab (Vectibix), and the like. x), everolimus (Afinitor), alemtuzumab (Campath), gemtuzumab (Mylotarg), temsirolimus (Torisel), pazopanib (Votrient), dasatinib (Sprycel), nilotinib (Tasigna), vatalanib (Ptk787, ZK222584), CEP-701, SU5614, MLN518, XL999, VX-322, Azd0530, BMS-354825, SKI-606 CP-690, AG-490, WHI-P154, WHI-P131, AC-220 or AMG888.

[0251] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetinib.

[0252] Exemplary microtubule targeting agents include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

[0253] Exemplary topoisomerase poisons include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0254] Exemplary taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0255] Exemplary general chemotherapeutic, antineoplastic, and antiproliferative agents include, but are not limited to, altretamine (Hexalen), isotretinoin (Accutane, Amnesteem, Claravis, Sotret), tretinoin (Vesanoid), azacytidine (Vidaza), bortezomib (Velcade) asparaginase (Elspar), levamisole (Ergamisol), mitotane (Lysodren), procarbazine (Matulane), pegaspargase (Oncaspar), dendritic cell carcinoma (Dennis), and cyclosporine (Cyclosporin). These include leukin diftitox (Ontak), porfimer (Photofrin), aldesleukin (Proleukin), lenalidomide (Revlimid), bexarotene (Targretin), thalidomide (Thalomid), temsirolimus (Torisel), arsenic trioxide (Trisenox), verteporfin (Visudyne), mimosine (Leucenol), (1M tegafur to 0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxonate) or lovastatin.

[0256] In some embodiments, combination treatment modalities are provided in which the additional therapeutic agent is a cytokine, e.g., G-CSF (granulocyte colony stimulating factor). In another aspect, the pharmaceutical compositions provided herein can be administered in combination with radiation therapy. Radiation therapy can also be administered in combination with the pharmaceutical compositions provided herein and another chemotherapeutic agent described herein as part of a multi-drug therapy. In yet another aspect, the pharmaceutical compositions provided herein can be administered in combination with CMF (cyclophosphamide, methotrexate, and 5-fluorouracil), CAF (cyclophosphamide, adriamycin, and 5-fluorouracil), AC (adriamycin and cyclophosphamide), FEC (5-fluorouracil, epirubicin, and cyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, and paclitaxel), rituximab, Xeloda (capecitabine), cisplatin (CDDP), carboplatin, TS-1 (tegafur in a molar ratio of 1:0.4:1, gibberellin, cyclophosphamide ... It may be administered in combination with standard chemotherapy combinations such as, but not limited to, gimestat and otastat potassium, camptothecin-11 (CPT-11, irinotecan or Camptosar™), CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin and prednisone or prednisolone), R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, Oncovin, prednisone or prednisolone) or CMFP (cyclophosphamide, methotrexate, 5-fluorouracil and prednisone).

[0257] In some embodiments, the pharmaceutical compositions provided herein can be administered with enzyme inhibitors, such as receptor or non-receptor kinases.Receptor and non-receptor kinases include, for example, tyrosine kinases or serine / threonine kinases.The kinase inhibitors described herein are small molecules, polynucleic acids, polypeptides or antibodies.

[0258] Exemplary kinase inhibitors include, but are not limited to, bevacizumab (targets VEGF), BIBW2992 (targets EGFR and Erb2), cetuximab / Erbitux (targets Erb1), imatinib / Gleevec (targets Bcr-Abl), trastuzumab (targets Erb2), gefitinib / Iressa (targets EGFR), ranibizumab (targets VEGF), pegaptanib (targets VEGF), and rituximab (targets Erb1). (targets VEGF), erlotinib / Tarceva (targets Erb1), nilotinib (targets Bcr-Abl), lapatinib (targets Erb1 and Erb2 / Her2), GW-572016 / lapatinib ditosylate (targets HER2 / Erb2), panitumumab / Vectibix (targets EGFR), vandetinib (targets RET / VEGFR), E7080 (targets RET and VEGFR including multiple targets), Herceptin (targets HER2 / Erb2), PKI-166 (targets EGFR), canertinib / CI-1033 (targets EGFR), sunitinib / SU-11464 / Sutent (targets EGFR and FLT3), matuzumab / Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR) (targets VEGR and FLT3), PKC-412 (targets VEGR and FLT3), vatalanib / Ptk787 / ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targets SRC), BMS-354825 ( SRC), SKI-606 (SRC), CP-690 (JAK), AG-490 (JAK), WHI-P154 (JAK), WHI-P131 (JAK), sorafenib / Nexavar (RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, KIT, FLT-3 and RET), dasatinib / Sp These include rycel (BCR / ABL and Src), AC-220 (targets Flt3), AC-480 (targets all HER proteins, "pan-HER"), motesanib diphosphate (targets VEGF1-3, PDGFR and c-kit), denosumab (targets RANKL and inhibits SRC), AMG888 (targets HER3) and AP24534 (multiple targets including Flt3).

[0259] In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same polypeptide disclosed herein is administered to a subject once per day. In some embodiments, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once every two days. In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once every three days. In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once every four days. In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once every five days. In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once every six days. In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once per week. In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once every seven to fourteen days. In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once every ten to twenty days. In another embodiment, an activatable proprotein or pharmaceutical composition comprising the same is administered to a subject once every five to fifteen days. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to a subject once every 15 to 30 days.

[0260] In one embodiment, the dose of the activatable proprotein of the present invention comprises 0.005-0.1 milligrams / kg in injectable solution. In another embodiment, the dose comprises 0.005-0.5 milligrams / kg of activatable proprotein. In another embodiment, the dose comprises 0.05-0.1 micrograms of activatable proprotein. In another embodiment, the dose comprises 0.005-0.1 milligrams / kg of activatable proprotein in injectable solution.

[0261] In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.0001 mg to 0.6 mg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.001 mg to 0.005 mg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.005 mg to 0.01 mg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to a subject at a dose ranging from 0.01 mg to 0.3 mg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to a subject at a dose ranging from 0.2 mg to 0.6 mg.

[0262] In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 1 to 100 mcg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 10 to 80 mcg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 20 to 60 mcg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 10 to 50 mcg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 40 to 80 mcg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 10 to 30 mcg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 30 to 60 mcg / kg.

[0263] In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.1 mcg / kg to 100 mg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.1 mcg / kg to 50 mg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.1 mcg / kg to 25 mg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.1 mcg / kg to 10 mg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.1 mcg / kg to 5 mg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject at a dose ranging from 0.1 mcg / kg to 1 mg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to a subject at a dose ranging from 0.1 mcg / kg to 0.1 mg / kg.

[0264] In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject in a dose ranging from 0.2 mg to 2 mg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject in a dose ranging from 2 mg to 6 mg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject in a dose ranging from 4 mg to 10 mg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising same is administered to the subject in a dose ranging from 5 mg to 15 mg.

[0265] In one embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 10 μg / kg to 1000 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 25 μg / kg to 600 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose ranging from 50 μg / kg to 400 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 25 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 50 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 100 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 100 μg / kg. The activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 200 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 300 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 400 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 500 μg / kg. In another embodiment, the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at a dose of about 600 μg / kg.

[0266] In one embodiment, a single dose of the activatable proprotein or pharmaceutical composition comprising the same is administered to the subject. In another embodiment, a total of two doses are administered to the subject. In another embodiment, a total of two or more doses are administered to the subject.

[0267] In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at least once a day. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at least once every two days. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at least once every two days or longer. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject every week, every two weeks or every three weeks. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at least once a week. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at least once every two weeks. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at least once every three weeks. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at least once every three weeks or longer. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered to the subject twice or more per week. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered to the subject twice or more per month. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered to the subject twice or more per year. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered to the subject twice or more per two years. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered to the subject twice or more per two years or more.

[0268] In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 36 hours. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 48 hours. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 60 hours. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 72 hours. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 84 hours. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 96 hours. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 5 days. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 6 days. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once every 7 days. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered at least once every 8-10 days. A dose of the activatable proprotein or pharmaceutical composition comprising same is administered at least once every 10-12 days. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered at least once every 12-15 days. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered at least once every 15-25 days. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising same is administered at least once every 20-30 days.

[0269] In one embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered to the subject at least once per month. In one embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once per two months. In one embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once per three months. In one embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once per four months. In one embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once per five months. In one embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once per six months. In one embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered at least once per six to twelve months. In another embodiment, a dose of the activatable proprotein or pharmaceutical composition comprising it is administered four times per year. In another embodiment, the dose is administered daily, weekly, biweekly, monthly or annually. In another embodiment, the dose is administered once, twice or more daily, weekly, monthly or yearly, hi another embodiment, the dose is administered every 2, 3, 4 or at least 5 years.

[0270] In one embodiment, repeated administrations (doses) of the compositions of the invention can be performed immediately following a first course of treatment, or after intervals of days, weeks or years, to achieve the desired effect, as further provided herein (e.g., for preventing or treating a cardiovascular disease or condition or a CNS-related disease or condition).

[0271] In one embodiment, the pharmaceutical composition is administered by intravenous, intraarterial, subcutaneous or intramuscular injection of a liquid preparation. In another embodiment, the liquid preparation comprises a solution, suspension, dispersion, emulsion, oil, etc. In one embodiment, the pharmaceutical composition is administered intravenously and is therefore formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intraarterially and is therefore formulated in a form suitable for intraarterial administration.

[0272] In some embodiments, compositions for use in the methods disclosed herein include solutions or emulsions, which in some embodiments are aqueous solutions or emulsions containing a safe, effective amount of a compound disclosed herein and optionally other compounds, intended for intravenous or subcutaneous administration.

[0273] In some embodiments, the various components of the composition are pre-measured and / or pre-packaged and / or ready for use without additional measurements. The present invention also includes kits for carrying out / using the methods and / or compositions of the present invention, if desired. In addition, such kits may also include suitable excipients (e.g., pharma- ceutically acceptable excipients) for carrying out the therapeutic and / or prophylactic treatments of the present invention. Such kits may also optionally contain additional components for assembly and / or use of the compositions of the present invention, including, but not limited to, diluents, etc.

[0274] The compositions described herein may, if desired, contain all (or nearly all) of the components necessary to carry out the methods or use the compositions of the invention. The kits are packaged to include components (optionally including, e.g., written instructions for use of the methods / compositions of the invention). For example, the kits may include components such as, e.g., buffers, reagents, serum proteins, antibodies, substrates, etc., as appropriate. In the case of prepackaged reagents, the kits include premeasured or predosed amounts, e.g., premeasured fluid aliquots or preweighed or premeasured solid reagents that can be easily reconstituted by the end user of the kit, ready to be incorporated into the method without measurement, as appropriate.

[0275] Such kits also typically include suitable instructions for carrying out the methods of the invention and / or for using the compositions of the invention. In some embodiments, the components of the kit / package are provided in a stable form to prevent degradation or other losses during long-term storage, e.g., leakage. Several stabilization processes / agents are widely used for reagents to be preserved, such as including chemical stabilizers (i.e., enzymatic inhibitors, bactericides / bacteriostats, anticoagulants). The actual dosage level of the active ingredient in the pharmaceutical composition of the invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for the individual patient, composition and mode of administration that is not toxic to the patient. The selected dosage level will vary depending on various pharmacokinetic factors, including the activity of the individual compositions of the invention used, the route of administration, the time of administration, the rate of excretion of the individual compounds used, the duration of treatment, other drugs, compounds and / or materials used in combination with the individual compositions used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0276] The composition must be sterile and fluid to the extent that the composition can be delivered by syringe.Besides water, the carrier is preferably an isotonic buffered saline solution.Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.In many cases, it is preferred to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride.

[0277] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration that is not toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0278] Although several inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision a variety of other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials and configurations described herein are exemplary, and that the actual parameters, dimensions, materials and / or configurations will vary depending on the specific application(s) in which the inventive techniques are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific inventive embodiments described herein. Thus, the foregoing embodiments are presented by way of example only, and it is to be understood that within the scope of the appended claims and equivalents thereof, inventive embodiments may be practiced other than as specifically described and claimed. It should be understood that the inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Moreover, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0279] All definitions as defined and used herein should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meaning of the defined terms.

[0280] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, in some cases including the entire document.

[0281] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0282] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjoint in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, whether related or unrelated to those elements specifically identified, may optionally be present other than the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may in one embodiment refer to only A (including, optionally, elements other than B), in another embodiment may refer to only B (including, optionally, elements other than A), in yet another embodiment may refer to both A and B (including, optionally, other elements), etc.

[0283] As used herein, and in the claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, but not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") may in one embodiment refer to at least one, and optionally more than one, A, and no B (optionally including elements other than B); in another embodiment it may refer to at least one, and optionally more than one, B, and no A (optionally including elements other than A); in yet another embodiment it may refer to at least one, and optionally more than one, A and at least one, and optionally more than one, B (optionally including other elements), etc.

[0284] The present invention provides a kit for preventing, treating or delaying a cardiovascular disease or condition in a human. The present invention further provides a kit comprising one or more doses of a pharmaceutical composition comprising an activatable proprotein disclosed herein for use in preventing, treating or delaying a cardiovascular disease or condition, and instructions on how to use the pharmaceutical preparation or composition.

[0285] The present invention further provides kits for preventing, treating or delaying a CNS-related disease or condition in a human, the kit comprising one or more doses of a pharmaceutical composition comprising an activatable proprotein disclosed herein for use in preventing, treating or delaying a cardiovascular disease or condition, and instructions on how to use the pharmaceutical preparation or composition.

[0286] The invention further provides kits for preventing, treating or delaying heart failure with preserved ejection fraction in humans, the kits comprising one or more doses of a pharmaceutical composition comprising an activatable proprotein disclosed herein used to prevent, treat or delay heart failure with preserved ejection fraction and instructions on how to use the pharmaceutical preparation or composition.

[0287] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention, but they should not be construed in any way as limiting the broad scope of the invention. EXAMPLES

[0288] Example 1 Engineering an Fc-IL-15-linker-IL-15Rα fusion protein To reduce the toxicity of IL-15-related therapeutics, Fc-IL-15-linker-IL-15Rα fusion proteins (referred to herein as Fc-ILR fusion proteins) were generated as prodrugs (i.e., activatable proproteins as described elsewhere herein). The prodrugs have very low activity. Full activity can be restored upon protease cleavage of a designed protease-specific linker sequence within the prodrug.

[0289] Plasmids encoding single chain IL-15-linker-IL-15Rα (ILR) with or without Fc fusion were constructed by standard gene synthesis followed by subcloning into the pTT5 expression vector. Schematic diagrams of exemplary Fc-ILR fusion protein formats are depicted in Figures 3E, 3F, 4A and 4F.

[0290] Exemplary proteins of the Fc-ILR format that do not have a protease cleavage site include P1187 (SEQ ID NO: 18), P1188 (SEQ ID NO: 19), P1250 (SEQ ID NO: 20), P1251 (SEQ ID NO: 21) and P1252 (SEQ ID NO: 22).

[0291] Exemplary proteins of the IL-15-linker-IL-15Rα-Fc (ILR-Fc) format ( FIG. 3B ) include P1186 (SEQ ID NO: 17) and P1331 (SEQ ID NO: 73).

[0292] Exemplary proteins of the IL-15-linker-IL-15Rα format with a His6-tag at the C-terminus of IL-15Rα (FIGS. 2C and 2D) include P1185 (SEQ ID NO: 16) and P1275 (SEQ ID NO: 39).

[0293] Exemplary proteins of the ILR-Fc-a format (Figure 3B) include P1332 (SEQ ID NO:74) and P1333 (SEQ ID NO:75).

[0294] The C-terminal serine (S162) of IL-15 in the IL-15 fusion protein is a potential glycosylation site in the IL-15 fusion protein, and glycosylation of S162 may affect the potency of the fusion protein. Point mutations were introduced into IL-15-linker-IL-15Rα-Fc (ILR-Fc), Fc-IL-15-linker-IL-15Rα (Fc-ILR) and IL-15-linker-IL-15Rα (ILR) by standard mutagenesis techniques. An exemplary protein of ILR-Fc with S162A is P1253 (SEQ ID NO: 23). An exemplary protein of Fc-ILR with S162A is P1254 (SEQ ID NO: 24), P1255 (SEQ ID NO: 25), P1279 (SEQ ID NO: 41) and P1280 (SEQ ID NO: 42). Exemplary proteins of ILR-His6 having S162A include P1274 (SEQ ID NO:38) and P1276 (SEQ ID NO:40). Production, purification and characterization

[0295] Fc fusion proteins were produced by transient transfection in Expi293 cells and purified by a two-step purification process including MabSelect SuRe chromatography (GE Healthcare) and size exclusion chromatography (Superdex 200, GE Healthcare).

[0296] His-tagged proteins were produced by transient transfection in Expi293 cells and purified by a two-step purification process including nickel affinity chromatography (GE Healthcare) and size-exclusion chromatography (Superdex 75 or Superdex 200, GE Healthcare).

[0297] The purified proteins were characterized by SDS-PAGE and high performance liquid chromatography (HPLC) for purity and homogeneity assessment. HPLC analysis was performed using a Nanofilm SEC-250 column (Sepax) and an Agilent 1260 according to the manufacturer's instructions. Representative SDS-PAGE and HPLC results are shown in Figures 9A-9F and 10A-10G. The purified proteins showed good purity and homogeneity. For the ILR-His formats, P1274 (SEQ ID NO: 38) with a 15 amino acid linker and P1276 (SEQ ID NO: 40) with a 26 amino acid linker, the theoretical MW of the monomer is approximately 24 KD and the dimer is approximately 50 KD. Based on the HPLC results shown in FIG. 10G, P1274 (SEQ ID NO: 38) appears to exist as a homodimer in solution, indicating interchain IL-15 / IL-15Rα complex formation, and P1276 (SEQ ID NO: 40) appears to exist as a monomer, indicating intrachain IL-15 / IL-15Rα complex formation. Functional Assays - Cell Signaling Transduction

[0298] The proteins were then tested in in vitro functional assays. In cell signaling assays using PBMCs (phospho-STAT5), purified fusion proteins (with or without Fc) were incubated with PBMCs for 15 minutes at 37°C. After incubation, cellular stimulation was terminated by the addition of fixation buffer, and cells were fixed for cell staining. Cells were then permeabilized by the addition of permeabilization buffer (BD Perm III buffer, Cat. No. 558050) according to the manufacturer's protocol. PBMCs were then stained, stained with anti-CD3-FITC (UCHT1, BD, Cat. No. 555916) and phospho-STAT5 antibody (pY694, Alexa647 conjugated, BD, Cat. No. 612599). Figures 11A and 11B represent the selection of cell populations following incubation with P1185 (SEQ ID NO: 16). Lymphocytes were first gated based on side scatter (SSC) and forward scatter (FSC) (Figure 11A). Lymphocytes were then screened for CD3 expression and It was gated based on STAT5 phosphorylation (FIG. 11B). The signaling assay results are summarized in FIG. 11C and FIG. 11D. ILR-His (P1185 having SEQ ID NO: 16, P1275 having SEQ ID NO: 39, P1274 having SEQ ID NO: 38 and P1276 having SEQ ID NO: 40) and ILR-Fc (P1186 having SEQ ID NO: 17, P1331 having SEQ ID NO: 73, P1253 having SEQ ID NO: 23, P1332 having SEQ ID NO: 74 and P1333 having SEQ ID NO: 75) exhibited cell signaling activity (phospho-STAT5), whereas Fc-ILR (P1187 having SEQ ID NO: 18, P1188 having SEQ ID NO: 19, P1250 having SEQ ID NO: 20, P1251 having SEQ ID NO: 21, P1252 having SEQ ID NO: 22, P1254 having SEQ ID NO: 24, P1255 having SEQ ID NO: 25, P1279 having SEQ ID NO: 41 and P1280 having SEQ ID NO: 42) are not active in stimulating STAT5 phosphorylation.

[0299] Cell signaling assays were also performed using human whole blood for some purified fusion proteins. Human whole blood samples were collected from healthy individuals and used according to guidelines as per signed consent agreement. Purified fusion proteins were incubated with fresh human whole blood for 15 min at 37°C. After incubation, cellular stimulation was terminated by addition of lysis and fixation buffer (BD, Cat. No. 558049) to remove red blood cells and fix remaining cells for cell staining. Anti-CD3-FITC (UCHT1, BD, Cat. No. 555916) was added to the cell suspension and incubated for 1 h at 37°C for staining, followed by removal by two washing steps using phosphate-buffered saline (PBS). Cells were then permeabilized by addition of permeabilization buffer (BD Perm III buffer, Cat. No. 558050) according to the manufacturer's protocol and stained for 1 hour using phospho-STAT5 antibody (pY694, Alexa647 conjugated, BD, Cat. No. 612599). The results are summarized in Figures 12A-12F and are consistent with the cell signaling assay results using PBMCs. ILR-His (P1274 with SEQ ID NO: 38) stimulated proliferation of PBMCs compared to the control group, whereas Fc-ILR (P1254 with SEQ ID NO: 24) did not. Functional assays – proliferation

[0300] The purified proteins were also tested in a proliferation assay. M-07e (IL-2R / c) cells were cultured in RPMI1640 supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA) and 10% 5637 cell culture supernatant. To measure cytokine-dependent cell proliferation, M-07e cells were harvested in their logarithmic growth phase and washed twice with PBS. 90 μl of cell suspension (2 × 10 4 Cells (10 cells / well) were seeded in 96-well plates and incubated at 37°C and 5% CO 2The wells were incubated for 4 h at 37 °C and 5% CO in assay medium (RPMI 1640 supplemented with 10% FBS and 1% NEAA) for cytokine starvation. IL-15 and purified protein samples used in the assay were prepared in assay medium to an initial concentration of 300 nM and subsequently diluted 1 / 3 serially. 10 μl of diluted protein was added to the corresponding wells and incubated at 37 °C and 5% CO 2 The cells were incubated at 4°C for 72 hours. A colorimetric assay using Cell Counting Kit-8 (CCK-8, Dojindo, CK04) was performed to measure the amount of viable cells, and the results are shown in Figures 13A and 13C. For the ILR-Fc format (Figure 13B), P1186 (SEQ ID NO: 17), P1331 (SEQ ID NO: 73) and P1333 (SEQ ID NO: 75) showed 3-4 fold lower activity compared to P1185 (SEQ ID NO: 16).

[0301] Constructs with the S162A mutation were compared for potency with the corresponding constructs with S162 (Figures 13D-13H). For the Fc-ILR format (P1254 with SEQ ID NO: 24 and P1255 with SEQ ID NO: 25), activity was not improved with the S162A mutation (Figures 13F-13G). For the ILR-Fc (P1254 with SEQ ID NO: 23), activity was not improved with the S162A mutation (Figures 13F-13G). For ILR-His (P1253 with SEQ ID NO: 38) and ILR-His (P1274 with SEQ ID NO: 38) with a 15 amino acid linker between IL-15 and IL-15Rα, the potency of the fusion protein improved 5-7 fold when the S162A mutation was used (Figures 13D-13E). For ILR-His (P1276 with SEQ ID NO: 40) with a 26 amino acid linker between IL-15 and IL-15Rα, the potency was not improved when the S162A mutation was used (Figure 13H), suggesting that the linker between IL-15 and IL15-Rα may not interfere with receptor βγ binding to IL-15 due to intrachain LR dimer formation.

[0302] Proliferation assays were also performed for several purified fusion proteins using human peripheral blood mononuclear cells (PBMCs). Human PBMCs were prepared by Ficoll centrifugation and stained with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE, Thermofisher, Cat. No. C34554) according to the manufacturer's instructions. Stained human PBMCs were seeded in 96-well plates (5×10 per well). 4 Human PBMC proliferation was assessed by flow cytometry and the results are summarized in Figures 14A-14F. The human PBMC proliferation results were consistent with other functional assay results, in which ILR-His (P1185 with SEQ ID NO: 16) and ILR-Fc (P1253 with SEQ ID NO: 23) failed to stimulate proliferation of PBMCs. Example 2 Engineering an Fc-IL-15-linker-IL-15Rα fusion protein with a protease cleavage site in the linker between Fc and IL-15

[0303] To restore the activity of IL-15, a TEV protease cleavage site was used as a proof of concept and introduced into the linker between Fc and IL-15 in the Fc-ILR construct.

[0304] Plasmids encoding Fc-TEV-ILR were constructed by standard gene synthesis and subcloned into pTT5 expression vectors with linkers of various lengths flanking the TEV cleavage site. A cartoon schematic of an exemplary Fc-TEV-ILR fusion protein format is depicted in FIG. 6. Exemplary proteins of the Fc-TEV-ILR format include P1256 (SEQ ID NO: 26), P1261 (SEQ ID NO: 32), P1281 (SEQ ID NO: 43), P1282 (SEQ ID NO: 49), P1283 (SEQ ID NO: 55), P1284 (SEQ ID NO: 61) and P1285 (SEQ ID NO: 67).

[0305] To form a more rigid IL-15 / IL-15Rα complex, a disulfide bond was introduced between IL-15 and IL-15Rα, with a L52C mutation in IL-15 and a S40C mutation in IL-15Rα. Exemplary proteins of this format include P1378 (SEQ ID NO:226) and P1379 (SEQ ID NO:232).

[0306] For activatable prodrug design, a real protease cleavage site (PS) was introduced into the linker between Fc and IL-15 of the Fc-ILR construct. A plasmid encoding Fc-PSs-ILR was constructed by standard gene synthesis and subcloned into the pTT5 expression vector with the linker flanking the protease cleavage sites. A cartoon schematic of an exemplary Fc-PSs-ILR fusion protein format is depicted in FIG. 6. Exemplary proteins in the Fc-PSs-ILR format include P1334 (SEQ ID NO:76), P1335 (SEQ ID NO:87), P1336 (SEQ ID NO:98), P1337 (SEQ ID NO:114), P1338 (SEQ ID NO:130), P1339 (SEQ ID NO:146), P1340 (SEQ ID NO:162), P1341 (SEQ ID NO:178), P1342 (SEQ ID NO:194), P1343 (SEQ ID NO:210), P1380 (SEQ ID NO:238), P1381 (SEQ ID NO:254), P1382 ​​(SEQ ID NO:270), P1383 (SEQ ID NO:286), P1423 (SEQ ID NO:338), P1424 (SEQ ID NO:339), P1425 (SEQ ID NO:340), P1426 (SEQ ID NO:341), P1347 (SEQ ID NO:342), P1348 (SEQ ID NO:343), P1349 (SEQ ID NO:344), P1350 (SEQ ID NO:351), P1351 (SEQ ID NO:352), P1352 (SEQ ID NO:353), P1353 (SEQ ID NO:354), P1354 (SEQ ID NO:355), P1355 (SEQ ID NO:356), P1356 (SEQ ID NO:357), P1357 (SEQ ID NO:358), P1358 (SEQ ID NO:359), P1359 (SEQ ID NO:360), P1359 (SEQ ID NO:361), P1350 (SEQ ID NO:362), P1 No. 341), P1427 (SEQ ID NO: 342) and P1428 (SEQ ID NO: 343).

[0307] The Fc fusion proteins were produced, purified and characterized as described in Example 1.

[0308] Purified proteins with TEV cleavage sites were tested by TEV cleavage (Figures 15A-15I). P1256 (SEQ ID NO: 26), which has the shortest linker adjacent to the TEV cleavage site, can be partially cleaved by TEV at both 4°C and 37°C at TEV to protein ratios of 1:5 and 1:10. For proteins with longer linkers than P1256 (SEQ ID NO: 26) adjacent to the TEV cleavage site (P1261 with SEQ ID NO: 32, P2181 with SEQ ID NO: 43, P1282 with SEQ ID NO: 49, P1283 with SEQ ID NO: 55, P1284 with SEQ ID NO: 61, and P1285 with SEQ ID NO: 67), most of the target proteins can be cleaved by TEV at both 4°C and 37°C at a TEV to protein ratio of 1:20. P1378 (SEQ ID NO: 226) and P1379 (SEQ ID NO: 232) can be partially cleaved by TEV at 4°C at a TEV to protein ratio of 1:1. The uncleaved and TEV cleaved proteins were then tested in in vitro functional assays using the same methods as described in Example 1. The cell signaling assay (phospho-STAT5) results are summarized in Figure 17A and the M-07e proliferation assay results are summarized in Figures 18A-18F. Based on the functional assay results, IL-15 activity was restored after TEV cleavage between the Fc and IL-15 of the Fc-TEV-ILR fusion protein, whereas TEV-treated P1187 (SEQ ID NO: 18) showed no function.

[0309] The purified Fc-PSs-ILR protein was tested for protease cleavage assays with proteases uPA (R&D, Cat. No. 1310-SE-010), matriptase (R&D, Cat. No. 3946-SEB-010), legumain (R&D, Cat. No. 2199-CY-010), MMP-2 (R&D, Cat. No. 902-MP-010) and MMP-9 (R&D, Cat. No. 911-MP-010), respectively, and the results are shown in Figures 16A-16G. The purified protein could be fully or partially cleaved by the above proteases.

[0310] The uncleaved and protease-cleaved proteins were then tested in in vitro functional assays using the same methods as described in Example 1. The cell signaling assay (phospho-STAT5) results are summarized in Figure 17B, the M-07e proliferation assay results are summarized in Figures 18G-18I, and the PBMC proliferation assay results are summarized in Figures 19A-19D. IL-15 activity was restored after protease cleavage between Fc and IL-15. Example 3 Engineering an Fc-IL-15-linker-IL-15Rα fusion protein with a protease cleavage site in the linker between IL-15 and IL-15Rα

[0311] To restore activity of IL-15, a TEV protease cleavage site was used as a proof of concept and introduced into the linker between IL-15 and IL-15Rα in the Fc-ILR construct.

[0312] Plasmids were constructed using the same methodology as described in Example 2. A cartoon schematic of an exemplary Fc-ILR fusion protein format is depicted in Figure 8. Exemplary proteins of the Fc-IL-15(S162A)-TEV-IL-15Rα format include P1393 (SEQ ID NO: 302), P1394 (SEQ ID NO: 308) and P1395 (SEQ ID NO: 314). Some examples include:

[0313] Exemplary proteins of the Fc-IL-15(S162A, L52C)-TEV-IL-15Rα(S40C) format include P1396 (SEQ ID NO: 320), P1397 (SEQ ID NO: 326) and P1398 (SEQ ID NO: 332).

[0314] The Fc fusion proteins were produced, purified and characterized as described in Example 1.

[0315] The purified proteins were tested by TEV cleavage at a 1:1 TEV to protein ratio and the results are shown in Figures 20A-20F. The results showed that all six proteins could be completely cleaved by TEV at 4°C at a 1:1 TEV to protein ratio.

[0316] The uncleaved and TEV cleaved proteins were then tested in in vitro functional assays using the same methods as described in Example 1. The cell signaling assay (phospho-STAT5) results are summarized in Figure 21 and the M-07e proliferation assay results are summarized in Figures 22A-22F. IL-15 activity was restored after protease cleavage of the linker sequence between IL-15 and IL-15Rα. Example 4 Optimization of cleavable linker sequences

[0317] For activatable prodrug design, various protease cleavage sequences were introduced into the linker between Fc and IL-15 in the Fc-IL-15-PS-IL-15Rα format. Plasmids encoding Fc-IL-15-PS-IL-15Rα were synthesized by standard gene synthesis and subcloned into pTT5 vector.

[0318] Exemplary proteins of Fc-PS-IL-15-stable linker-IL-15Rα include P1423, P1424, P1425, P1426, P1427, P1428, P1471, P1472, P1473, P1474, P1475, P1476, P1477, P1478, P1479, P1480, P1481, P1482, P1483, P1484, P1485, P1486, P1487, P1488, P1489, P1490, P1491, P1492, P1493, P1494, P1495, P1496, P1497, P1498, P1499, P1500, P1501, P1502, P1503, P1504, P1505, P1506, P1507, P1508, P1509, P1510, P1511, P1512, P1513, P1514, P1515, P1516, P1517, P1518, P1519, P1610, P1611, P1612, P1613, P1614, P1615, P1616, P1617, P1618, P1619, P1620, P1621, P1622, P1623, P1624, P1625, P1626, P1627, P1628, P1629, P1700, P1701, P1702, P1703, P1704, P17 487, P1488, P1489, P1490, P1491, P1492, P1493, P1494, P1495, P1496, P1497, P1498, P1499, P1500, P1501, P1502, P1503, P1504, P1505, P1506, P1511, P1540, P1541, P1637, P1638, P1639, P1658 and P1659.

[0319] Exemplary proteins of Fc-PS-IL-15mut-stable linker-IL-15Rα include P1652, P1653, P1654, P1655, P1656 and P1657.

[0320] Exemplary proteins of Fc-PS-IL-15-stable linker-IL-15Rα with codon optimized for IL-15 and IL-15Rα include P1660, P1661 and P1663.

[0321] Exemplary proteins of Fc-PS-IL-15-stable linker-IL-15Rα with different stable linker lengths include P2162, P2163, P2164, P2165, P2166, P2167, P2168 and P2169.

[0322] Exemplary proteins for Fc-stable linker-IL-15-PS-IL-15Rα include P1542, P1636, P1696, P1697, P1698, P1699, P170 0, P1701, P1702, P1703, P1704, P1705, P1706 and P1707.

[0323] Exemplary proteins of Fc-stable linker-IL-15-PS-IL-15Rα with a disulfide bond between two IL-15Rα molecules include P1973, P1974 and P1975.

[0324] Exemplary proteins for Fc-PS-IL-15-stable linker-IL-15Rα-sushi include P1682 and P1683.

[0325] The Fc fusion proteins were produced, purified and characterized as described in Example 1. Representative SDS-PAGE and HPLC results are summarized in Figures 23A-23F, 24A-24F and 25A-25X. The purified proteins showed good purity based on the SDS-PAGE results. Most of the purified proteins showed good homogeneity based on the HPLC results. Dimers and / or polymers were observed for fusion proteins with shorter linkers between IL-15 and IL-15Rα, e.g., P2163, P2164, P2166 and P2167.

[0326] The purified proteins were tested for protease cleavage assays with proteases uPA (R&D, Cat. No. 1310-SE-010), matriptase (R&D, Cat. No. 3946-SEB-010), legumain (R&D, Cat. No. 2199-CY-010), MMP-2 (R&D, Cat. No. 902-MP-010), MMP-9 (R&D, Cat. No. 911-MP-010), KLK5 (R&D, Cat. No. 1108-SE) or / and KLK7 (R&D, Cat. No. 2624-SE), respectively, and representative results are shown in Figures 26A-26P.

[0327] The uncleaved and protease-cleaved proteins were then tested in the M-07e proliferation assay using the same method as described in Example 1. The results are summarized in Figures 27A-27N and Figures 28A-28W.

[0328] Protein in vitro serum stability was tested with serum from human, cynomolgus monkey (cyno), mouse and rat. Equal amounts of protein were incubated with fresh serum for various times at 37°C. Proteins were run on 12% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with nonfat dry milk or BSA in TBST and probed with biotinylated anti-IL-15 antibody (R&D, Cat. No. BAF247) followed by streptavidin-HRP. Alternatively, membranes were probed with HRP-anti-human IgG antibody. Representative western blot results are shown in Figures 29A-29F. Example 5 Engineering anti-FAP-IL-15 prodrugs

[0329] An IL-15 prodrug with a protease cleavable sequence between Fc and IL-15 or / and a cleavable protease sequence between IL-15 and IL-15Rα was fused to the C-terminus of an anti-FAP antibody. Plasmids encoding the anti-FAP-IL-15 prodrug were synthesized by standard gene synthesis and subcloned into the pTT5 vector.

[0330] Exemplary proteins that have a protease cleavage site between Fc and IL-15 include P15431450 and P16401450.

[0331] Exemplary proteins having a protease cleavage site between IL-15 and IL-15Rα include P18121450, P18131453, P18141563, P18151450, P18161453, P18171563, P19681450 and P19691450.

[0332] Exemplary proteins having a protease cleavage site between Fc and IL-15 and a protease cleavage site between IL-15 and IL-15Rα include P24872158, P24882158, P24892158, P24902158, P24912158, P25162158 and P25172158.

[0333] Anti-FAP-IL-15 fusion proteins were produced, purified, and characterized as described in Example 1. Representative SDS-PAGE and HPLC results are summarized in Figures 30A-30B and 31A-31F. The purified proteins showed good purity based on SDS-PAGE results. Most of the purified proteins showed good homogeneity based on HPLC results.

[0334] The purified proteins were tested in protease cleavage assays with proteases uPA (R&D, Catalog No. 1310-SE-010), matriptase (R&D, Catalog No. 3946-SEB-010), legumain (R&D, Catalog No. 2199-CY-010), MMP-2 (R&D, Catalog No. 902-MP-010), MMP-9 (R&D, Catalog No. 911-MP-010), KLK5 (R&D, Catalog No. 1108-SE) or / and KLK7 (R&D, Catalog No. 2624-SE), respectively, and the results are shown in FIG. 32.

[0335] The uncleaved and protease-cleaved proteins were then tested in the M-07e proliferation assay using the same method as described in Example 1. The results are summarized in Figures 33A-33J.

[0336] Protein in vitro serum stability was tested in serum obtained from humans, cynomolgus monkeys (cyno), mice and rats as described in Example 4. Representative results are shown in Figures 34A-34B. Exemplary Sequences

[0337] Human IL-15 precursor sequence (SEQ ID NO:1) >Human IL-15 LSP(P40933) [ka]

[0338] Human IL-15 mature form sequence (SEQ ID NO:2) >P40933(N49-S162)(L) [ka]

[0339] Human IL-15 mature form sequence with S162A point mutation (SEQ ID NO:3) >IL-15_v1(49-162 with S162A)(L) [ka]

[0340] Human IL-15Rα full length sequence (SEQ ID NO:4) >Human IL-15Rα FL (Q13261) [ka]

[0341] Human IL-15Rα extracellular domain (SEQ ID NO:5) >Human IL-15Rα-ECD [ka]

[0342] Human IL-15Rα, sushi+ (SEQ ID NO:6) >Q13261 31-108(R) [ka]

[0343] Human IL-15Rα, sushi (SEQ ID NO: 7) >Human IL-15Rα-sushi [ka]

[0344] Fc-IL-15-IL-15Rα Scaffold 1 (SEQ ID NO: 8) >Human IgG1 Fc [ka] [ka]

[0345] Fc-IL-15-IL-15Rα Scaffold 2 (SEQ ID NO: 9) >Human IgG1 Fc-a [ka]

[0346] Fc-IL-15-IL-15Rα Scaffold 3 (SEQ ID NO: 10) >Human IgG1 Fc-b_L234A / L235A / P329A / M252Y / S254T / T256E [ka]

[0347] Fc-IL-15-IL-15Rα Scaffold 4 (SEQ ID NO: 11) >Human IgG1 Fc-c [ka]

[0348] IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 12) >L15R [ka]

[0349] IL-15 (L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 13) >L15R_(L:L52C)_(R:S40C) [ka]

[0350] IL-15-(GGS)2-EPKSSDKTHT-(GGS)2-IL-15Rα (SEQ ID NO: 14) >L22R [ka]

[0351] IL-15-(GGS)2-EPKSSDKTHT-(GGS)2GGGS-IL-15Rα (SEQ ID NO: 15) >L26R [ka]

[0352] IL-15-(GGS)5-IL-15Rα-G4S-HHHHHH homodimer (SEQ ID NO: 16) >P1185_L15R-G4S-his6_strands 1 and 2 [ka]

[0353] IL-15-(GGS)5-IL-15Rα-G4S-Fc homodimer (SEQ ID NO: 17) >P1186_L15R-stable-Fc_chains 1 and 2 [ka]

[0354] Fc-GGGSKTHTGGGS-IL-15-(GGS)5-IL-15Rα homodimer (SEQ ID NO: 18) >P1187_Fc-stable S-L15R_chains 1 and 2 [ka]

[0355] Fc-GGGSKTHTGGGS-IL-15-(GGS)2-EPKSSDKTHT-(GGS)2-IL-15Rα homodimer (SEQ ID NO: 19) >P1188_Fc-stable-L22R_chains 1 and 2 [ka]

[0356] Fc-GGGS-IL-15-(GGS)5-IL-15Rα homodimer (SEQ ID NO: 20) >P1250_Fc-4-L15R_Chains 1 and 2 [ka] [ka]

[0357] Fc-GGGS-IL-15-(GGS)2-EPKSSDKTHT-(GGS)2-IL-15Rα homodimer (SEQ ID NO: 21) >P1251_Fc-4-L22R_Chains 1 and 2 [ka]

[0358] Fc-GGGSKTHTGGGS-IL-15-(GGS)2-EPKSSDKTHT-(GGS)2-GGGS-IL-15Rα homodimer (SEQ ID NO: 22) >P1252_Fc-stable-L26R_chains 1 and 2 [ka]

[0359] IL-15 (S162A)-(GGS)5-IL-15Rα-GGGGS-Fc homo Dimer (SEQ ID NO:23) >P1253_L15R(IL15-S162A)-stable-Fc)_chains 1 and 2 [ka]

[0360] Fc-GGGSKTHTGGGS-IL-15(S162A)-(GGS)5-IL-15Rα homodimer (SEQ ID NO: 24) >P1254_Fc-stable S-L15R (IL15-S162A)_chains 1 and 2 [ka] [ka]

[0361] Fc-GGGSKTHTGGGS-IL-15 (S162A)-(GGS)2-EPKSSDKTHT-(GGS)2-IL15Rα homodimer (SEQ ID NO: 25) >P1255_Fc-stable-L22R(IL15-S162A)_chains 1 and 2 [ka]

[0362] Fc-GS-ENLYFQG-GS-IL-15-(GGS)5-IL-15Rα homodimer (SEQ ID NO: 26) >P1256_Fc-TEV-L15R_Chains 1 and 2 Strands 1 and 2 [ka]

[0363] Predicted cleavage products from TEV cleavage of P1256

[0364] 1) Fc-GSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 27)

[0365] Strands 1 and 2

[0366] [ka]

[0367] 2) GGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 28)

[0368] Strands 1 and 2

[0369] [ka]

[0370] 3) Fc-GS-ENLYFQG-GS-IL-15-(GGS)5-IL-15Rα / Fc-GSENLYFQ / GGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0371] Chain 1: Fc-GS-ENLYFQG-GS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 29)

[0372] [ka]

[0373] Chain 2: Fc-GSENLYFQ (SEQ ID NO: 30)

[0374] [ka]

[0375] Chain 3: GGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 31)

[0376] [ka]

[0377] Fc-GGGGSENLYFQGGGGGS-IL-15-(GGS)5-IL-15Rα homodimer (SEQ ID NO: 32) >P1261_15LR-13_Fc-linker-TEV-linker-L15R chains 1 and 2 [ka]

[0378] Predicted cleavage products from TEV cleavage of P1261:

[0379] 1) Fc-GGGGSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 33)

[0380] Strands 1 and 2

[0381] [ka]

[0382] 2) GGGGGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 34)

[0383] Strands 1 and 2

[0384] [ka]

[0385] 3) Fc-GGGGS-ENLYFQG-GGGGS-IL-15-(GGS)5-IL-15Rα / Fc-GGGGSENLYFQ / GGGGGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0386] Chain 1: Fc-GGGGS-ENLYFQG-GGGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 35)

[0387] [ka]

[0388] Chain 2: Fc-GGGGSENLYFQ (SEQ ID NO: 36)

[0389] [ka]

[0390] Chain 3: GGGGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 37)

[0391] [ka]

[0392] IL-15(S162A)-(GGS)5-IL-15Rα-GGGGSHHHHHH (SEQ ID NO: 38)

[0393] >P1274_L15R(IL15-S162A)-G4S-his6 strands 1 and 2

[0394] [ka]

[0395] IL-15-(GGS)2-EPKSSDKTHT-(GGS)2-GGGS-IL-15Rα-GGGGSHHHHHH (SEQ ID NO: 39) >P1275_L26R-G4S-his6 [ka]

[0396] IL-15(S162A)-(GGS)2-EPKSSDKTHT-(GGS)2-GGGS-IL-15Rα-GGGGSHHHHHH (SEQ ID NO: 40) >P1276_L26R(IL15-S162A)-G4S-his6 [ka]

[0397] Fc-G-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 41) >F1279__Fc-1-L15R (IL15-S162A) chains 1 and 2 [ka]

[0398] Fc-G-IL-15(S162A)-(GGS)2-EPKSSDKTHT-(GGS)2GGGS-IL-15Rα (SEQ ID NO: 42) >F1280__Fc-1-L26R (IL15_S162A) chains 1 and 2 [ka]

[0399] Fc-GSGGGGSENLYFQGGGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 43) >F1281__Fc-7-TEV-7-L15R chains 1 and 2 [ka]

[0400] Predicted cleavage products from TEV cleavage of P1281:

[0401] 1) Fc-GSGGGGSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 44)

[0402] Strands 1 and 2

[0403] [ka] [ka]

[0404] 2) GGGGGSGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 45)

[0405] Strands 1 and 2

[0406] [ka]

[0407] 3) Fc-GSGGGGS-ENLYFQG-GGGGSGS-IL-15-(GGS)5-IL-15Rα / Fc-GSGGGGSENLYFQ / GGGGGSGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0408] Chain 1: Fc-GSGGGGS-ENLYFQG-GGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 46)

[0409] [ka]

[0410] Chain 2: Fc-GSGGGGSENLYFQ (SEQ ID NO: 47)

[0411] [ka]

[0412] Chain 3: GGGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 48)

[0413] [ka]

[0414] Fc-GGGSGGGGSENLYFQGGGGGSGGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 49) >F1282_Fc-9-TEV-9-L15R chains 1 and 2 [ka] [ka]

[0415] Predicted cleavage products from TEV cleavage of P1282:

[0416] 1) Fc-GGGSGGGGSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 50)

[0417] Strands 1 and 2

[0418] [ka]

[0419] 2) GGGGGSGGGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO:51)

[0420] Strands 1 and 2

[0421] [ka]

[0422] 3) Fc-GGGSGGGGS-ENLYFQG-GGGGSGGGS-IL-15-(GGS)5-IL-15Rα / Fc-GGGSGGGGSENLYFQ / GGGGGSGGGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0423] Chain 1: Fc-GGGSGGGGS-ENLYFQG-GGGGSGGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 52)

[0424] [ka]

[0425] Chain 2: Fc-GGGSGGGGSENLYFQ (SEQ ID NO:53)

[0426] [ka]

[0427] Chain 3: GGGGGSGGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO:54)

[0428] [ka]

[0429] Fc-GSGGGSGGGGSENLYFQGGGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 55) >F1283_Fc-11-TEV-11-L15R chains 1 and 2

[0430] [ka]

[0431] Predicted cleavage products from TEV cleavage of P1283:

[0432] 1) Fc-GSGGGSGGGGSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 56)

[0433] Strands 1 and 2

[0434] [ka]

[0435] 2) GGGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 57)

[0436] Strands 1 and 2

[0437] [ka]

[0438] 3)Fc-GSGGGSGGGS-ENLYFQG-GGGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα / Fc-GSGGGSGGGSENLYFQ / GGGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα (from partial cleavage (product, activity)

[0439] Chain 1: Fc-GSGGGSGGGGS-ENLYFQG-GGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 58)

[0440] [ka] [ka]

[0441] Chain 2: Fc-GSGGGSGGGGSENLYFQ (SEQ ID NO:59)

[0442] [ka]

[0443] Chain 3: GGGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 60)

[0444] [ka]

[0445] Fc-GGGSGGGSGGGSENLYFQGGGGGSGGGSGGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 61) >F1284_Fc-13-TEV-13-L15R chains 1 and 2 [ka]

[0446] Predicted cleavage products from TEV cleavage of P1284:

[0447] 1) Fc-GGGSGGGSGGGGSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 62)

[0448] Strands 1 and 2

[0449] [ka]

[0450] 2) GGGGGSGGGSGGGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 63)

[0451] Strands 1 and 2

[0452] [ka]

[0453] 3) Fc-GGGSGGGSGGGGS-ENLYFQG-GGGGSGGGSGGGS-IL-15-(GGS)5-IL-15Rα / Fc-GGGSGGGSGGGGSENLYFQ / GGGGGSGGGSGGGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0454] Chain 1: Fc-GGGSGGGSGGGGS-ENLYFQG-GGGGSGGGSGGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 64)

[0455] [ka]

[0456] Chain 2: Fc-GGGSGGGSGGGGSENLYFQ (SEQ ID NO: 65)

[0457] [ka]

[0458] Chain 3: GGGGGSGGGSGGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 66)

[0459] [ka]

[0460]

[0461] Fc-GSGGGSGGGSGGGSENLYFQGGGGGSGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 67) >F1285_Fc-15-TEV-15-L15R chains 1 and 2 [ka]

[0462] Predicted cleavage products from TEV cleavage of P1285:

[0463] 1) Fc-GSGGGSGGGSGGGGSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 68)

[0464] Strands 1 and 2

[0465] [ka]

[0466] 2) GGGGGSGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 69)

[0467] Strands 1 and 2

[0468] [ka]

[0469] 3) Fc-GSGGGSGGGSGGGGS-ENLYFQG-GGGGSGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα / Fc-GSGGGSGGGSGGGGSENLYFQ / GGGGGSGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0470] Chain 1: Fc-GSGGGSGGGSGGGS-ENLYFQG-GGGGGSGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 70)

[0471] [ka]

[0472] Chain 2: Fc-GSGGGSGGGSGGGGSENLYFQ (SEQ ID NO: 71)

[0473] [ka]

[0474] Chain 3: GGGGGSGGGSGGGSGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 72)

[0475] [ka]

[0476] IL-15-GGSGGSEPKSSDKTHTGGSGGSGGGS-IL-15Rα-G4S-Fc (SEQ ID NO: 73) >P1331_L26R-stable-Fc chains 1 and 2 [ka] [ka]

[0477] IL-15-(GGS)5-IL-15Rα-Fc-a (SEQ ID NO: 74) >P1332_L15R-Fc-a chains 1 and 2

[0478] [ka]

[0479] IL-15-GGSGGSEPKSSDKTHTGGSGGSGGGS-IL-15Rα-Fc-a (SEQ ID NO: 75) >P1333_L26R-Fc-a chains 1 and 2

[0480] [ka]

[0481] Fc-b-GSLGGSGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 76) >P1334_Fc-b-PS1-L15R-S162A chains 1 and 2 [ka]

[0482] Predicted cleavage products from uPA or matriptase cleavage of P1334:

[0483] 1) Fc-b-GSLGGSGR homodimer (product from complete cleavage) (SEQ ID NO: 77)

[0484] Strands 1 and 2

[0485] [ka] [ka]

[0486] 2) SANAGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 78)

[0487] Strands 1 and 2

[0488] [ka]

[0489] 3) Fc-b-GSLGGSGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GSLGGSGR / SANAGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0490] Chain 1: Fc-b-GSLGGSGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 79)

[0491] [ka]

[0492] Chain 2: Fc-b-GSLGGSGR (SEQ ID NO: 80)

[0493] [ka]

[0494] Chain 3: SANAGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 81)

[0495] [ka]

[0496] Predicted cleavage products from legumain cleavage of P1334:

[0497] 1) Fc-b-GSLGGSGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 82)

[0498] Strands 1 and 2

[0499] [ka]

[0500] 2) AGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 83)

[0501] Strands 1 and 2

[0502] [ka]

[0503] 3) Fc-b-GSLGGSGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GSLGGSGRSAN / AGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active

[0504] Chain 1: Fc-b-GSLGGSGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 84)

[0505] [ka]

[0506] Chain 2: Fc-b-GSLGGSGRSAN (SEQ ID NO: 85)

[0507] [ka]

[0508] Chain 3: AGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 86)

[0509] [ka]

[0510]

[0511] Fc-b-GGSLSGRSANAGGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 87)

[0512] >P1335_Fc-b-PS2-L15R-S162A chains 1 and 2

[0513] [ka]

[0514] Predicted cleavage products from uPA or matriptase cleavage of P1335:

[0515] 1) Fc-b-GGSLSGR homodimer (product from complete cleavage) (SEQ ID NO: 88)

[0516] Strands 1 and 2

[0517] [ka]

[0518] 2) SANAGGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 89)

[0519] Strands 1 and 2

[0520] [ka]

[0521] 3) Fc-b-GGSLSGRSANAGGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GGSLSGR / SANAGGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0522] Chain 1: Fc-b-GGSLSGRSANAGGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 90)

[0523] [ka]

[0524] Chain 2: Fc-b-GGSLSGR (SEQ ID NO: 91)

[0525] [ka]

[0526] Chain 3: SANAGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 92)

[0527] [ka] [ka]

[0528] Predicted cleavage products from legumain cleavage of P1335:

[0529] 1) Fc-b-GGSLSGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 93)

[0530] Strands 1 and 2

[0531] [ka]

[0532] 2) AGGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 94)

[0533] Strands 1 and 2

[0534] [ka]

[0535] 3) Fc-b-GGSLSGRSANAGGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GGSLSGRSAN / AGGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0536] Chain 1: Fc-b-GGSLSGRSANAGGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 95)

[0537] [ka]

[0538] Chain 2: Fc-b-GGSLSGRSAN (SEQ ID NO: 96)

[0539] [ka]

[0540] Chain 3: AGGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 97)

[0541] [ka]

[0542]

[0543] Fc-b-GPLGLAGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 98)

[0544] >P1336_Fc-b-PS3-L15R-S162A chains 1 and 2

[0545] [ka]

[0546] Predicted cleavage products from uPA or matriptase cleavage of P1336:

[0547] 1) Fc-b-GPLGLAGR homodimer (product from complete cleavage) (SEQ ID NO: 99)

[0548] Strands 1 and 2

[0549] [ka]

[0550] 2) SANAGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 100)

[0551] Strands 1 and 2

[0552] [ka]

[0553] 3) Fc-b-GPLGLAGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLGLAGR / SANAGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0554] Chain 1: Fc-b-GPLGLAGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 101)

[0555] [ka]

[0556] Chain 2: Fc-b-GPLGLAGR (SEQ ID NO: 102)

[0557] [ka]

[0558] Chain 3: SANAGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 103)

[0559] [ka]

[0560] Predicted cleavage products from legumain cleavage of P1336:

[0561] 1) Fc-b-GPLGLAGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 104)

[0562] Strands 1 and 2

[0563] [ka]

[0564] " 2) AGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 105)

[0565] Strands 1 and 2

[0566] [ka]

[0567] 3) Fc-b-GPLGLAGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLGLAGRSAN / AGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0568] Chain 1: Fc-b-GPLGLAGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 106)

[0569] [ka]

[0570] Chain 2: Fc-b-GPLGLAGRSAN (SEQ ID NO: 107)

[0571] [ka]

[0572] Chain 3: AGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 108)

[0573] [ka]

[0574] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1336:

[0575] 1) Fc-b-GPLG homodimer (product from complete cleavage) (SEQ ID NO: 109)

[0576] Strands 1 and 2

[0577] [ka]

[0578] 2) LAGRSANAGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 110)

[0579] Strands 1 and 2

[0580] [ka]

[0581] 3) Fc-b-GPLGLAGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLG / LAGRSANAGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0582] Chain 1: Fc-b-GPLGLAGRSANAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 111)

[0583] [ka]

[0584] Chain 2: Fc-b-GPLG (SEQ ID NO: 112)

[0585] [ka]

[0586] Chain 3: LAGRSANAGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 113)

[0587] [ka]

[0588]

[0589] Fc-b-PLGLSGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 114) >P1337_Fc-b-PS4-L15R-S162A chains 1 and 2 [ka]

[0590] Predicted cleavage products from uPA or matriptase cleavage of P1337:

[0591] 1) Fc-b-PLGLSGR homodimer (product from complete cleavage) (SEQ ID NO: 115)

[0592] Strands 1 and 2

[0593] [ka]

[0594] 2) SANAGPA-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 116)

[0595] Strands 1 and 2

[0596] [ka]

[0597] 3) Fc-b-PLGLSGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-PLGLSGR / SANAGPA-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0598] Chain 1: Fc-b-PLGLSGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 117)

[0599] [ka]

[0600] Chain 2: Fc-b-PLGLSGR (SEQ ID NO: 118)

[0601] [ka]

[0602] Chain 3: SANAGPA-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 119)

[0603] [ka]

[0604] Predicted cleavage products from legumain cleavage of P1337:

[0605] 1) Fc-b-PLGLSGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 120)

[0606] Strands 1 and 2

[0607] [ka]

[0608] 2) AGPA-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 121)

[0609] Strands 1 and 2

[0610] [ka]

[0611] 3) Fc-b-PLGLSGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-PLGLSGRSAN / AGPA-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0612] Chain 1: Fc-b-PLGLSGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 122)

[0613] [ka]

[0614] Chain 2: Fc-b-PLGLSGRSAN (SEQ ID NO: 123)

[0615] [ka]

[0616] Chain 3: AGPA-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 124)

[0617] [ka]

[0618] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1337:

[0619] 1) Fc-b-PLG homodimer (product from complete cleavage) (SEQ ID NO: 125)

[0620] Strands 1 and 2

[0621] [ka]

[0622] 2) LSGRSANAGPA-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 126)

[0623] Strands 1 and 2

[0624] [ka]

[0625] 3) Fc-b-PLGLSGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-PLG / LSGRSANAGPA-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0626] Chain 1: Fc-b-PLGLSGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 127)

[0627] [ka]

[0628] Chain 2: Fc-b-PLG (SEQ ID NO: 128)

[0629] [ka]

[0630] Chain 3: LSGRSANAGPA-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 129)

[0631] [ka]

[0632] Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 130) >P1338_Fc-b-PS5-L15R-S162A chains 1 and 2 [ka]

[0633] Predicted cleavage products from uPA or matriptase cleavage of P1338:

[0634] 1) Fc-b-PLGLAGR homodimer (product from complete cleavage) (SEQ ID NO: 131)

[0635] Strands 1 and 2

[0636] [ka] [ka]

[0637] 2) SANAGPA-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 132)

[0638] Strands 1 and 2

[0639] [ka]

[0640] 3) Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-PLGLAGR / SANAGPA-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0641] Chain 1: Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 133)

[0642] [ka]

[0643] Chain 2: Fc-b-PLGLAGR (SEQ ID NO: 134)

[0644] [ka]

[0645] Chain 3: SANAGPA-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 135)

[0646] [ka]

[0647] Predicted cleavage products from legumain cleavage of P1338:

[0648] 1) Fc-b-PLGLAGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 136)

[0649] Strands 1 and 2

[0650] [ka] [ka]

[0651] 2) AGPA-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 137)

[0652] Strands 1 and 2

[0653] [ka]

[0654] 3) Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-PLGLAGRSAN / AGPA-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0655] Chain 1: Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 138)

[0656] [ka]

[0657] Chain 2: Fc-b-PLGLAGRSAN (SEQ ID NO: 139)

[0658] [ka]

[0659] Chain 3: AGPA-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 140)

[0660] [ka]

[0661] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1338:

[0662] 1) Fc-b-PLG homodimer (product from complete cleavage) (SEQ ID NO: 141)

[0663] Strands 1 and 2

[0664] [ka] [ka]

[0665] 2) LAGRSANAGPA-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 142)

[0666] Strands 1 and 2

[0667] [ka]

[0668] 3) Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-PLG / LAGRSANAGPA-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0669] Chain 1: Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 143)

[0670] [ka]

[0671] Chain 2: Fc-b-PLG (SEQ ID NO: 144)

[0672] [ka]

[0673] Chain 3: LAGRSANAGPA-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 145)

[0674] [ka]

[0675] Fc-b-GPLGLSGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 146) >P1339_Fc-b-PS6-L15R-S162A chains 1 and 2 [ka] [ka]

[0676] Predicted cleavage products from uPA or matriptase cleavage of P1339:

[0677] 1) Fc-b-GPLGLSGR homodimer (product from complete cleavage) (SEQ ID NO: 147)

[0678] Strands 1 and 2

[0679] [ka]

[0680] 2) SANAGPASG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 148)

[0681] Strands 1 and 2

[0682] [ka]

[0683] 3) Fc-b-GPLGLSGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLGLSGR / SANAGPASG-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0684] Chain 1: Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 149)

[0685] [ka]

[0686] Chain 2: Fc-b-GPLGLSGR (SEQ ID NO: 150)

[0687] [ka]

[0688] Chain 3: SANAGPASG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 151)

[0689] [ka]

[0690] Predicted cleavage products from legumain cleavage of P1339:

[0691] 1) Fc-b-GPLGLSGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 152)

[0692] Strands 1 and 2

[0693] [ka]

[0694] 2) AGPASG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 153)

[0695] Strands 1 and 2

[0696] [ka]

[0697] 3)Fc-b-GPLGLSGRSANGPASG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLGLSGRSAN / AGPA

[0698] Chain 1: Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 154)

[0699] [ka]

[0700] Chain 2: Fc-b-GPLGLSGRSAN (SEQ ID NO: 155)

[0701] [ka]

[0702] Chain 3: AGPASG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 156)

[0703] [ka]

[0704] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1339:

[0705] 1) Fc-b-GPLG homodimer (product from complete cleavage) (SEQ ID NO: 157)

[0706] Strands 1 and 2

[0707] [ka]

[0708] 2) LSGRSANAGPASG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 158)

[0709] Strands 1 and 2

[0710] [ka]

[0711] 3) Fc-b-GPLGLSGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLG / LSGRSANAGPASG-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0712] Chain 1: Fc-b-PLGLAGRSANAGPA-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 159)

[0713] [ka]

[0714] Chain 2: Fc-b-GPLG (SEQ ID NO: 160)

[0715] [ka]

[0716] Chain 3: LSGRSANAGPASG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 161)

[0717] [ka]

[0718] Fc-b-GPLGLAGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 162) >P1340_Fc-b-PS7-L15R-S162A chains 1 and 2 [ka]

[0719] Predicted cleavage products from uPA or matriptase cleavage of P1340:

[0720] 1) Fc-b-GPLGLAGR homodimer (product from complete cleavage) (SEQ ID NO: 163)

[0721] Strands 1 and 2

[0722] [ka]

[0723] 2) SANAGPASG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 164)

[0724] Strands 1 and 2

[0725] [ka]

[0726] 3) Fc-b-GPLGLAGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLGLAGR / SANAGPASG-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0727] Chain 1: Fc-b-GPLGLAGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 165)

[0728] [ka] [ka]

[0729] Chain 2: Fc-b-GPLGLAGR (SEQ ID NO: 166)

[0730] [ka]

[0731] Chain 3: SANAGPASG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 167)

[0732] [ka]

[0733] Predicted cleavage products from legumain cleavage of P1340:

[0734] 1) Fc-b-GPLGLAGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 168)

[0735] Strands 1 and 2

[0736] [ka]

[0737] 2) AGPASG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 169)

[0738] Strands 1 and 2

[0739] [ka]

[0740] 3) Fc-b-GPLGLAGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLGLAGRSAN / AGPASG-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0741] Chain 1: Fc-b-GPLGLAGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 170)

[0742] [ka] [ka]

[0743] Chain 2: Fc-b-GPLGLAGRSAN (SEQ ID NO: 171)

[0744] [ka]

[0745] Chain 3: AGPASG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 172)

[0746] [ka]

[0747] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1340:

[0748] 1) Fc-b-GPLG homodimer (product from complete cleavage) (SEQ ID NO: 173)

[0749] Strands 1 and 2

[0750] [ka]

[0751] 2) LAGRSANAGPASG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 174)

[0752] Strands 1 and 2

[0753] [ka]

[0754] 3) Fc-b-GPLGLAGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GPLG / LAGRSANAGPASG-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0755] Chain 1: Fc-b-GPLGLAGRSANAGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 175)

[0756] [ka] [ka]

[0757] Chain 2: Fc-b-GPLG (SEQ ID NO: 176)

[0758] [ka]

[0759] Chain 3: LAGRSANAGPASG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 177)

[0760] [ka]

[0761] Fc-b-SGPLGLAGRSANAGPAS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 178) >P1341_Fc-b-PS8-L15R-S162A) chains 1 and 2 [ka]

[0762] Predicted cleavage products from uPA or matriptase cleavage of P1341:

[0763] 1) Fc-b-SGPLGLAGR homodimer (product from complete cleavage) (SEQ ID NO: 179)

[0764] Strands 1 and 2

[0765] [ka]

[0766] 2) SANAGPAS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 180)

[0767] Strands 1 and 2

[0768] [ka] [ka]

[0769] 3) Fc-b-SGPLGLAGRSANAGPAS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-SGPLGLAGR / SANAGPAS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0770] Chain 1: Fc-b-SGPLGLAGRSANAGPAS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 181)

[0771] [ka]

[0772] Chain 2: Fc-b-SGPLGLAGR (SEQ ID NO: 182)

[0773] [ka]

[0774] Chain 3: SANAGPAS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 183)

[0775] [ka]

[0776] Predicted cleavage products from legumain cleavage of P1341:

[0777] 1) Fc-b-SGPLGLAGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 184)

[0778] Strands 1 and 2

[0779] [ka]

[0780] 2) AGPAS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 185)

[0781] Strands 1 and 2

[0782] [ka]

[0783] 3) Fc-b-SGPLGLAGRSANAGPAS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-SGPLGLAGRSAN / AGPAS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0784] Chain 1: Fc-b-SGPLGLAGRSANAGPAS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 186)

[0785] [ka]

[0786] Chain 2: Fc-b-SGPLGLAGRSAN (SEQ ID NO: 187)

[0787] [ka]

[0788] Chain 3: AGPAS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 188)

[0789] [ka]

[0790] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1341:

[0791] 1) Fc-b-SGPLG homodimer (product from complete cleavage) (SEQ ID NO: 189)

[0792] Strands 1 and 2

[0793] [ka]

[0794] 2) LAGRSANAGPAS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 190)

[0795] Strands 1 and 2

[0796] [ka]

[0797] 3) Fc-b-SGPLGLAGRSANAGPAS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-SGPLG / LAGRSANAGPAS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0798] Chain 1: Fc-b-SGPLGLAGRSANAGPAS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 191)

[0799] [ka]

[0800] Chain 2: Fc-b-SGPLG (SEQ ID NO: 192)

[0801] [ka]

[0802] Chain 3: LAGRSANAGPAS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 1953)

[0803] [ka]

[0804] Fc-b-SGPASGRSANAPLGLAG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 194) >P1342_Fc-b-PS9-L15R-S162A chains 1 and 2 [ka]

[0805] Predicted cleavage products from uPA or matriptase cleavage of P1342:

[0806] 1) Fc-b-SGPASGR homodimer (product from complete cleavage) (SEQ ID NO: 195)

[0807] Strands 1 and 2

[0808] [ka]

[0809] 2) SANAPLGLAG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 196)

[0810] Strands 1 and 2

[0811] [ka]

[0812] 3) Fc-b-SGPASGRSANAPLGLAG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-SGPASGR / SANAPLGLAG-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0813] Chain 1: Fc-b-SGPASGRSANAPLGLAG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 197)

[0814] [ka]

[0815] Chain 2: Fc-b-SGPASGR (SEQ ID NO: 198)

[0816] [ka]

[0817] Chain 3: SANAPLGLAG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 199)

[0818] [ka]

[0819] Predicted cleavage products from legumain cleavage of P1342:

[0820] 1) Fc-b-SGPASGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 200)

[0821] Strands 1 and 2

[0822] [ka]

[0823] 2) APLGLAG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 201)

[0824] Strands 1 and 2

[0825] [ka]

[0826] 3) Fc-b-SGPASGRSANAPLGLAG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-SGPASGRSAN / APLGLAG-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0827] Chain 1: Fc-b-SGPASGRSANAPLGLAG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 202)

[0828] [ka]

[0829] Chain 2: Fc-b-SGPASGRSAN (SEQ ID NO: 203)

[0830] [ka]

[0831] Chain 3: APLGLAG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 204)

[0832] [ka]

[0833] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1342:

[0834] 1) Fc-b-SGPASGRSANAPLG homodimer (product from complete cleavage) (SEQ ID NO: 205)

[0835] Strands 1 and 2

[0836] [ka]

[0837] 2) LAG-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 206)

[0838] Strands 1 and 2

[0839] [ka]

[0840] 3) Fc-b-SGPASGRSANAPLGLAG-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-SGPASGRSANAPLG / LAG-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0841] Chain 1: Fc-b-SGPASGRSANAPLGLAG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 207)

[0842] [ka]

[0843] Chain 2: Fc-b-SGPASGRSANAPLG (SEQ ID NO: 208)

[0844] [ka]

[0845] Chain 3: LAG-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 209)

[0846] [ka]

[0847] Fc-b-GSGPASGRSANAPLGLAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 210) >P1343_Fc-b-PS10-L15R-S162A chains 1 and 2 [ka]

[0848] Predicted cleavage products from uPA or matriptase cleavage of P1343:

[0849] 1) Fc-b-GSGPASGR homodimer (product from complete cleavage) (SEQ ID NO: 211)

[0850] Strands 1 and 2

[0851] [ka]

[0852] 2) SANAPLGLAGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 212)

[0853] Strands 1 and 2

[0854] [ka]

[0855] 3) Fc-b-GSGPASGRSANAPLGLAGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GSGPASGR / SANAPLGLAGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0856] Chain 1: Fc-b-GSGPASGRSANAPLGLAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 213)

[0857] [ka]

[0858] Chain 2: Fc-b-GSGPASGR (SEQ ID NO: 214)

[0859] [ka]

[0860] Chain 3: SANAPLGLAGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 215)

[0861] [ka]

[0862] Predicted cleavage products from legumain cleavage of P1343:

[0863] 1) Fc-b-GSGPASGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 216)

[0864] Strands 1 and 2

[0865] [ka]

[0866] 2) APLGLAGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 217)

[0867] Strands 1 and 2

[0868] [ka]

[0869] 3) Fc-b-GSGPASGRSANAPLGLAGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GSGPASGRSAN / APLGLAGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0870] Chain 1: Fc-b-GSGPASGRSANAPLGLAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 218)

[0871] [ka]

[0872] Chain 2: Fc-b-GSGPASGRSAN (SEQ ID NO: 219)

[0873] [ka]

[0874] Chain 3: APLGLAGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 220)

[0875] [ka]

[0876] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1343:

[0877] 1) Fc-b-GSGPASGRSANAPLG homodimer (product from complete cleavage) (SEQ ID NO: 221)

[0878] Strands 1 and 2

[0879] [ka]

[0880] 2) LAGS-IL-15-(GGS)5-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 222)

[0881] Strands 1 and 2

[0882] [ka]

[0883] 3) Fc-b-GSGPASGRSANAPLGLAGS-IL-15(S162A)-(GGS)5-IL-15Rα / Fc-b-GSGPASGRSANAPLG / LAGS-IL-15-(GGS)5-IL-15Rα (product from partial cleavage, active)

[0884] Chain 1: Fc-b-GSGPASGRSANAPLGLAGS-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 223)

[0885] [ka]

[0886] Chain 2: Fc-b-GSGPASGRSANAPLG (SEQ ID NO: 224)

[0887] [ka]

[0888] Chain 3: LAGS-IL-15-(GGS)5-IL-15Rα (SEQ ID NO: 225)

[0889] [ka]

[0890]

[0891] Fc-GSENLYFQGGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 226) >P1378_Fc-TEV-L15R_(L:L52C)_(R:S40C) chains 1 and 2 [ka]

[0892] Predicted cleavage products from TEV cleavage of P1378:

[0893] 1) Fc-GSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 227)

[0894] Strands 1 and 2

[0895] [ka]

[0896] 2) GGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 228)

[0897] Strands 1 and 2

[0898] [ka]

[0899] 3) Fc-GSENLYFQGGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) / Fc-GSENLYFQ / GGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[0900] Chain 1: Fc-GSENLYFQGGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 229)

[0901] [ka]

[0902] Chain 2: Fc-GSENLYFQ (SEQ ID NO: 230)

[0903] [ka]

[0904] Chain 3: GGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 231)

[0905] [ka]

[0906] SSFc-GSENLYFQGGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 232)

[0907] >P1379_SSFc-TEV-L15R_(L:L52C)_(R:S40C) strands 1 and 2

[0908] [ka]

[0909] Predicted cleavage products from TEV cleavage of P1379:

[0910] 1) SSFc-GSENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 233)

[0911] Strands 1 and 2

[0912] [ka]

[0913] 2) GGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 234)

[0914] Strands 1 and 2

[0915] [ka]

[0916] 3) SSFc-GSENLYFQGGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) / SSFc-GSENLYFQ / GGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[0917] Chain 1: SSFc-GSENLYFQGGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 235)

[0918] [ka]

[0919] Strand 2: SSFc-GSENLYFQ (SEQ ID NO: 236)

[0920] [ka]

[0921] Chain 3: GGS-IL-15(L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 237)

[0922] [ka]

[0923]

[0924] Fc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 238) >P1380_Fc-b-PS4-L15R-S162A_(L:L52C)_(R:S40C) chains 1 and 2 [ka]

[0925] Predicted cleavage products from uPA or matriptase cleavage of P1380:

[0926] 1) Fc-b-PLGLSGR homodimer (product from complete cleavage) (SEQ ID NO: 239)

[0927] Strands 1 and 2

[0928] [ka]

[0929] 2) SANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 240)

[0930] Strands 1 and 2

[0931] [ka]

[0932] 3) Fc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLGLSGR / SANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[0933] Chain 1: Fc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 241)

[0934] [ka]

[0935] Chain 2: Fc-b-PLGLSGR (SEQ ID NO: 242)

[0936] [ka]

[0937] Chain 3: SANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 243)

[0938] [ka] [ka]

[0939] Predicted cleavage products from legumain cleavage of P1380:

[0940] 1) Fc-b-PLGLSGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 244)

[0941] Strands 1 and 2

[0942] [ka]

[0943] 2) AGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 245)

[0944] Strands 1 and 2

[0945] [ka]

[0946] 3) Fc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLGLSGRSAN / AGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[0947] Chain 1: Fc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 246)

[0948] [ka]

[0949] Chain 2: Fc-b-PLGLSGRSAN (SEQ ID NO: 247)

[0950] [ka]

[0951] Chain 3: AGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 248)

[0952] [ka]

[0953] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1380:

[0954] 1) Fc-b-PLG homodimer (product from complete cleavage) (SEQ ID NO: 249)

[0955] Strands 1 and 2

[0956] [ka]

[0957] 2) LSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 250)

[0958] Strands 1 and 2

[0959] [ka]

[0960] 3) Fc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLG / LSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[0961] Chain 1: Fc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 251)

[0962] [ka]

[0963] Chain 2: Fc-b-PLG (SEQ ID NO: 252)

[0964] [ka]

[0965] Chain 3: LSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 253)

[0966] [ka]

[0967] SSFc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 254) >P1381_SSFc-b-PS4-L15R-S162A_(L:L52C)_(R:S40C) strands 1 and 2 [ka]

[0968] Predicted cleavage products from uPA or matriptase cleavage of P1381:

[0969] 1) SSFc-b-PLGLSGR homodimer (product from complete cleavage) (SEQ ID NO: 255)

[0970] Strands 1 and 2

[0971] [ka]

[0972] 2) SANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 256)

[0973] Strands 1 and 2

[0974] [ka]

[0975] 3) SSFc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / SSFc-b-PLGLSGR / SANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[0976] Chain 1: SSFc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 257)

[0977] [ka]

[0978] Strand 2: SSFc-b-PLGLSGR (SEQ ID NO: 26058)

[0979] [ka]

[0980] Chain 3: SANAGPA-IL-15(S162A,L52C)-(GGS)5-IL- 15Rα(S40C) (SEQ ID NO:259)

[0981] [ka]

[0982] Predicted cleavage products from legumain cleavage of P1381:

[0983] 1) SSFc-b-PLGLSGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 260)

[0984] Strands 1 and 2

[0985] [ka]

[0986] 2) AGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 261)

[0987] Strands 1 and 2

[0988] [ka]

[0989] 3) SSFc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / SSFc-b-PLGLSGRSAN / AGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[0990] Chain 1: SSFc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 262)

[0991] [ka]

[0992] Strand 2: SSFc-b-PLGLSGRSAN (SEQ ID NO: 263)

[0993] [ka]

[0994] Chain 3: AGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 264)

[0995] [ka]

[0996] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1381:

[0997] 1) SSFc-b-PLG homodimer (product from complete cleavage) (SEQ ID NO: 265)

[0998] Strands 1 and 2

[0999] [ka]

[1000] 2) LSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 266)

[1001] Strands 1 and 2

[1002] [ka]

[1003] 3) SSFc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / SSFc-b-PLG / LSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[1004] Chain 1: SSFc-b-PLGLSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 267)

[1005] [ka]

[1006] Strand 2: SSFc-b-PLG (SEQ ID NO: 268)

[1007] [ka]

[1008] Chain 3: LSGRSANAGPA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 269)

[1009] [ka]

[1010]

[1011] Fc-b-PLGLSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 270) >P1382_Fc-b-PS4-L15R-S162A_(L:L52C)_(R:S40C)(PLGLSGRSANAGPA→PLGLSGRSANAG) chains 1 and 2 [ka]

[1012] Predicted cleavage products from uPA or matriptase cleavage of P1382:

[1013] 1) Fc-b-PLGLSGR homodimer (product from complete cleavage) (SEQ ID NO: 271)

[1014] Strands 1 and 2

[1015] [ka]

[1016] 2) SANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 272)

[1017] Strands 1 and 2

[1018] [ka]

[1019] 3) Fc-b-PLGLSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLGLSGR / SANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[1020] Chain 1: Fc-b-PLGLSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 273)

[1021] [ka]

[1022] Chain 2: Fc-b-PLGLSGR (SEQ ID NO: 274)

[1023] [ka]

[1024] Chain 3: SANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 275)

[1025] [ka]

[1026] Predicted cleavage products from legumain cleavage of P1382:

[1027] 1) Fc-b-PLGLSGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 276)

[1028] Strands 1 and 2

[1029] [ka]

[1030] 2) AG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 277)

[1031] Strands 1 and 2

[1032] [ka]

[1033] 3) Fc-b-PLGLSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLGLSGRSAN / AG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[1034] Chain 1: Fc-b-PLGLSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 278)

[1035] [ka]

[1036] Chain 2: Fc-b-PLGLSGRSAN (SEQ ID NO: 279)

[1037] [ka]

[1038] Chain 3: AG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 280)

[1039] [ka]

[1040] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1382:

[1041] 1) Fc-b-PLG homodimer (product from complete cleavage) (SEQ ID NO: 281)

[1042] Strands 1 and 2

[1043] [ka]

[1044] 2) LSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 282)

[1045] Strands 1 and 2

[1046] [ka]

[1047] 3) Fc-b-PLGLSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLG / LSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[1048] Chain 1: Fc-b-PLGLSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 283)

[1049] [ka]

[1050] Chain 2: Fc-b-PLG (SEQ ID NO: 284)

[1051] [ka]

[1052] Chain 3: LSGRSANAG-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 285)

[1053] [ka]

[1054] Fc-b-PLGLSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 286)

[1055] >P1383_Fc-b-PS4-L15R-S162A_(L:L52C)_(R:S40C)(PLGLSGRSANAGPA→PLGLSGRSANA) chains 1 and 2

[1056] [ka]

[1057] Predicted cleavage products from uPA or matriptase cleavage of P1383:

[1058] 1) Fc-b-PLGLSGR homodimer (product from complete cleavage) (SEQ ID NO: 287)

[1059] Strands 1 and 2

[1060] [ka]

[1061] 2) SANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 288)

[1062] Strands 1 and 2

[1063] [ka]

[1064] 3)Fc-b-PLGLSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLGLSGR / SANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (partial cleavage (product from, activity)

[1065] Chain 1: Fc-b-PLGLSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 289)

[1066] [ka]

[1067] Chain 2: Fc-b-PLGLSGR (SEQ ID NO: 290)

[1068] [ka] [ka]

[1069] Chain 3: SANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 291)

[1070] [ka]

[1071] Predicted cleavage products from legumain cleavage of P1383:

[1072] 1) Fc-b-PLGLSGRSAN homodimer (product from complete cleavage) (SEQ ID NO: 292)

[1073] Strands 1 and 2

[1074] [ka]

[1075] 2) A-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from complete cleavage, active) (SEQ ID NO: 293)

[1076] Strands 1 and 2

[1077] [ka]

[1078] 3) Fc-b-PLGLSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLGLSGRSAN / A-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[1079] Chain 1: Fc-b-PLGLSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 294)

[1080] [ka]

[1081] Chain 2: Fc-b-PLGLSGRSAN (SEQ ID NO: 295)

[1082] [ka] [ka]

[1083] Chain 3: A-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 296)

[1084] [ka]

[1085] Predicted cleavage products from MMP-2 or MMP-9 cleavage of P1383:

[1086] 1) Fc-b-PLG homodimer (product from complete cleavage) (SEQ ID NO: 297)

[1087] Strands 1 and 2

[1088] [ka]

[1089] 2) LSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 298)

[1090] Strands 1 and 2

[1091] [ka]

[1092] 3) Fc-b-PLGLSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) / Fc-b-PLG / LSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (product from partial cleavage, active)

[1093] Chain 1: Fc-b-PLGLSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 299)

[1094] [ka]

[1095] Chain 2: Fc-b-PLG (SEQ ID NO: 300)

[1096] [ka]

[1097] Chain 3: LSGRSANA-IL-15(S162A,L52C)-(GGS)5-IL-15Rα(S40C) (SEQ ID NO: 301)

[1098] [ka]

[1099] Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα (SEQ ID NO: 302) >P1393_Fc-1-L26-PS-R(IL15_S162A) chains 1 and 2

[1100] [ka]

[1101] Predicted cleavage products from TEV cleavage of P1393:

[1102] 1) Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQ homodimer (product from full cleavage) (SEQ ID NO: 303)

[1103] Strands 1 and 2

[1104] [ka]

[1105] 2) GGGSGGSGGGS-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 304)

[1106] Strands 1 and 2

[1107] [ka]

[1108] 3) Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα / Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQ / GGGSGGSGGGS-IL-15Rα (product from partial cleavage, Active)

[1109] Chain 1: Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα (SEQ ID NO: 305)

[1110] [ka]

[1111] Chain 2: Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQ (SEQ ID NO: 306)

[1112] [ka]

[1113] Chain 3: GGGSGGSGGGS-IL-15Rα (SEQ ID NO: 307)

[1114] [ka]

[1115]

[1116] Fc-GGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα (SEQ ID NO: 308) >P1394_Fc-6-L26-PS-R(IL15_S162A) chains 1 and 2 [ka]

[1117] Predicted cleavage products from TEV cleavage of P1394:

[1118] 1) Fc-GGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQ homodimer (product from full cleavage) (SEQ ID NO: 309)

[1119] Strands 1 and 2

[1120] [ka] [ka]

[1121] 2) GGGSGGSGGGS-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 310)

[1122] Strands 1 and 2

[1123] [ka]

[1124] 3) Fc-GGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα / Fc-GGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQ / GGGSGGSGGGS-IL-15Rα (product from partial cleavage, active)

[1125] Chain 1: Fc-GGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα (SEQ ID NO: 311)

[1126] [ka]

[1127] Chain 2: Fc-GGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQ (SEQ ID NO: 312)

[1128] [ka]

[1129] Chain 3: GGGSGGSGGGS-IL-15Rα (SEQ ID NO: 313)

[1130] [ka]

[1131]

[1132] Fc-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGSEPKENLYFQGGGSGGGSGGGS-IL-15Rα (SEQ ID NO: 314) >P1395_Fc-12-L26-PS-R (IL15_S162A) chains 1 and 2 [ka] [ka]

[1133] Predicted cleavage products from TEV cleavage of P1395:

[1134] 1) Fc-GGSGGSGGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQ homodimer (product from full cleavage) (SEQ ID NO: 315)

[1135] Strands 1 and 2

[1136] [ka]

[1137] 2) GGGSGGSGGGS-IL-15Rα homodimer (product from full cleavage, active) (SEQ ID NO: 316)

[1138] Strands 1 and 2

[1139] [ka]

[1140] 3) Fc-GGSGGSGGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα / Fc-GGSGGSGGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQ / GGGSGGSGGGS-IL-15Rα (product from partial cleavage, active)

[1141] Chain 1: Fc-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGSEPKENLYFQGGGSGGGSGGGS-IL-15Rα (SEQ ID NO: 317)

[1142] [ka]

[1143] Chain 2: Fc-GGSGGSGGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQ (SEQ ID NO: 318)

[1144] [ka]

[1145] Chain 3: GGGSGGSGGGS-IL-15Rα (SEQ ID NO: 319)

[1146] [ka]

[1147] Fc-G-IL-15(S162A,L52C)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(S40C) (SEQ ID NO: 320)

[1148] >P1396_Fc-1-L26-PS-R (IL15:S162A, L52C_IL15R:S40C) chains 1 and 2

[1149] [ka]

[1150] Predicted cleavage products from TEV cleavage of P1396:

[1151] 1) Fc-G-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ homodimer (product from full cleavage) (SEQ ID NO: 321)

[1152] Strands 1 and 2

[1153] [ka]

[1154] 2) GGGSGGSGGGS-IL-15Rα (S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 322)

[1155] Strands 1 and 2

[1156] [ka]

[1157] 3) Fc-G-IL-15(S162A,L52C)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(S40C) / Fc-G-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ / GGGSGGSGGGS-IL-15Rα(S40C) (product from partial cleavage, active)

[1158] Chain 1: Fc-G-IL-15(S162A,L52C)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(S40C) (SEQ ID NO: 323)

[1159] [ka] [ka]

[1160] Chain 2: Fc-G-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ (SEQ ID NO: 324)

[1161] [ka]

[1162] Chain 3: GGGSGGSGGGS-IL-15Rα (S40C) (SEQ ID NO: 325)

[1163] [ka]

[1164] Fc-GGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(S40C) (SEQ ID NO: 326) >P1397_Fc-6-L26-PS-R (IL15:162A, L52C_IL15R:S40C) chains 1 and 2

[1165] [ka]

[1166] Predicted cleavage products from TEV cleavage of P1397:

[1167] 1) Fc-GGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ homodimer (product from full cleavage) (SEQ ID NO: 327)

[1168] Strands 1 and 2

[1169] [ka]

[1170] 2) GGGSGGSGGGS-IL-15Rα (S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 328)

[1171] Strands 1 and 2

[1172] [ka]

[1173] 3) Fc-GGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(S40C) / Fc-GGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ / GGGSGGSGGGS-IL-15Rα(S40C) (product from partial cleavage, active)

[1174] Chain 1: Fc-GGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(S40C) (SEQ ID NO: 329)

[1175] [ka]

[1176] Chain 2: Fc-GGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ (SEQ ID NO: 330)

[1177] [ka]

[1178] Chain 3: GGGSGGSGGGS-IL-15Rα (S40C) (SEQ ID NO: 331)

[1179] [ka]

[1180]

[1181] Fc-GGSGGSGGSGGS-IL-15(S162A,L52C)-GGGSGGSEPKENLYFQGGGSGGGSGGGS-IL-15Rα(S40C) (SEQ ID NO: 332)

[1182] >P1398_Fc-12-L26-PS-R (IL15:S162A, L52C_IL15R:S40C) chains 1 and 2

[1183] [ka]

[1184] Predicted cleavage products from TEV cleavage of P1398:

[1185] 1) Fc-GGSGGSGGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ homodimer (product from complete cleavage) (SEQ ID NO: 333)

[1186] Strands 1 and 2

[1187] [ka]

[1188] 2) GGGSGGSGGGS-IL-15Rα (S40C) homodimer (product from full cleavage, active) (SEQ ID NO: 334)

[1189] Strands 1 and 2

[1190] [ka]

[1191] 3) Fc-GGSGGSGGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(S40C) / Fc-GGSGGSGGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ / GGGSGGSGGGS-IL-15Rα(S40C) (product from partial cleavage, active) (

[1192] Chain 1: Fc-GGSGGSGGSGGS-IL-15(S162A,L52C)-GGGSGGSEPKENLYFQGGGSGGGSGGGS-IL-15Rα(S40C) (SEQ ID NO: 335)

[1193] [ka]

[1194] Chain 2: Fc-GGSGGSGGSGGS-IL-15(S162A,L52C)-GGSGGSEPKENLYFQ (SEQ ID NO: 336)

[1195] [ka]

[1196] Chain 3: GGGSGGSGGGS-IL-15Rα (S40C) (SEQ ID NO: 337)

[1197] [ka]

[1198] Fc-b-GPLGLAGRSANPGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 3438) >P1423_Fc-b-PS11-L15R-S162A chains 1 and 2 [ka]

[1199] Fc-b-GPLGLAGRSDNHGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 339) >P1424_Fc-b-PS12-L15R-S162A chains 1 and 2 [ka]

[1200] Fc-b-GPLGLAGRSDNPGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 340) >P1425_Fc-b-PS13-L15R-S162A chains 1 and 2 [ka]

[1201] Fc-b-GPLGLAGRSENPGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 341) >P1426_Fc-b-PS14-L15R-S162A chains 1 and 2 [ka] [ka]

[1202] Fc-b-GPLGLAGRSDNLGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 342) >P1427_Fc-b-PS15-L15R-S162A chains 1 and 2 [ka]

[1203] Fc-b-GPLGLAGRNAQVGPASG-IL-15(S162A)-(GGS)5-IL-15Rα (SEQ ID NO: 343) >P1428_Fc-b-PS16-L15R-S162A chains 1 and 2 [ka]

[1204] TEV cleavage site (SEQ ID NO: 344)

[1205] ENLYFQG

[1206] Linker sequence (SEQ ID NO:345)

[1207] GGSGGSGGSGGSGGS

[1208] Exemplary cleavable linker (SEQ ID NO:346)

[1209] GSENLYFQGGS

[1210] Fc-GSLSGRSDNAGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 347)

[1211] >P1471_Fc-PS17-L15R chains 1 and 2

[1212] [ka]

[1213] Fc-GSLSGRSDNDGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 348)

[1214] >P1472_Fc-PS18-L15R chains 1 and 2

[1215] [ka]

[1216] Fc-GSLSGRSDNEGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 349)

[1217] >P1473_Fc-PS19-L15R chains 1 and 2

[1218] [ka]

[1219] Fc-GSLSGRSDNFGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 350)

[1220] >P1474_Fc-PS20-L15R chains 1 and 2

[1221] [ka]

[1222] Fc-GSLSGRSDNGGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 351)

[1223] >P1475_Fc-PS21-L15R chains 1 and 2

[1224] [ka]

[1225] Fc-GSLSGRSDNIGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 352)

[1226] >P1476_Fc-PS22-L15R chains 1 and 2

[1227] [ka]

[1228] Fc-GSLSGRSDNKGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 353)

[1229] >P1477_Fc-PS23-L15R chains 1 and 2

[1230] [ka]

[1231] Fc-GSLSGRSDNLGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 354)

[1232] >P1478_Fc-PS24-L15R chains 1 and 2

[1233] [ka]

[1234] Fc-GSLSGRSDNMGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 355)

[1235] >P1479_Fc-PS25-L15R

[1236] [ka]

[1237] Fc-GSLSGRSDNNGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 356)

[1238] >P1480_Fc-PS26-L15R

[1239] [ka] [ka]

[1240] Fc-GSLSGRSDNPGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 357)

[1241] >P1481_Fc-PS27-L15R

[1242] [ka]

[1243] Fc-GSLSGRSDNQGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 358)

[1244] >P1482_Fc-PS28-L15R

[1245] [ka]

[1246] Fc-GSLSGRSDNRGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 359)

[1247] >P1483_Fc-PS29-L15R

[1248] [ka]

[1249] Fc-GSLSGRSDNSGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 360)

[1250] >P1484_Fc-PS30-L15R

[1251] [ka] [ka]

[1252] Fc-GSLSGRSDNTGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 361)

[1253] >P1485_Fc-PS31-L15R

[1254] [ka]

[1255] Fc-GSLSGRSDNVGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 362)

[1256] >P1486_Fc-32-L15R

[1257] [ka]

[1258] Fc-GSLSGRSDNWGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 363)

[1259] >P1487_Fc-PS33-L15R

[1260] [ka]

[1261] Fc-GSLSGRSDNYGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 364)

[1262] >P1488_Fc-PS34-L15R

[1263] [ka] [ka]

[1264] Fc-GSLSGRSANDGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 365)

[1265] >P1489_Fc-PS35-L15R

[1266] [ka]

[1267] Fc-GSLSGRSANEGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 366)

[1268] >P1490_Fc-PS36-L15R

[1269] [ka]

[1270] Fc-GSLSGRSANFGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 367)

[1271] >P1491_Fc-PS37-L15R

[1272] [ka]

[1273] Fc-GSLSGRSANGGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 368)

[1274] >P1492_Fc-PS38-L15R

[1275] [ka]

[1276] Fc-GSLSGRSANHGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 369)

[1277] >P1493_Fc-PS39-L15R

[1278] [ka]

[1279] Fc-GSLSGRSANIGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 370)

[1280] >P1494_Fc-PS40-L15R

[1281] [ka]

[1282] Fc-GSLSGRSANKGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 371)

[1283] >P1495_Fc-PS41-L15R

[1284] [ka]

[1285] Fc-GSLSGRSANLGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 372)

[1286] >P1496_Fc-PS42-L15R

[1287] [ka]

[1288] Fc-GSLSGRSANMGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 373)

[1289] >P1497_Fc-PS43-L15R

[1290] [ka]

[1291] Fc-GSLSGRSANNGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 374)

[1292] >P1498_Fc-PS44-L15R

[1293] [ka]

[1294] Fc-GSLSGRSANPGs-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 375)

[1295] >P1499_Fc-PS45-L15R

[1296] [ka]

[1297] Fc-GSLSGRSANQGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 376)

[1298] >P1500_Fc-PS46-L15R

[1299] [ka]

[1300] Fc-GSLSGRSANRGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 377)

[1301] >P1501_Fc-PS47-L15R

[1302] [ka]

[1303] Fc-GSLSGRSANSGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 378)

[1304] >P1502_Fc-PS48-L15R

[1305] [ka]

[1306] Fc-GSLSGRSANTGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 379)

[1307] >P1503_Fc-PS49-L15R

[1308] [ka]

[1309] Fc-GSLSGRSANVGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 380)

[1310] >P1504_Fc-PS50-L15R

[1311] [ka]

[1312] Fc-GSLSGRSANWGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 381)

[1313] >P1505_Fc-PS51-L15R

[1314] [ka] [ka]

[1315] Fc-GSLSGRSANYGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 382)

[1316] >P1506_Fc-PS52-L15R

[1317] [ka]

[1318] Fc-b-GPLGLAGRSDNHSG-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 383)

[1319] >P1511_Fc-b-PS12b-L15R-S162A

[1320] [ka]

[1321] Fc-PLGLAGSGRSDNR-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 384)

[1322] >P1540_Fc-PS53-L15R

[1323] [ka]

[1324] Fc-PLGLAGSGRSDNRGS-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 385)

[1325] >P1541_Fc-PS54-L15R

[1326] [ka] [ka]

[1327] Fc-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGRSDNRGS-IL-15Rα (SEQ ID NO: 386)

[1328] >P1542_Fc-Stable SL-PS55-R

[1329] [ka]

[1330] Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGRSDNRGS-IL-15Rα (SEQ ID NO: 387)

[1331] >P1636_(Fc)SPG-Stable_Linker-L-PS55-R

[1332] [ka]

[1333] Fc-GGGSAPTSSGSLSGRSDNHGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 388)

[1334] >P1637_(Fc)SPGK-GGGSAPTSS-GSLSGRSDNHGS-L15R

[1335] [ka]

[1336] Fc-GSLSGRSDNRGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 389)

[1337] >P1638_(Fc)SPGK-GSLSGRSDNRGS-L15R

[1338] [ka] [ka]

[1339] Fc-delK-GSLSGRSDNHGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 390)

[1340] >P1639_(Fc)SPG-GSLSGRSDNHGS-L15R

[1341] [ka]

[1342] Fc-delK-PLGLAGSGRSDNRGA-IL-15(D8N,S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 391)

[1343] >P1652_Fc_delK_PS103_L15R(IL15-D8N-S162A)

[1344] [ka]

[1345] Fc-delK-PLGLAGSGRSDNRGA-IL-15(E64Q,S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 392)

[1346] >P1653_Fc_delK_PS103_L15R(IL15-E64Q-S162A)

[1347] [ka]

[1348] Fc-delK-PLGLAGSGRSDNRGA-IL-15(E64K,S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 393)

[1349] >P1654_Fc_delK_PS103_L15R(IL15-E64K-S162A)

[1350] [ka] [ka]

[1351] Fc-delK-PLGLAGSGRSDNRGA-IL-15(N65A, S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 394)

[1352] >P1655_Fc_delK_PS103_L15R(IL15-N65A-S162A)

[1353] [ka]

[1354] Fc-delK-PLGLAGSGRSDNRGA-IL-15(N65D,S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 395)

[1355] >P1656_Fc_delK_PS103_L15R(IL15-N65D-S162A)

[1356] [ka]

[1357] Fc-delK-PLGLAGSGRSDNRGA-IL-15(I68D,S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 396)

[1358] >P1657_Fc_delK_PS103_L15R(IL15-I68D-S162A)

[1359] [ka]

[1360] Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 397)

[1361] >P1658_Fc_delK_PS104_L15R(IL15-S162A)

[1362] [ka]

[1363] Fc-delK-PLGLAGSGRSDNYGA-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 398)

[1364] >P1659_Fc_delK_PS105_L15R(IL15-S162A)

[1365] [ka]

[1366] Fc-delK-PLGLAGSGRSDNYGA-IL-15(S162A)-(GGS)3-IL-15Rα (IL-15opt1, IL-15Ropt) (SEQ ID NO: 399)

[1367] >P1660_1659a_Fc_delK-PS105-L15R(IL-15-S162A, IL-15opt1, IL-15Ropt)

[1368] [ka]

[1369] [ka] [ka]

[1370] Fc-delK-PLGLAGSGRSDNYGA-IL-15(S162A)-(GGS)3-IL-15Rα(IL-15opt2, IL-15Ropt) (SEQ ID NO: 401)

[1371] >P1661_1659b_Fc_delK-PS105-L15R(IL-15-S162A, IL-15opt2, IL-15Ropt)

[1372] [ka]

[1373] [ka]

[1374] Fc-delK-PLGLAGSGRSDNYGA-IL-15(S162A)-(GGS)3-IL-15Rα(IL-15co,IL-15Raco) (SEQ ID NO: 403)

[1375] >P1663_1659c_Fc_delK-PS105-L15R(IL-15-S162A, IL15co, IL15Raco)

[1376] [ka]

[1377] [ka]

[1378] Fc-delK-PLGLAGSGRSDNYGA-IL-15(S162A)-(GGS)3-IL-15Rα-sushi(T2A) (SEQ ID NO: 405)

[1379] >P1682_Fc_delK-PS105-L15Rsushi (IL15_S162A-IL15Rsushi_T2A)

[1380] [ka]

[1381] Fc-delK-PLGLAGSGRSDNYGA-IL-15(L52C,S162A)-(GGS)3-IL-15Rα-sushi(T2A,S40C) (SEQ ID NO: 40 6)

[1382] >P1683_Fc_delK-PS105-L15Rsushi(IL15_L52C_S162A-IL15Rsushi_T2A_S40C)

[1383] [ka]

[1384] Fc-delK-GG-IL-15(S162A)-GPLGLAGSGRSDNQG-IL-15Rα(T2A) (SEQ ID NO: 407)

[1385] >P1696_Fc_delK-GG-L_S162A-GPLGLAGSGRSDNQG-R_T2A(2 / 15Q)

[1386] [ka]

[1387] Fc-delK-GG-IL-15(S162A)-GSPLGLAGSGRSDNQGA-IL-15Rα(T2A) (SEQ ID NO: 408)

[1388] >P1697_Fc_delK-GG-L_S162A-GSPLGLAGSGRSDNQGA-R_T2A(2 / 17Q)

[1389] [ka]

[1390] Fc-delK-GG-IL-15(S162A)-GGSPLGLAGSGRSDNQGGA-IL-15Rα(T2A) (SEQ ID NO: 409)

[1391] >P1698_Fc_delK-GG-L_S162A-GGSPLGLAGSGRSDNQGGA-R_T2A (2 / 19Q)

[1392] [ka] [ka]

[1393] Fc-delK-GG-IL-15(S162A)-GGGSPLGLAGSGRSDNQGGGA-IL-15Rα(T2A) (SEQ ID NO: 410)

[1394] >P1699_15LR-119_Fc_delK-GG-L_S162A-GGGSPLGLAGSGRSDNQGGGA-R_T2A(2 / 21Q)

[1395] [ka]

[1396] Fc-delK-GG-IL-15(S162A)-GGSGSPLGLAGSGRSDNQGGGGA-IL-15Rα(T2A) (SEQ ID NO: 411)

[1397] >P1700_Fc_delK-GG-L_S162A-GGSGSPLGLAGSGRSDNQGGGGA-R_T2A(2 / 23Q)

[1398] [ka]

[1399] Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) (SEQ ID NO: 412)

[1400] >P1701_Fc_delK-GG-L_S162A-GGSGGSPLGLAGSGRSDNQGGSGGA-R_T2A(2 / 25Q)

[1401] [ka]

[1402] Fc-delK-GG-IL-15(S162A)-GPLGLAGSGRSDNRG-IL-15Rα(T2A) (SEQ ID NO: 413)

[1403] >P1702_Fc_delK-GG-L_S162A-GPLGLAGSGRSDNRG-R_T2A(2 / 15R)

[1404] [ka] [ka]

[1405] Fc-delK-GG-IL-15(S162A)-GSPLGLAGSGRSDNRGA-IL-15Rα(T2A) (SEQ ID NO: 414)

[1406] >P1703_Fc_delK-GG-L_S162A-GSPLGLAGSGRSDNRGA-R_T2A(2 / 17R)

[1407]

Chem.

[1408] Fc-delK-GG-IL-15(S162A)-GGSPLGLAGSGRSDNRGGA-IL-15Rα(T2A)(SEQ ID NO: 415)

[1409] >P1704_Fc_delK-GG-L_S162A-GGSPLGLAGSGRSDNRGGA-R_T2A(2 / 19R)

[1410]

Chem.

[1411] Fc-delK-GG-IL-15(S162A)-GGGSPLGLAGSGRSDNRGGGA-IL-15Rα(T2A)(SEQ ID NO: 416)

[1412] >P1705_Fc_delK-GG-L_S162A-GGGSPLGLAGSGRSDNRGGGA-R_T2A(2 / 21R)

[1413]

Chem.

[1414] Fc-delK-GG-IL-15(S162A)-GGSGSPLGLAGSGRSDNRGGGGA-IL-15Rα(T2A)(SEQ ID NO: 417)

[1415] >P1706_Fc_delK-GG-L_S162A-GGSGSPLGLAGSGRSDNRGGGGA-R_T2A (2 / 23R)

[1416] [ka]

[1417] Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNRGGSGGA-IL-15Rα(T2A) (SEQ ID NO: 418)

[1418] >P1707_Fc_delK-GG-L_S162A-GGSGGSPLGLAGSGRSDNRGGSGGA-R_T2A (2 / 25R)

[1419] [ka]

[1420] Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(A50C,T58C) (SEQ ID NO: 419)

[1421] >P1973_Fc-1-L26-PS-R (IL15_S162A, IL15Rα_A50C_T58C)

[1422] [ka]

[1423] Fc-GGSGGS-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(A50C,T58C) (SEQ ID NO: 420)

[1424] >P1974_Fc-6-L26-PS-R (IL15_S162A, IL15Rα_A50C_T58C)

[1425] [ka]

[1426] Fc-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGSEPKENLYFQGGGSGGGSGGGS-IL-15Rα(A50C,T58C) (SEQ ID NO: 421)

[1427] >P1975_Fc-12-L26-PS-R (IL15_S162A, IL15Rα_A50C_T58C)

[1428] [ka]

[1429] Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-GGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 422)

[1430] >P2162_Fc_delK-PS104-L12R(IL15-S162A)

[1431] [ka]

[1432] Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-GGSGGSGGS-IL-15Rα (SEQ ID NO: 423)

[1433] >P2163_Fc_delK-PS104-L9R(IL15-S162A)

[1434] [ka]

[1435] Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-GGSGGS-IL-15Rα (SEQ ID NO: 424)

[1436] >P2164_Fc_delK-PS104-L6R(IL15-S162A)

[1437] [ka] [ka]

[1438] Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-GGS-IL-15Rα (SEQ ID NO: 425)

[1439] >P2165_Fc_delK-PS104-L3R(IL15-S162A)

[1440] [ka]

[1441] Fc-delK-GSPLGLAGSGRSDNQGSGA-IL-15(S162A)-GGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 426)

[1442] >P2166_Fc_delK-PS106-L12R(IL15-S162A)

[1443] [ka]

[1444] Fc-delK-GSPLGLAGSGRSDNQGSGA-IL-15(S162A)-GGSGGSGGS-IL-15Rα (SEQ ID NO: 427)

[1445] >P2167_Fc_delK-PS106-L9R(IL15-S162A)

[1446] [ka]

[1447] Fc-delK-GSPLGLAGSGRSDNQGSGA-IL-15(S162A)-GGSGGS-IL-15Rα (SEQ ID NO: 428)

[1448] >P2168_Fc_delK-PS106-L6R(IL15-S162A)

[1449] [ka] [ka]

[1450] Fc-delK-GSPLGLAGSGRSDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (SEQ ID NO: 429)

[1451] >P2169_Fc_delK-PS106-L3R(IL15-S162A)

[1452] [ka]

[1453] Examples of protease cleavage products

[1454] Predicted cleavage products from uPA / matriptase cleavage of P1482:

[1455] 1) Fc-GSLSGR (product from complete cleavage) (SEQ ID NO: 430)

[1456] Strands 1 and 2

[1457] [ka]

[1458] 2) SDNQGS-IL-15-(GGS)3-IL-15Rα (product from full cleavage, active) (SEQ ID NO: 431)

[1459] Strands 1 and 2

[1460] [ka]

[1461] 3) Fc-GSLSGRSDNQGS-IL-15-(GGS)3-IL-15Rα / Fc-GSLSGR / SDNQGS-IL-15-(GGS)3-IL-15Rα (product from partial cleavage, active)

[1462] Chain 1: Fc-GSLSGRSDNQGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 432)

[1463] [ka] [ka]

[1464] Chain 2: Fc-GSLSGR (SEQ ID NO: 433)

[1465] [ka]

[1466] Chain 3: SDNQGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 434)

[1467] [ka]

[1468] Predicted cleavage products from legumain cleavage of P1482:

[1469] 1) Fc-GSLSGRSDN (product from complete cleavage) (SEQ ID NO: 435)

[1470] Strands 1 and 2

[1471] [ka]

[1472] 2) QGS-IL-15-(GGS)3-IL-15Rα (product from complete cleavage, active) (SEQ ID NO: 436)

[1473] Strands 1 and 2

[1474] [ka]

[1475] 3) Fc-GSLSGRSDNQGS-IL-15-(GGS)3-IL-15Rα / Fc-GSLSGRSDN / QGS-IL-15-(GGS)3-IL-15Rα (product from partial cleavage, active)

[1476] Chain 1: Fc-GSLSGRSDNQGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 437)

[1477] [ka]

[1478] Chain 2: Fc-GSLSGRSDN (SEQ ID NO: 438)

[1479] [ka] [ka]

[1480] Chain 3: QGS-IL-15-(GGS)3-IL-15Rα (SEQ ID NO: 439)

[1481] [ka]

[1482] Predicted cleavage products from uPA / matriptase cleavage of P1540:

[1483] 1) Fc-PLGLAGSGR (product from complete cleavage) (SEQ ID NO: 440)

[1484] Strands 1 and 2

[1485] [ka]

[1486] 2) SDNR-IL-15(S162A)-(GGS)3-IL-15Rα (product from full cleavage, active) (SEQ ID NO: 441)

[1487] Strands 1 and 2

[1488] [ka]

[1489] 3) Fc-PLGLAGSGRSDNR-IL-15(S162A)-(GGS)3-IL-15Rα / Fc-PLGLAGSGR / SDNR-IL-15(S162A)-(GGS)3-IL-15Rα (product from partial cleavage, active)

[1490] Chain 1: Fc-PLGLAGSGRSDNR-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 442)

[1491] [ka]

[1492] Chain 2: Fc-PLGLAGSGR (SEQ ID NO: 443)

[1493] [ka]

[1494] Chain 3: SDNR-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 44)

[1495] [ka]

[1496] Predicted cleavage products from MMP-2 / MMP-9 cleavage of P1540:

[1497] 1) Fc-PLG (product from complete cleavage) (SEQ ID NO: 445)

[1498] Strands 1 and 2

[1499] [ka]

[1500] 2) LAGSGRSDNR-IL-15(S162A)-(GGS)3-IL-15Rα (product from full cleavage, active) (SEQ ID NO: 446)

[1501] Strands 1 and 2

[1502] [ka]

[1503] 3) Fc-PLGLAGSGRSDNR-IL-15(S162A)-(GGS)3-IL-15Rα / Fc-PLG / LAGSGRSDNR-IL-15(S162A)-(GGS)3-IL-15Rα (product from partial cleavage, active)

[1504] Chain 1: Fc-PLGLAGSGRSDNR-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 447)

[1505] [ka]

[1506] Chain 2: Fc-PLG (SEQ ID NO: 448)

[1507] [ka]

[1508] Chain 3: LAGSGRSDNR-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 449)

[1509] [ka]

[1510] Predicted cleavage products from uPA / matriptase cleavage of P1636:

[1511] 1) Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGR (product from complete cleavage) (SEQ ID NO: 450)

[1512] Strands 1 and 2

[1513] [ka]

[1514] 2) SDNRGS-IL-15Rα (product from full cleavage, active) (SEQ ID NO: 451)

[1515] Strands 1 and 2

[1516] [ka]

[1517] 3) Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGRSDNRGS-IL-15Rα / Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGR / DNRGS-IL-15Rα (product from partial cleavage, active)

[1518] Chain 1: Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGRSDNRGS-IL-15Rα (SEQ ID NO: 452)

[1519] [ka]

[1520] Chain 2: Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGR (SEQ ID NO: 453)

[1521] [ka] [ka]

[1522] Chain 3: SDNRGS-IL-15Rα (SEQ ID NO: 454)

[1523] [ka]

[1524] Predicted cleavage products from MMP-2 / MMP-9 cleavage of P1636:

[1525] 1) Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLG (product from complete cleavage) (SEQ ID NO: 455)

[1526] Strands 1 and 2

[1527] [ka]

[1528] 2) LAGSGRSDNRGS-IL-15Rα (product from full cleavage, active) (SEQ ID NO: 456)

[1529] Strands 1 and 2

[1530] [ka]

[1531] 3) Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGRSDNRGS-IL-15Rα / Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLG / LAGSGRSDNRGS-IL-15Rα (product from partial cleavage, active)

[1532] Chain 1: Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLGLAGSGRSDNRGS-IL-15Rα (SEQ ID NO: 457)

[1533] [ka]

[1534] Chain 2: Fc-delK-GGGSKTHTGGGS-IL-15(S162A)-GSPLG (SEQ ID NO: 458)

[1535] [ka] [ka]

[1536] Chain 3: LAGSGRSDNRGS-IL-15Rα (SEQ ID NO: 459)

[1537] [ka]

[1538] Predicted cleavage products from uPA / matriptase cleavage of P1658:

[1539] 1) Fc-delK-PLGLAGSGR (product from complete cleavage) (SEQ ID NO: 460)

[1540] Strands 1 and 2

[1541] [ka]

[1542] 2) SDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (product from complete cleavage, active) (SEQ ID NO: 461)

[1543] Strands 1 and 2

[1544] [ka]

[1545] 3) Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα / Fc-delK-PLGLAGSGR / SDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (product from partial cleavage, active)

[1546] Chain 1: Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 462)

[1547] [ka]

[1548] Chain 2: Fc-delK-PLGLAGSGR (SEQ ID NO: 463)

[1549] [ka]

[1550] Chain 3: SDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 464)

[1551] [ka]

[1552] Predicted cleavage products from MMP-2 / MMP-9 cleavage of P1658:

[1553] 1) Fc-delK-PLG (product from complete cleavage) (SEQ ID NO: 465)

[1554] Strands 1 and 2

[1555] [ka]

[1556] 2) LAGSGRSDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (product from full cleavage, active) (SEQ ID NO: 466)

[1557] Strands 1 and 2

[1558] [ka]

[1559] 3) Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα / Fc-delK-PLG / LAGSGRSDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (product from partial cleavage, active)

[1560] Chain 1: Fc-delK-PLGLAGSGRSDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 467)

[1561] [ka]

[1562] Chain 2: Fc-delK-PLG (SEQ ID NO: 468)

[1563] [ka]

[1564] Chain 3: LAGSGRSDNQGA-IL-15(S162A)-(GGS)3-IL-15Rα (SEQ ID NO: 469)

[1565] [ka]

[1566] Predicted cleavage products from uPA / matriptase cleavage of P1701:

[1567] 1) Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGR (product from complete cleavage) (SEQ ID NO: 470)

[1568] Strands 1 and 2

[1569] [ka]

[1570] 2) SDNQGGSGGA-IL-15Rα (T2A) (product from full cleavage, active) (SEQ ID NO: 471)

[1571] Strands 1 and 2

[1572] [ka]

[1573] 3) Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) / Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGR / SDNQGGSGGA-IL-15Rα(T2A) (product from partial cleavage, active)

[1574] Chain 1: Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) (SEQ ID NO: 472)

[1575] [ka]

[1576] Chain 2: Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGR (SEQ ID NO: 473)

[1577] [ka]

[1578] Chain 3: SDNQGGSGGA-IL-15Rα (T2A) (SEQ ID NO: 474)

[1579] [ka]

[1580] Predicted cleavage products from MMP-2 / MMP-9 cleavage of P1701:

[1581] 1) Fc-delK-GG-IL-15(S162A)-GGSGGSPLG (product from complete cleavage) (SEQ ID NO: 475)

[1582] Strands 1 and 2

[1583] [ka]

[1584] 2) LAGSGRSDNQGGSGGA-IL-15Rα (T2A) (product from full cleavage, active) (SEQ ID NO: 476)

[1585] Strands 1 and 2

[1586] [ka]

[1587] 3) Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) / Fc-delK-GG-IL-15(S162A)-GGSGGSPLG / LAGSGRSDNQGGSGGA-IL-15Rα(T2A) (product from partial cleavage, active)

[1588] Chain 1: Fc-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) (SEQ ID NO: 477)

[1589] [ka]

[1590] Chain 2: Fc-delK-GG-IL-15(S162A)-GGSGGSPLG (SEQ ID NO: 478)

[1591] [ka]

[1592] Chain 3: LAGSGRSDNQGGSGGA-IL-15Rα (T2A) (SEQ ID NO: 479)

[1593] [ka]

[1594] Predicted cleavage products from TEV cleavage of P1973:

[1595] 1) Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQ (product from full cleavage) (SEQ ID NO: 480)

[1596] Strands 1 and 2

[1597] [ka]

[1598] 2) GGGSGGSGGGS-IL-15Rα (A50C, T58C) (product from full cleavage, active) (SEQ ID NO: 481)

[1599] Strands 1 and 2

[1600] [ka]

[1601] 3) Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(A50C,T58C) / Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQ / GGGSGGSGGGS-IL-15Rα(A50C,T58C) (product from partial cleavage, active)

[1602] Chain 1: Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQGGGSGGSGGGS-IL-15Rα(A50C,T58C) (SEQ ID NO: 482)

[1603] [ka]

[1604] Chain 2: Fc-G-IL-15(S162A)-GGSGGSEPKENLYFQ (SEQ ID NO: 483)

[1605] [ka]

[1606] Chain 3: GGGSGGSGGGS-IL-15Rα (A50C, T58C) (SEQ ID NO: 484)

[1607] [ka]

[1608] Predicted cleavage products from uPA / matriptase cleavage of P2169:

[1609] 1) Fc-delK-GSPLGLAGSGR (product from complete cleavage) (SEQ ID NO: 485)

[1610] Strands 1 and 2

[1611] [ka]

[1612] 2) SDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (product from complete cleavage, active) (SEQ ID NO: 486)

[1613] Strands 1 and 2

[1614] [ka]

[1615] 3) Fc-delK-GSPLGLAGSGRSDNQGSGA-IL-15(S162A)-GGS-IL-15Rα / Fc-delK-GSPLGLAGSGR / SDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (product from partial cleavage, active)

[1616] Chain 1: Fc-delK-GSPLGLAGSGRSDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (SEQ ID NO: 487)

[1617] [ka]

[1618] Chain 2: Fc-delK-GSPLGLAGSGR (SEQ ID NO: 488)

[1619] [ka]

[1620] Chain 3: SDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (SEQ ID NO: 489)

[1621] [ka]

[1622] Predicted cleavage products from MMP-2 / MMP-9 cleavage of P2169:

[1623] 1) Fc-delK-GSPLG (product from complete cleavage) (SEQ ID NO: 490)

[1624] Strands 1 and 2

[1625] [ka]

[1626] 2) LAGSGRSDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (product from complete cleavage, active) (SEQ ID NO: 491)

[1627] Strands 1 and 2

[1628] [ka]

[1629] 3) Fc-delK-GSPLGLAGSGRSDNQGSGA-IL-15(S162A)-GGS-IL-15Rα / Fc-delK-GSPLG / LAGSGRSDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (product from partial cleavage, active)

[1630] Chain 1: Fc-delK-GSPLGLAGSGRSDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (SEQ ID NO: 492)

[1631] [ka]

[1632] Chain 2: Fc-delK-GSPLG (SEQ ID NO: 493)

[1633] [ka]

[1634] Chain 3: LAGSGRSDNQGSGA-IL-15(S162A)-GGS-IL-15Rα (SEQ ID NO: 494)

[1635] [ka]

[1636] P15431450: FAP-H1L1-huIgG1-PLGLAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 495)

[1637] >Strands 1 and 2: FAP-H1-hG1-PS53-L15R

[1638] [ka]

[1639] >Strands 3 and 4: FAP-L1-huIgKLC (SEQ ID NO: 496)

[1640] [ka]

[1641] P16401450: FAP-H1L1-huIgG1-delK-PLGLAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 497)

[1642] >Strands 1 and 2: FAP-H1-hG1-delK-PS53-L15R

[1643] [ka]

[1644] >Strands 3 and 4: FAP-L1-huIgKLC (SEQ ID NO: 498)

[1645] [ka]

[1646] P18121450:FAP-H1L1-huIgG1(L234A,L235A,P329A)-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A)

[1647] > Chains 1 and 2: FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-R_T2A (SEQ ID NO: 499)

[1648] [ka]

[1649] > Chains 3 and 4: FAP-L1-huIgKLC (SEQ ID NO: 500)

[1650] [ka]

[1651] P18131453:FAP-H2L2-huIgG1(L234A,L235A,P329A)-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A)

[1652] > Chains 1 and 2: FAP-H2-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-R_T2A (SEQ ID NO: 501)

[1653] [ka]

[1654] >Strands 3 and 4: FAP-L2-huIgKLC (SEQ ID NO: 502)

[1655] [ka]

[1656] P18141563:FAP-H4L4-huIgG1(L234A,L235A,P329A)-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A)

[1657] > Chains 1 and 2: FAP-H4-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-R_T2A (SEQ ID NO: 503)

[1658] [ka]

[1659] >Strands 3 and 4: FAP-L4-huIgKLC (SEQ ID NO: 504)

[1660] [ka]

[1661] P18151450:FAP-H1L1-huIgG1(L234A,L235A,P329A)-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNHGGSGGA-IL-15Rα(T2A)

[1662] > Chains 1 and 2: FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNHGGSGGA-R_T2A (SEQ ID NO: 505)

[1663] [ka]

[1664] >Strands 3 and 4: FAP-L1-huIgKLC (SEQ ID NO: 506)

[1665] [ka]

[1666] P18161453:FAP-H2L2-huIgG1(L234A,L235A,P329A)-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNHGGSGGA-IL-15Rα(T2A)

[1667] > Chains 1 and 2: FAP-H2-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNHGGSGGA-R_T2A (SEQ ID NO: 507)

[1668] [ka]

[1669] >Strands 3 and 4: FAP-L1-huIgKLC (SEQ ID NO: 508)

[1670] [ka]

[1671] P18171563:FAP-H4L4-huIgG1(L234A,L235A,P329A)-delK-GG-IL-15(S162A)-GGSGGSPLGLAGSGRSDNHGGSGGA-IL-15Rα(T2A)

[1672] > Chains 1 and 2: FAP-H4-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNHGGSGGA-R_T2A (SEQ ID NO: 509)

[1673] [ka]

[1674] >Strands 3 and 4: FAP-L4-huIgKLC (SEQ ID NO: 510)

[1675] [ka]

[1676] P19681450:FAP-H1L1-huIgG1(L234A,L235A,P329A)-delK-GG-IL-15(K86A,S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A)

[1677] > Chains 1 and 2: FAP-H1-huIgG1(AAA)_delK-GG-L_(K86A,S162A)-GGSGGS PLGLAGSGRSDNQGGSGGA-R_T2A (SEQ ID NO: 511)

[1678] [ka]

[1679] >Strands 3 and 4: FAP-L1-huIgKLC (SEQ ID NO: 512)

[1680] [ka]

[1681] P19691450:FAP-H1L1-huIgG1(L234A,L235A,P329A)-delK-GG-IL-15(K86R,S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A)

[1682] > Chains 1 and 2: FAP-H1-huIgG1(AAA)_delK-GG-L_(K86R,S162A)-GGSGGS PLGLAGSGRSDNQGGSGGA-R_T2A (SEQ ID NO: 513)

[1683] [ka]

[1684] >Strands 3 and 4: FAP-L1-huIgKLC (SEQ ID NO: 514)

[1685] [ka]

[1686] P24872158:FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A)

[1687] > Chains 1 and 2: FAP-H6_huIgG1(AAA)_delK-GGS-PLGLAG-GGS-L_(S162A)-GGGSGGG-SGRSDNQ-GGGSG-R_T2A (SEQ ID NO: 515)

[1688] [ka]

[1689] >Strands 3 and 4: FAP-L7-huIgKLC (SEQ ID NO: 516)

[1690] [ka]

[1691] P24882158:FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSGGG-IL-15Rα(T2A)

[1692] > Chains 1 and 2: FAP-H6_huIgG1(AAA)_delK-GGS-PLGLAG-GGS-L_(S162A)-GGGSGGG-SGRSDNQ-GGGSGGG-R_T2A (SEQ ID NO: 517)

[1693] [ka]

[1694] >Strands 3 and 4: FAP-L7-huIgKLC (SEQ ID NO: 518)

[1695] [ka]

[1696] P24892158:FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSGGGSG-IL-15Rα(T2A)

[1697] > Chains 1 and 2: FAP-H6_huIgG1(AAA)_delK-GGS-PLGLAG-GGS-L_(S162A)-GGGSGGG-SGRSDNQ-GGGSGGGSG-R_T2A (SEQ ID NO: 519)

[1698] [ka]

[1699] >Strands 3 and 4: FAP-L7-huIgKLC (SEQ ID NO: 520)

[1700] [ka]

[1701] P24902158:FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGPLGLAGG-IL-15(S162A)-GGGSGGGSGRSDNQGGGSGGSG-IL-15Rα(T2A)

[1702] > Chains 1 and 2: FAP-H6_huIgG1(AAA)_delK-GG-PLGLAG-G-L_(S162A)-GGGSGGG-SGRSDNQ-GGGSGGGSG-R_T2A (SEQ ID NO: 521)

[1703] [ka]

[1704] >Strands 3 and 4: FAP-L7-huIgKLC (SEQ ID NO: 522)

[1705] [ka]

[1706]

[1707] P24912158:FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSGGSG-IL-15Rα(T2A)

[1708] > Chains 1 and 2: FAP-H6_huIgG1(AAA)_delK-GGS-GGSGGS-GGS-L_(S162A)-GGGSGGG-SGRSDNQ-GGGSGGGSG-R_T2A (SEQ ID NO: 523)

[1709] [ka]

[1710] >Strands 3 and 4: FAP-L7-huIgKLC (SEQ ID NO: 524)

[1711] [ka]

[1712] Examples of protease cleavage products

[1713] Predicted cleavage products from uPA / matriptase cleavage of P15431450:

[1714] 1) FAP-H1L1-huIgG1-PLGLAGSGR / SDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα / FAP-L1-huIgKLC (product from complete cleavage)

[1715] Chains 1 and 2: FAP-H1L1-huIgG1-PLGLAGSGR (SEQ ID NO: 525)

[1716] [ka]

[1717] Chains 3 and 4: SDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 526)

[1718] [ka]

[1719] Chains 5 and 6: FAP-L1-huIgKLC (SEQ ID NO:527)

[1720] [ka]

[1721] 2. product, activity)

[1722] Chain 1: FAP-H1L1-huIgG1-PLGLAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 528)

[1723] [ka]

[1724] Chain 2: FAP-H1L1-huIgG1-PLGLAGSGR (SEQ ID NO: 529)

[1725] [ka]

[1726] Chain 3: SDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 530)

[1727] [ka]

[1728] Chains 4 and 5: FAP-L1-huIgKLC (SEQ ID NO:531)

[1729] [ka]

[1730] Predicted cleavage products from MMP-2 / MMP-9 cleavage of P15431450:

[1731] 1) FAP-H1L1-huIgG1-PLG / LAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα / FAP-L1-huIgKLC (product from complete cleavage)

[1732] Chains 1 and 2: FAP-H1L1-huIgG1-PLG (SEQ ID NO: 532)

[1733] [ka]

[1734] Chains 3 and 4: LAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 533)

[1735] [ka]

[1736] Chains 5 and 6: FAP-L1-huIgKLC (SEQ ID NO:534)

[1737] [ka]

[1738] 2) FAP-H1L1-huIgG1-PLGLAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα / FAP-H1L1-huIgG1-PLG / LAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα / FAP-L1-huIgKLC (product from partial cleavage, active)

[1739] Chain 1: FAP-H1L1-huIgG1-PLGLAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 535)

[1740] [ka]

[1741] Chain 2: FAP-H1L1-huIgG1-PLG (SEQ ID NO: 536)

[1742] [ka]

[1743] Chain 3: LAGSGRSDNR-IL-15(S162A)-GGSGGSGGSGGSGGS-IL-15Rα (SEQ ID NO: 537)

[1744] [ka]

[1745] Chains 4 and 5: FAP-L1-huIgKLC (SEQ ID NO:538)

[1746] [ka]

[1747] Predicted cleavage products from uPA / matriptase cleavage of P18121450:

[1748] 1) FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGR / SDNQGGSGGA-IL-15Rα(T2A) / FAP-L1-huIgKLC (product from complete cleavage)

[1749] Chains 1 and 2: FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGR (SEQ ID NO: 539)

[1750] [ka]

[1751] Chains 3 and 4: SDNQGGSGGA-IL-15Rα (T2A) (SEQ ID NO: 540)

[1752] [ka]

[1753] Chains 5 and 6: FAP-L1-huIgKLC (SEQ ID NO:541)

[1754] [ka]

[1755] 2) FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) / FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGR / SDNQGGSGGA-IL-15Rα(T2A) / FAP-L1-huIgKLC (product from partial cleavage, active)

[1756] Chain 1: FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) (SEQ ID NO: 542)

[1757] [ka]

[1758] Chain 2: FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGR (SEQ ID NO: 543)

[1759] [ka]

[1760] Chain 3: SDNQGGSGGA-IL-15Rα (T2A) (SEQ ID NO: 544)

[1761] [ka]

[1762] Chains 4 and 5: FAP-L1-huIgKLC (SEQ ID NO:545)

[1763] [ka]

[1764] Predicted cleavage products from MMP-2 / MMP-9 cleavage of P18121450:

[1765] 1) FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLG / LAGSGRSDNQGGSGGA-IL-15Rα(T2A) / FAP-L1-huIgKLC (product from complete cleavage)

[1766] Chains 1 and 2: FAP-H1-huIgG1(AAA)_delK-GG-L_(S1 62A)-GGSGGSP LG (SEQ ID NO: 546)

[1767] [ka]

[1768] Chains 3 and 4: LAGSGRSDNQGGSGGA-IL-15Rα (T2A) (SEQ ID NO: 547)

[1769] [ka]

[1770] Chains 5 and 6: FAP-L1-huIgKLC (SEQ ID NO:548)

[1771] [ka]

[1772] 2) FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) / FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLG / LAGSGRSDNQGGSGGA-IL-15Rα(T2A) / FAP-L1-huIgKLC (product from partial cleavage, active)

[1773] Chain 1: FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLGLAGSGRSDNQGGSGGA-IL-15Rα(T2A) (SEQ ID NO: 549)

[1774] [ka]

[1775] Chain 2: FAP-H1-huIgG1(AAA)_delK-GG-L_(S162A)-GGSGGSPLG (SEQ ID NO: 550)

[1776] [ka]

[1777] Chain 3: LAGSGRSDNQGGSGGA-IL-15Rα (T2A) (SEQ ID NO: 551)

[1778] [ka]

[1779] Chains 4 and 5: FAP-L1-huIgKLC (SEQ ID NO: 552)

[1780] [ka]

[1781] Predicted cleavage products from uPA / matriptase cleavage of P24872158:

[1782] 1) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from complete cleavage)

[1783] Chains 1 and 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:553)

[1784] [ka]

[1785] Chains 3 and 4: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 554)

[1786] [ka]

[1787] Chains 5 and 6: FAP-L7-huIgKLC (SEQ ID NO:555)

[1788] [ka]

[1789] 2) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1790] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:556)

[1791] [ka] [ka]

[1792] Chain 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:557)

[1793] [ka]

[1794] Chain 3: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 558)

[1795] [ka]

[1796] Chains 4 and 5: FAP-L7-huIgKLC (SEQ ID NO:559)

[1797] [ka]

[1798] Predicted cleavage products from MMP-2 / MMP-9 cleavage of P24872158:

[1799] 1)FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC(complete (products from cleavage)

[1800] Chains 1 and 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG (SEQ ID NO:560)

[1801] [ka]

[1802] Chains 3 and 4: LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:561)

[1803] [ka] [ka]

[1804] Chains 5 and 6: FAP-L7-huIgKLC (SEQ ID NO:562)

[1805] [ka]

[1806] 2) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1807] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:563)

[1808] [ka]

[1809] Chain 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG (SEQ ID NO:564)

[1810] [ka]

[1811] Chain 3: LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:565)

[1812] [ka] [ka]

[1813] Chains 4 and 5: FAP-L7-huIgKLC (SEQ ID NO:566)

[1814] [ka]

[1815] Predicted cleavage products from uPA / matriptase and MMP-2 / MMP-9 cleavage of P24872158:

[1816] 1)FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from complete cleavage)

[1817] Chains 1 and 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG (SEQ ID NO:567)

[1818] [ka]

[1819] Chains 3 and 4: LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:568)

[1820] [ka]

[1821] Chains 5 and 6: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 569)

[1822] [ka]

[1823] Chains 7 and 8: FAP-L7-huIgKLC (SEQ ID NO: 570)

[1824] [ka]

[1825] 2) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1826] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:571)

[1827] [ka]

[1828] Chain 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG (SEQ ID NO:572)

[1829] [ka]

[1830] Chain 3: LAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:573)

[1831] [ka]

[1832] Chain 4: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 574)

[1833] [ka]

[1834] Chains 5 and 6: FAP-L7-huIgKLC (SEQ ID NO:575)

[1835] [ka]

[1836] 3) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1837] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:576)

[1838] [ka]

[1839] Chain 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:577)

[1840] [ka]

[1841] Chain 3: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 578)

[1842] [ka]

[1843] Chains 4 and 5: FAP-L7-huIgKLC (SEQ ID NO:579)

[1844] [ka]

[1845] 4) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1846] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:580)

[1847] [ka]

[1848] Chain 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG (SEQ ID NO:581)

[1849] [ka]

[1850] Chain 3: LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:582)

[1851] [ka]

[1852] Chains 4 and 5: FAP-L7-huIgKLC (SEQ ID NO:583)

[1853] [ka]

[1854] 5) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1855] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG (SEQ ID NO:584)

[1856] [ka]

[1857] Chain 2: LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:585)

[1858] [ka]

[1859] Chain 3: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:586)

[1860] [ka]

[1861] Chain 4: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 587)

[1862] [ka]

[1863] Chains 5 and 6: FAP-L7-huIgKLC (SEQ ID NO:588)

[1864] [ka]

[1865] 6) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1866] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG (SEQ ID NO:589)

[1867] [ka]

[1868] Chain 2: LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO:590)

[1869] [ka]

[1870] Chain 3: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:591)

[1871] [ka]

[1872] Strand 4: LAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:592)

[1873] [ka]

[1874] Chain 5: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 593)

[1875] [ka]

[1876] Chains 6 and 7: FAP-L7-huIgKLC (SEQ ID NO:594)

[1877] [ka]

[1878] 7)FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2 A) / FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T 2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1879] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:595)

[1880] [ka]

[1881] Chain 2: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 596)

[1882] [ka]

[1883] Chain 3: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:597)

[1884] [ka]

[1885] Strand 4: LAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO:598)

[1886] [ka]

[1887] Chain 5: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 599)

[1888] [ka]

[1889] Chains 6 and 7: FAP-L7-huIgKLC (SEQ ID NO: 600)

[1890] [ka]

[1891] 8) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1892] Chains 1 and 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLGLAGGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO: 601)

[1893] [ka]

[1894] Chains 3 and 4: SDNQGGGSG-IL-15Rα (T2A) (SEQ ID NO: 602)

[1895] [ka]

[1896] Chains 5 and 6: FAP-L7-huIgKLC (SEQ ID NO: 603)

[1897] [ka]

[1898] 9)FAP-H6L7-huIgG1(L234A,L235A,P329A)-de lK-GGSPLG / LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, active)

[1899] Chains 1 and 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSPLG (SEQ ID NO: 604)

[1900] [ka]

[1901] Chains 3 and 4: LAGGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSG-IL-15Rα(T2A) (SEQ ID NO: 605)

[1902] [ka]

[1903] Chains 5 and 6: FAP-L7-huIgKLC (SEQ ID NO: 606)

[1904] [ka]

[1905] Predicted cleavage products from uPA / matriptase cleavage of P24912158:

[1906] 1) FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from complete cleavage)

[1907] Chains 1 and 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO: 607)

[1908] [ka]

[1909] Chains 3 and 4: SDNQGGGSGGGSG-IL-15Rα (T2A) (SEQ ID NO: 608)

[1910] [ka]

[1911] Chains 5 and 6: FAP-L7-huIgKLC (SEQ ID NO: 609)

[1912] [ka]

[1913] 2)FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSGGSG-IL-15Rα(T2A) / FAP-H6L7-huIg G1(L234A,L235A,P329A)-delK-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGGSGR / SDNQGGGSGGGSG-IL-15Rα(T2A) / FAP-L7-huIgKLC (product from partial cleavage, activity)

[1914] Chain 1: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGGSGRSDNQGGGSGGSG-IL-15Rα(T2A) (SEQ ID NO: 610)

[1915] [ka] [ka]

[1916] Chain 2: FAP-H6L7-huIgG1(L234A,L235A,P329A)-delK-GGSGGSGGSGGS-IL-15(S162A)-GGGSGGGSGR (SEQ ID NO: 611)

[1917] [ka]

[1918] Chain 3: SDNQGGGSGGGSG-IL-15Rα (T2A) (SEQ ID NO: 612)

[1919] [ka]

[1920] Chains 4 and 5: FAP-L7-huIgKLC (SEQ ID NO: 613)

[1921] [ka]

[1922] Although the embodiments of the present invention have been described with reference to the accompanying drawings, it should be understood that the present invention is not limited to the precise embodiments, and various changes and modifications can be made thereto by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims. The present invention provides, for example, the following items. (Item 1) An activatable proprotein comprising a first polypeptide and a second polypeptide, wherein said first and second polypeptides each comprise a masking moiety operably linked to IL-15 or a variant thereof via a first linker at their C-terminus, said IL-15 or a variant thereof being linked to IL-15Rα or a variant thereof via a second linker at their C-terminus, said masking moiety masking an active portion of said proprotein. (Item 2) 2. The activatable proprotein according to claim 1, wherein the first linker on the first or second polypeptide, or on both the first and second polypeptides, is a cleavable linker. (Item 3) 3. The activatable proprotein according to any one of items 1 to 2, wherein the second linker on the first or second polypeptide, or on both the first and second polypeptide, is a cleavable linker. (Item 4) 4. The activatable proprotein of any one of items 1 to 3, wherein said IL-15 or variant thereof and said IL-15Rα or variant thereof in said first polypeptide and said IL-15 or variant thereof and said IL-15Rα or variant thereof in said second polypeptide comprise one or more Cys substitution mutations. (Item 5) The IL-15 or variant thereof in the first polypeptide comprising one or more Cys substitution mutations forms a disulfide bond with the IL-15Rα or variant thereof comprising one or more Cys substitution mutations in the second polypeptide. or the IL-15 or variant thereof in the second polypeptide comprising one or more Cys substitution mutations forms a disulfide bond with the IL-15Rα or variant thereof comprising one or more Cys substitution mutations in the first polypeptide. (Item 6) 6. The activatable proprotein according to any one of items 1 to 5, which is a dimeric proprotein. (Item 7) 7. The activatable proprotein of item 6, wherein the dimeric proprotein comprises, from N-terminus to C-terminus, a first polypeptide and a second polypeptide each comprising the masking moiety linked to the IL-15 or variant thereof via the first linker, the IL-15 or variant thereof being linked to the IL-15Rα or variant thereof via the second linker, and the masking moiety of the first polypeptide forms one or more covalent disulfide bonds or one or more non-covalent bonds with the masking moiety of the second polypeptide. (Item 8) 8. The activatable proprotein according to any one of items 1 to 7, wherein the masking moiety in the first polypeptide comprises one or more protein domains. (Item 9) 9. The activatable proprotein according to any one of items 1 to 8, wherein the masking moiety in the second polypeptide comprises one or more protein domains. (Item 10) 10. The activatable proprotein according to any one of items 1 to 9, wherein the masking moiety in the first polypeptide does not bind to an antigen. (Item 11) 10. The activatable proprotein according to any one of items 1 to 9, wherein the masking moiety in the first polypeptide binds to an antigen. (Item 12) 10. The activatable proprotein according to any one of items 1 to 9, wherein the masking moiety in the second polypeptide does not bind to an antigen. (Item 13) 10. The activatable proprotein according to any one of items 1 to 9, wherein the masking moiety in the second polypeptide binds to an antigen. (Item 14) 14. The activatable proprotein according to any one of items 1 to 13, wherein the masking moiety in the first polypeptide comprises the CH1, CH2, CH3, CH2CH3 or CH1CH2CH3 domain of an antibody constant (Fc) region. (Item 15) 15. The activatable proprotein of any one of items 1 to 14, wherein the masking moiety in the first polypeptide comprises one or more heterodimers of at least two different CH3 variant domains of an antibody constant (Fc) region. (Item 16) 12. The activatable proprotein according to any one of items 1 to 9 and 11, wherein the masking moiety in the first polypeptide comprises a fusion of an antigen-binding domain with a CH1, CH2, CH3, CH2CH3 or CH1CH2CH3 domain of an antibody constant (Fc) region. (Item 17) 14. The activatable proprotein according to any one of items 1 to 13, wherein the masking moiety in the second polypeptide comprises the CH1, CH2, CH3, CH2CH3 or CH1CH2CH3 domain of an antibody constant (Fc) region. (Item 18) 18. The activatable proprotein of any one of items 1 to 13 or 17, wherein the masking moiety in the second polypeptide comprises one or more heterodimers of at least two different CH3 variant domains of an antibody constant (Fc) region. (Item 19) 14. The activatable proprotein according to any one of items 1 to 9 and 13, wherein the masking moiety in the second polypeptide comprises a fusion of an antigen-binding domain with a CH1, CH2, CH3, CH2CH3 or CH1CH2CH3 domain of an antibody constant (Fc) region. (Item 20) 20. The activatable proprotein of any one of items 14 to 19, wherein the antibody Fc region is derived from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA or IgM. (Item 21) 13. The activatable proprotein of any one of items 10 or 12, wherein the masking moiety comprises a constant domain of an antibody light chain (CL), said light chain being a lambda or kappa chain. (Item 22) 12. The activatable proprotein of claim 11, wherein the masking moiety in the first polypeptide comprises an antigen-binding domain. (Item 23) 14. The activatable proprotein of claim 13, wherein the masking moiety in the second polypeptide comprises an antigen-binding domain. (Item 24) 24. The activatable proprotein according to any one of items 22 to 23, wherein the antigen-binding domain is a variable heavy domain (VH), a variable domain derived from a heavy chain antibody (VHH) or an antigen-specific peptide. (Item 25) 24. The activatable proprotein of any one of items 22 to 23, wherein the antigen-binding domain comprises an antibody light chain (LC) and an antibody heavy chain (HC). (Item 26) 26. The activatable proprotein according to any one of items 2 to 25, wherein the cleavable linker comprises a cleavage site for a protease. (Item 27) 27. The activatable proprotein according to item 26, wherein the protease is selected from a metalloprotease, a serine protease, a cysteine ​​protease, an aspartic protease, or any combination thereof. (Item 28) 28. The activatable proprotein according to any one of items 26 to 27, wherein the protease cleavage site is cleavable by an MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV, matriptase, uPA, FAP, legumain, PSA, kallikrein, cathepsin A or cathepsin B protease. (Item 29) 29. The activatable proprotein according to any one of items 1 to 28, wherein the masking moiety in the first and / or second polypeptide prevents in vitro or in vivo binding of the IL-15 / IL-15Rα complex in the first and / or second polypeptide to IL-15Rβ / γC present on the surface of lymphocytes or blood cells. (Item 30) 30. The activatable proprotein according to any one of items 2 to 29, wherein cleavage of the masking moiety in either the first and / or second polypeptide in the activatable proprotein results in partial activation of the activatable proprotein. (Item 31) 31. The activatable proprotein according to any one of items 2 to 30, wherein cleavage of the masking moieties in both the first and second polypeptides in the activatable proprotein results in full activation of the activatable proprotein into an active complex of IL-15 / IL-15Rα or a variant thereof. (Item 32) 31. The activatable proprotein of any one of items 1 to 30, wherein the IL-15 or variant thereof is human IL-15 or an amino acid mutant derived therefrom. (Item 33) 31. The activatable proprotein of any one of items 1 to 30, wherein the IL-15Rα or variant thereof comprises human IL-15Rα, a truncated human IL-15Rα or an amino acid mutant derived from human IL-15Rα. (Item 34) 32. The activatable proprotein according to any one of items 1 to 31, wherein the IL-15 / IL-15Rα or variant thereof comprises human IL-15 or an amino acid mutant derived therefrom and human IL-15Rα, a truncated human IL-15Rα or an amino acid mutant derived from human IL-15Rα. (Item 35) 35. The activatable proprotein according to any one of the preceding claims, wherein the activatable proprotein comprises the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 228 or a sequence in the sequence listing comprising a variant thereof having at least 80, 85, 90, 95, 97, 98, 99% identity to the sequence in the sequence listing. (Item 36) 36. A recombinant nucleic acid molecule encoding the activatable proprotein of any one of items 1 to 35. (Item 37) A vector comprising the recombinant nucleic acid molecule of item 36. (Item 38) 36. A pharmaceutical composition comprising an activatable proprotein according to any one of items 1 to 35 and a pharma- ceutically acceptable carrier. (Item 39) An activatable proprotein comprising a fusion of a first polypeptide and a second polypeptide, wherein said first and second polypeptides each comprise IL-15 or a variant thereof operably linked to IL-15Rα or a variant thereof via a first linker at their C-terminus, said IL-15Rα or a variant thereof linked via a second linker at their C-terminus to a masking moiety present on each of said first and second polypeptides, said masking moiety masking an active portion of said proprotein. (Item 40) 40. The activatable proprotein of item 39, wherein the first linker on the first or second polypeptide, or on both the first and second polypeptides, is a cleavable linker. (Item 41) 41. The method according to claim 39, wherein the second linker on the first or second polypeptide or on both the first and second polypeptide is a cleavable linker. 2. An activatable proprotein...

Claims

[Claim 1] The invention described in this specification.

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