HK40002287A

HK40002287A - Compositions and methods for targeted cytokine delivery

Info

Publication number: HK40002287A
Application number: HK19125552.0A
Authority: HK
Inventors: Alexander Sasha KRUPNICK; Eric Reed LAZEAR; John Westwick; Daved Henry FREMONT
Original assignee: Washington University
Priority date: 2016-02-05
Filing date: 2017-02-06
Publication date: 2020-03-20

Description

Compositions and methods for targeted cytokine delivery

Government rights

The invention was made with government support from AI073552, AI019687, AI109948, HHSN272201200026C and HL113931 sponsored by National Institutes of Health. The government has certain rights in this invention.

Reference to related applications

The present application claims benefit from U.S. provisional application No. 62/292,046 filed on 5/2/2016, U.S. provisional application No. 62/342,630 filed on 27/5/2016, U.S. provisional application No. 62/350,056 filed on 14/6/2016, and U.S. provisional application No. 62/419,146 filed on 8/11/2016; the disclosure of each of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure includes compositions and methods for targeted delivery of cytokines and for recruitment of immune cells to target cells. Through specific delivery of cytokines and other agents, the compositions disclosed herein can improve immunotherapy and, in some cases, limit side effects associated with immunotherapy.

Background

Systemic administration of high doses of interleukin 2 (IL 2) is one of the most effective forms of immunotherapy and is currently approved by the FDA for the treatment of several malignancies. The efficacy of this treatment depends on the activation of cytotoxic lymphocytes (CTL), such as natural killer cells (NK) and CD8⁺T lymphocyte (CD 8)⁺CTL). Clinical trials have demonstrated partial or complete tumor responses in about 15% and long-lasting long-term responses with similar cures in up to 5% of patients. Despite these encouraging results in a small number of patients, most patients do not receive benefit or prematurely discontinue IL2 treatment due to complications such as blood pressure changes and pulmonary or systemic capillary leakage. It is believed that the direct effect of IL2 on the vascular endothelium leads to most of these side effects. The efficacy of IL2 is also limited by CD4⁺Foxp3⁺Regulatory T cells (T)_regs) Which reduces tumor immune response. For these reasons, high doses of IL are used2 treatment has become no longer clinically favored.

due to the high affinity trimeric α β γ IL2 receptor (IL 2R), it is composed of vascular endothelial cells and T at baseline_regsExpression, and therefore the side effects and reduced efficacy of IL2 therapy. Thus, CD4⁺Foxp3⁺T_regsand vascular endothelium is activated at much lower doses of IL2 than NK cells, which express the lower affinity β γ chain of IL2R at rest, NK cells do express the high affinity α chain of IL2R after activation and are dependent on the peak cytolytic capacity of the trimeric receptor_regsAnd endothelial cells. Such IL2 derivatives may overcome this clinical obstacle and result in more effective immunotherapy with fewer side effects.

Summary of The Invention

In one aspect, the disclosure provides compositions comprising a cytokine linked to an NKG2D ligand. In a specific embodiment, the NKG2D ligand is an anti-NKG 2D antibody.

In another aspect, the present disclosure provides a composition comprising a ligand for the NKG2D receptor and a targeting molecule. The targeting molecule directs the composition to a binding partner on the target cell and recruits the immune cell upon specific binding of the ligand to the NKG2D receptor on the immune cell. In one example, the ligand is an orthopoxvirus major histocompatibility complex class I-like protein (OMCP). The targeting molecule may be linked or unlinked to the ligand and present with the ligand in a single composition, or administered simultaneously in separate compositions.

In another aspect, the present disclosure provides a method of delivering a cytokine to a target cell comprising contacting the target cell with a composition comprising a cytokine linked to an NKG2D ligand. In yet another aspect, the present disclosure provides a method of activating an immune cell comprising contacting an immune cell with a composition comprising a pro-inflammatory cytokine linked to an NKG2D ligand. The ligand specifically binds to a receptor on an immune cell, thereby activating the cell.

In yet another aspect, the present disclosure provides methods of recruiting and activating immune cells at specific target cells comprising providing a composition comprising a ligand for the NKG2D receptor and a targeting molecule.

In yet another aspect, the present disclosure provides a method of treating a tumor comprising identifying a subject having a tumor and administering to the subject a therapeutically effective amount of a composition comprising a pro-inflammatory cytokine linked to an NKG2D ligand.

In a different aspect, the present disclosure provides a method of treating a viral infection comprising administering to a subject a therapeutically effective amount of a composition comprising a pro-inflammatory cytokine linked to an NKG2D ligand. In other aspects, the disclosure provides chimeric peptides comprising a cytokine peptide and an NKG2D ligand peptide.

In certain aspects, the disclosure provides chimeric peptides comprising a cytokine peptide and an anti-NKG 2D antibody.

In another aspect, the present disclosure provides a composition comprising a cytokine linked to a programmed cell death protein 1 (PD 1) ligand. In a specific embodiment, the PD1 ligand is programmed cell death ligand 1 (PDL 1). In another specific embodiment, the PD1 ligand is programmed cell death ligand 2 (PDL 2).

In another aspect, the present disclosure provides a method of delivering a cytokine to a target cell comprising contacting the target cell with a composition comprising the cytokine linked to a PD1 ligand. In yet another aspect, the present disclosure provides a method of activating an immune cell comprising contacting the immune cell with a composition comprising a pro-inflammatory cytokine linked to a PD1 ligand. The ligand specifically binds to a receptor on an immune cell, thereby activating the cell.

In yet another aspect, the present disclosure provides a method of treating a tumor comprising identifying a subject having a tumor and administering to the subject a therapeutically effective amount of a composition comprising a pro-inflammatory cytokine linked to a PD1 ligand.

In a different aspect, the present disclosure provides a method of treating a viral infection comprising administering to a subject a therapeutically effective amount of a composition comprising a pro-inflammatory cytokine linked to a PD1 ligand. In other aspects, the disclosure provides chimeric peptides comprising a cytokine peptide and a PD1 ligand peptide.

In certain aspects, the present disclosure provides chimeric peptides comprising a cytokine peptide and an anti-PD 1 antibody.

In another different aspect, the present disclosure provides a nucleic acid molecule comprising a sequence encoding a chimeric peptide of the disclosure.

In yet another different aspect, the present disclosure provides a pharmaceutical composition comprising a chimeric peptide of the present disclosure.

In yet another different aspect, the present disclosure provides a method of treating a subject diagnosed with cancer, comprising administering to the subject a pharmaceutical composition of the present disclosure.

Another aspect is a method of treating a tumor by (1) identifying a subject having a tumor; and (2) administering to the subject a therapeutically effective amount of a combination therapy described herein.

Another aspect is a method of treating a viral infection by administering to a subject a therapeutically effective amount of a combination therapy described herein.

In some embodiments of the various methods provided herein, the pharmaceutical compositions of the present disclosure are administered in combination with a PD-1 inhibitor. In certain embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In some embodiments, the PD-1 inhibitor is selected from nivolumab, pembrolizumab, pidilizumab, REGN2810, PDR001, and MEDI 0680.

In other embodiments of the various methods provided herein, the pharmaceutical compositions of the present disclosure are administered in combination with a PD-L1 inhibitor. In certain embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is an antagonistic antibody. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of Duvacizumab, Avermezumab, Atuzumab or BMS-936559, STI-A1010, STI-A1011, STI-A1012, STI-A1013, STI-A1014, and STI-A1015.

Brief Description of Drawings

The specification contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E and FIG. 1F depict graphs, immunoblots and graphs showing production and in vitro evaluation of OMCP-mutIL 2. (FIG. 1A) schematic structure of OMCP-mutIL 2. (FIG. 1B) molecular weight of OMCP-mutIL2 compared to mutIL2 and wild-type IL 2. IL2, mutIL2 and OMCP-mutIL2 are produced in mammalian cells and have higher molecular weights due to glycosylation. The lower mobility band of mutIL2 corresponds to the unglycosylated protein, probably due to lysis of the producing cells. Based on the difference in molecular weight, all cytokines and constructs were administered on a molar basis with 1 μ l of 4.4 μ M solution as defined herein as 1000IU equivalents (IUe). This effectively allows an equimolar comparison between IL2, mutIL2 and OMCP-mutIL2 despite the difference in molecular weight. (FIG. 1C, FIG. 1D) in vitro activation of A/J lymphocyte subpopulations after 36 hours of culture in 100 IUe cytokine or OMCP-mutIL2 construct. (FIG. 1E, FIG. 1F) proliferation of a subpopulation of B6 lymphocytes after 5 days of culture in 1000IUe/ml cytokine or OMCP-mutIL2 construct. The graph represents 3-6 replicates for each condition. Black = saline; blue = wtIL2, red = OMCP-mutIL2, green = mutIL 2.

Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K, 2L, 2M, 2N and 2O depict diagrams and images showing in vivo administration of IL2 and IL2 constructs. Mortality of animals after administration of wtIL2 (fig. 2A) and incidence assessed by weight loss (fig. 2B), ascites and pleural fluid accumulation (representative syringe-fig. 2C; mean values from all mice in the group-fig. 2D) and (fig. 2E) organ inflammation. Animal mortality (fig. 2F, fig. 2H, fig. 2J) and morbidity assessed by weight loss (fig. 2G, fig. 2I, fig. 2K) following administration of high doses of wtIL2 (fig. 2F, fig. 2G), OMCP-mutIL2 (fig. 2H, fig. 2I) and mutIL2 (fig. 2J, fig. 2K) in anti-AsialoGM 1 (solid line) or rabbit IgG treated (dashed line) a/J mice. Weight loss in mice treated with wtIL2, OMCP-mutIL2 or mutIL2 of 200,000 IUe (fig. 2L), ascites (representative syringe-fig. 2M; mean value of all mice in group-fig. 2N) and organ inflammation (fig. 2O). All graphs represent 46 animals per treatment condition. ns p.05, p.01, p.001; black = saline; blue = wtIL2, red = OMCP-mutIL2, green = mutIL 2.

Fig. 3A, fig. 3B, fig. 3C, fig. 3D, fig. 3E, fig. 3F, fig. 3G, fig. 3H, fig. 3I and fig. 3J depict graphs and images showing immunological changes associated with administration of IL2 and IL2 constructs in vivo. (FIG. 3A, FIG. 3B) Total splenocyte counts after the 5 day course of IL2 (blue), mutIL2 (green) and OMCP-mutIL2 (red) at 200,000 IUe. (FIG. 3C) NK cell expansion and activation following IL2, mutIL2, OMCP-mutIL2, high dose IL2, high dose mutIL2 and IL 2/anti-IL 2 complex as measured by cell count in the spleen (upper panel) and KLRG1 upregulation (lower panel). (FIG. 3D) as by cell count in spleen (top panel) and ICOS upregulation (bottom panel) and (FIG. 3E) NK/T in spleen_regRatiometric CD4⁺Foxp3⁺T_regAmplification and activation. Expansion of splenocytes (fig. 3F, fig. 3G) and NK cells (fig. 3H) in B6 mice treated with cytokine or construct of 750,000 IUe. B6T in mouse spleen_regAmplification and activation (FIG. 3I) and NK: T_regRatio (fig. 3J). All ofThe graph represents the mean cell counts. + -. SEM for 5-10 mice per group. ns p>.05; * p<.05; ** p<.01; *** p<001; black = saline; blue = wtIL2, red = OMCP-mutIL2, green = mutIL 2.

Fig. 4A, 4B, 4C, 4D, and 4E depict graphs and images showing cytokine-mediated tumor immunotherapy. (FIG. 4A) in vivo cytotoxicity of YAC-1 lymphoma after intravenous injection. (FIG. 4B, FIG. 4C) LLC tumor growth after the 5 day course of 750,000 IUe cytokine treatment given at 10 doses on days 5-10 post tumor injection. LLC tumor growth in NK cell depleted mice (fig. 4D) or NKG2D deficient mutant mice (fig. 4E). Data represent 5-6 mice per group. ns p.05, p.01, p.001; black = saline; blue = wtIL2, red = OMCP-mutIL2, green = mutIL 2.

Fig. 5A, fig. 5B, fig. 5C, fig. 5D, fig. 5E, fig. 5F, fig. 5G, fig. 5H, fig. 5I, fig. 5J, and fig. 5K depict graphs and schematic diagrams showing IL2 signaling in NK cells. (FIG. 5A, FIG. 5B) i.v. injection 1X10⁶IUe fluorescent dye-labeled cytokine or serum levels after construction. (FIG. 5C) degranulation of NK cells in the presence of cytokines and pentameric OMCP mediated cross-linking of NKG2D, as measured by surface CD107a expression at 1000 IUe/ml. STAT5 phosphorylation in isolated NK cells from a/J (fig. 5D) or B6 mice (fig. 5E) by increasing cytokine dose. STAT5 phosphorylation was attenuated 15 minutes after stimulation by IL2 or OMCP-mutIL2 at 1000IUe/ml (FIG. 5F) or 100 IUe/ml (FIG. 5G). (FIG. 5H) proposed model of competition between NK cells and stromal cells for IL-2. (FIG. 5I) STAT5 phosphorylation of B5 NK cells by wtIL2 and OMCP-mutIL2 in the presence of other splenocytes. (FIG. 5J) wild-type or NKG2D by wtIL2 and OMCP-mutIL2 in the presence of competitive splenocytes^-/-STAT5 phosphorylation by NK cells. (FIG. 5K) STAT5 phosphorous on wild type NK cells in the presence of competitive splenocytes treated with saturating concentrations of rat anti-mouse CD25 (clone 3C 7) or rat IgG isotype control, as measured by fold change relative to saline treated controlsAnd (4) acidifying.

Figure 6 depicts a graph showing that B6NK cells are preferentially activated by low-dose OMCP-mutIL2, but the selectivity disappears at the highest dose of cytokines or in the absence of NKG2D expression by NK cells. The two panels on the left show B6NK cells, and the two panels on the right show BK NKG2D^-/-NK cells.

Fig. 7A, 7B, and 7C depict images showing that inspection of the viscera confirmed limited food consumption after a5 day course of wtIL2 of 200,000 or 750,000 IUe. Figure 7D depicts a graph showing that, unlike the a/J strain, B6 mice were able to tolerate higher doses of wtIL2 and had only modest weight loss after 750,000 IUe. Higher doses of 1,500,000IUe IL2 resulted in increased weight loss. Doses above this regimen resulted in death of the animals.

Figure 8A depicts a graph showing that a/J mice treated with IL 2/anti-IL 2 antibody or high dose mutIL2 lost significant weight during treatment. Most IL 2/anti-IL 2 treated mice failed to survive after complete 200,000 IUe dosing and were sacrificed 4 days after starting treatment thus receiving 160,000-180,000 IUe. FIG. 8B depicts a graph and flow cytometry plot showing NK expansion in A/J spleen with ULBP3-mutIL2 and lower dose of OMCP-mutIL2 (upper panel). NK activation as measured by surface KLRG1 expression on NK cells treated with mutIL2 (green) or ULBP3-mutIL2 (purple) of 200,000 IUe in A/J spleen (lower panel). FIGS. 8C and 8D depict graphs showing CD8 in IL2, OMCP-mut-IL2 or mutIL2 treated mice, unlike in the case of NK cells⁺Or CD4⁺Foxp^3-Little expansion of T cells was evident. Figure 8E depicts a graph showing weight loss in B6 mice treated with high dose mutIL2 or IL 2/anti-IL 2 antibody complex. FIGS. 8F and 8G depict CD8 in B6 mice that showed cytokine treatment⁺Or CD4⁺Foxp3^-Graph of expansion of T cells. The graph represents 5-10 mice per group.

Fig. 9A and 9B depict graphs showing in vitro lysis of a large number of splenocytes against a/J tumors, e.g., LM2 lung adenocarcinoma (fig. 9A) or YAC-1 lymphoma (fig. 9B), after a5 day course of 200,000 IUe cytokine administered at 10 doses. Fig. 9C shows in vitro lysis of LLC lung cancer by B6 splenocytes treated with 750,000 IUe cytokine or construct administered at 10 doses over 5 days.

Figure 10A depicts a flow cytometry plot showing enhanced NK degranulation mediated by a plate-bound anti-NKG 2D antibody (clone a 10), with cytokines added at 1000 IUe/ml. FIG. 10B depicts a flow cytometry plot showing CD69 levels on NK cells cultured under 100 IUe/ml OMCP-mut-IL2 or mutIL2 with pentameric OMCP.

Fig. 11A, 11B, and 11C depict schematic diagrams of differential IL2 binding and activation in vivo. (FIG. 11A) conventional wild-type IL2 preferentially binds to cells, e.g., CD4⁺Foxp3⁺T_regsand vascular endothelium, both of which express the high affinity α chain of the IL2 receptor and signaling beta and gamma chains (fig. 11B) mutations at R38A and F42K in IL2 reduce the affinity for the IL2 receptor α chain (fig. 11C) by linking R38A/F42K IL2 to the high affinity NKG2D ligand OMCP, which delivers and binds to CTLs expressing NKG2D (e.g., NK cells and activated CD 8)⁺T cells) increase. The width of the arrow represents proposed IL2 binding and/or signaling strength.

Fig. 12 depicts a schematic of the experimental design of an immunotherapy experiment.

Fig. 13 depicts a schematic of the experimental design of the vaccination experiment.

Fig. 14A and 14B depict graphs showing lung cancer susceptible and resistant strains of mice. (FIG. 14A) the AJ and 129 mouse strains were sensitive to lung cancer as evidenced by tumor burden, while the B6 and C3H mouse strains were resistant to lung cancer as evidenced by tumor burden. (fig. 14B) B6 and C3H NK cells produced significantly more LM2 lung cancer cell lysis than AJ and 129 NK cells when incubated with freshly isolated NK cells from various mouse strains.

figure 15 depicts a graph showing that a greater percentage of NK cells appear to produce TNF α in "resistant" patients relative to "susceptible" patients in human males.

Figure 16 depicts a graph showing ex vivo (ex vivo) cytokine activation can reverse natural killer cell dysfunction. Mouse NK cells that did not show significant lysis of cancer cells (NK cells from 129 and AJ strains) were much more potent in lysis when treated with IL 2. NK cells from anti-cancer lines also showed an increase in the percentage of specific lysis.

Fig. 17A, 17B, 17C, 17D, 17E, and 17F depict graphs showing binding of fluorescently labeled constructs tested in a large number of splenocytes in vitro at 37 degrees. This construct appears to bind only NK cells (expressing NKG 2D). The red line is the OMCP-IL2 construct. (FIG. 17A) DX5+ CD 3-NK cells; (fig. 17B) CD4+ CD3+ T cells; (fig. 17C) CD8+ CD3+ T cells; (FIG. 17D) CD11C + CD11 b-DCs; (FIG. 17E) CD11c-CD11b + Macs; (FIG. 17F) CD19+ CD 3-B cells.

Fig. 18 depicts a schematic dosing regimen for an IL2 or IL2 construct.

Fig. 19 depicts a schematic dosing regimen of the IL2 or IL2 constructs after irradiation.

FIGS. 20A, 20B, and 20C depict images and alignments of OMCP structure (FIG. 20A) a strip chart of CPXV OMCP showing secondary structural elements, S representing a β chain and H representing a helix.the α 1/α 2 portion of the plateau domain is represented in cyan and magenta colors, respectively, (FIG. 20B) a strip chart of the α 1/α 2 domain of MICA (PDB identifier 1 HYR), and the α 3 domain is removed for clarity the residues in contact with NKG2D are shown in yellow (FIG. 20C) structural alignment of OMCP with NKG2DLs_BR(CPXV-BR-018; GenBank accession number NP-619807; PDB identifier 4 FFE) and OMCP_MPX(MPXV-ZAR-1979-005-198; N3R; GenBank accession No. AAY 97396) aligned with the ectodomain sequence of MICA (1 HYR), MICB (1 JE 6), ULBP3 (1 KCG) and RAE-1 β (1 JFM) the known NKG2D contact residue of NKG2DL is represented in yellow, by the black box in FIG. C and the black boxes in FIGS. A and BThe side chain shows Asn residues that may be glycosylated. OMCPbr = SEQ ID NO:13, OMCPmpx = SEQ ID NO:14, MICA = SEQ ID NO:15, MICB = SEQ ID NO:16, ULBP3= SEQ ID NO:17, and RAE-1B = SEQ ID NO: 18.

Figure 21 depicts a graph showing OMCP targeted delivery of IL 15. Levels of CD25 were significantly higher when IL15 was delivered by OMCP compared to equimolar doses of naked cytokine alone.

Figure 22 depicts a graph showing that the D132R mutation in OMCP significantly reduced its NKG2D binding. NK amplification and activation in the presence of mutIL2, OMCP-mutIL2 and D132ROMCP-mutIL2 were tested. The D132R mutation improves the advantage of natural killer cell activation over cytokine alone.

Fig. 23 depicts various embodiments of the present invention. 1. OMCP helix 2 linked to cytokines is depicted. 2. Pegylation of the composition is described. 3. Compositions comprising engineered glycans are depicted. 4. Various joint lengths and compositions are described. 5. Antibodies linked to cytokines are depicted. For example, Fab-specific NKG2D antibodies. 6. NKG2DL linked to cytokines is depicted. Such as MIC or ULBP. Optional OMCP linked to cytokines is depicted at 7. For example, OMCP max may represent gain of function for NKG2D binding, while mutant OMCP may represent loss of function for NKG2D binding. 8. Retargeting of OMCP in the composition is depicted. For example, OMCP may be directed against NKG2A, NKG2C, NKG2E, and the like. 9. Other viral proteins linked to cytokines are depicted. For example, other viral proteins may also bind to receptors on immune cells. 10. OMCP linked to the mutant cytokine is depicted. It will be appreciated that the OMCP sequence may be from a variety of sources, for example vaccinia or monkeypox. In addition, Fc chimeras of OMCP and IL2, and variants thereof, may be used.

Fig. 24A and 24B depict the structure of OMCP complexed with NKG 2D. (FIG. 24A) OMCP binding to NKG 2D. OMCP is colored in magenta, and the promoter of NKG2D is colored in cyan ("a") and yellow ("B"). NKG2D^AMainly in helical contact with H2a, while NKG2D^BWith H2 b. To facilitate alternate crystal packingthe introduced mutations are shown in red, the S193-S194 bond is shown as a sphere on each NKG2D promoter, asparagine of the putative hNKG2D glycosylation site is shown in orange, asparagine of the confirmed N-glycan site of OMCP is shown in green (data not shown) (fig. 24B) a view of the interface between OMCP-NKG 2D. the α 2 domain of OMCP is shown in front and the α 1 domain is shown in back, OMCP and NKG2D are shown, and the cartoon indicates the backbone, the side chains of the contact residues are shown as rods.

Fig. 25A, 25B, and 25C depict the interfaces of OMCP and NKG 2D. (FIG. 25A) local environment of OMCP-NKG2D binding interface around residues D132R. The D132R mutation abrogated OMCP-NKG2D binding. (FIG. 25B) representative experiments of WT and (D132R) OMCP binding to NKG2D by SPR. 100nM OMCP or (D132R) OMCP was injected at 50. mu.l/min on a flow cell containing immobilized biotinylated murine NKG 2D. (FIG. 25C) Ba/F3 cells transduced with NKG2D, FCRL5 or empty vector were stained with OMCP tetramer (solid line), D132R tetramer (dashed line) or WNV DIII tetramer control (grey histogram). Representative results of three independent experiments.

FIGS. 26A, 26B, 26C and 26D depict the differences in the β 5'- β 5 loop (L2) of human and murine NKG2D (FIG. 26A, 26B) mNKG2D (gray) (PDBID: 1HQ 8) and the structure of OMCP-hNKG2D (yellow and cyan). core binding residues Y152 (Y168) and Y199 (Y215) are position-conserved, whereas core binding residues M184 (I200) are not (FIG. 26C) the surface of OMCP (magenta) interacting with the β 5' - β 5 loop shows the conservation of M184 and Q185 (FIG. 26D). mouse, rat, guinea pig and flying fox NKG2D differs (not shown). the protection score is as calculated by the Conrf server.

Figure 27A, figure 27B, figure 27C, figure 27D, figure 27E, figure 27F, figure 27G, figure 27H and figure 27I depict novel NKG2D binding adaptations. Surface representation of NKG2D and surface and cartoon representations of OMCP, MICA and ULBP 3. NKG2D^AAnd NKG2D^Bthe buried surface regions of OMCP (magenta) and MICA (green) are shown in cyan and yellow, respectively, the buried surface region by NKG2D of OMCP, MICA (green) and ULBP3 (orange), the core binding residues of NKG2D and the NKG2D binding element of NKG2DL are shown, NKG2D (FIG. 27A) and OMCP (FIG. 27B, FIG. 27C) bind the interaction NKG2D (FIG. 27D) and MICA (FIG. 27E, FIG. 27F) bind the interaction, NKG2D (FIG. 27G) and ULBP3 (FIG. 27H, FIG. 27I) bind the interaction as shown by the secondary structure alignment of NKG2DL (PDBID: OMCP (4 annotated FFE), MICA (SEQ ID 1 SEQ R), MICB (1 6), ULBP 539 7 (1 KCG) and RAE-1 β (JJJJJJJJJJJJG) through the oblique fold-locus of the open helix (SEQ ID = 17), the oblique helix contact of the open-NO-1 [ 18 ] and black ". 17. alpha.: SEQ ID = 17, the oblique fold-SEQ ID, indicated by arrows, SEQ ID = 17, SEQ ID, respectively, SEQ ID, and SEQ ID, see the oblique fold, SEQ ID, SEQ.

Fig. 28A, 28B, 28C, 28D, and 28E depict activation of NK cells by cell-associated OMCP. Models of NKG2D interaction with (figure 28A) host, (figure 28B) cancer-induced, (figure 28C) virus, or (figure 28D) chimeric ligand are depicted. Tyrosine phosphorylation of DAP10 (red filled circles) was shown to result in NKG 2D-mediated signaling binding interactions. (FIG. 28E) IL2 activated splenocytes were used as cytotoxic effectors against the stably transduced Ba/F3 cell line. Splenocytes were activated with 200U/ml IL2 for 24 hours. Labeled target cells were mixed with activated splenocytes at effector ratios of 10:1, 20:1 and 40: 1: target ratio was co-incubated for 4 hours. Killing was measured by incorporation of 7AAD by CFSE labeled target cells using flow cytometry. Representative data from five independent experiments are shown.

FIGS. 29A and 29B depict the electron density supporting the cis-peptide conformation, a perspective view of the β 5- β 6 loop of hNKG2D residues 193-Ala-Ser-Ser-Phe-Lys-197 (SEQ ID NO:33) showing the structure of OMCP-hNKG2D (yellow) and the structure of hNKG2D alone (grey). the 2Fo-Fc diagram of OMCP-hNKG2D is shown at 2 σ.

FIGS. 30A and 30B depict graphs showing survival curves of C57BI/6J mice after West Nile Virus (WNV) infection. After infection with WNV, mice were treated with OMCP-IL2, OMCP (D132R) -IL2, IL2, IL (38R/42A) or PBS. Infection with OMCP-IL2 and IL2(38R/42A) resulted in 40% of mice surviving for more than 21 days compared to 0 mice treated with PBS or OMCP (D132R) -IL 2.

Fig. 31A, fig. 31B, fig. 31C, and fig. 31D depict flow cytometry data showing that OMCP-mutant IL2 activates NK and CD8+ T cells. Figure 31A shows that a relatively high proportion of NK cells is evident in the OMCP mutant IL2 group. Figure 31B shows higher perforin levels in OMCP-mutant IL2 treated NK cells (red) compared to saline (black), IL2 (blue) or mutant IL2 (green) treatment. Figure 31C shows that similar to NK cells, higher intracellular levels of perforin were evident in CD8+ T cells treated with OMCP-mutant IL2 compared to other conditions. Figure 31D shows that a relatively higher proportion of activated CD25+ CD 127-regulatory T cells in IL2 treated peripheral blood lymphocyte cultures compared to other conditions was evident when gated on CD4+ Foxp3+ CD45 RA-T cells.

FIG. 32 depicts a schematic of various IL18-OMCP constructs. Three versions were prepared, each with OMCP linked to WT human IL-18, WT murine IL-18, or mutant human IL-18, which inhibits its interaction with IL-18 BP.

FIG. 33 depicts a flow cytometry plot showing IL18-OMCP activation of NK cells. Peripheral blood lymphocytes were cultured in 4.4. mu.M wild-type IL18 (blue), OMCP-IL18 (red) or saline (black) for 48 hours. Activation of CD56+ CD 3-natural killer cells by OMCP-IL18 was superior compared to wild-type IL18 as measured by surface CD69 expression.

Figure 34 depicts lungs of a group of mice treated with isotype antibodies, anti-PD-1 antibodies, OMCP-IL2 and isotype antibodies, and anti-PD-1 antibodies and OMCP.

Figure 35 shows lung weights as measured from the lungs of the mouse cohort of figure 34.

Fig. 36 depicts various embodiments of the present invention. 1. Depicted is a composition comprising full-length PDL1 or PDL2 linked to a cytokine. 2. Depicted is a composition comprising a PDL1 or PDL2 derived peptide linked to a cytokine. 3. Depicted are compositions comprising cytokine-linked PDL1 or PDL2, wherein the composition is pegylated. 4. Depicted are compositions comprising cytokine-linked PDL1 or PDL2, wherein the composition comprises N-glycans. 5. Depicted are compositions comprising PDL1 or PDL2 linked to cytokines, wherein the linkers include various sequences and various lengths. 6. Depicted is a composition comprising a Fab-specific antibody to PD1 linked to a cytokine. 7. Depicted is a composition comprising various PD1 ligands linked to cytokines, including mutant forms of PDL1 or PDL 2. PDL1 or PDL2 may be mutated to have an improved binding affinity or a weaker binding affinity. 8. Depicted are compositions comprising PDL1 or PDL2 linked to a mutated cytokine. It is understood that the PDL1 and PDL2 sequences may be from a variety of sources, such as human, mouse or monkey. In addition, Fc chimeras of PDL1 or PDL2 and IL2 and variants thereof may be used.

FIGS. 37A, 37B, 37C, 37D and 37E depict graphs showing NK cell physiology after in vitro expansion with wild-type IL-2 (blue) or OMCP-mutIL-2 (red). Figure 37A depicts NK cell expansion. Figure 37B depicts PD1 expression on NK cells. Figure 37C depicts NK cell proliferation. Fig. 37C depicts NK cell viability. Fig. 37E depicts flow cytometry plots of Tim3 and Lag3 expression on NK cells.

FIGS. 38A, 38B, 38C, 38D and 38E depict graphs showing T cell physiology after in vitro expansion with wild-type IL-2 (blue) or OMCP-mutIL-2 (red). Fig. 38A depicts expansion of T cells. Fig. 38B depicts PD1 expression on T cells. Fig. 38C depicts T cell proliferation. Fig. 38C depicts the viability of T cells. Fig. 38E depicts flow cytometry plots of Tim3 and Lag3 expression on T cells.

FIG. 39A, FIG. 39B, FIG. 39C and FIG. 39D depict graphs showing anti-NKG 2D antibody-mediated delivery of the R38A/F42K mutant IL-2. At 10U/ml, OMCP-mutant IL-2 showed a trend of increased perforin levels relative to antibody-mediated delivery, but it did not reach statistical significance (fig. 39A). At 100U/ml, NK cells treated with 2HL2 and 2LH2 antibodies synthesized as much perforin as OMCP-mutIL-2 treated cells, but lower levels of perforin were evident in 1HL2 and 1LH2 treated NK cells (fig. 39B). Higher levels of CD25 were evident in wild-type IL-2 treated cultures for all constructs (fig. 39C, fig. 39D). Data are representative of 4-7 independent experiments. P <0.05 and ns = p > 0.05.

Detailed Description

3 certain 3 compositions 3 and 3 methods 3 described 3 herein 3 provide 3 for 3 cytokine 3 delivery 3 to 3 established 3 cells 3 by 3 NKG 32 3D 3 ligands 3 fusion 3 of 3 the 3 cytokine 3 to 3 NKG 32 3D 3 ligands 3 that 3 specifically 3 bind 3 the 3 NKG 32 3D 3 receptor 3 on 3 target 3 cells 3 creates 3 an 3 " 3 address 3" 3 for 3 cytokine 3 delivery 3, 3 specifically 3, 3 using 3 the 3 invention 3 disclosed 3 herein 3, 3 IL 32 3 is 3 directly 3 targeted 3 to 3 lymphocytes 3 by 3 anti 3- 3 NKG 32 3D 3 antibodies 3, 3 such 3 as 3 natural 3 killer 3 ( 3 NK 3) 3 cells 3 and 3 CD 38 3+ 3 cytotoxic 3 T 3 lymphocytes 3 ( 3 CTLs 3) 3, 3 however 3, 3 other 3 NKG 32 3D 3 ligands 3, 3 including 3 but 3 not 3 limited 3 to 3 OMCP 3 ligands 3, 3 ULBP 31 3, 3 ULBP 32 3, 3 ULBP 33 3, 3H 3 60 3, 3 Rae 3- 31 3 α 3, 3 Rae 3- 31 3 β 3, 3 Rae 3- 31 3 δ 3, 3 Rae 3- 31 3 γ 3, 3 MICA 3, 3 MICB 3, 3H 3- 3 HLA 3- 3a 3, 3 may 3 also 3 be 3 used 3 in 3 place 3 of 3 anti 3- 3 NKG 32 3D 3 antibodies 3. 3

Other compositions and methods described herein provide for the activation of NK cells and CTLs and recruitment to specific cells or tissues through the combination of ligands with NKG2D receptors and targeting molecules. In particular, in certain aspects, using the invention disclosed herein, NK cells and CTLs are recruited to target cells by a composition comprising an OMCP ligand or portion thereof and a targeting molecule. The targeting molecules allow recruitment of NK cells and CTLs to specific target cells, with OMCP ligands or portions thereof providing recruitment, and in some cases activation, of NK cells and CTLs, resulting in a site-specific response. Targeting molecules can include any molecule capable of binding a target specific for a cell in a disease state or the extracellular matrix surrounding the diseased cell, including but not limited to receptor ligands and antibodies. Specific aspects of the invention are described in detail below.

I. Composition comprising a metal oxide and a metal oxide

In one aspect, the invention includes a composition comprising a cytokine linked to an immune cell surface protein targeting ligand. In a particular aspect, the cytokine is linked to an NKG2D ligand. In another aspect, the cytokine is linked to a ligand that targets a PD1 surface protein. The composition may further comprise a linker connecting the cytokine to the ligand. Cytokines, ligands, and linkers are described in more detail below. It is to be understood that any cytokine described in detail below may be linked to any ligand described in detail below in the absence or presence of any linker described below. In another aspect, the invention provides nucleic acid molecules encoding a cytokine, a ligand, and optionally a linker.

(a) Cytokine

As used herein, "cytokines" are small proteins (-5-20 kDa) important in cell signaling. Cytokines are released by cells and affect the behavior of other cells and/or cells that release cytokines. Non-limiting examples of cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, monokines, and colony stimulating factors. Cytokines can be produced by a variety of cells including, but not limited to, immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells and monocytes, endothelial cells, fibroblasts and stromal cells. Cytokines may be produced by more than one type of cell. Cytokines act through receptors, are particularly important in the immune system, regulating the balance between humoral and cell-based immune responses, and regulating the maturation, growth, and response of cell populations. Cytokines are important in host responses to infection, immune responses, inflammation, trauma, sepsis, cancer, and reproduction. The cytokine of the present invention may be a naturally occurring cytokine or may be a mutant form of a naturally occurring cytokine. As used herein, "naturally occurring," which may also be referred to as wild-type, includes allelic variations. A mutant form or "mutant" of a naturally occurring cytokine refers to a specific mutation made to a naturally occurring sequence to alter the function, activity and/or specificity of the cytokine. In one embodiment, the mutation may enhance the function, activity and/or specificity of the cytokine. In another embodiment, the mutation may reduce the function, activity and/or specificity of the cytokine. Mutations may include deletions or additions of one or more amino acid residues of the cytokine.

cytokines may be classified based on structure, for example, cytokines may be classified into four types, the four- α -helix bundle family, the IL1 family, the IL17 family, and the cysteine knot cytokines members of the four- α -helix bundle family have a three-dimensional structure with a four-strand α -helix, the family is further classified into three subfamilies, the IL2 subfamily, the Interferon (IFN) subfamily, and the IL10 subfamily the IL2 subfamily is largest and comprises several non-immune cytokines including, but not limited to, Erythropoietin (EPO) and Thrombopoietin (TPO), in certain embodiments, the cytokine of the composition is a cytokine from the four- α -helix bundle family or a mutant thereof.

in another embodiment, the cytokines of the present invention are IL1 family cytokines or mutants thereof, the IL1 family is a group of 11 cytokines that play an important role in the regulation of immune and inflammatory responses.typically, the IL1 family of cytokines are pro-inflammatory cytokines that regulate and elicit inflammatory responses.non-limiting examples of IL1 family cytokines include IL1 α, IL1 β, IL1Ra, IL18, IL36Ra, IL36 α, IL37, IL36 β, IL36 γ, IL38, and IL33. IL1 family members have similar genetic structures.

in other embodiments, the cytokines of the composition are interferon subfamily cytokines or mutants thereof IFN also has other functions in that they activate immune cells such as natural killer cells and macrophages by up-regulating antigen presentation by increasing expression of Major Histocompatibility Complex (MHC) antigens, and based on the type of receptor through which they signal, human interferons have been classified into three major types-type I IFN, type II and type III IFN.type I IFN in combination with a specific cell surface receptor complex known as IFN- α/β receptor (IFNAR) consisting of IFNAR1 and IFNAR2 chain based on the receptor type through which they signal, non-limiting examples of type I interferons present in humans are IFN- α, IFN- β, IFN- ε, IFN- κ and IFN- ω. thus, in certain embodiments, the cytokines of the composition are type 1 IFN cytokines or mutants thereof, including but not limited to IFN- α, IFN- β, IFN- λ 3, and IFN- λ - β, and IFN- β -IFN- β -IFN, thus, and, in certain embodiments, the composition, including but also wild type IFN-type 3, wild type IFN- β -3, wild- β -type IFN-3, wild- β -3, wild-type IFN- β -3, wild- β -3, wild-type IFN- β -IFN- β -IFN- β -IL, and IFN- β.

in other embodiments, the cytokine of the composition is a member of the Tumor Necrosis Factor Superfamily (TNFSF) or a mutant thereof, the TNFSF member is a pro-inflammatory cytokine that is predominantly expressed by immune cells, which induces an inflammatory state and stimulates immune cell function there are at least 18 TNFSF homologs, including but not limited to TNF (TNF α), CD40 (TNFSF), CD (TNFSF; CD 27), EDA, FASL (TNFSF; Fas ligand), LTA (TNFSF; lymphotoxin- α), LTB (TNFSF; lymphotoxin- β), TNFSF (OX 40), TNFSF (CD153), TNFSF (4-1BBL), TNFSF (TRAIL), TNFSK (RANKL; receptor activator of nuclear factor κ -B ligand), TNFSF (TWEAK), TNFSF13, TNFSF 18.

In certain embodiments, the cytokine of the composition is OX40L, a fragment thereof, or a mutant thereof. Sequence information for the full-length human OX40L amino acid sequence can be found using, for example, GenBank accession numbers XP _016857719.1, XP _016857718.1, XP _016857717.1, XP _011508266.2, NP _001284491.1, NP _003317.1, CAG 46830.1. Sequence information for the full-length human OX40L mRNA sequence can be found using, for example, GenBank accession numbers XR _001737396.1, XR _001737395.1, XR _001737394.1, XR _001737393.1, XM _017002230.1, XM _017002229.1, XM _017002228.1, XM _011509964.2, NM _001297562.1, NM _ 003326.4. The skilled artisan will appreciate that OX40L may be found in a variety of species, and that methods of identifying analogs or homologs of OX40L are known in the art, as described in detail below.

The skilled artisan will appreciate that OX40L may be present in a variety of species. Non-limiting examples include mouse (NP _ 033478.1), pig (NP _ 001020388.1), cow (NP _ 001192644.1), rat (NP _ 446004.1), rabbit (NP _ 001075454.1), goat (XP _ 013825644.1), sheep (XP _ 012042680.1), chicken (XP _ 430147.2), hamster (XP _ 007610839.1), and dog (XP _ 003639215.1). It is to be understood that the present invention relates to analogs of OX40L in other organisms, and is not limited to human analogs. Homologues may be found in other species by methods known in the art. For example, sequence similarity can be determined by conventional algorithms, which typically allow the introduction of a small number of gaps to achieve the best match. Specifically, the "percent identity" of two polypeptides or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1993). This algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al (J. mol. biol.215:403-410, 1990). BLAST nucleotide searches can be performed using the BLASTN program to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. Similarly, a BLAST protein search can be performed using the BLASTX program to obtain amino acid sequences homologous to the polypeptides of the present invention. To obtain gap alignments for comparison purposes, Gapped BLAST was used as described by Altschul et al (Nucleic Acids Res. 25:3389-3402, 1997). When BLAST and Gapped BLAST programs are used, the default parameters for each program (e.g., BLASTX and BLASTN) are employed. See www.ncbi.nlm.nih.gov for more details. Typically, homologues will have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89% homology. In another embodiment, the sequence may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous to OX 40L.

In a particular embodiment, the cytokine of the composition is the wild-type sequence of OX 40L. In a particular embodiment, the cytokine may comprise a wild-type fragment of OX40L, e.g., SEQ ID NO:57

The sequence shown in (a). In certain embodiments, these fragments may be joined into a continuous peptide by a linker fragment. In a particular embodiment, the cytokine may be a fragment of OX40L linked by a linker peptide, e.g.

Shown inThe sequence of (a). In an alternative embodiment, the cytokine of the composition is a mutated sequence of OX 40L. In one embodiment, the mutation is a mutation that results in OX40L binding but inhibits signaling by tumor necrosis factor receptor superfamily member 4 (TNFRSF 4, also known as OX40, also known as CD 134). For example, the mutation can be one or more mutations selected from N166A and F180A relative to the full length OX40L sequence in SEQ ID NO: 56. In a particular embodiment, the mutant form of OX40L comprises at least one mutation selected from the group consisting of N166A and F180A relative to the full length OX40L sequence in SEQ ID NO 56. In a particular embodiment, the cytokine may contain a mutated OX40L fragment, e.g.

The sequence shown in (a). In certain embodiments, these fragments may be joined into a continuous peptide by a linker fragment. In a particular embodiment, the cytokine may be a fragment of mutated and unmutated OX40L linked by a linker peptide, e.g.

The sequence shown in (a). In another specific embodiment, the cytokine may be a fragment of mutated and unmutated OX40L linked by a linker peptide, e.g.

The sequence shown in (a).

In certain embodiments, the cytokine of the composition is 4-1BBL, a fragment thereof, or a mutant thereof. Sequence information for the full-length human 4-1BBL amino acid sequence can be found using, for example, GenBank accession No. NP _ 003802.1. Sequence information for the full-length human 4-1BBL mRNA sequence can be found using, for example, GenBank accession No. NM _ 003811.3. The skilled artisan will appreciate that 4-1BBL can be found in a variety of species, and methods of identifying analogs or homologs of 4-1BBL are known in the art, as described in detail below.

The skilled artisan will appreciate that 4-1BBL can be found in a variety of species. Non-limiting examples include mouse (NP _ 033430.1), pig (XP _ 003480863.1), cow (NP _ 001306831.1), rat (NP _ 852049.1), rabbit (XP _ 008251123.1), goat (XP _ 013820683.1), sheep (XP _ 014951136.1), hamster (XP _ 007627369.1), and dog (XP _ 005633029.1). It is to be understood that the present invention relates to analogs of 4-1BBL in other organisms, and is not limited to human analogs. Homologues may be found in other species by methods known in the art. For example, sequence similarity can be determined by conventional algorithms, which typically allow the introduction of a small number of gaps to achieve the best match. In particular, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268,1993) is used to determine the "percent identity" of two polypeptides or of two nucleic acid sequences. This algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al (J. mol. biol.215:403-410, 1990). BLAST nucleotide searches can be performed using the BLASTN program to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. Similarly, a BLAST protein search can be performed using the BLASTX program to obtain amino acid sequences homologous to the polypeptides of the present invention. To obtain gap alignments for comparison purposes, Gapped BLAST was used as described by Altschul et al (nucleic acids Res. 25:3389-3402, 1997). When BLAST and Gapped BLAST programs are used, the default parameters for each program (e.g., BLASTX and BLASTN) are employed. See www.ncbi.nlm.nih.gov for more details. Typically, homologues will have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89% homology. In another embodiment, the sequence may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous to 4-1 BBL.

In a specific embodiment, the cytokine of the composition is a wild-type sequence of 4-1 BBL. In one embodiment, the cytokine may comprise a wild-type 4-1BBL fragment, e.g.

The sequence shown in (a). In certain embodiments, these fragments may be joined into a continuous peptide by a linker fragment. In one embodiment, the cytokine may be a 4-1BBL fragment linked by a linker peptide, e.g.

The sequence shown in (a). In an alternative embodiment, the cytokine of the composition is a mutated sequence of 4-1 BBL. In one embodiment, the mutation is one that affects the binding affinity between 4-1BBL and its receptor, tumor necrosis factor receptor superfamily member 9 (TNFRSF 9, also known as 4-1BB, also known as CD 137).

In certain embodiments, the cytokine of the invention is an interleukin or a mutant thereof. Most interleukins are synthesized by helper CD 4T lymphocytes as well as by monocytes, macrophages and endothelial cells. Interleukins promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Non-limiting examples of interleukins include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL 36. Thus, in certain embodiments, the cytokine of the composition is an interleukin or a mutant thereof, including, but not limited to, wild-type and mutant forms of IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, or IL 36. In a specific embodiment, the cytokine of the composition is IL2 or a mutant thereof. IL2 is a lymphokine that induces the proliferation of responsive T cells. In addition, it acts on some B cells through receptor specific binding as a stimulator of growth factor and antibody production. The IL2 protein is secreted as a mono-glycosylated polypeptide, and cleavage of the signal sequence is necessary for its activity. The structure of IL2 comprises a bundle of 4 helices (called A-D) flanked by 2 shorter helices and several loops of undefined definition. Residues in helix a and residues in the loop region between helices a and B are important for receptor binding. Secondary structure analysis indicated similarity to IL4 and granulocyte-macrophage colony stimulating factor (GMCSF). In a specific embodiment, the cytokine of the composition is IL2 or a variant thereof. Variants may be truncated or mutated IL 2. Sequence information for the full-length human IL2 amino acid sequence can be found using, for example, GenBank accession No. AAA59140.1 or AAH 70338.1. Sequence information for the full-length human IL2 mRNA sequence can be found using, for example, GenBank accession numbers BC070338.1 or M22005.1.

The skilled artisan will appreciate that IL2 may be found in a variety of species. Non-limiting examples include mouse (AAI 16874.1), pig (NP _ 999026.1), cow (AAQ 10670.1), rat (EDM 01295.1), rabbit (AAC 23838.1), goat (AAQ 10671.1), sheep (ABK 41601.1), chicken (AAV 35056.1), hamster (ERE 88380.1), and dog (AAA 68969.1). It is to be understood that the present invention relates to analogs of IL2 in other organisms, and is not limited to human analogs. Homologues may be found in other species by methods known in the art. For example, sequence similarity can be determined by conventional algorithms, which typically allow the introduction of a small number of gaps to achieve the best match. In particular, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268,1993) is used to determine the "percent identity" of two polypeptides or of two nucleic acid sequences. This algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al (J. mol. biol.215:403-410, 1990). BLAST nucleotide searches can be performed using the BLASTN program to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. Similarly, a BLAST protein search can be performed using the BLASTX program to obtain amino acid sequences homologous to the polypeptides of the present invention. To obtain gap alignments for comparison purposes, Gapped BLAST was used as described by Altschul et al (nucleic acids Res. 25:3389-3402, 1997). When BLAST and Gapped BLAST programs are used, the default parameters for each program (e.g., BLASTX and BLASTN) are employed. See www.ncbi.nlm.nih.gov for more details. Typically, homologues will have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89% homology. In another embodiment, the sequence may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous to IL 2.

In a particular embodiment, the cytokine of the composition is the wild-type sequence of IL2, e.g.

in one embodiment, the mutation is one or more mutations selected from the group consisting of R38A, F42K, and/or C125S relative to SEQ ID No. 5. in another embodiment, the mutation of IL2 comprises at least one mutation selected from the group consisting of R38A, F42K, and C125S relative to SEQ ID No. 5. in one embodiment, the mutation of IL2 comprises a mutation of R38A, F42K, and C125 2 relative to SEQ ID No. 5

The sequence shown in (a).

In an alternative aspect, the toxin replaces a cytokine. The term "toxin" refers to a toxic substance or product of a plant, animal, microorganism (including but not limited to bacteria, viruses, fungi, rickettsia or protozoa), or infectious substance, or a recombinant or synthetic molecule, regardless of its origin and method of production. Toxins may be small molecules, peptides or proteins that are capable of causing disease when contacted with or absorbed by body tissue that interacts with biological macromolecules (e.g., enzymes or cellular receptors). The toxin may be a "biotoxin" that is used to specifically identify toxins from biological sources. Biotoxins can be further classified as mycotoxins, or short mycotoxins, microbial biotoxins, plant biotoxins, short plant toxins, and animal biotoxins. Non-limiting examples of biological toxins include: cyanobacterial toxins produced by cyanobacteria, such as microcystins, nodulotoxin, denatured toxin-a, coluocytoxin, sphingomyelintoxin-a, saxitoxin, lipopolysaccharides, dolastatin, BMAA; dinoxins produced by dinoflagellates, such as saxitoxin and gonyatoxin; necrotic toxins, e.g. from the spider of the Cryptotaenia arachnoids or the "Violet dorsum", most of the Crotalus viridis and venomous snake, the drum belly venomous snake, Streptococcus pyogenes ((S. pyogenes))Streptococcus pyogenes) Generating; neurotoxins, for example produced by the black widow spider, most scorpions, boxfish, cobra, cardiospira, octopus, poisonous fish, frogs, sargassum, corals, various types of algae, cyanobacteria, and dinoflagellates, such as botulinum toxin (e.g., botulinum toxin), tetanus toxin, tetrodotoxin, chlorotoxin, conotoxin, modified toxin-a, bungarotoxin, caramboxin, curare; muscle toxins, such as found in snake and lizard venom; and cytotoxins, such as ricin (from castor bean), bee venom (from bee venom)Bees) and T-2 mycotoxins (from some virulent mushrooms). In certain embodiments, the toxin is a cytotoxin. In one embodiment, the cytotoxin is selected from the group consisting of ricin, bee venom, and T-2 mycotoxin. In a particular embodiment, the toxin is ricin.

In certain embodiments, the cytokines or toxins of the present invention can be pegylated for improved systemic half-life and reduced dosage frequency. In one embodiment, PEG may be added to the cytokine or toxin. Thus, the compositions of the invention may comprise a cytokine or toxin comprising PEG. In one embodiment, the PEG can be selected from PEG-10K, PEG-20K and PEG-40K. Methods for conjugating PEG to proteins are standard in the art. See, for example, Kolate et al,Journal of Controlled Release2014, 192(28) 67-81, which are incorporated herein by reference in their entirety. Still further, the cytokines or toxins of the present invention may be modified to remove T cell epitopes. T cell epitopes may be responsible for immunogenicity issues when administering compositions to subjects. Through their presentation to T cells, they activate the process of anti-drug antibody development. Preclinical screening of T cell epitopes can be performed in silico, followed by in vitro and in vivo validation. T cell epitope mapping tools such as EpiMatrix can be highly accurate predictors of immune responses. Deliberate removal of T cell epitopes may reduce immunogenicity. Other methods of improving the safety and efficacy of the compositions of the invention by reducing their immunogenicity include humanization and pegylation.

(b) Ligands

As used herein, a "ligand" is a protein that specifically binds to a receptor on a target cell and is not the corresponding binding partner of a cytokine linked to the ligand. The ligand may be from a eukaryote, prokaryote, or virus. In certain embodiments, the ligand may be from a virus. The phrase "specifically binds" refers herein to the affinity (K) with which a ligand binds to a target protein_d) In the range of at least 0.1 mM to 1 pM, or in the range of at least 0.1 pM to 200 nMAt least in the range of 0.1 pM to 10 nM. Dissociation constant (K)_d) The tendency of larger objects to reversibly separate (dissociate) into smaller components is measured. The dissociation constant is the inverse of the association constant. The dissociation constant can be used to describe the affinity between the ligand (L) and the target protein (P). Thus, K_d=（[P]x[L]）/[C]Wherein C is a ligand-target protein complex, and wherein [ P]、[L]And [ C]Representing the molar concentrations of protein, ligand and complex, respectively. Methods for determining whether a ligand binds to a target protein are known in the art. See, for example, Rossi and Taylor,Nature Protocols2011; 6: 365-387。

the ligand may trigger a signal by binding to a receptor on the target cell. Receptors are protein molecules that can intercalate into the plasma membrane surface of cells that receive chemical signals from outside the cell. When these chemical signals bind to receptors, they cause some form of cellular/tissue reaction. In a preferred embodiment, the target cell is an immune cell. Thus, the ligand of the composition binds to a receptor expressed on the immune cell. Non-limiting examples of immune cells include macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, neutrophils. Thus, in certain embodiments, immune cells include, but are not limited to, macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, neutrophils. In a specific embodiment, the immune cell is a natural killer cell or a T lymphocyte. Non-limiting examples of receptors expressed on immune cells include the major histocompatibility complex (MHC; e.g., MHCI, MHCII, and MHCIII), toll-like receptors (TLRs; e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR 13), CD94/NKG2 family of receptors, endothelin receptors, Signaling Lymphocyte Activation Molecule (SLAM) family of receptors. Thus, in certain embodiments, receptors on target cells include, but are not limited to, major histocompatibility complex (MHC; e.g., MH)CI. MHCII and MHCIII), toll-like receptors (TLRs; such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and TLR 13), CD94/NKG2 family receptors, endothelin receptors, Signaling Lymphocyte Activating Molecule (SLAM) family of receptors. In a specific embodiment, the receptor on the target cell is a CD94/NKG2 family receptor. In another specific embodiment, the ligand of the composition specifically binds to a receptor expressed on Natural Killer (NK) cells and CD8+ Cytotoxic T Lymphocytes (CTLs). In a preferred embodiment, the ligand of the composition does not specifically bind to vascular endothelial cells or regulatory T cells (T cells)_regs) (ii) a receptor of (i).

The receptor expressed on NK cells and CTLs may be CD94/NKG2 family receptors or KLRG 1. KLRG1 (killer lectin-like receptor subfamily G member 1) is a protein encoded by the KLRG1 gene in humans. The CD94/NKG2 family of receptors is the C-type lectin receptor family, which is expressed predominantly on the NK cell surface and in a CD8+ T lymphocyte subset. These receptors stimulate or inhibit the cytotoxic activity of NK cells, and thus they are classified into activating receptors and inhibitory receptors according to their functions. CD94/NKG2 recognizes MHC class I-related glycoproteins. The CD94/NKG2 family includes seven members: NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F and NKG 2H. Thus, in certain embodiments, the ligands of the invention specifically bind to a receptor selected from the group consisting of NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, and NKG 2H. The NKG2 receptor is a type II transmembrane protein that dimerizes with the CD94 molecule. CD94 contains a short cytoplasmic domain and is responsible for signal transduction. Thus, the NKG2 receptor forms disulfide-bonded heterodimers. NKG2D represents an exception, it is a homodimer. NKG2A and NKG2B receptors transmit inhibitory signals. NKG2C, NKG2E and NKG2H are activating receptors. NKG2D is also an activating receptor, but it is coupled to the adapter protein DAP10, which adapter protein DAP10 carries the signaling motif YINM (SEQ ID NO: 34). Src or Jak kinases phosphorylate DAP10, which can then associate with the p85 subunit of PI (3) K or the adaptor molecule Grb 2. This signaling triggers actin reorganization (cell polarization) and degranulation. The function of the NKG2F receptor has not been elucidated.

in a particular embodiment, the ligand of the composition specifically binds the NKG2D receptor NKG2D is an activating receptor found on NK cells and CD8+ T cells (α β and γ δ) the structure of NKG2D consists of two disulfide-linked type II transmembrane proteins with short intracellular domains incapable of transducing signals, NKG2D functions on CD8+ T cells to send co-stimulatory signals to activate them in one embodiment, the ligand that binds to NKG2D may be an anti-NKG 2D antibody, "anti-NKG 2D" includes all antibodies that specifically bind to epitopes within NKG 2D. the term "antibody" includes the term "monoclonal antibody" refers to antibodies derived from a single copy or clone, including for example any eukaryotic, prokaryotic or phage clone_HH fragment) or cartilaginous fish (V)_NARFragments). As used herein, "humanized antibody" includes anti-NKG 2D antibodies that are composed partially or entirely of amino acid sequence sequences derived from the germline of human antibodies by altering the sequence of an antibody having non-human complementarity determining regions ("CDRs"). The simplest such alteration may consist simply of substituting the murine constant region with the constant region of a human antibody, thereby producing a human/murine chimera that may have sufficiently low immunogenicity to be pharmaceutically acceptable for use. Preferably, however, the variable regions and even the CDRs of the antibodies are also passed through the artHumanization is carried out by well known techniques. The framework regions of the variable regions are replaced by the corresponding human framework regions such that the non-human CDRs are substantially intact, or even replaced with sequences derived from the human genome. CDRs can also be randomly mutated such that binding activity and affinity to NKG2D is maintained or enhanced in the context of fully human germline framework regions or substantially human framework regions. In certain embodiments, the anti-NKG 2D antibody is a Fab, Fab ', or F (ab') 2 fragment.

In a specific embodiment, the anti-NKG 2D antibody is KYK-1 or KYK-2, e.g., Kwong et al,J Mol Biol2008 Dec 31;384(5): 1143-56. The light chain of KYK-1 comprises

The amino acid sequence shown in (a) and the heavy chain of KYK-1 comprise

The amino acid sequence shown in (a). The light chain of KYK-2 comprises

The amino acid sequence shown in (a) and the heavy chain of KYK-2 comprise

The amino acid sequence shown in (a).

In another specific embodiment, the anti-NKG 2D antibody is a scFv derived from KYK-1. For example, the KYK-1scFv comprises

The amino acid sequence shown in (a). Alternatively, the KYK-1scFv comprises

The amino acid sequence shown in (a).

In another specific embodiment, the anti-NKG 2D antibody is a scFv derived from KYK-2. For example, the KYK-2scFv comprises

The amino acid sequence shown in (a). Alternatively, the KYK-2scFv comprises

The amino acid sequence shown in (a).

As noted above, the various KYK-1 and KYK-2 antibodies or scfvs thereof may be combined with any of the cytokines disclosed herein, in the absence or presence of any linker described herein, to provide the compositions or chimeric peptides of the invention. It is also understood that KYK-1 and KYK-2 antibodies are examples of antibodies suitable for use in the present compositions and that one of skill in the art would understand based on the present disclosure that other anti-NKG 2D antibodies are also suitable.

3 in 3 another 3 embodiment 3, 3 ligands 3 that 3 bind 3 NKG 32 3 share 3 an 3 MHC 3 class 3I 3- 3 related 3 α 31 3 α 32 3 suprastructural 3 domain 3 that 3 constitutes 3a 3 common 3 site 3 for 3 interacting 3 with 3 NKG 32 3. 3 non 3- 3 limiting 3 examples 3 of 3 ligands 3 that 3 bind 3 NKG 32 3 include 3 MHC 3 class 3I 3- 3 related 3 glycoproteins 3, 3 such 3 as 3 MIC 3 family 3 proteins 3 ( 3 i.e. 3, 3 MICA 3, 3 MICB 3) 3, 3 UL 3 binding 3 family 3 proteins 3 ( 3 i.e. 3, 3 ULBP 3, 3 ULPB 3, 3 ULBP 3) 3, 3 retinoic 3 acid 3 early 3- 3 inducible 3 gene 31 3 ( 3 Rael 3) 3- 3 like 3 proteins 3 ( 3 i.e. 3, 3 Rae 3 α 3, 3 Rae 3 β 3, 3 Rae 3 γ 3, 3 Rae 3 δ 3, 3 Rae 3e 3 ∈ 3) 3, 3H 3 protein 3 family 3 members 3 ( 3 i.e. 3, 3H 3 60 3) 3, 3H 3- 3 HLA 3- 3a 3, 3 and 3 mult 3 and 3 omcp 3 in 3 mice 3, 3 which 3 ligands 3 are 3 MHC 3 class 3I 3- 3 related 3 glycoproteins 3, 3 in 3 certain 3 embodiments 3, 3 the 3 ligands 3 of 3 the 3 present 3 invention 3 are 3 selected 3 from 3 the 3 group 3 consisting 3 of 3 MICA 3, 3 MICB 3, 3 ULBP 3, 3 rabp 3, 3 Rae 3 α 3, 3 rab 3 β 3, 3 and 3 the 3 binding 3 of 3 human 3 proteins 3 with 3a 3 low 3 affinity 3, 3 such 3 as 3 about 3 no 3, 3 no 3. 3

the structure of OMCP consists of MHC class I-like α 1/α 2 platform domains (fig. 20A) the platform domain of OMCP has been tailored to have only a six-chain β -fold and has a shorter flanking helix the helix of OMCP α 01 domain (H1) is continuous, whereas the helix of α 12 domain is split into two regions (H2 a and H2B) the helices flank the six-chain β -fold and together form a characteristic platform defining MHC proteins as with other NKG2DL (fig. 20B), the α 2 helices of OMCP are close together and thus do not have a groove for binding peptides or other ligands such as antigen presenting MHC platform domains [ OMCP contains a disulfide bond between S5 and H2B and this disulfide bond is conserved in most NKG2DL (fig. 20℃) in certain embodiments, the ligand of the invention comprises one or more α helices of MHC class I-related glycoproteins (MHC class I-related glycoproteins) (H734, H2B), in other embodiments the invention consists of one MHC class I-like α 1/α 2-related glycoprotein, H2-related domains (H462H 9), in more specific embodiments the invention, H2H-related glycoproteins (H7342), in certain embodiments the invention consists of one MHC class I-related glycoprotein I-related domains, H1, H2H-related domains (H-related domains) or H-related glycoproteins (H-related domains thereof, 9) or H2H-related to bind proteins of the invention, in combination of the inventioncomprising the α 2 domain of an MHC class I-associated glycoprotein (H2) in another specific embodiment, the ligand of the invention consists of the α 2 domain of an MHC class I-associated glycoprotein (H2) the skilled person will be able to determine the position of the α helix in other MHC class I-associated glycoproteins, e.g. using sequence alignment (see figure 20C, which is derived from Lazear et alJ Virol2013; 87(2): 840-850, which article is incorporated herein by reference in its entirety.) in one embodiment, the ligands of the invention comprise one or more α helices of OMCP in another embodiment, the ligands of the invention comprise the α 1 domain (H1), the α 2 domain (H2), H2a, H2b, or a combination thereof in yet another embodiment, the ligands of the invention comprise the α 2 domain (H2) of OMCP in a particular embodiment, the ligands of the invention consist of one or more α helices of OMCP in yet another particular embodiment, the ligands of the invention consist of the α 1 domain (H1), the α 2 domain (H2), H2a, H2b, or a combination thereof in yet another particular embodiment, the ligands of the invention consist of the α 2 domain (H2) of OMCP.

Sequence information for the full-length OMCP amino acid sequence can be found using, for example, GenBank accession numbers 4FFE _ Z, 4FFE _ Y or 4FFE _ X. The skilled person will appreciate that homologues of OMCP may be found in other species or viruses. See, for example, Lefkowitz et al,Nucleic Acidsres 2005; 33: D311-316 (which is incorporated herein by reference in its entirety), which describes eighteen OMCP variants between vaccinia and monkeypox virus strains. In one embodiment, OMCP is from an orthopoxvirus. In a specific embodiment, the OMCP is from a vaccinia virus or a monkeypox virus. In another specific embodiment, OMCP is from the brayton red strain of vaccinia virus. Homologues may be found in other species by methods known in the art. For example, sequence similarity can be determined by conventional algorithms, which typically allow the introduction of a small number of gaps to achieve the best match. Specifically, the "percent identity" of two polypeptides or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1993). Such a calculationThe method is incorporated into the BLASTN and BLASTX programs of Altschul et al (J.mol. biol.215:403-410, 1990). BLAST nucleotide searches can be performed using the BLASTN program to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. Similarly, a BLAST protein search can be performed using the BLASTX program to obtain amino acid sequences homologous to the polypeptides of the present invention. To obtain gap alignments for comparison purposes, GappedBLAST was used as described by Altschul et al (Nucleic Acids Res. 25:3389-3402, 1997). When BLAST and Gapped BLAST programs are used, the default parameters for each program (e.g., BLASTX and BLASTN) are employed. See www.ncbi.nlm.nih.gov for more details. Typically, homologues will have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89% homology. In another embodiment, the sequence may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to OMCP.

The skilled person will appreciate that structural homologues of OMCP may be found in other species or viruses. A structural homologue may be a protein that is structurally related but whose sequence is a distal homologue. For example, OMCP has low sequence identity to endogenous NKG2D, but OMCP was found to bind NKG2D based on structural homology. Structural homologues may be found in other species by methods known in the art. For example, protein structure predictions can be determined from various databases, such as Phyre and Phyre 2. Such databases produce reliable protein models that can be used to determine structural homologues. The main results table in Phyre2 provides confidence estimates, links to images and three-dimensional prediction models, and information derived from protein structural taxonomy databases (SCOP) or Protein Databases (PDB), depending on the source of the detected template. For each match, the link takes the user to a detailed view of the alignment between the user sequence and the sequence of the known three-dimensional structure. See www.sbg.bio.ic.ac.uk/phyre2/, for more details. Typically, a structural homologue will have at least 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59% confidence with OMCP. In one embodiment, the structural homologue and OMCP will have a confidence of at least 60, 61, 62, 63, 64,65, 66, 67, 68 or 69%. In another embodiment, the structural homologue will have a confidence of at least 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79% with OMCP. In yet another embodiment, the structural homologue has a confidence of at least 80, 81, 82, 83, 64, 85, 86, 87, 88 or 89% with OMCP. In yet another embodiment, the structural homologue may have a confidence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% with OMCP. PDB ID can be used: 4PDC found structural information of OMCP-human NKG 2D.

In a particular embodiment, the ligand of the composition is a sequence of OMCP, e.g.

The sequence shown in (a). In one embodiment, the ligand of the composition is an OMCP sequence having at least 80% identity to SEQ ID No. 7. For example, the ligand may be 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID No. 7.

In another specific embodiment, the ligand of the composition is a sequence of OMCP, e.g.

The sequence shown in (a). In one embodiment, the ligand of the composition is an OMCP sequence having at least 80% identity to SEQ ID No. 13. For example, the ligand may be 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID No. 13.

In yet another embodiment, the ligand of the composition is an OMCP sequence, e.g.

The sequence shown in (a). In one embodiment, the ligand of the composition is an OMCP sequence having at least 80% identity to SEQ ID No. 14. For example, the ligand may have about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID No. 14.

In an alternative aspect, the receptor expressed on the immune cell may be PD 1. PD1, also known as programmed cell death protein 1 and CD279 (cluster of differentiation 279), is a protein encoded by the PDCD1 gene in humans. PD1 is a cell surface receptor belonging to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD1 binds two ligands PDL1 and PDL 2. PD1 functions as an immune checkpoint and plays an important role in down-regulating the immune system by preventing activation of T cells. In certain embodiments, the ligand of the composition specifically binds PD 1. In one embodiment, the ligand that specifically binds PD1 may be an anti-PD 1 antibody. "anti-PD 1" includes all antibodies that specifically bind to an epitope within PD 1. The term "antibody" is as described above. In another embodiment, the ligand that specifically binds PD1 may be PDL1 or PDL 2. PDL1 (programmed death ligand 1 also known as cluster of differentiation 274 (CD 274)) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene in humans. PDL1 binds to its receptor PD1 found on activated T cells, B cells and bone marrow cells to modulate activation or inhibition. E.g. from dissociation constant K_dThe defined affinity between PDL1 and PD1 is 770 nM. PDL2 (programmed death ligand 2, also known as cluster of differentiation 273 (CD 273) or B7 DC) is a protein encoded by the PDCD1LG2 gene in humans. PDL2 also binds to the PD1 receptor. E.g. from dissociation constant K_dThe defined affinity between PDL2 and PD1 is 590 nM.

For example, sequence information for full-length PDL1 mRNA may be found using NCBI accession numbers NM _014143, NM _001267706, NR _052005, NM _001314029, and full-length amino acid sequence may be found using, for example, NCBI accession numbers NP _001300958, NP _001254635, NP _ 054862. The skilled person will appreciate that homologues of PDL1 may be found in other species. In one embodiment, the PDL1 is derived from homo sapiens (homo sapiens). Sequence similarity may be determined by conventional algorithms, such as described above for OMCP. Specifically, the "percent identity" of two polypeptides or two nucleic acid sequences is determined using the BLASTN, BLASTX and Gapped BLAST programs using default parameters. See www.ncbi.nlm.nih.gov for more details. Typically, homologues will have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89% homology. In another embodiment, the sequence may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous to PDL 1.

In one embodiment, the ligand of the composition is a sequence of PDL1, for example

The sequence shown in (a). In one embodiment, the ligand of the composition is the sequence of PDL1, which has at least 80% identity to SEQ ID NO: 51. For example, the ligand may have about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID No. 51.

In yet another embodiment, the ligand of the composition is a sequence of PDL1, e.g.

The sequence shown in (a). In one embodiment, the ligand of the composition is the sequence of PDL1, which has at least 80% identity to SEQ ID No. 52. For example, the ligand may have about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID No. 52.

The sequence shown in (a). In one embodiment, the ligand of the composition is the sequence of PDL1, which has at least 80% identity to SEQ ID NO: 53. For example, the ligand may have about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID No. 53.

For example, sequence information for full-length PDL2 mRNA can be found using NCBI accession numbers NM _025239 and XM _005251600, and full-length amino acid sequences can be found using, for example, NCBI accession numbers NP _079515 and XP _ 005251657. The skilled person will appreciate that homologues of PDL1 may be found in other species. In one embodiment, the PDL2 is derived from homo sapiens (homo sapiens). Sequence similarity may be determined by conventional algorithms, such as described above for OMCP. Specifically, the "percent identity" of two polypeptides or two nucleic acid sequences is determined using the BLASTN, BLASTX and Gapped BLAST programs using default parameters. See www.ncbi.nlm.nih.gov for more details. Typically, homologues will have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89% homology. In another embodiment, the sequence may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% homologous to PDL 2.

In one embodiment, the ligand of the composition is a sequence of PDL2, for example

The sequence shown in (a). In one embodiment, the ligand of the composition is the sequence of PDL2, which has at least 80% identity to SEQ ID NO: 54. For example, the ligand may have about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to SEQ ID No. 54.

In another specific embodiment, the ligand of the composition is a sequence of PDL2, for example

In another aspect, the ligand of the composition can be a glucocorticoid-induced TNFR-related (GITR) ligand (GITRL). GITR activation by GITRL affects the activity of effector T cells and regulatory T cells, thereby participating in the development of immune responses against tumors and infectious agents as well as autoimmune and inflammatory diseases. GITR triggers stimulation of T effector activity and suppresses Treg activity. GITR inhibition can ameliorate autoimmune/inflammatory diseases, while GITR activation can treat viral, bacterial and parasitic infections, as well as enhance the immune response against tumors. GITRL is a type II transmembrane protein that is expressed at high levels on Antigen Presenting Cells (APC) and endothelial cells.

In certain embodiments, the ligands of the invention are modified for improved systemic half-life and reduced dosing frequency. In one embodiment, N-glycans can be added to the ligands. While biological function is generally determined by protein components, carbohydrates can play a role in molecular stability, solubility, in vivo activity, serum half-life, and immunogenicity. Sialic acid components, particularly carbohydrates, can extend the serum half-life of protein therapeutics. Thus, a novel N-linked glycosylation consensus sequence can be introduced at a desired position in the peptide backbone to produce a protein with increased sialic acid containing carbohydrates, thereby increasing in vivo activity due to longer serum half-life. In another embodiment, PEG may be added to the ligand. Methods for conjugating PEG to proteins are standard in the art. See, for example, Kolate et al,Journal of Controlled Release2014, 192(28) 67-81, which are incorporated herein by reference in their entirety. In one embodiment, the compositions of the invention may comprise a ligand comprising PEG and/or one or more N-glycans. In one embodiment, the PEG is selected from the group consisting of PEG-10K, PEG-20K and PEG-40K. Still further, the ligands of the invention may be modified to remove T cell epitopes. T cell epitopes may be responsible for immunogenicity issues when administering compositions to subjects. Through their presentation to T cells, they activate the process of anti-drug antibody development. Preclinical screening of T cell epitopes can be performed in silico, followed by in vitro and in vivo validation. T cell epitope mapping tools such as EpiMatrix can be highly accurate predictors of immune responses. Deliberate removal of T cell epitopes may reduce immunogenicity. Other methods of improving the safety and efficacy of the compositions of the invention by reducing their immunogenicityIncluding humanization and pegylation.

(c) Joint

In one aspect, the compositions of the invention further comprise a linker. Linkers can be used to link the cytokine to the ligand. It is understood that linking the cytokine to the ligand does not adversely affect the function of the cytokine or ligand. Suitable linkers include amino acid chains for coupling cytokines and ligands and alkyl chains functionalized with reactive groups or combinations thereof.

In one embodiment, the linker may comprise amino acid side chains, referred to as peptide linkers. An amino acid residue linker is typically at least one residue, and can be 50 or more residues, but does not specifically bind to the target protein alone. In one embodiment, the linker may be from about 1 to about 10 amino acids. In another embodiment, the linker may be from about 10 to about 20 amino acids. In yet another embodiment, the linker may be about 20 to about 30 amino acids. In yet another embodiment, the linker may be from about 30 to about 40 amino acids. In various embodiments, the linker may be about 40 to about 50 amino acids. In other embodiments, the linker may be more than 50 amino acids. For example, a linker can be 1, 2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In one embodiment, the linker is from about 20 to about 30 amino acids. In another embodiment, the linker is about 26 amino acids.

Any amino acid residue can be used for the linker, provided that the linker does not specifically bind to the target protein. Typical amino acid residues for attachment are glycine, serine, alanine, leucine, tyrosine, cysteine, lysine, glutamic acid, aspartic acid and the like. For example, the joint may be (AAS)_n、(AAAL)_n(SEQ ID NO:68)、(G_nS)_nOr (G)₂S)_nWhereinA is alanine, S is serine, L is leucine, and G is glycine, and wherein n is an integer from 1 to 20, or from 1 to 10, or from 3 to 10. Thus, n may be 1, 2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Thus, in certain embodiments, linkers include, but are not limited to (AAS)_n、(AAAL)_n(SEQ ID NO:68)、(G_nS)_nOr (G)₂S)_nWherein A is alanine, S is serine, L is leucine, and G is glycine, and wherein n is an integer from 1 to 20, or from 1 to 10, or from 3 to 10. The linker may comprise one or more epitope tags. For example, the linker may comprise 1, 2,3, 4,5, 6, 7, or 8 epitope tags. In a specific embodiment, the linker comprises 2 epitope tags. Non-limiting examples of epitope tags include a FLAG tag (DYKDDDK epitope (SEQ ID NO: 9)), an HA tag (YPYDVPDYA epitope (SEQ ID NO: 10)), a His tag (6 x-His or 8 x-His), a Myc tag (EQKLISEEDL epitope (SEQ ID NO: 11)), and a V5 tag (GKPIPNPLLGLDST epitope (SEQ ID NO: 12)). In one embodiment, the linker may comprise at least one tag selected from a FLAG tag and a His tag. In a specific embodiment, the linker comprises a FLAG tag and a His tag. In another specific embodiment, the linker comprises the sequence shown in SEQ ID NO 8 (GSSGSSDYKDDDDKHHHHHHHHGSSGSS).

In another embodiment, the alkyl chain linking group may be coupled to the cytokine by reacting the terminal amino or carboxyl group with a functional group on the alkyl chain (e.g., carboxyl or activated ester). Subsequently, the ligand is attached to the alkyl chain by reacting a second functional group on the alkyl chain with the appropriate group on the ligand to complete the formation of the complex. The second functional group on the alkyl chain is selected from substituents that react with a functional group on the ligand but not with a cytokine. For example, when the ligand incorporates a functional group such as a carboxyl group or an activated ester, the second functional group of the alkyl chain linking group may be an amino group, or vice versa. It will be appreciated that the formation of the conjugate may require protection and deprotection of functional groups present to avoid the formation of undesirable products. Protection and deprotection is accomplished using protecting groups, reagents and protocols common in the art of organic synthesis. In particular, protection and deprotection techniques employed in solid phase peptide synthesis may be used. It will be appreciated that the linking group may alternatively be coupled first to the ligand and then to the cytokine.

An alternative chemical linking group for the alkyl chain is polyethylene glycol (PEG), which is functionalized in the same manner as the alkyl chain described above. Such linkers may be referred to as heterobifunctional PEG linkers or homobifunctional PEG linkers. Non-limiting examples of heterobifunctional PEG linkers include: o- (2-aminoethyl) -O' - [2- (biotinylamino) ethyl](ii) octaethylene glycol; o- (2-aminoethyl) -O' - (2-carboxyethyl) polyethylene glycol hydrochloride M_p3000A; o- (2-aminoethyl) -O' - (2-carboxyethyl) polyethylene glycol 5,000 hydrochloride salt M_p5,000; o- (2-aminoethyl) polyethylene glycol 3,000 Mp 3,000; o- (2-aminoethyl) -O' - (2- (succinylamino) ethyl) polyethylene glycol hydrochloride M_p10,000; o- (2-azidoethyl) heptaethylene glycol; o- [2- (biotinylamino) ethyl]-O' - (2-carboxyethyl) undecanediol; 21- [ D (+) -bio-amino]-4,7,10,13,16, 19-hexaoxaheneicosanoic acid; o- (2-carboxyethyl) -O' - [2- (Fmoc-amino) -ethyl]Heptacosyl glycol; o- (2-carboxyethyl) -O' - (2-mercaptoethyl) heptaethylene glycol; o- (3-carboxypropyl) -O' - [2- (3-mercaptopropionylamino) ethyl]Polyethylene glycol M_w3000A; o- (3-carboxypropyl) -O' - [2- (3-mercaptopropionylamino) ethyl]Polyethylene glycol M_w5000; o- [ N- (3-maleimidopropanoyl) aminoethyl group]-O' - [3- (N-succinimidyloxy) -3-oxopropyl]Heptacosyl glycol; and O- [2- (3-triphenylmethylthiopropylamino) ethyl group]Polyethylene glycol M_p3,000. Non-limiting examples of homobifunctional PEG linkers include: MAL-PEG-MAL (bifunctional maleimide PEG maleimide); OPSS-PEG-OPSS (OPSS: orthopyridyl disulfide; PDP-PEG-PDP); HS-PEG-SH (bifunctional thiol PEG thiol); SG-PEG-SG (bifunctional PEG succinimidyl glutarate NHS ester); SS-PEG-SS (bifunctional PEG succinimidyl succinate NHS ester); GAS-PEG-GAS (bifunctional PEG succinimidyl ester NHS-PEG-NHS); SAS-PEG-SAS (bifunctional PEG succinimidyl ester NHS-PEG-NHS); amine-PEG-amine (bifunctional PEG amine NH2-PEG-NH 2); AC-PEG-AC (bifunctional acrylate PE)G acrylates); ACA-PEG-ACA (bifunctional polymerizable PEG acrylate acrylamide); epoxide-PEG-epoxide (bifunctional PEG epoxide or EP); NPC-PEG-NPC (bifunctional NPC PEG, nitrophenylcarbonate); aldehyde-PEG-aldehyde (ALD-PEG-ALD, bifunctional PEG propionaldehyde); AA-PEG-AA (acid-PEG-acid, AA-acetic acid or carboxymethyl); GA-PEG-GA (acid-PEG-acid, GA: glutaric acid); SA-PEG-SA (bifunctional PEG carboxylic acid-succinic acid); GAA-PEG-GAA (bifunctional PEG carboxylic acid, glutaramic acid); SAA-PEG-SAA (bifunctional PEG carboxylic acid, succinamic acid); azide-PEG-azide (bifunctional PEG azide, N3-PEG-N3); alkyne-PEG-alkyne (bifunctional alkyne or acetylene PEG); biotin-PEG-biotin (bifunctional biotin PEG linker); silane-PEG-silane (bifunctional silane PEG); hydrazide-PEG-hydrazide (bifunctional PEG hydrazide); tosylate-PEG-tosylate (bifunctional PEG p-tosyl); and chloride-PEG-chloride (bifunctional PEG halide).

In certain embodiments, linkers of the invention can be modified for improved systemic half-life and reduced dosing frequency. In one embodiment, an N-glycan is added to the linker. While biological function is generally determined by protein components, carbohydrates can play a role in molecular stability, solubility, in vivo activity, serum half-life, and immunogenicity. Sialic acid components, particularly carbohydrates, can extend the serum half-life of protein therapeutics. Thus, a novel N-linked glycosylation consensus sequence can be introduced at a desired position in the peptide backbone to produce a protein with increased sialic acid containing carbohydrates, thereby increasing in vivo activity due to longer serum half-life. In another embodiment, PEG is added to the linker. Methods for conjugating PEG to proteins are standard in the art. See, for example, Kolate et al,Journal of Controlled Release2014, 192(28) 67-81, which are incorporated herein by reference in their entirety. In one embodiment, the composition of the invention comprises a ligand comprising PEG and/or one or more N-glycans. In one embodiment, the PEG is selected from the group consisting of PEG-10K, PEG-20K and PEG-40K.

in another aspect, the invention relates to crosslinking the peptides of the invention to improve their pharmacokinetic, immunogenic, diagnostic and/or therapeutic properties, crosslinking involves linking two molecules by covalent bonds via a chemical reaction at suitable sites (e.g., primary amines, thiols) on the cytokines and ligands of the invention, in one embodiment, the cytokines and ligands may be crosslinked together, the crosslinking agent may form a cleavable or non-cleavable linker between the cytokine and ligand, the crosslinking agent forming a non-cleavable linker between the cytokine and ligand may comprise maleimide-based or haloacetyl-based moieties according to the invention, such non-cleavable linker is said to be derived from maleimide-based or haloacetyl-based moieties, the crosslinking agent comprising maleimide-based moieties includes N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate), which is a "long chain" analogue of SMCC (LC-cc), kappa-maleimidocaproic acid N-succinimidyl-ester (undecamido- γ -succinimidyl) -cyclohexan-1-carboxy- (6-amidohexanoate) which is a cross-linked to a cross-linker derived from the non-succinimidyl-2- (maleimide-carbonyl-butyrimidoyl) ester (SBA), the cross-2- (maleimide-carbonyl-2-carbonyl-butyrimido-2- (sbc), the cross-linker may be derived from the maleimide-2- (maleimide-succinimidyl) and the cross-2- (maleimide-succinimidyl) linker, the cross-2- (maleimide-carbonyl-2-carbonyl-succinimidyl) linker, the cross-2-carbonyl-succinimidyl) linker, the cross-2-carbonyl-succinimide-carbonyl-butyrimidobutyrate (sbc, the cross-2-carbonyl-2-succinimidyl) cross-2-carbonyl-linker, the cross-2-succinimidyl-carbonyl-succinimidyl-2-carbonyl-2-carbonyl-succinimidyl-2-carbonyl-2-carbonyl-amino-succinimidyl-2-carbonyl-2-carbonyl.

(d) Chimeric peptides

in another aspect, the invention includes chimeric peptides comprising a cytokine peptide and a NKG2D ligand peptide, in an alternative aspect, the invention includes chimeric peptides comprising a cytokine peptide and a PD1 ligand peptide it is understood that "ligand peptide" may be used interchangeably with "ligand" and "cytokine peptide" may be used interchangeably with "cytokine" for the purposes of describing various cytokines and ligands suitable for use in the compositions and methods of the invention herein, in certain embodiments, the cytokine peptide is in the IL2 subfamily, more specifically, the cytokine peptide is selected from IL8, IL7, IL15 and IL21 in a specific embodiment, the cytokine peptide is IL15 or a mutant thereof in another specific embodiment, the cytokine peptide is IL2 or a mutant thereof in another specific embodiment, the cytokine peptide is mutant IL2 comprising at least one mutation selected from the group consisting of R38A, F42K and C125S in a specific embodiment, the cytokine peptide comprises IL peptide of SEQ ID 585, IL 6348, IL 6324, IL 4148, IL 4142, IL 639, IL 465, IL 6324, IL 4624, IL 5923, IL 6324, and IL 5923.

in certain embodiments, the cytokine peptide is in the tumor necrosis factor ligand superfamily (TNFSF). more specifically, the cytokine peptide is selected from the group consisting of TNF- α, OX40L, 4-1BB ligand, TRAIL, Fas ligand, lymphotoxin- α, lymphotoxin- β, CD30L, CD40L, CD27L, and RANKL.in one embodiment, the cytokine peptide is OX40L or a mutant thereof.in another embodiment, the cytokine peptide comprises an OX40L fragment.in one embodiment, the OX40L fragment comprises the amino acid sequence shown in SEQ ID NO: 57. in certain embodiments, the OX40L fragment may be joined together by a linker peptide into a continuous construct.in one embodiment, the construct comprising an OX40L fragment comprises the amino acid sequence shown in SEQ ID NO: 58. in certain embodiments, the cytokine peptide is an OXX40894 mutant comprising at least one mutant of the amino acid sequence shown in the sequence No. 36 and F180A. in one embodiment, the construct comprises a BBL-BBL linker peptide sequence shown in another embodiment, the amino acid sequence shown in the BBL-linker variant of SEQ ID NO: 584, the BBL-mutant of the BBL-mutant-polypeptide is shown in one embodiment, the BBL-mutant of the BBL-mutant of the polypeptide comprises the amino acid sequence shown in the BBL-mutant of SEQ ID NO: No. 40, in one embodiment, in embodiments, the BBL-mutant of the BBL-mutant of the amino acid sequence No. 40, the BBL-mutant of the amino acid sequence shown in embodiments, the BBL-mutant of the BBL-mutant.

In certain embodiments, the NKG2D ligand peptide is an anti-NKG 2D antibody. In another embodiment, the NKG2D ligand peptide is an MHC class I-related glycoprotein. In another embodiment, the ligand peptide is OMCP, a portion thereof, or a mutant thereof. In one embodiment, the ligand peptide binds to receptors expressed on NK cells and CD8+ CTLs. In a specific embodiment, the ligand peptide binds the NKG2D receptor. In certain embodiments, the ligand peptide comprises the amino acid sequence set forth in SEQ ID No. 7 or a portion thereof capable of binding to the NKG2D receptor.

In certain embodiments, the PD1 ligand peptide is an anti-PD 1 antibody. In another embodiment, the PD1 ligand peptide is PDL1, a portion thereof, or a mutant thereof. In yet another embodiment, the PD1 ligand peptide is PDL2, a portion thereof, or a mutant thereof. In one embodiment, the ligand peptide binds to a receptor expressed on T cells, NK cells and macrophages. In a specific embodiment, the ligand peptide binds to the PD1 receptor. In certain embodiments, the ligand peptide comprises the amino acid sequence shown in SEQ ID No. 48 or SEQ ID No. 50 or a portion thereof capable of binding to the PD1 receptor.

In other embodiments, the chimeric peptide further comprises a linker peptide. In certain embodiments, the linker peptide comprises a peptide selected from (AAS)_n、(AAAL)_n(SEQ ID NO:68)、(G_nS)_nOr (G)₂S)_nWherein A is alanine, S serine, L is leucine, and G is glycine, and wherein n is an integer from 1 to 20, or from 1 to 10, or from 3 to 10. In various embodiments, the linker peptide comprises at least one tag selected from a FLAG tag and a His tag. In one embodiment, the linker peptide is from about 20 to about 30 amino acids. In one embodiment, the linker peptide comprises the amino acid sequence shown in SEQ ID NO 8.

The invention also includes nucleic acid molecules encoding the chimeric peptides as described herein. In addition, the invention includes pharmaceutical compositions comprising the chimeric peptides as described herein. Pharmaceutical compositions are described in more detail in section I (h).

The chimeric peptides of the present disclosure may optionally comprise a signal peptide and/or a purification moiety. When present, typically the signal peptide is located at the N-terminus of the chimeric peptide and the purification moiety is located at the C-terminus of the chimeric peptide. Alternatively, both the signal peptide and the purification moiety are at the N-terminus of the chimeric peptide. The choice of signal peptide can and will vary depending on a number of factors, including but not limited to the desired cell location and cell type. Suitable polynucleotide sequences encoding signal peptides are known in the art, as are the polypeptide sequences encoded thereby. In one embodiment, the signal peptide comprises SEQ ID NO:69 (MGILPSPGMPALLSLVSLLSVLLMGCVAETG). Similarly, the choice of purification moiety can and will vary. Suitable purification moieties are known in the art, as are the polynucleotide sequences encoding them. Typically, the signal peptide and/or the purified portion is cleaved off during processing and is not included in the final chimeric peptide for use in a pharmaceutical composition.

The present disclosure also encompasses vectors comprising nucleic acid sequences capable of encoding the chimeric peptides of the disclosure. As used herein, "vector" is defined as a nucleic acid molecule that serves as a vehicle for transferring genetic material. Vectors include, but are not limited to, plasmids, phages, cosmids, transposable elements, viruses (phages, animal viruses and plant viruses) and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors, including replication-competent, replication-defective, and enteromorphic (gut form) vectors thereof, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, EB virus, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor vectors. Expression vectors encoding the chimeric peptides of the disclosure can be delivered to cells using viral vectors or by non-viral transfer methods. Viral vectors suitable for introducing nucleic acids into cells include retroviruses, adenoviruses, adeno-associated viruses, rhabdoviruses, and herpes viruses. Non-viral nucleic acid transfer methods include naked nucleic acids, liposomes, and protein/nucleic acid conjugates. The expression construct encoding the chimeric peptide of the present disclosure introduced into the cell may be linear or circular, may be single-stranded or double-stranded, and may be DNA, RNA, or any modification thereof, or a combination thereof. The present disclosure also encompasses cell lines comprising a vector comprising a nucleic acid sequence capable of encoding the chimeric peptides of the disclosure. In some embodiments, the cell line is an immortalized cell line.

(e) Targeting molecules

As used herein, a "targeting molecule" is a molecule that is capable of binding a target specific to a cell in a disease state or to the extracellular matrix surrounding the diseased cell.

(f) Combination therapy

As used herein, "combination" is meant to include therapies that can be administered alone, e.g., formulated separately for administration alone (e.g., as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., "co-formulation"). The combination of a polypeptide provided herein and one or more active therapeutic agents can be administered or applied sequentially (e.g., where one agent is administered prior to one or more other agents) or simultaneously (e.g., where two or more agents are administered at or about the same time). In some embodiments, the administration is sequential. In other embodiments, the administration is simultaneous. Whether two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

Thus, the methods and uses of the polypeptides described herein may be performed before, substantially simultaneously with, or after another treatment, and may be supplemented with other forms of treatment.

In one aspect, provided herein is a combination therapy comprising a composition as described herein and a PD-1 inhibitor. "PD-1 inhibitor" refers to a moiety (e.g., a compound, nucleic acid, polypeptide, antibody) that reduces, inhibits, blocks, eliminates or interferes with the activity or expression of PD-1 (e.g., programmed cell death protein 1; PD-1 (CD 279); G1: 145559515) (including variants, isoforms, species homologs (e.g., mouse) of human PD-1 and analogs having at least one common epitope with PD-1). PD-1 inhibitors include molecules and macromolecules such as, for example, compounds, nucleic acids, polypeptides, antibodies, peptibodies, diabodies, minibodies, nanobodies, single chain variable fragments (scFv), and functional fragments or variants thereof. In particular embodiments described herein, the PD-1 inhibitor is an anti-PD-1 antibody. PD-1 inhibitors (including anti-PD-1 antibodies) may antagonize PD-1 activity or expression. The anti-PD-1 antibody can be a monoclonal or polyclonal antibody as described herein. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody. In other embodiments, the anti-PD-1 antibody is a polyclonal antibody. 0).

In one embodiment, the PD-1 inhibitor is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, REGN2810, PDR001, and MEDI 0680. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is pidilizumab. In some embodiments, the PD-1 inhibitor is AMP-224. In some embodiments, the PD-1 inhibitor is REGN 2810. In some embodiments, the PD-1 inhibitor is PDR 001. In some embodiments, the PD-1 inhibitor is MEDI 0680.

in one aspect, the invention includes a combination therapy comprising a PD-1 inhibitor as described herein and a composition comprising a cytokine as provided herein linked to an NKG2D ligand as provided herein the composition may further comprise a linker as described herein to link the cytokine to the ligand as provided herein, e.g., the cytokine may be an IL1 family cytokine including those described herein (e.g., IL1 α, IL1 β, IL1Ra, IL36Ra, IL Ra α, IL Ra β, IL Ra γ, IL Ra, and IL 33. e.g., the cytokine may be an IL Ra subfamily cytokine, e.g., IL Ra, and IL 21. in some embodiments, the cytokine is IL 2. in other embodiments, the cytokine is a mutant of IL Ra, e.g., a TNF p 38/F42, e.g., the cytokine may be an IFN-IL 72, TNF-p, IL-p, TNF-p, IL-p, TNF-p, IL-p, TNF-p, IL-p, TNF-p, IL-p, TNF-p, IL-p, TNF-p.

in one embodiment, the combination therapy comprises a PD-1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a targeting molecule OMCP may be linked to a targeting molecule, or a portion of OMCP may be linked to a targeting molecule in one embodiment, the combination therapy comprises a PD-1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a Tumor Necrosis Factor (TNF) family member in one embodiment, the combination therapy comprises a PD-1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a TNF-related apoptosis-inducing targeting molecule in one embodiment, the combination therapy comprises a PD-1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a 4-1BB ligand in one embodiment, the combination therapy comprises a PD-1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a TNF-inducing targeting molecule in one embodiment, the combination therapy comprises a TNF-1 inhibitor as provided herein and a TNF-1 antagonist as provided herein and a composition comprising OMCP or a TNF-1B ligand, a TNF-1 inhibitor as provided herein and a composition comprising OMCP or a TNF-1 inhibitor as provided herein and a TNF-1-B, a composition comprising OMCP or a targeting molecule, a TNF-1 inhibitor as provided herein and a composition as provided herein and a TNF-1-B, a composition comprising OMCP or a TNF-1 inhibitor as provided herein and a composition, a TNF-B, a composition comprising OMCP or a TNF-2, a c-1-2, a c-B, a composition, a targeting molecule, a composition.

In one embodiment, the combination therapy comprises a PD-1 inhibitor described herein and a cytokine linked to an NKG2D ligand (e.g., a scFv of KYK-1, KYK-2, or a scFv of KYK-2). In one embodiment, the combination therapy comprises a PD-1 inhibitor described herein and a cytokine linked to a NKG2D ligand, wherein the NKG2D ligand has the amino acid sequence set forth in SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, or SEQ ID No. 42.

In one embodiment, the combination therapy comprises a PD-1 inhibitor described herein and a fusion protein described herein (e.g., NKG2D ligand and cytokine). Combination therapy can include the PD-1 inhibitors described herein and fusion proteins having the amino acid sequence set forth in SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, or SEQ ID NO 46.

Further provided herein are combination therapies comprising a PD-1 inhibitor described herein and a chimeric peptide. In one embodiment, the combination therapy comprises a PD-1 inhibitor as described herein and a chimeric peptide comprising a cytokine peptide as described herein and an NKG2D ligand peptide as described herein. In certain instances, the cytokine peptide may be selected from IL2, IL7, IL15, IL18, IL21, and mutants thereof. In one embodiment, the cytokine peptide of the combination therapy is IL or a mutant thereof (e.g., SEQ ID NO:5 or 6). The NKG2D ligand of the chimeric peptide in the combination therapies described herein include those ligands provided herein (e.g., KYK-1, an scFv of KYK-1, KYK-2, or an scFv of KYK-2). In another example, the NKG2D ligand of the chimeric peptide of the combination therapy has the amino acid sequence shown in SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 or SEQ ID NO 42.

In another embodiment, provided herein is a combination therapy comprising a PD-1 inhibitor provided herein and a chimeric peptide comprising a cytokine peptide and an anti-NKG 2D antibody. The cytokine peptide is a cytokine as described above. anti-NKG 2D antibodies were as described above.

In one embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and an anti-PD-1 antibody. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2.

In one embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and an anti-PD-1 antibody. In some embodiments, the chimeric protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2.

In other embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D antibody is KYK-1. In other embodiments, the anti-NKG 2D antibody is KYK-2. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2.

In some embodiments, the combination therapy comprises an anti-NKG 2D scFv, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises anti-NKG 2D scFv, IL2, and anti-PD 1 antibodies. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D scFv is a KYK-1 scFv. In other embodiments, the anti-NKG 2DscFv is KYK-2 scFv. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2.

In another aspect of the invention, provided herein is a combination therapy comprising a composition as described herein and a PD-L1 inhibitor. The term "PD-L1 inhibitor" refers to a moiety (e.g., a compound, nucleic acid, polypeptide, antibody) that reduces, inhibits, blocks, eliminates or interferes with the activity of PD-L1 (e.g., programmed cell death 1 ligand; PD-L1 (CD 274); G1: 30088843), including variants, isoforms, species homologs (e.g., mouse) of human PD-L1 and analogs having at least one common epitope with PD-L1, binding of PD-L1 to its receptor, or expression of PD-L1. PD-L1 inhibitors include molecules and macromolecules such as compounds (small molecule compounds), nucleic acids, polypeptides, antibodies, peptibodies, diabodies, minibodies, single domain antibodies or nanobodies, single chain variable fragments (ScFv), and fragments or variants thereof. In a specific embodiment, the inhibitor of PD-L1 is an anti-PD-L1 antibody. PD-L1 inhibitors (including anti-PD-L1 antibodies) can antagonize PD-L1 activity, its binding to PD-1, or its expression. Exemplary PD-L1 inhibitors include, but are not limited to, Durvalumab, Avermemab, Atlantizumab, BMS-936559, STI-A1010, STI-A1011, STI-A1012, STI-A1013, STI-A1014, and STI-A1015.

In one aspect, the invention includes a combination therapy comprising a PD-L1 inhibitor as described herein and a composition comprising a cytokine as provided herein linked to an NKG2D ligand as provided herein. In some embodiments, the PD-L1 inhibitor is dolvacizumab. In some embodiments, the PD-L1 inhibitor is avizumab. In some embodiments, the PD-L1 inhibitor is atelizumab. In some embodiments, the PD-L1 inhibitor is BMS-936559. In some embodiments, the PD-L1 inhibitor is STI-A1010, STI-A1011, STI-A1012, STI-A1013, STI-A1014, or STI-A1015.

the composition can further comprise a linker as described herein to link a cytokine to a ligand provided herein, the cytokine is a cytokine as described herein, e.g., the cytokine can be an IL1 family cytokine, including those described herein (e.g., IL1 α, IL1 β, IL1Ra, IL18, IL36Ra, IL36 α, IL36 β, IL36 γ, IL36, and IL 33. e.g., the cytokine can be an IL36 subfamily cytokine, e.g., IL36, and IL 21. in some embodiments, the cytokine is IL 2. in other embodiments, the cytokine is a mutant R38 36/F42 36 form of IL36, e.g., the cytokine can be an interferon as described herein (e.g., IFN- α, IFN- β, IFN- κ, IFN- ω, IFN-10, TNF-p, TNF-p.

in one embodiment, the combination therapy comprises a PD-L1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a targeting molecule OMCP may be linked to a targeting molecule or a portion of OMCP may be linked to a targeting molecule in one embodiment, the combination therapy comprises a PD-L1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a Tumor Necrosis Factor (TNF) family member in one embodiment, the combination therapy comprises a PD-L1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a TNF-related apoptosis inducing targeting molecule in one embodiment, the combination therapy comprises a PD-L1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a 4-1BB ligand in one embodiment, the combination therapy comprises a PD-L1 inhibitor as described herein and a composition comprising OMCP or a portion thereof as provided herein and a TNF- α -c receptor antagonist as provided herein and a TNF-B antagonist as provided herein and a composition comprising OMCP or a TNF-L-B ligand, a TNF-B antagonist, a CD-L-B-c.

In one embodiment, the combination therapy comprises a PD-L1 inhibitor described herein and a cytokine linked to an NKG2D ligand (e.g., KYK-1, an scFv of KYK-1, KYK-2, or an scFv of KYK-2). In one embodiment, the combination therapy comprises a PD-L1 inhibitor described herein and a cytokine linked to a NKG2D ligand, wherein the NKG2D ligand has the amino acid sequence set forth in SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41 or SEQ ID NO 42.

In one embodiment, the combination therapy comprises a PD-L1 inhibitor described herein and a fusion protein described herein (e.g., NKG2D ligand and cytokine). Combination therapy may include the PD-L1 inhibitors described herein and fusion proteins having the amino acid sequence set forth in SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, or SEQ ID NO 46.

Further provided herein are combination therapies comprising a PD-L1 inhibitor described herein and a chimeric peptide. In one embodiment, the combination therapy comprises a PD-L1 inhibitor as described herein and a chimeric peptide comprising a cytokine peptide as described herein and an NKG2D ligand peptide as described herein. In certain instances, the cytokine peptide may be selected from IL2, IL7, IL15, IL18, IL21, and mutants thereof. In one embodiment, the cytokine peptide of the combination therapy is IL or a mutant thereof (e.g., SEQ ID NO:5 or 6). The NKG2D ligand of the chimeric peptide in the combination therapies described herein includes those ligands provided herein (e.g., the scFv of KYK-1, KYK-2 or KYK-2 in another example, the NKG2D ligand of the chimeric peptide of the combination therapy has the amino acid sequence set forth in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO: 42.

In another embodiment, provided herein is a combination therapy comprising a PD-L1 inhibitor provided herein and a chimeric peptide comprising a cytokine peptide and an anti-NKG 2D antibody. The cytokine peptide is a cytokine as described above. anti-NKG 2D antibodies were as described above.

In one embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and a PD-L1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and an anti-PD-L1 antibody. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2.

In one embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and a PD-L1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and an anti-PD-L1 antibody. In some embodiments, the chimeric protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2.

In other embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-L1 antibody is an antagonistic antibody. In certain embodiments, the anti-NKG 2D antibody is KYK-1. In other embodiments, the anti-NKG 2D antibody is KYK-2. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2.

In some embodiments, the combination therapy comprises an anti-NKG 2D scFv, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D scFv, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-L1 antibody is an antagonistic antibody. In certain embodiments, the anti-NKG 2D scFv is a KYK-1 scFv. In other embodiments, the anti-NKG 2DscFv is KYK-2 scFv. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2.

(g) Description of the preferred embodiments

by way of non-limiting example, several preferred compositions of the invention are depicted in fig. 23.1. depicts a composition comprising the α 2 domain of OMCP linked to a cytokine (H2). 2. depicts a composition comprising OMCP linked to a cytokine, wherein the composition is pegylated.3. depicts a composition comprising OMCP linked to a cytokine, wherein the composition comprises N-glycans.4. depicts a composition comprising OMCP linked to a cytokine, wherein the linker comprises various sequences and various lengths.5. depicts a composition comprising Fab-specific antibodies to NKG2D linked to a cytokine.6. depicts a composition comprising various NKG2D ligands linked to a cytokine.7. depicts a composition comprising OMCP linked to a cytokine in mutant form, wherein OMCP can be mutated to have improved or weaker binding affinity.8. depicts a composition comprising mutant form OMCP linked to a cytokine, wherein OMCP can be mutated to have improved binding affinity or weaker binding affinity to other OMCP, or can be linked to a chimeric protein comprising OMCP from mhcp.

In preferred embodiments, the composition comprises IL2, IL15, or IL18 linked to OMCP. In another preferred embodiment, the composition comprises IL2, IL15, or IL18 linked to OMCP by a peptide linker. In yet another preferred embodiment, the composition comprises IL2, IL15, or IL18 linked to OMCP through a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to OMCP through a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, IL2 may be a mutant form of IL2 comprising mutations R38A and F42K.

In preferred embodiments, the composition comprises IL2, IL15 or IL18 linked to an anti-NKG 2D antibody. In another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to an anti-NKG 2D antibody by a peptide linker. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to an anti-NKG 2D antibody by a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to an anti-NKG 2D antibody by a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, IL2 may be a mutant form of IL2 comprising mutations R38A and F42K.

In various preferred embodiments, the composition comprises IL2 linked to OMCP. In another preferred embodiment, the composition comprises IL2 linked to OMCP through a peptide linker. In yet another preferred embodiment, the composition comprises IL2 linked to OMCP through a peptide linker comprising from about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises IL2 linked to OMCP through a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, IL2 may be a mutant form of IL2 comprising mutations R38A and F42K.

In various preferred embodiments, the composition comprises IL2 linked to an anti-NKG 2D antibody. In another preferred embodiment, the composition comprises IL2 linked to an anti-NKG 2D antibody by a peptide linker. In yet another preferred embodiment, the composition comprises IL2 linked to an anti-NKG 2D antibody by a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises IL2 linked to an anti-NKG 2D antibody by a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, IL2 may be a mutant form of IL2 comprising mutations R38A and F42K.

In an exemplary embodiment, the NKG2D ligand is an anti-NKG 2D antibody or scFv thereof, e.g., a KYK-1 antibody, a KYK-2 antibody, a KYK-1scFv or a KYK-2 scFv. In a specific exemplary embodiment, chimeric peptides are provided wherein the anti-NKG 2D antibody is KYK-1 linked to mutIL2 and comprises the amino acid sequence set forth in SEQ ID NO:43

Or alternatively, comprises the amino acid sequence set forth in SEQ ID NO:44

。

In another specific exemplary embodiment, chimeric peptides are provided wherein the anti-NKG 2D antibody is KYK-2 linked to mutIL2 and comprises the amino acid sequence set forth in SEQ ID NO:45

Or alternatively, comprises the amino acid sequence set forth in SEQ ID NO 46

。

In an exemplary embodiment, the composition comprises the DNA sequence set forth in SEQ ID NO 10.

In another exemplary embodiment, the composition comprises the amino acid sequence set forth in SEQ ID NO 20.

In a preferred embodiment, the composition comprises OX40L or 4-1BBL or a fragment construct thereof linked to OMCP. In another preferred embodiment, the composition comprises OX40L or 4-1BBL or a fragment construct thereof linked to OMCP by a peptide linker. In yet another preferred embodiment, the composition comprises OX40L or 4-1BBL or a fragment construct thereof linked to OMCP through a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises an OX40L or 4-1BBL or fragment thereof construct linked to OMCP through a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, the OX40L or fragment construct thereof may be a mutant form of OX40L comprising the mutations N166A and F180A.

In a preferred embodiment, the composition comprises an OX40L or 4-1BBL or fragment construct thereof linked to an anti-NKG 2D antibody. In another preferred embodiment, the composition comprises an OX40L or 4-1BBL or fragment construct thereof linked by a peptide linker to an anti-NKG 2D antibody. In yet another preferred embodiment, the composition comprises an OX40L or 4-1BBL or fragment construct thereof linked to an anti-NKG 2D antibody by a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises an OX40L or 4-1BBL or fragment construct thereof linked to an anti-NKG 2D antibody by a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, the OX40L or fragment construct thereof may be a mutant form of OX40L comprising the mutations N166A and F180A.

In various preferred embodiments, the compositions comprise OX40L or a fragment construct thereof linked to OMCP. In another preferred embodiment, the composition comprises OX40L or a fragment construct thereof linked to OMCP by a peptide linker. In yet another preferred embodiment, the composition comprises OX40L or a fragment construct thereof linked to OMCP through a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises OX40L or a fragment thereof linked to OMCP through a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, the OX40L or fragment construct thereof may be a mutant form of OX40L comprising the mutations N166A and F180A.

In a preferred embodiment, the composition comprises an OX40L or fragment construct thereof linked to an anti-NKG 2D antibody. In another preferred embodiment, the composition comprises an OX40L or fragment construct thereof linked to an anti-NKG 2D antibody by a peptide linker. In yet another preferred embodiment, the composition comprises an OX40L or fragment construct thereof linked to an anti-NKG 2D antibody by a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises an OX40L or fragment construct thereof linked to an anti-NKG 2D antibody by a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, the OX40L or fragment construct thereof may be a mutant form of OX40L comprising the mutations N166A and F180A.

In exemplary embodiments, the NKG2D ligand is OMCP. In an exemplary embodiment, the cytokine is OX 40L. In a specific exemplary embodiment, the cytokine is a construct comprising a fragment of OX 40L. In a specific exemplary embodiment, fragments of OX40L are combined into a contiguous construct. In a specific exemplary embodiment, chimeric peptides are provided in which OMCP is linked to the OX40L construct by a linker peptide and comprises the amino acid sequence shown in SEQ ID NO:62

。

In a specific exemplary embodiment, the cytokine is a construct comprising a mutated OX40L fragment containing mutations at amino acid positions N166A and F180A. In a specific exemplary embodiment, a mutated OX40L fragment is combined into a contiguous construct, with or without an unmutated OX40L fragment. In a specific exemplary embodiment, chimeric peptides are provided in which OMCP is linked to a mutant OX40L construct by a linker peptide and comprises SEQ ID NO 63

The amino acid sequence shown in (a).

In various preferred embodiments, the compositions comprise a 4-1BBL or fragment construct thereof linked to OMCP. In another preferred embodiment, the composition comprises a 4-1BBL or fragment construct thereof linked to OMCP by a peptide linker. In yet another preferred embodiment, the composition comprises a 4-1BBL or fragment thereof construct linked to OMCP through a peptide linker comprising from about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises a 4-1BBL or fragment thereof construct linked to OMCP through a peptide linker comprising a FLAG tag and a His tag.

In various preferred embodiments, the compositions comprise a 4-1BBL or fragment thereof construct linked to an anti-NKG 2D antibody. In another preferred embodiment, the composition comprises a 4-1BBL or fragment thereof construct linked to an anti-NKG 2D antibody by a peptide linker. In yet another preferred embodiment, the composition comprises a 4-1BBL or fragment thereof construct linked to an anti-NKG 2D antibody by a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises a 4-1BBL or fragment thereof construct linked to an anti-NKG 2D antibody by a peptide linker comprising a FLAG tag and a His tag.

In exemplary embodiments, the NKG2D ligand is OMCP. In an exemplary embodiment, the cytokine is 4-1 BBL. In a specific exemplary embodiment, the cytokine is a construct comprising a 4-1BBL fragment. In a specific exemplary embodiment, the 4-1BBL fragments are combined into a contiguous construct. In a specific exemplary embodiment, chimeric peptides are provided in which OMCP is linked to the 4-1BBL construct by a linker peptide and comprises the amino acid sequence shown in SEQ ID NO 67

。

In another preferred embodiment, the composition comprises OMCP or a portion thereof and a targeting molecule. In another preferred embodiment, the OMCP or portion thereof is linked to the targeting molecule via a linker. In yet another preferred embodiment, the portion of OMCP comprises the activated portion of H2b OMCP. In a particularly preferred embodiment, the activating moiety of OMCP comprises the H2B helix.

In another non-limiting example, several preferred compositions of the invention that bind to the PD1 receptor are depicted in fig. 36.

In preferred embodiments, the composition comprises IL2, IL15 or IL18 linked to PDL 1. In another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to PDL1 by a peptide linker. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to PDL1 by a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to PDL1 by a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, IL2 may be a mutant form of IL2 comprising mutations R38A and F42K.

In another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to PDL 2. In another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to PDL2 by a peptide linker. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to PDL2 by a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to PDL2 by a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, IL2 may be a mutant form of IL2 comprising mutations R38A and F42K.

In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to an anti-PD 1 antibody. In another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to an anti-PD 1 antibody by a peptide linker. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to an anti-PD 1 antibody by a peptide linker comprising about 20 to about 30 amino acids. In yet another preferred embodiment, the composition comprises IL2, IL15 or IL18 linked to an anti-PD 1 antibody by a peptide linker comprising a FLAG tag and a His tag. In each of the foregoing embodiments, IL2 may be a mutant form of IL2 comprising mutations R38A and F42K.

In an exemplary embodiment, the PD1 ligand is PDL 1. In a specific exemplary embodiment, a chimeric peptide is provided wherein the PD1 ligand is PDL1 linked to mutIL2 and comprises

The amino acid sequence shown in (a). In a specific exemplary embodiment, the composition comprises the DNA sequence shown in SEQ ID NO 47

。

In an exemplary embodiment, the PD1 ligand is PDL 2. In a specific exemplary embodiment, a chimeric peptide is provided wherein the PD1 ligand is PDL2 linked to mutIL2 and comprises

The amino acid sequence shown in (a). In a specific exemplary embodiment, the composition comprises the DNA sequence shown in SEQ ID NO 49

(h) Pharmaceutical composition

The present disclosure also provides pharmaceutical compositions. The pharmaceutical composition may comprise as active ingredient the composition of the invention as detailed above and at least one pharmaceutically acceptable excipient. The pharmaceutical compositions provided herein may further comprise a combination therapy as described herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor. In other embodiments, the combination therapy comprises a PD-L1 inhibitor.

The pharmaceutically acceptable excipient may be a diluent, binder, filler, buffer, pH adjuster, disintegrant, dispersing agent, preservative, lubricant, taste masking agent, flavoring agent, or coloring agent. The amount and type of excipients used to form the pharmaceutical composition can be selected according to known principles of pharmaceutical science.

In one embodiment, the excipient may be a diluent. The diluent may be compressible (i.e. plastically deformable) or abrasive brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powders, cellulose esters (i.e., mixed esters of acetate and butyrate), ethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, maltitol (malitol), sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable attrition brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate, calcium carbonate, and magnesium carbonate.

In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyl oxazolidinone, polyvinyl alcohol, C₁₂-C₁₈Fatty acid alcohols, polyethylene glycols, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.

In another embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of non-limiting example, the filler may be calcium sulfate (di-and tri-basic), starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, lactose, sucrose, mannitol, or sorbitol.

In yet another embodiment, the excipient may be a buffer. Representative examples of suitable buffers include, but are not limited to, phosphate, carbonate, citrate, Tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).

In various embodiments, the excipient may be a pH adjuster. As non-limiting examples, the pH adjusting agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

In a further embodiment, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch (which pregelatinized and modified starches), sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar gum, locust bean gum, karaya gum, peptin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

In yet another embodiment, the excipient may be a dispersant or dispersion enhancer. Suitable dispersing agents may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isomorphous silicate (isoamophorus silicate), and microcrystalline cellulose.

In another alternative embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin a, vitamin C, vitamin E or retinyl palmitate, citric acid, sodium citrate; chelating agents such as EDTA or EGTA; and antimicrobial agents such as parabens, chlorobutanol, or phenol.

In a further embodiment, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate or stearic acid.

In yet another embodiment, the excipient may be a taste-masking agent. The taste masking material comprises a cellulose ether; polyethylene glycol; polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers; a monoglyceride or triglyceride; an acrylic polymer; a mixture of an acrylic polymer and a cellulose ether; cellulose acetate phthalate; and combinations thereof.

In an alternative embodiment, the excipient may be a flavoring agent. The flavoring agent can be selected from synthetic flavor oils and flavoring aromatics and/or natural oils, extracts from plants, leaves, flowers, fruits and combinations thereof.

In yet a further embodiment, the excipient may be a colorant. Suitable color additives include, but are not limited to, food, pharmaceutical and cosmetic pigments (FD & C), pharmaceutical and cosmetic pigments (D & C), or external pharmaceutical and cosmetic pigments (Ext. D & C).

The weight fraction of an excipient or combination of excipients in a composition can be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2% or about 1% or less of the total weight of the composition.

The compositions can be formulated in a variety of dosage forms and administered by a number of different methods that will deliver a therapeutically effective amount of the active ingredient. These compositions may be administered orally, parenterally or topically in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles as required. Topical administration may also involve the use of transdermal administration, such as a transdermal patch or iontophoresis device. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques. The formulation of drugs is discussed in, for example, Gennaro, A. R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18 th edition, 1995) and Liberman, H.A. and Lachman, L.editors, Pharmaceutical laboratory Forms, Marcel Dekker Inc., New York, N.Y. (1980).

Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets and granules. In such solid dosage forms, the active ingredient is typically combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral formulations may also be administered as aqueous suspensions, elixirs or syrups. For these, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.

For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, and intraperitoneal), the formulation may be an aqueous or oil-based solution. Aqueous solutions may include sterile diluents such as water, saline solution, pharmaceutically acceptable polyols such as glycerol, propylene glycol or other synthetic solvents; antibacterial and/or antifungal agents, such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and/or agents for adjusting tonicity, for example sodium chloride, glucose or polyols such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. The oil-based solution or suspension may further comprise sesame oil, peanut oil, olive oil or mineral oil.

For topical (e.g., transdermal or transmucosal) administration, penetrants appropriate to the permeation barrier will generally be included in the formulation. Transmucosal administration can be accomplished through the use of nasal sprays, aerosol sprays, tablets, or suppositories, and transdermal administration can be via ointments, salves, gels, patches, or creams as is well known in the art.

In certain embodiments, compositions comprising a compound of the present invention are encapsulated in a suitable vehicle to facilitate delivery of the compound to a target cell, to increase the stability of the composition, or to minimize potential toxicity of the composition. As will be appreciated by those skilled in the art, various vehicles are suitable for delivering the compositions of the present invention. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating the compositions into a delivery vehicle are known in the art.

In an alternative embodiment, a liposomal delivery vehicle may be employed. Depending on the embodiment, liposomes are suitable for delivery of the compounds of the invention in view of their structural and chemical properties. In general, liposomes are spherical vesicles with a phospholipid bilayer membrane. The lipid bilayer of the liposome can be fused with other bilayers (e.g., cell membranes) to deliver the contents of the liposome to the cell. In this manner, the compounds of the invention can be selectively delivered to cells by encapsulation in liposomes fused to the target cell membrane.

Liposomes can be composed of many different types of phospholipids having different hydrocarbon chain lengths. Phospholipids typically comprise two fatty acids linked to one of various polar groups through glycerophosphate. Suitable phospholipids include Phosphatidic Acid (PA), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidylglycerol (PG), Diphosphatidylglycerol (DPG), Phosphatidylcholine (PC) and Phosphatidylethanolamine (PE). The fatty acid chains comprising the phospholipid may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated. Suitable fatty acid chains include (common names are shown in parentheses) n-dodecanoate (laurate), n-tetradecanoate (myristate), n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecanoate (palmitoleate), cis-9-octadecenoate (oleate), cis-9, 12-octadecadienoate (linoleate), all cis-9, 12, 15-octadecatrienoate (linoleate), and all cis-5, 8,11, 14-eicosatetraenoate (arachidonate). The two fatty acid chains of the phospholipid may be the same or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linolenyl PS, and the like.

The phospholipids may be derived from any natural source and may therefore comprise a mixture of phospholipids. For example, egg yolk is rich in PC, PG and PE, soybean contains PC, PE, PI and PA, and animal brain or spinal cord is rich in PS. Phospholipids may also be derived from synthetic sources. Mixtures of phospholipids with various phospholipids in different ratios can be used. Mixtures of different phospholipids can produce liposome compositions with advantageous activity or activity stability characteristics. The above-mentioned phospholipids may be mixed with a cationic lipid such as N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride, 1 '-dioctadecyl-3, 3,3',3 '-tetramethylindocyanine perchlorate, 3,3' -diheptyloxycarbocyanine iodide, 1 '-dodecyl-3, 3,3',3 '-tetramethylindocyanine perchlorate, 1' -dioleyl-3, 3,3',3' -tetramethylindocyanine methanesulfonate, N-4- (dioleylaminostyryl) -N-methylpyridinioide (N-4- (delinorylaminostyryl) -N-methylpyridinioide) or 1 in an optimum ratio, 1-dioleyl-3, 3,3',3' -tetramethylindocyanine perchlorate.

Liposomes can optionally contain sphingolipids (where sphingosine is the structural counterpart of glycerol) and one of the fatty acids of phosphoglycerides, or cholesterol (the major component of animal cell membranes). Liposomes can optionally contain pegylated lipids, which are lipids covalently linked to a polymer of polyethylene glycol (PEG). The PEG can range in size from about 500 to about 10,000 daltons.

The liposomes may further comprise a suitable solvent. The solvent may be an organic solvent or an inorganic solvent. Suitable solvents include, but are not limited to, dimethyl sulfoxide (DMSO), methyl pyrrolidone, N-methyl pyrrolidone, acetonitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof.

Liposomes carrying a compound of the invention (i.e., having at least one methionine compound) can be prepared by any known method of preparing liposomes for drug delivery, for example, as detailed in U.S. patent nos. 4,241,046, 4,394,448, 4,529,561, 4,755,388, 4,828,837, 4,925,661, 4,954,345, 4,957,735, 5,043,164, 5,064,655, 5,077,211, and 5,264,618, the disclosures of which are incorporated herein by reference in their entirety. For example, liposomes can be prepared by sonication of lipids in aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze-drying by repeated freezing and thawing. In a preferred embodiment, the liposomes are formed by sonication. Liposomes can be multilamellar (which has many layers like onions) or unilamellar. Liposomes can be large or small. Continued high shear sonication tends to form smaller unilamellar liposomes.

It will be apparent to the skilled person that all parameters controlling liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of methionine compound, concentration and composition of lipid, concentration of multivalent cation, mixing rate, presence and concentration of solvent.

In another embodiment, the compositions of the present invention may be delivered to cells as a microemulsion. Microemulsions are generally clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and an "oil". The "oil" in this case is the supercritical fluid phase. The surfactant stays at the oil-water interface. Any of a variety of surfactants are suitable for use in the microemulsion formulation, including those described herein or otherwise known in the art. Aqueous microdomains suitable for use in the present invention typically have a characteristic structural dimension of from about 5nm to about 100 nm. Aggregates of this size are poor scatterers of visible light, and therefore, these solutions are optically transparent. As understood by the skilled artisan, the microemulsions may and will have a variety of different microstructures including spherical, rod-shaped, or disk-shaped aggregates. In one embodiment, the structure may be a micelle, which is the simplest microemulsion structure, which is a generally spherical or cylindrical object. Micelles resemble oil droplets in water and reverse micelles resemble water droplets in oil. In an alternative embodiment, the microemulsion structure is a sheet. It comprises successive layers of water and oil separated by a layer of surfactant. The "oil" of the microemulsion optimally comprises phospholipids. Any of the phospholipids detailed above for liposomes are suitable for embodiments involving microemulsions. The composition of the present invention may be encapsulated in the microemulsion by any method known in the art.

In yet another embodiment, the compositions of the invention may be delivered in a dendrimer or dendrimer. In general, a dendrimer is a branched dendrimer in which each branch is a molecularly interconnected chain that, after a certain length, splits into two new branches (molecules). This branching continues until the branches (molecules) become so densely packed that the crowns form a sphere. Generally, the nature of a dendrimer is determined by the functional groups on its surface. For example, hydrophilic end groups (e.g., carboxyl groups) typically form water-soluble dendrimers. Alternatively, phospholipids may be incorporated into the surface of the dendrimer to facilitate absorption across the skin. Any phospholipid detailed for use in liposome embodiments is suitable for use in dendrimer embodiments. Dendrimers can be prepared and the compositions of the present invention encapsulated therein using any method known in the art. For example, dendrimers can be generated by a sequence of iterations of reaction steps, with each additional iteration generating a higher level dendrimer. Thus, they have a regular, highly branched 3D structure and have almost uniform size and shape. Furthermore, the final size of the dendrimer is typically controlled by the number of iterative steps used during synthesis. Various dendrimer sizes are suitable for use in the present invention. Typically, dendrimers can range in size from about 1nm to about 100 nm.

Process II

in one aspect, the invention includes a method of delivering a cytokine to a target cell, the method including contacting the target cell with a composition including a cytokine linked to a ligand, wherein the ligand specifically binds to a receptor on the target cell, further, the method includes contacting the target cell with a composition including a chimeric peptide as described in section I the target cell can be any cell including a target receptor to which the ligand specifically binds, the ligand and the specific binding are described in section I in certain embodiments, the target cell can be an immune cell non-limiting examples of immune cells include macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, neutrophils in certain embodiments, the immune cells are selected from macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils and neutrophils, in a particular embodiment, the target cell is a Natural Killer (NK) PD) cell and/or CD8+ T cells, the method including the method of administering a chimeric peptide of the invention to a target cell expressing a ligand of a CD 2-expressing a cytokine, CD8+ T-CD 9, the invention includes the embodiments where the ligand is a chimeric peptide of a ligand-CD 2-CD-9, the invention includes embodiments where the invention includes administering a chimeric peptide of a chimeric peptide-CD-9, where the invention includes embodiments where the invention as described in embodiments where the invention includes a ligand, where the invention includes a ligand is specifically binds to a ligand, where the invention includes a ligand is.

In some embodiments, the combination therapy comprises a PD-L1 inhibitor. In other embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, provided herein is a method of delivering a cytokine to a target cell comprising administering a composition provided herein. In one embodiment, provided herein is a method of delivering a cytokine to a target cell comprising administering a combination therapy provided herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor provided herein and a chimeric peptide comprising a cytokine peptide and an anti-NKG 2D antibody. In certain embodiments, the cytokine peptide is a cytokine as described above. In some embodiments, the anti-NKG 2D antibody is as described above. In one embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and an anti-PD-1 antibody. In some embodiments, the fusion protein further comprises a linker. In one embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and an anti-PD-1 antibody. In some embodiments, the chimeric protein further comprises a linker. In other embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D antibody is KYK-1. In other embodiments, the anti-NKG 2D antibody is KYK-2. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the combination therapy comprises an anti-NKG 2D scFV, IL2, and PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2 dsfv, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D scFv is a KYK-1 scFv. In other embodiments, the anti-NKG 2D scFv is KYK-2 scFv. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2. In some embodiments, the target cell is a target cell of a subject. In certain embodiments, the subject has its need. In certain embodiments, an effective amount of a combination therapy is administered to a subject. The term "effective amount" as used herein refers to an amount of a pharmaceutical composition provided herein sufficient to result in a desired result. In a specific embodiment, the subject is a human. In certain embodiments, the subject is a subject having a cancer or tumor. In a specific embodiment, the cancer or tumor is a lung cancer or tumor.

the present invention also provides a method for treating a tumor, which comprises administering an anti-tumor cell, a pro-inflammatory factor, a pro-inflammatory protein, a pro-inflammatory factor, a pro-inflammatory protein, a pro-tumor cell, a pro-inflammatory factor, a cytokine, a pro-cytokine, a cell expressing PD, a natural killer cell, a neutrophil, and a neutrophil, or a CD + T cell, which comprises an anti-tumor cell, a ligand, a chimeric antibody, a ligand, a chimeric-tumor cell, a pro-tumor-factor, a pro-tumor-cell, a pro-tumor-factor, a cell, a pro-tumor-factor, a pro-cell, a pro-tumor-factor, a cell, a pro-tumor-cytokine, a pro-tumor-cell, a pro-tumor-factor, a cell, a pro-tumor-cell, a pro-tumor-cytokine, a cell, a pro-tumor-factor, a cell, a pro-tumor-cell, a pro-tumor-cytokine, a cell.

In yet another aspect, the invention includes a method of treating a tumor. The method comprises identifying a subject having a tumor and administering to the subject a composition comprising a cytokine linked to a ligand, wherein the ligand specifically binds to a receptor on a target cell. In addition, the method comprises administering to the subject a composition comprising a chimeric peptide as described in section I. In particular, the present inventors have shown that delivery of cytokines to target cells activates the cells bound by the composition, wherein the activated cells specifically lyse tumor cells, thereby reducing the amount of cancer cells. In a specific embodiment, the cytokine is a pro-inflammatory cytokine as described in the preceding paragraph. Thus, the compositions of the present invention are useful for treating, stabilizing and preventing cancer and related diseases in a subject. "treating, stabilizing or preventing cancer" refers to causing a reduction in tumor size or number of cancer cells, slowing or preventing an increase in tumor size or proliferation of cancer cells, increasing disease-free survival time between disappearance and recurrence of a tumor or other cancer, preventing initial or subsequent development of a tumor or other cancer, or reducing adverse symptoms associated with a tumor or other cancer. The inventors have shown that the compositions of the present invention activate Natural Killer (NK) cells bound by the composition, wherein the activated NK cells specifically lyse tumor cells, thereby reducing the amount of tumor cells. For example, when cancer cells are "stressed," NKG2D ligand becomes upregulated, rendering the cells susceptible to NK cell-mediated lysis. In desirable embodiments, the percentage of tumor or cancer cells that survive treatment is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% less than the initial number of tumor or cancer cells, as measured using any standard assay (e.g., caspase assay, TUNEL and DNA fragmentation assay, cell permeability assay, and Annexin V assay). Desirably, the reduction in the number of tumor or cancer cells induced by administration of the composition of the invention is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 fold less than the reduction in the number of non-tumor or non-cancer cells. Ideally, the methods of the invention result in a 20, 30, 40, 50, 60, 50, 80, 90 or 100% reduction in tumor size or number of cancer cells, as determined using standard methods. Ideally, at least 20, 30, 40, 50, 60, 70, 80, 90 or 95% of treated subjects have complete remission in which all evidence of the tumor or cancer has disappeared. Ideally, the tumor or cancer does not recur or recurs after at least 1, 2,3, 4,5, 10, 15, or 20 years. In some embodiments, the methods further comprise administering a chimeric peptide comprising a PD1 ligand and a cytokine. In another embodiment, the chimeric peptide further comprises a linker. In one embodiment, the PD1 ligand is PDL 1. In another embodiment, the PD1 ligand is PDL 2. In yet another embodiment, the PD1 ligand is an antibody specific for PD 1. In another embodiment, the cytokine is IL 2. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2. In some embodiments, the method further comprises administering a combination therapy as provided herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the combination therapy comprises a PD-L1 inhibitor. In other embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, provided herein is a method of treating a tumor in a subject comprising administering to the subject a composition provided herein. In one embodiment, provided herein is a method of treating a tumor in a subject comprising administering to the subject a combination therapy provided herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor provided herein and a chimeric peptide comprising a cytokine peptide and an anti-NKG 2D antibody. In certain embodiments, the cytokine peptide is a cytokine as described above. In some embodiments, the anti-NKG 2D antibody is as described above. In one embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and an anti-PD-1 antibody. In some embodiments, the fusion protein further comprises a linker. In one embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and an anti-PD-1 antibody. In some embodiments, the chimeric protein further comprises a linker. In other embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D antibody is KYK-1. In other embodiments, the anti-NKG 2D antibody is KYK-2. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the combination therapy comprises an anti-NKG 2 dsfv, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises anti-NKG 2D scFV, IL2, and anti-PD 1 antibodies. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D scFv is a KYK-1 scFv. In other embodiments, the anti-NKG 2D scFv is KYK-2 scFv. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2. In certain embodiments, the subject has its need. In certain embodiments, an effective amount of a combination therapy is administered to a subject. In a specific embodiment, the subject is a human. In a specific embodiment, the tumor is a lung tumor.

in another aspect, the invention includes a method of inhibiting immune cells comprising contacting immune cells with a composition comprising a cytokine linked to a ligand, wherein the ligand specifically binds to a receptor on an immune cell, thereby inhibiting the cell, in addition, the method includes contacting immune cells with a composition comprising a chimeric peptide as described in section I a non-limiting example of an immune cell including a macrophage, B lymphocyte, T lymphocyte, mast cell, monocyte, dendritic cell, eosinophil, natural killer cell, basophil granulocyte, neutrophil, in some embodiments, the immune cell is selected from the group consisting of macrophage, B lymphocyte, T lymphocyte, mast cell, monocyte, dendritic cell, eosinophil, natural killer cell, basophil, and neutrophil, in a particular embodiment, the immune cell is a Natural Killer (NK) cell and/or CD8+ T cell, in a particular embodiment, the immune cell expresses nkg2d, in an alternative embodiment, the immune cell expresses pd1, is an anti-inflammatory cytokine inhibitory factor, or a chimeric peptide, or an anti-inflammatory cytokine-tnf-receptor-agonist antibody, which is an anti-inflammatory factor, a chimeric peptide, or an anti-inflammatory factor-tnf-CD-or a human, or a human-or a human.

In yet another aspect, the invention includes a method of treating an infection comprising administering a composition comprising a cytokine linked to a ligand. For example, a composition comprising a cytokine linked to a ligand can specifically bind to an immune cell, which is then activated to target and lyse the infected host cell. In addition, the method comprises administering to the subject a composition comprising a chimeric peptide as described in section I. The term "infection" as used herein includes the presence of a pathogen in or on a subject which, if growth of the pathogen is inhibited, results in a benefit to the subject. Thus, in addition to reference to the presence of pathogens, the term "infection" also refers to an unwanted normal flora. The term "pathogen" as used herein refers to an infectious agent that can produce a disease. Non-limiting examples of infectious agents include viruses, bacteria, prions, fungi, viroids, or parasites that cause disease in a subject. In a particular embodiment, the infection is caused by a pathogen, such as a bacterium or virus. In certain embodiments, the infection is an intracellular infection. In one embodiment, the infection is a viral infection. In another embodiment, the viral infection is caused by a flavivirus. The flavivirus genus is a genus of viruses in the flaviviridae family. Non-limiting examples of flaviviruses include Gadget ' S Gully virus, Khatag virus, Quadry forest disease virus, Langat virus, Omsk hemorrhagic fever virus, tick-borne encephalitis virus, sheep jump disease virus, Alloav virus, dengue virus 1-4, Kadu gu virus, Casepari virus, Kutango virus, Murray Valley encephalitis virus, St.Louis encephalitis virus, Sousu picture virus, West Nile virus, Yawend virus, Cokebaia virus group, Cokebaia virus, Bagaza virus, Ile.g., Irish meningitis virus, Entaya virus, Tabusu virus, Zika virus, Spanish virus, Bobo virus, Bigara virus, Zhugela virus, Sawoya virus, Sepitek ' S virus, Uganda S virus, Wesselsbraun virus, yellow fever virus, bat ' S, Besse virus, Apoay virus, Ribes nigella virus, Trita virus, Motork virus, Salicohol virus, St Palita virus, Bukala saturvirus, Kalima island virus, Dacalco bat virus, Mengdana batu encephalitis virus, Jinbiangpau virus, Rijowa virus, hepatitis C virus, such as hepatitis C virus genotypes 1-6, and GB viruses A and B. In a certain embodiment, the flavivirus may be selected from the group consisting of west nile virus, dengue virus, japanese encephalitis virus, and yellow fever virus. In a specific embodiment, the viral infection is caused by west nile virus. In certain embodiments, a pathogen, more specifically a virus, may induce the expression of NKG 2D-binding proteins. Thus, a composition comprising a cytokine linked to a ligand can specifically bind NK cells, which are then activated to target and lyse infected host cells expressing NKG 2D. In another embodiment, a composition comprising a cytokine linked to a ligand can activate cytotoxic T lymphocytes that recognize infected cells via other mechanisms of targeted killing. In some embodiments, the methods further comprise administering a chimeric peptide comprising a PD1 ligand and a cytokine. In another embodiment, the chimeric peptide further comprises a linker. In one embodiment, the PD1 ligand is PDL 1. In another embodiment, the PD1 ligand is PDL 2. In yet another embodiment, the PD1 ligand is an antibody specific for PD 1. In another embodiment, the cytokine is IL 2. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2. In some other embodiments, the method further comprises administering a combination therapy as provided herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the combination therapy comprises a PD-L1 inhibitor. In other embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, provided herein is a method of treating an infection in a subject comprising administering to the subject a composition provided herein. In one embodiment, provided herein is a method of treating an infection in a subject comprising administering to the subject a combination therapy provided herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor provided herein and a chimeric peptide comprising a cytokine peptide and an anti-NKG 2D antibody. In certain embodiments, the cytokine peptide is a cytokine as described above. In some embodiments, the anti-NKG 2D antibody is as described above. In one embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and an anti-PD-1 antibody. In some embodiments, the fusion protein further comprises a linker. In one embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and an anti-PD-1 antibody. In some embodiments, the chimeric protein further comprises a linker. In other embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D antibody is KYK-1. In other embodiments, the anti-NKG 2D antibody is KYK-2. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the combination therapy comprises an anti-NKG 2D scFV, IL2, and PD1 inhibitor. In some embodiments, the combination therapy comprises anti-NKG 2D scFV, IL2, and anti-PD 1 antibodies. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D scFv is a KYK-1 scFv. In other embodiments, the anti-NKG 2D scFv is KYK-2 scFv. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL 2. In certain embodiments, the subject has its need. In certain embodiments, an effective amount of a combination therapy is administered to a subject. In a specific embodiment, the subject is a human.

In various aspects, the invention includes methods of slowing immunosuppression associated with radiation exposure or lymphotoxic substances comprising administering a composition comprising a cytokine linked to a ligand. Further, the method comprises administering a composition comprising a chimeric peptide as described in section I. In addition, the compositions of the present invention may be used to increase CD4 counts in HIV positive subjects. For example, the compositions of the present invention may be used to activate immune cells that may help restore the immune system of a subject. In some embodiments, the methods further comprise administering a chimeric peptide comprising a PD1 ligand and a cytokine. In another embodiment, the chimeric peptide further comprises a linker. In one embodiment, the PD1 ligand is PDL 1. In another embodiment, the PD1 ligand is PDL 2. In yet another embodiment, the PD1 ligand is an antibody specific for PD 1. In another embodiment, the cytokine is IL2. In some embodiments, IL2 is a mutant R38A/F42K form of IL2.

In some embodiments, the method further comprises administering a combination therapy as provided herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor. In other embodiments, the PD-1 inhibitor is an anti-PD-1 antibody.

In some embodiments, the combination therapy comprises a PD-L1 inhibitor. In other embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, provided herein is a method of reducing immunosuppression associated with radiation exposure or lymphotoxic substances in a subject, comprising administering to the subject a composition provided herein. In one embodiment, provided herein is a method of reducing immunosuppression associated with radiation exposure or lymphotoxic substances in a subject comprising administering to the subject a combination therapy provided herein. In one embodiment, the immunosuppression is associated with radiation exposure. In another embodiment, the immunosuppression is associated with a lymphotoxic substance. In some embodiments, the combination therapy comprises a PD-1 inhibitor provided herein and a chimeric peptide comprising a cytokine peptide and an anti-NKG 2D antibody. In certain embodiments, the cytokine peptide is a cytokine as described above. In some embodiments, the anti-NKG 2D antibody is as described above. In one embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and an anti-PD-1 antibody. In some embodiments, the fusion protein further comprises a linker. In one embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and an anti-PD-1 antibody. In some embodiments, the chimeric protein further comprises a linker. In other embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D antibody is KYK-1. In other embodiments, the anti-NKG 2D antibody is KYK-2. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the combination therapy comprises an anti-NKG 2D scFV, IL2, and PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2 dsfv, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D scFv is a KYK-1 scFv. In other embodiments, the anti-NKG 2D scFv is KYK-2 scFv. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL2. In certain embodiments, the subject has its need. In certain embodiments, an effective amount of a combination therapy is administered to a subject. In a specific embodiment, the subject is a human. In certain embodiments, the subject is a subject having a cancer or tumor. In a specific embodiment, the cancer or tumor is a lung cancer or tumor.

In an alternative aspect, the invention includes methods of using adjuvants in vaccine compositions. In one embodiment, provided herein is a method of vaccination in a subject comprising administering to the subject a composition provided herein. In some embodiments, the method of vaccination in a subject comprises administering a chimeric peptide comprising a PD1 ligand and a cytokine. In another embodiment, the chimeric peptide further comprises a linker. In one embodiment, the PD1 ligand is PDL 1. In another embodiment, the PD1 ligand is PDL 2. In yet another embodiment, the PD1 ligand is an antibody specific for PD 1. In another embodiment, the cytokine is IL2. In some embodiments, IL2 is a mutant R38A/F42K form of IL2. In one embodiment, provided herein is a method of vaccination in a subject comprising administering to the subject a combination therapy provided herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor provided herein and a chimeric peptide comprising a cytokine peptide and an anti-NKG 2D antibody. In certain embodiments, the cytokine peptide is a cytokine as described above. In some embodiments, the anti-NKG 2D antibody is as described above. In one embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and an anti-PD-1 antibody. In some embodiments, the fusion protein further comprises a linker. In one embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and an anti-PD-1 antibody. In some embodiments, the chimeric protein further comprises a linker. In other embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D antibody is KYK-1. In other embodiments, the anti-NKG 2D antibody is KYK-2. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the combination therapy comprises an anti-NKG 2D scFV, IL2, and PD1 inhibitor. In some embodiments, the combination therapy comprises anti-NKG 2D scFV, IL2, and anti-PD 1 antibodies. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D scFv is a KYK-1 scFv. In other embodiments, the anti-NKG 2D scFv is KYK-2 scFv. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL2. In certain embodiments, the subject has its need. In certain embodiments, an effective amount of a combination therapy is administered to a subject. In a specific embodiment, the subject is a human.

In other aspects, provided herein are compositions of the invention for expanding CD8+ memory cells. In one embodiment, provided herein is a method of expanding CD8+ T cells in a subject comprising administering to the subject a composition provided herein.

In one embodiment, provided herein is a method of expanding CD8+ T cells in a subject, the method further comprising administering a chimeric peptide comprising a PD1 ligand and a cytokine. In another embodiment, the chimeric peptide further comprises a linker. In one embodiment, the PD1 ligand is PDL 1. In another embodiment, the PD1 ligand is PDL 2. In yet another embodiment, the PD1 ligand is an antibody specific for PD 1. In another embodiment, the cytokine is IL2. In some embodiments, IL2 is a mutant R38A/F42K form of IL2. In one embodiment, provided herein is a method of expanding CD8+ T cells in a subject comprising administering to the subject a combination therapy provided herein. In some embodiments, the combination therapy comprises a PD-1 inhibitor provided herein and a chimeric peptide comprising a cytokine peptide and an anti-NKG 2D antibody. In certain embodiments, the cytokine peptide is a cytokine as described above. In some embodiments, the anti-NKG 2D antibody is as described above. In one embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 fusion protein and an anti-PD-1 antibody. In some embodiments, the fusion protein further comprises a linker. In one embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and a PD-1 inhibitor. In another embodiment, the combination therapy comprises an OMCP-IL2 chimeric protein and an anti-PD-1 antibody. In some embodiments, the chimeric protein further comprises a linker. In other embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and a PD1 inhibitor. In some embodiments, the combination therapy comprises an anti-NKG 2D antibody, IL2, and an anti-PD 1 antibody. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D antibody is KYK-1. In other embodiments, the anti-NKG 2D antibody is KYK-2. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D antibody and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, the combination therapy comprises an anti-NKG 2D scFV, IL2, and PD1 inhibitor. In some embodiments, the combination therapy comprises anti-NKG 2D scFV, IL2, and anti-PD 1 antibodies. In some embodiments, the anti-PD-1 antibody is an antagonist antibody. In certain embodiments, the anti-NKG 2D scFv is a KYK-1 scFv. In other embodiments, the anti-NKG 2D scFv is KYK-2 scFv. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as chimeric polypeptides. In some embodiments, the chimeric polypeptide further comprises a linker. In some embodiments, the anti-NKG 2D scFv and IL2 are provided as fusion proteins. In some embodiments, the fusion protein further comprises a linker. In some embodiments, IL2 is a mutant R38A/F42K form of IL2. In certain embodiments, the subject has its need. In certain embodiments, an effective amount of a combination therapy is administered to a subject. In a specific embodiment, the subject is a human. In certain embodiments, the subject is a subject having a cancer or tumor. In a specific embodiment, the cancer or tumor is a lung cancer or tumor.

In other aspects, the disclosure provides methods of expanding cytotoxic lymphocytes ex vivo. The method comprises culturing lymphocytes in the presence of a composition provided herein. Lymphocytes can be derived from publicly available cell lines, e.g., ATCC^TMA cell line. Alternatively, lymphocytes may be isolated from the subject. Lymphocytes can be obtained from a single subject or from multiple subjects. Plural refers to at least two (e.g., more than one) bodies. When the lymphocytes obtained are from multiple subjects, their relationship may be autologous, syngeneic, allogeneic or xenogeneic. In particular, lymphocytes can be cultured in the presence of the chimeric peptide described in section I. In certain embodiments, the chimeric peptide comprises OMCP or a fragment thereof linked to IL2 or a mutant thereof. In other embodiments, the chimeric peptide comprises OMCP linked to mutant IL2. In another aspect, the present disclosure provides methods of improving adoptive cellular immunotherapy in a subject. The method comprises administering to the subject a therapeutic composition comprising isolated cytotoxic lymphocytes that have been cultured in the presence of a composition provided herein. As used herein, "adoptive cellular immunotherapy," also referred to as "ACI," is a lymphocyte-based immunotherapy in which lymphocytes are taken from a subject and stimulated and/or genetically manipulated. After population expansion, lymphocytes are then transferred back into the host. Thus, the methods of the present disclosure may be used to treat diseases or conditions in which an increase in lymphocyte numbers is desired. For example, cancer and chronic viral infections. Of virus-specific T cells in connection with viral infectionACI can restore virus-specific immunity in a subject to prevent or treat viral diseases. Thus, virus-specific T cells can be used to reconstitute antiviral immunity after transplantation and/or to treat active viral infections. In one embodiment, the subject receiving the T cells for treatment or prevention of a viral infection may be immunodeficient.

(a) Administration of

In certain aspects, a pharmacologically effective amount of a composition of the present invention may be administered to a subject. Administration using standard effective techniques includes peripheral (i.e., not by administration to the central nervous system) or local to the central nervous system. Peripheral administration includes, but is not limited to, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Local administration, including direct to the Central Nervous System (CNS), includes, but is not limited to, controlled release formulations via lumbar, intraventricular or intraparenchymal catheters or using surgical implants. Apheresis (Pheresis) may be used to deliver the compositions of the present invention. In certain embodiments, the compositions of the present invention may be administered by infusion (continuous or bolus).

Pharmaceutical compositions for effective administration are intentionally designed to be appropriate for the mode of administration selected, and pharmaceutically acceptable excipients such as compatible dispersants, buffers, surfactants, preservatives, solubilizers, isotonicity agents, stabilizers and the like are suitably used. Remington's Pharmaceutical Sciences, Mack Publishing co., easton pa., 16Ed ISBN: 0-912734-04-3, latest edition (incorporated herein by reference in its entirety) provides a summary of formulation technology commonly known to practitioners.

Effective peripheral systemic delivery by intravenous or intraperitoneal or subcutaneous injection is the preferred method of administration to living patients. Suitable vehicles for such injections are straightforward. Alternatively, however, administration may be by mucosal application via a nasal aerosol or suppository. Suitable formulations for such administration are well known and typically include surfactants that promote transmembrane transport. Such surfactants are typically derived from steroids or cationic lipids, such as N- [1- (2, 3-dioleoyl) propyl ] -N, N-trimethylammonium chloride (DOTMA) or various compounds such as cholesterol hemisuccinate, phosphatidyl glycerol, and the like.

For therapeutic use, a therapeutically effective amount of a composition of the invention is administered to a subject. A "therapeutically effective amount" is an amount of the therapeutic composition sufficient to produce a measurable response (e.g., immune stimulation, anti-angiogenic response, cytotoxic response, tumor regression, immunosuppression, reduction of infection). The actual dosage level of the active ingredient in the therapeutic compositions of the present invention can be varied so as to administer an amount of the active compound effective to achieve the desired therapeutic response in a particular subject. The selected dosage level will depend upon a variety of factors including the activity of the therapeutic composition, the formulation, the route of administration, combination with other drugs or treatments, the size and longevity of the tumor in the subject being treated, autoimmune diseases, infection and physical condition, and prior medical history. In some embodiments, a minimum dose is administered and the dose is increased in the absence of dose limiting toxicity. Determining and adjusting a therapeutically effective dose, and assessing when and how such adjustments are made, are known to those of ordinary skill in the medical arts. In one aspect, a typical dose contains from about 10 IU/kg to about 1,000,000 IU/kg of a cytokine as described herein. In one embodiment, a typical dose contains from about 10 IU/kg to about 100 IU/kg. In another embodiment, a typical dose contains from about 100IU/kg to about 1,000 IU/kg. In yet another embodiment, a typical dose contains from about 1,000 IU/kg to about 10,000 IU/kg. In yet another embodiment, a typical dose contains from about 10,000IU/kg to about 100,000 IU/kg. In various embodiments, a typical dose contains from about 100,000 IU/kg to about 1,000,000 IU/kg. In certain embodiments, a typical dose contains from about 500,000 IU/kg to about 1,000,000 IU/kg. In other embodiments, a typical dose contains from about 100,000 IU/kg to about 500,000 IU/kg. Alternatively, a typical dose contains from about 50,000IU/kg to about 100,000 IU/kg. In another embodiment, a typical dose contains from about 10,000IU/kg to about 50,000 IU/kg. In yet another embodiment, a typical dose contains from about 5,000 IU/kg to about 10,000 IU/kg. In one embodiment, a typical dose contains from about 5,000 IU/kg to about 200,000 IU/kg. In another embodiment, a typical dose contains from about 5,000 IU/kg to about 500,000 IU/kg. In yet another embodiment, a typical dose contains from about 50,000IU/kg to about 500,000 IU/kg. In yet another embodiment, a typical dose contains from about 250,000IU/kg to about 750,000 IU/kg.

The frequency of administration may be once, twice, three or more times per day, or once, twice, three or more times per week or per month, as needed to effectively treat the symptoms or disease. In certain embodiments, the frequency of administration may be once, twice, or three times daily. For example, one dose may be administered every 24 hours, every 12 hours, or every 8 hours. In a particular embodiment, the frequency of administration may be twice daily.

The duration of treatment can range from a single dose administered at a time to a lifetime of treatment with the therapeutic agent. The duration of treatment may and will vary depending on the subject and the cancer or autoimmune disease or infection to be treated. For example, the duration of treatment may be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. Alternatively, the duration of treatment may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. Alternatively, the duration of treatment may be 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In yet another embodiment, the duration of treatment may be 1 year, 2 years, 3 years, 4 years, 5 years, or greater than 5 years. It is also contemplated that the administration may be frequent over a period of time and then the administration may be spaced apart over a period of time. For example, the duration of treatment may be 5 days, followed by 9 days without treatment, followed by 5 more days of treatment.

The timing of the administration of the treatment relative to the disease itself and the duration of the treatment will be determined by the circumstances surrounding the case. Treatment may be initiated immediately, for example at diagnosis, or may be initiated post-operatively. Treatment may be initiated in the hospital or clinic itself, or at a later time after discharge or seen in an outpatient clinic.

In addition, treatment with a composition as described above can be initiated in an administration regimen with (e.g., sequentially or simultaneously with) administration of a PD-1 inhibitor or a PD-L1 inhibitor as described herein. In some embodiments, the PD-L1 inhibitor is present in an amount that is a measure for the body weight of a patient in need thereof. For example, in some embodiments, the PD-L1 inhibitor is present in an amount of about: 0.1mg/kg to about 50 mg/kg, 0.1mg/kg to about 40 mg/kg, 0.1mg/kg to about 30mg/kg, 0.1mg/kg to about 25mg/kg, 0.1mg/kg to about 20mg/kg, 0.1mg/kg to about 15mg/kg, 0.1mg/kg to about 10mg/kg, 0.1mg/kg to about 7.5 mg/kg, 0.1mg/kg to about 5mg/kg, 0.1mg/kg to about 2.5 mg/kg, or about 0.1mg/kg to about 1 mg/kg. In some embodiments, the PD-L1 inhibitor is present in an amount of about: 0.5mg/kg to about 50 mg/kg, 0.5mg/kg to about 40 mg/kg, 0.5mg/kg to about 30mg/kg, 0.5mg/kg to about 25mg/kg, 0.5mg/kg to about 20mg/kg, 0.5mg/kg to about 15mg/kg, 0.5mg/kg to about 10mg/kg, 0.5mg/kg to about 7.5 mg/kg, 0.5mg/kg to about 5mg/kg, 0.5mg/kg to about 2.5 mg/kg, or about 0.5mg/kg to about 1 mg/kg. In some embodiments, the PD-L1 inhibitor is present in an amount from about 0.5mg/kg to about 5mg/kg or from about 0.1mg/kg to about 10 mg/kg. In some embodiments, the PD-L1 inhibitor is present in an amount from about 0.1mg/kg to about 20mg/kg or from about 0.1mg/kg to about 30 mg/kg.

In still other embodiments, in some embodiments, the PD-L1 inhibitor is present in an amount of about: 0.1mg/kg, 0.5mg/kg, 1mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg or 50 mg/kg. The PD-L1 antibody can be present in an amount of about: 1mg/kg, 2 mg/kg, 3mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg or 30 mg/kg. In some embodiments, the PD-L1 inhibitor is present in an amount of about: 3mg/kg, 10mg/kg, 20mg/kg or 30 mg/kg.

In some embodiments, the PD-L1 inhibitor is present in the combination therapy in an amount of about: 1mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 40 mg, 50mg, 60 mg, 70mg, 75 mg, 80 mg, 90 mg, 100mg, 150 mg or 200 mg. In some embodiments, the PD-L1 inhibitor is present in the combination therapy in an amount of about: 250mg, 300mg, 400mg, 500mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000mg, 1100 mg, 1200 mg, 1300mg, 1400 mg, 1500mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg or 2000 mg. In some embodiments, the PD-L1 inhibitor is present in an amount of about 1000mg to about 2000mg in the combination therapy. In some embodiments, the PD-L1 inhibitor is present in the combination therapy in an amount of about: 1mg to about 10mg, 10mg to about 20mg, 25mg to about 50mg, 30mg to about 60 mg, 40 mg to about 50mg, 50mg to about 100mg, 75 mg to about 150 mg, 100mg to about 200 mg, 200 mg to about 500mg, 500mg to about 1000mg, 1000mg to about 1200 mg, 1000mg to about 1500mg, 1200 mg to about 1500mg, or 1500 to about 2000 mg.

In some embodiments, the PD-L1 inhibitor is present in the combination therapy in an amount of about: 0.1 mg/mL, 0.5mg/mL, 1mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 400mg/mL, or 500 mg/mL. In one embodiment, the PD-L1 inhibitor is present in the combination therapy in an amount of about: 1mg/mL to about 10 mg/mL, 5mg/mL to about 15 mg/mL, 10 mg/mL to about 25 mg/mL, 20 mg/mL to about 30 mg/mL, 25 mg/mL to about 50 mg/mL, or 50 mg/mL to about 100 mg/mL.

In certain instances, a therapeutically effective amount of a PD-L1 inhibitor is determined as the amount provided in the package insert provided with the PD-L1 inhibitor. The term package insert refers to an insert typically contained in a commercial pharmaceutical package approved by the FDA or similar regulatory agency in a country other than the united states, which contains information regarding, for example, usage, dosage, administration, contraindications and/or warnings associated with the use of these drugs.

In some embodiments, the PD-1 inhibitor is present in an amount that is a measure of the body weight of the patient in need thereof. For example, in some embodiments, the PD-1 inhibitor is present in an amount of about: 0.1mg/kg to about 30mg/kg, 0.1mg/kg to about 25mg/kg, 0.1mg/kg to about 20mg/kg, 0.1mg/kg to about 15mg/kg, 0.1mg/kg to about 10mg/kg, 0.1mg/kg to about 7.5 mg/kg, 0.1mg/kg to about 5mg/kg, 0.1mg/kg to about 2.5 mg/kg, or about 0.1mg/kg to about 1 mg/kg. In some embodiments, the PD-1 inhibitor is present in an amount of about: 0.5mg/kg to about 30mg/kg, 0.5mg/kg to about 25mg/kg, 0.5mg/kg to about 20mg/kg, 0.5mg/kg to about 15mg/kg, 0.5mg/kg to about 10mg/kg, 0.5mg/kg to about 7.5 mg/kg, 0.5mg/kg to about 5mg/kg, 0.5mg/kg to about 2.5 mg/kg, or about 0.5mg/kg to about 1 mg/kg. In some embodiments, the PD-1 inhibitor is present in an amount of about 0.5mg/kg to about 5mg/kg or about 0.1mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is present in an amount of about 0.5mg/kg to about 15mg/kg or about 0.1mg/kg to about 20 mg/kg.

In some embodiments, the PD-1 inhibitor is present in an amount of about: 0.1mg/kg, 0.5mg/kg, 1mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg or 30 mg/kg. In some embodiments, the PD-1 inhibitor is present in an amount of about: 1mg/kg, 2 mg/kg, 3mg/kg or 5 mg/kg.

In some embodiments, the PD-1 inhibitor is present in the combination therapy in an amount of about: 1mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 40 mg, 50mg, 60 mg, 70mg, 75 mg, 80 mg, 90 mg, 100mg, 150 mg, 200 mg, 250mg, 300mg, 400mg, 500mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000mg, 1100 mg, 1200 mg, 1300mg, 1400 mg, 1500mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg. In some embodiments, the PD-1 inhibitor is present in the combination therapy in an amount of about: 1mg to about 10mg, 10mg to about 20mg, 25mg to about 50mg, 30mg to about 60 mg, 40 mg to about 50mg, 50mg to about 100mg, 75 mg to about 150 mg, 100mg to about 200 mg, 200 mg to about 500mg, 500mg to about 1000mg, 1000mg to about 1200 mg, 1000mg to about 1500mg, 1200 mg to about 1500mg, or 1500mg to about 2000 mg.

In some embodiments, the PD-1 inhibitor is present in the combination therapy in an amount of about: 0.1 mg/mL, 0.5mg/mL, 1mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 400mg/mL, or 500 mg/mL. In one embodiment, the PD-1 inhibitor is present in the combination therapy in an amount of about: 1mg/mL to about 10 mg/mL, 5mg/mL to about 15 mg/mL, 10 mg/mL to about 25 mg/mL, 20 mg/mL to about 30 mg/mL, 25 mg/mL to about 50 mg/mL, or 50 mg/mL to about 100 mg/mL.

In certain instances, a therapeutically effective amount of a PD-1 inhibitor is determined as the amount provided in the package insert provided with the PD-1 inhibitor.

The synergistic effect of the combination therapies described herein may allow for the use of lower doses of one or more of the components of the combination (e.g., the composition described herein and PD-1 or PD-L1 inhibitor). The synergistic effect may allow for less frequent administration of at least one administered treatment (e.g., a composition described herein and a PD-1 or PD-L1 inhibitor) to a subject having a disease, disorder, or condition described herein. Such lower doses and reduced frequency of administration can reduce the toxicity associated with administering at least one treatment (e.g., a composition described herein and a PD-1 or PD-L1 inhibitor) to a subject without reducing the efficacy of the treatment. The synergistic effect as described herein may avoid or reduce adverse or unwanted side effects associated with the use of any of the therapies described herein.

Although the foregoing methods appear to be the most convenient and appropriate and effective for administering the compositions or combination therapies of the present invention, other effective administration techniques, such as intraventricular administration, transdermal administration, and oral administration, may be employed with appropriate modification, so long as the appropriate formulation is used herein.

Furthermore, it may be desirable to employ controlled release formulations using biodegradable membranes and matrices, or osmotic minipumps, or delivery systems based on dextran beads, alginate or collagen.

(b) Tumor(s)

the composition of the invention can be used in a method for treating or identifying a tumor derived from a neoplasm or cancer, the neoplasm may be malignant or benign, the cancer may be primary or metastatic, the neoplasm or cancer may be early or late stage, the neoplasm or cancer that may be treated includes acute lymphoblastic leukemia, acute myelogenous leukemia, adrenal cortex cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma (of the cerebellum or brain of a child), basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain stem glioma, brain tumor (cerebellar astrocytoma, cerebral astrocytoma/glioblastoma, ependymoma, medulloblastoma, epididymoblastoma, epididymal or cerebrovateleocytoma, prostatic hyperplasia, neuroblastoma, splenomegaloblastoma, neuroblastoma, melanoma, neuroblastoma, melanoma, neuroblastoma, melanoma, neuroblastoma, melanoma, neuroblastoma, melanoma, neuroblastoma, melanoma, neuroblastoma, melanoma.

(c) Main body

Suitable subjects include humans, livestock animals, companion animals, laboratory animals, or zoo animals. In one embodiment, the subject may be a rodent, such as a mouse, rat, guinea pig, or the like. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cattle, horses, goats, sheep, llamas, and alpacas. In yet another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals can include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoo animal. As used herein, "zoo animal" refers to an animal that can be found in a zoo. These animals may include non-human primates, large felines, wolves, and bears. In a particular embodiment, the animal is a laboratory animal. Non-limiting examples of experimental animals can include rodents, canines, felines, and non-human primates. In certain embodiments, the animal is a rodent. Non-limiting examples of rodents can include mice, rats, guinea pigs, and the like. In a preferred embodiment, the subject is a human.

Examples

The following examples are included to demonstrate the preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples 1 to 6

the IL2R α chain is used to capture IL2 on the cell surface to facilitate subsequent association with the signaling portion of the receptor, the IL2R β gamma chain, resting cytotoxic lymphocytes, such as Natural Killer (NK) and CD8⁺T cells, which do not express significant IL2R α on the cell surfaceAnd therefore are not activated by low levels of IL2¹however, IL2R α is expressed on this population after initial activation and is essential for maximal cytotoxic lymphocyte expansion². High doses of IL2 induce activation of all cytotoxic lymphocytes and are approved for the treatment of several malignancies with about 15% partial or complete tumor response^3-5. Due to regulatory T cells (T)_regs) Such as severe blood pressure changes, general capillary leakage and end organ failure due to activation of the vascular endothelium, most patients cannot benefit from this treatment^6,3,7. Vascular endothelium and T_regsall express IL2R α and are therefore preferentially activated by IL2 relative to cytotoxic lymphocytes⁸mutant forms of IL2, e.g., those having alanine for arginine at position 38 (R38A) and/or lysine for phenylalanine at position 42 (F42K), reduce the affinity of IL2 for IL2R α, thereby eliminating many side effects⁹. Such IL2a mutants may also reduce the efficacy of immunotherapy²a form of IL2 that can preferentially activate cytotoxic lymphocytes in the absence of IL2R α reactivity would be highly advantageous for clinical use.

NKG2D recognizes MHC class I-associated stress ligands expressed by malignant or virally transformed cells¹⁰. Among all activated immunoreceptors, NKG2D has the highest specificity for cytotoxic lymphocytes, as it is found on murine and human NK cells as well as activated CD8⁺Constitutive expression on T cells¹¹. Thus, it has been argued that tumor and virus infected cells utilize shed NKG2D ligand as a mechanism for immune evasion^12,13. Smallpox major histocompatibility complex class I-like protein or OMCP is an shed NKG2D ligand decoy by monkeypox and vaccinia virus infected cells. It is not expressed by smallpox or vaccinia virus and is therefore not recognized by those immunized with the smallpox vaccine. Since OMCP binds human and murine NKG2D with the highest affinity of any known ligand, we believe it may function as an ideal targeting vector to optimally targetDelivery of IL2 to cytotoxic lymphocytes^14,15Here we describe the construction and function of fusion proteins designed to deliver IL2R α mutants to NKG2D expressing lymphocytes¹⁵we demonstrate that this construct overcomes the reduced efficacy associated with IL2R α binding region mutations while maintaining a favorable safety profile systemic administration of this fusion protein improves immunotherapy against solid and liquid tumors therefore, targeted delivery of IL2 can be safely used to maximally activate NKG 2D-expressing lymphocytes, such as NK cells, to optimize immunotherapy without systemic side effects.

Example 1 targeted delivery of the NKG2D mutant IL2 preferentially activates cytotoxic lymphocytes in vitro.

to overcome the preferential activation of cells expressing IL2R α, we designed an IL2 fusion protein that could directly target cytotoxic lymphocytes through the NKG2D receptor, this fusion protein combines high affinity NKG2D ligand OMCP with IL2 mutated to reduce IL2R α reactivity our construct, called OMCP-mutIL2, consisting of 152 residue OMCP protein fused via a flexible 30 residue linker to the N-terminus of the 133 amino acid R38A/F42K mutant form of human IL2 (mutIL 2) (fig. 1A-B), first evaluated the in vitro binding capacity of the construct, binding of fluorescently labeled constructs was tested in vitro at 37 ℃ in a large number of splenocytes, fig. 17 shows that this construct only appears to bind to NK cells expressing NKG2D, this construct does not show binding to CD4+ CD3+ T cells, CD8+ CD 4611, CD 4611 + CD 4611-CD 11 + CD 4611, CD 573 + CD 11-CD 4611.

We have previously demonstrated strain-specific differences in murine NK cell cytotoxicity and immune surveillance for lung cancer¹⁶(and example 7). Therefore, we began to study the efficacy of OMCP-mut-IL2 in NK cell activation from two different mouse strains (i.e., a/J and B6 with poor and robust NK function, respectively). OMCP-mutIL2 was as fine as 100IUe/ml compared to wild-type IL2 (wtIL 2) or mutIL2Cytokine co-culture for 36 hours strongly up-regulated CD69 on NK cells of both lines (FIG. 1C, left; FIG. 6, left panels)^16,17. At high concentrations, similar increases in CD69 expression were observed with OMCP-mut-IL2, wtIL2, or mutIL2 (fig. 6). CD4 measured by ICOS upregulation⁺Foxp3⁺T_regsActivation of (D) was only evident with wtII2, but not with mutIL2 or OMCP-mutIL2 (FIGS. 1C-D). CD8, on the other hand⁺And CD4⁺Foxp3^-Effector T cells did not show up-regulation of CD69 after 36 hours, even at the highest dose of cytokines (fig. 1C-D and data not shown). Longer exposure over a 5 day period resulted in exposure to both NK and CD8 of wtIL2 and OMCP-mutIL2⁺Proliferation of T cells (FIGS. 1E-F). Importantly, OMCP-mutIL2 activates CD8⁺T cells and NK cells are equivalent to NKG2D^-/-mutIL2 in splenocytes, suggesting that increased activation is due to the effect of OMCP targeting on NKG 2D-bearing cells (FIG. 1F; FIG. 6, right panels). Incubation with wtIL2 alone resulted in CD4⁺Foxp3⁺T_regsAnd CD4⁺Foxp3^-The effector cells proliferated (FIGS. 1E-F). Thus, exposure to OMCP-mutIL2 resulted in preferential NK activation that was superior to or equivalent to wtIL2 in a dose-dependent manner. CD8⁺T cells can also be activated, but require prolonged exposure to higher doses of OMCP-mutIL 2.

Example 2. low dose cytokine therapy provides an advantageous safety profile.

Dose-dependent toxicity may limit the administration of cytokines in vivo. To mimic the human immunotherapy protocol, we next administered wtIL2 at 10 doses over a 5 day period to treat A/J mice¹⁸. Although a/J mice tolerated wtIL2 at 750,000 IUe, significant mortality was evident at higher doses (fig. 2A-B). Even after 750,000 IUe doses, mice exhibited extreme pain, weight loss, reduced food consumption, ascites, and liver inflammation (fig. 2A-E; fig. 7A-C). These side effects reflect high doses in humansIL2 treatment-related capillary leakage and distress⁷. Treatment with anti-Asialo-GM 1 improved mortality induced by high doses of wtIL2 (1,500,000 IUe) in a/J mice, but did not improve weight loss, confirming that the side effects of this therapy can occur independently of NK cells (fig. 2F-K). Unlike the case of wtIL2, there was no significant animal death after 1,500,000 IUe of OMCP-mutIL2 or mutIL2 in the presence or absence of NK cells. Only in NK-sufficient mice the animal weight loss after OMCP-mutIL2 of 1,500,000 IUe occurred, suggesting that the toxicity of our construct was due solely to immune activation (FIG. 2F-K). In A/J mice, the regimen of 200,000 IUe was well tolerated with minimal weight loss, pain or organ inflammation for all cytokines (FIG. 2L-O). However, at this dose, accumulation of pleural effusion and ascites after wtIL2 still indicates capillary leakage, but not OMCP-mutIL2 or mutIL 2. B6 mice were able to tolerate higher doses of wtIL2, but still had significant mortality above 750,000 IUe (fig. 7D).

Example 3 OMCP-mutIL2 preferentially expands and activates NK cells in vivo compared to wtIL2 or mutIL 2.

To assess the immunological changes associated with cytokine therapy, a/J mice received 200,000 IUe of the cytokine or construct, which was administered at 10 equal doses over 5 days. Splenic lymphocytes were assessed by flow cytometry on day 6. Both wtIL2 and OMCP-mutIL2 increased lymphocyte content and spleen size compared to saline-treated controls (FIGS. 3A-B). OMCP-mutIL2 resulted in significant expansion and activation of NK cells as measured by cellular and surface KLRG1 levels (fig. 3C). In OMCP-mutIL2 treated mice, NK cells accounted for nearly half of all splenic lymphocytes, paralleling or even exceeding the total lymphocyte count of saline or mutIL2 treated mice (fig. 3A vs fig. 3C). NK amplification by OMCP-mutIL2 of 200,000 IUe outperformed either a near toxic dose of wtIL2 (750,000 IU), a high dose of mutIL2 (3,500,000 IUe), or wtIL2 complexed with anti-IL2 antibody (clone MAB 602)¹⁹(FIG. 3C). In fact, most mice were unable to tolerate the wtIL 2/anti-IL2 antibody of all 200,000 IUe and due to animal pain and rapid weight loss, injections had to be terminated at 160,000 or 180,000 IUe with the necessary animal death (fig. 8A). WtIL2 resulted in a significant expansion of CD4⁺Foxp3⁺T_regsICOS, particularly in A/J mice⁺Subgroup of⁶Even when complexed with anti-IL2 antibody (fig. 3D). Importantly, predictors which have been described as success in immunotherapy compared to all other treatment conditions²⁰NK/T of_regThe ratio was significantly increased in OMCP-mutIL2 treated mice (fig. 3E). Excellent expansion of NK cells by OMCP-mutIL2 could be achieved even at 1/2 dose of wtIL2 (fig. 8B). However, targeting NKG2D with the 500-fold lower affinity NKG2D ligand ULBP3 improved the amplification efficacy of the fusion construct, but still provided superior NK activation compared to mutII2 alone (fig. 8B). CD4 following wtIL2 or OMCP-mutIL2 treatment⁺Foxp3^-Or CD8⁺There was apparently no statistically significant increase in T lymphocytes, despite CD8⁺The tendency of T cell expansion is evident (FIGS. 8C-D). These data are consistent with the prevalence of naive T lymphocytes, expressing low levels of IL2 receptor and NKG2D in mice without a specific pathogen.

Unlike the a/J strain, a small amount of immune activation of lymphocytes was evident in B6 mice treated with wtIL2 at 200,000 IUe (data not shown). OMCP-mutIL2 was more active than wtIL2 in expanding NK cells at higher doses of 750,000 IUe in this line (FIGS. 3F-H). IL2/anti-IL2 antibody complex prevents T_regExpanded, but similar to the a/J strain, this treatment was toxic, and most B6 mice were unable to tolerate the full 750,000 IUe dose (fig. 3I). However, OMCP-mutIL2 tolerated well at this dose and resulted in high NK/T_regRatio (fig. 3J). B6NKG2D treated in OMCP-mutIL2^-/-There was no significant NK cell expansion in the mutants, confirming the need for NKG2D in our construct function (data not shown). Although CD8 was observed after administration of wtIL2⁺The tendency to expand T cells is clear, but at any treatmentNone of the B6CD8 in the group⁺Or CD4⁺Foxp3^-Statistically significant expansion of T cells was evident (fig. 8F-G). The same data for lung resident lymphocytes were obtained in the a/J and B6 lines (data not shown).

Example 4 OMCP-mutIL2 preferentially expands and activates NK cells in human peripheral blood lymphocytes compared to wtIL2 or mutIL 2.

To demonstrate the efficacy of OMCP-mutIL2 in human lymphocytes, human peripheral blood lymphocytes were co-cultured for 36 hours in 100IUe of wild-type IL2, the R38A/F42K mutant form of IL2, or OMCP-mutant IL2.

NK cells: cells were analyzed by flow cytometry and the relative prevalence of CD56+ CD 3-NK cells was compared between conditions. A relatively high proportion of NK cells was evident in the OMCP-mutant IL2 group (fig. 31A). Perforin levels were higher in OMCP-mutant IL 2-treated NK cells (red) compared to saline (black), IL2 (blue) or mutant IL2 (green) treated NK cells (fig. 31B).

CD8+ T cells: similar to NK cells, CD8 treated with OMCP-mutant IL2 compared to other conditions⁺Higher intracellular levels of perforin in T cells were evident (fig. 31C).

Tregs: when CD4+ Foxp3+ CD45 RA-T cells were gated, a relatively higher proportion of activated CD25+ CD 127-regulatory T cells was evident in IL2 treated peripheral blood lymphocyte cultures compared to the other conditions (fig. 31D). Taken together, this data indicates that OMCP-mutIL2 preferentially expands and activates NK cells and CD8+ cells in human peripheral blood lymphocytes compared to wtIL2 or mutIL 2. Importantly, OMCP-mutIL2 did not significantly activate regulatory T cells relative to IL2.

Example 5 treatment with OMCP-mutIL2 provides excellent immune control of malignant tumors in vivo.

Unlike T lymphocytes that require prior antigen encounter to achieve optimal antigen-specific tumor cytotoxicity, NK cells can mediate native cytotoxicity without prior sensitization. NK cells also form a major obstacle for the expansion of selected malignancies, such as lymphoma and lung cancer^16,17,21,22. Treatment of A/J mice with OMCP-mutIL2 resulted in increased clearance in vivo and lysis of YAC-1 cells in vitro by large numbers of splenocytes, compared to wtIL2 or mutIL2 (FIG. 4A, FIG. 9A-B). The reduction in growth of the highly invasive Lewis Lung Carcinoma (LLC) cell line was evident in B6 mice after OMCP-mutIL2 at 750,000 IUe compared to wtIL2 or mutIL 2. Increased cytotoxicity was also evident in OMCP-mutIL 2-treated splenocytes for the LLC cell line (FIGS. 4B-C; FIGS. 9A-C). At NKG2D^-/-Enhanced immunotherapy was lost in mice or following NK depletion (fig. 4D-E). In the absence of host NKG2D, mutIL2 actually increased the rate of LLC growth. Thus, OMCP-mediated targeting of mutIL2 provides a safer and more effective form of immunotherapy for solid and liquid tumors in various mouse strains.

Example 6. Effect of NKG2D targeting on IL2 signaling.

antibody-IL 2 conjugate or IL2/anti-IL2 antibody complex show improved biological activity relative to purified cytokines by extending the duration of serum half-life^23,24. To investigate whether the linkage of IL2 to OMCP increased serum half-life, we injected 500,000 IUe of fluorescently labeled wtIL2, mutIL2 or OMCP-mutIL2 into a/J and B6 mice and monitored serum clearance by continuous blood draw. Although OMCP-mutIL2 had slightly higher serum concentrations at the early time points, not all constructs were detected in the blood one hour after injection (fig. 5A-B). This is significantly shorter than the 11-14 hour serum half-life of antibody-IL 2 conjugate²³. Interestingly, despite the same amount of cytokine injected, at all time points, lower levels were detected in B6 mice as compared to A/J miceCytokine levels. This data indicates a strain-specific difference in IL2 clearance and may explain why B6 mice are able to tolerate and require higher doses of cytokines for NK expansion. However, based on this data, prolonged cycling of the construct is unlikely to result in increased activity of OMCP-mutIL2 relative to wtIL 2.

We next considered the possibility that the superiority of OMCP-mutIL2 is a consequence of signaling through NKG2D, since antibody-mediated cross-linking of this receptor can activate NK cells (FIG. 10A)²⁵. While the addition of purified OMCP to mutIL2 did not increase NK activation or amplification in vitro or in vivo (data not shown), we did not expect the monomeric ligand to cross-link NKG2D. Therefore, we directly compared NK cell activation in the presence of 1000IUe OMCP-mutIL2, mutIL2 and mutIL2 in combination with equimolar concentrations of pentameric OMCP. In the presence of pentameric OMCP, NK activation was not increased as measured by CD69 upregulation or degranulation (fig. 5C, fig. 10B). This suggests that NKG2D cross-linking is not responsible for the enhanced NK cell activation of OMCP-mutIL2 at physiological concentrations.

to assess IL2 signaling, we next quantified STAT5 phosphorylation after 15 min cytokine stimulation on freshly isolated NK cells in vitro, compared to B6NK cells, lower levels of STAT5 phosphorylation in a/J were evident at all tested concentrations (fig. 5D-E), indicating that lymphocyte dysfunction in a/J mice may be at least partially the result of ineffective IL2 signaling, surprisingly, wtIL2 and OMCP-mutIL2 showed the same dose-dependent STAT5 phosphorylation pattern for B6 and a/J NK cells (fig. 5D-E), in the absence of NKG2D reactivity OMCP-mutIL2 failed to increase STAT5 over mutIL2 alone, taken together, these data indicate that IL2 α reactivity is important for peak IL2 signaling in resting NK cells, and that IL 638 binding can effectively replace IL 2-mediated phosphorylation of STAT 582 in vivo in signaling, whereas in vivo, these data are not interpreted by the presence of NK 3-mediated phosphorylation of IL 638 in vivo (fig. 5D-g 638).

to test this, we stimulated freshly isolated NK cells for 15 minutes, replaced the medium with cytokine-free medium, and monitored STAT5 phosphorylation for 4 hours the same attenuation of phospho-STAT 5 was evident for wtIL2 and OMCP-mutIL2 (fig. 5F-G). therefore, altering the duration of IL2 signaling was not responsible for excellent NK activation by OMCP-mutIL 2.

We next considered the possibility that excellent NK activation of OMCP-mutIL2 is the result of altered cytokine interaction with competing stromal cells (FIG. 5H). indeed, OMCP-mutIL2 demonstrated dose-dependent enhancement of NK STAT5 phosphorylation over wtIL2 in the presence of other splenocytes (FIG. 5I). We next explored the interplay of IL2R α expression by stromal cells with NKG2D expression by NK cells on IL2 signaling^-/-NK cell-depleted splenocytes were treated with saturating concentrations of IL2R α blocking antibody (clone 3C 7) prior to recombination with wild-type NK cells for some cultures, then the cultures were stimulated with 1000IUe of wtIL2 or OMCP-mutIL2 for 15 minutes STAT5 phosphorylates in NKG2D in the presence of wtIL2^-/-Or wild-type NK cells (fig. 5J, left two columns). Wild-type NK cells cultured with OMCP-mutIL2 showed excellent STAT5 phosphorylation relative to cultures with wtIL 2. NKG2D in culture with OMCP-mutIL2^-/-in NK cells, there was little apparent STAT5 phosphorylation (fig. 5J, right two panels) — in the presence of IL2R α blockade of competing splenocyte stromal cells, NK cell STAT5 phosphorylation by wtIL2 increased to levels comparable to OMCP-mutIL2 (fig. 5K)IL2-Ra expression limited NK cell activation by wtIL2, and this competition could be abrogated by the impaired OMCP-mutIL2 construct targeting IL2R α binding of NKG2D.

Discussion of examples 1-6.

Although IL2 treatment initially showed great promise, it was subjected to T_regsone strategy is to generate mutants with increased affinity for IL2R β to eliminate the preference for IL2R α^26,27importantly, these IL2 mutants retained wild-type binding of IL2R α and thus could still be bound by T_regour results also indicate that IL2-R α is expressed⁺The bioavailability of wtIL2 to cytotoxic lymphocytes is limited by the competition of cells.

Another promising therapy involves anti-IL2 antibodies that sterically inhibit the binding of wtIL2 to IL2R α^1,28,29due to the Fc region of the antibody and potentially due to the reduced competition of wtIL2 from IL2R α expressing cells, this treatment can prolong serum half-life²⁴. antibody-IL 2 fusion proteins were also designed to target IL2 to specific tumor antigens^30,31. While offering potential for personalized therapy, this antibody-mediated delivery of IL2 to tumors depends on the expression of known tumor-associated antigens, a condition that is not normally present. This approach may potentially be further limited by tumor-mediated alteration of the target antigen.

finally, IL2 mutants with reduced affinity for IL2R α have been extensively tested these mutants can be administered at supratherapeutic doses compared to wtIL2 without IL2R α mediated capillary leakage or systemic toxicity³². Although these mutants have an excellent safety profile, they activate poorly cytotoxic lymphocytes (FIGS. 5C-E)³³. Our method combines the above concepts to secure IL2this is achieved by replacing the normal targets of IL2 and IL2R α with NKG2D the combination of IL2R α deficient IL2 fused to a high affinity NKG2D ligand expands NK cells specifically without any apparent T-cell_regsActivation or capillary leakage improves previous strategies. These findings provide the prospect of a potentially safe and efficient form of IL2.

One limitation of transforming the results of inbred experimental animals into humans is the natural diversity of cytokine reactivity and the environmentally dependent threshold of lymphocyte activation. Previous studies have demonstrated a correlation between ex vivo killing of tumor cells and enhanced long-term cancer immunity³⁴. Thus, any potential treatment requires consideration of populations with different levels of cytotoxic lymphocyte activity. Therefore, we have attempted to mimic this natural variation by using two strains of mice known to be highly resistant (B6) or susceptible (a/J) to carcinogenesis. For example, NK cells from B6 mice were activated by wtIL2 and an extreme dose of mutIL 2. In contrast, the IL2/anti-IL2 antibody complex resulted in the expansion of NK cells in A/J but not B6 mice. These changes highlight the limitation of converting results from a single mouse strain to immunologically distinct humans. Importantly, the OMCP-mutIL2 construct was able to expand NK cells in both mouse strains, suggesting that this therapy may be effective in populations with different NK functions and cytokine responsiveness.

Since OMCP is described as an evolutionary antagonist of NKG2D³⁵Blockade of this immunoreceptor during tumor therapy may be considered counterproductive. Nevertheless, native cytotoxicity and tumor clearance were enhanced in OMCP-mutIL 2-treated mice even in the presence of established tumors. This indicates minimal or transient NKG2D receptor occupancy and preservation function. Alternatively, recent reports have demonstrated that shed NKG2D ligand can actually promote tumor immunity by reversing NK desensitization imposed by chronic agonist engagement³⁶. Although we did not detect NK activation or amplification by monomeric or even pentameric OMCP, in tumorsThis competitive antagonism in bed may play a conflicting role in NK activation. In addition, IL2 may upregulate receptors necessary for NK migration and tumor infiltration. Thus, OMCP-mutIL 2-mediated anti-tumor immunity may rely on NK cells located outside the tumor bed without experiencing local tumor-specific tolerance or inefficacy. Furthermore, OMCP may be an ideal "targeting vector" because of its high affinity and long half-life for binding to human NKG2D.

Although NK cells from two independent mouse strains were activated by OMCP-mutIL2, we did not detect our construct pair CD8⁺Global expansion of T cell activation. This is likely due to NKG2D being only in the selected CD8⁺The fact that it is expressed on a subpopulation of T cells (i.e. memory or activated cytotoxic lymphocytes). Based on the absence of this cell population in mice cultured in a specific pathogen-free environment, OMCP-mutIL 2-mediated activation was limited in our system by NK cells. For this reason, we focused on immunotherapy of lung cancer and lymphoma, whose growth is mainly regulated by NK cells^16,17,22,37. However, OMCP-mutIL2 was able to amplify CD8 when administered at high concentrations in vitro⁺T cells (FIGS. 1E-F). Thus, it is likely that targeted delivery of NKG2D of immunostimulatory cytokines may result in antigen-specific CD8⁺Expansion and/or activation of memory cells for long-term tumor immunity under normal immune conditions.

The method of examples 1-6.

Cytokine and construct production: cloning of sequences encoding human IL2 (1-133; C125S) and mutant IL2 (1-133; R38A, F42K, C125S) into pFMM 1.2R with an N-terminal FLAG/hexahistidine tag³⁸In (1). The chimeric OMCP-mutIL2 molecule contained the full-length OMCP (1-152) coding sequence cloned into pFM1.2R vector in-frame with the C-terminal FLAG/hexahistidine tag-mutant IL2 (1-133; R38A, F42K, C125S). Proteins were expressed by transient transfection into HEK293F (Life Technologies). Supernatants were recovered at 72 and 144 hours post-transfection. The supernatant was supplemented with 5mM imidazole and 0.02% sodium azide and purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (Qiagen). Purified protein buffer was exchanged into saline and snap frozen in liquid nitrogen. Equivalent in vitro and in vivo activity was recorded for wild type IL2 produced internally and Tecoeukin (TecinTM) available from the NCI depot (Frederick National Laboratory for Cancer Research). Thus, for some experiments, the two IL2 preparations were used interchangeably.

Wild type IL2 has a 15x10⁶Specific activity IU/mg³⁹. Thus, based on a molecular weight of 15.5kDa, a 4.4. mu.M solution corresponds to 1000 IU/. mu.l. Based on this calculation, all cytokines and constructs were administered on a molar basis of 1 μ l of a 4.4 μ M solution, defined as 1000IU equivalents (IUe from here on). This system allows an equimolar comparison between IL2, mutIL2 and OMCP-mutIL2 despite the difference in molecular weight.

Animals: A/J (8-12 weeks) and C57BL/6J (6-9 weeks) mouse strains were purchased from Jackson Laboratory (BarHarbor, Maine). NKG2D against B6 background^-/-Mice were kindly supplied and bred internally by Wayne Yokoyama (Howard Hughes Institute of Medicine in Washington University, St. Louis). Animals were housed in a barrier facility in air filtration cages and allowed free access to food and water. For some experiments, A/J mice were treated with depleted concentrations of anti-Asialo-GM 1 (50. mu.l day-2; 25. mu.l day-1) or control rabbit IgG (Wako Chemical Company). Animal procedures were approved by the animal research committee of the University School of Medicine, St. Louis, MO, Washington.

Tissue harvesting and in vitro culture: single cell suspensions of splenocytes were obtained by ACK buffer (Lonza, walker ville, MD) crushing the entire spleen through a 70 μm cell filter and refiltering through a 40 μm filter before RBC lysis. Before treatment in the same manner as the spleen, the lungs were digested in 1mg/ml collagenase II (Fisher scientific) and 5U/ml DNaseI (Sigma-Aldridge) for 90 minutes at 37 ℃.

For in vitro culture, from A/J, B6 or NKG2D^-/-Splenocytes from mice were extracted aseptically and inoculated in complete medium (RPMI 1640, supplemented with 10% FBS, 100U/ml penicillin and streptomycin, 2mM L-glutamine and 50 μ M2-mercaptoethanol) in 12-well plates at5 million cells per ml per well. Cells were treated with increasing doses of human recombinant IL2, mutIL2, OMCP-mutIL2 or OMCP for 36 hours as described in the literature. For some experiments, large numbers of splenocytes were labeled with CFSE and cultured in 1000IUe/ml cytokine for 5 days prior to flow cytometry analysis. For NK isolation experiments, a large number of splenocytes were treated with NK cell isolation kit II or CD49b (DX 5) positive magnetic bead selection (both from Miltenyi Biotech). For STAT5 phosphorylation experiments, isolated NK cells were stimulated at 100,000 cells/500 μ Ι for 15 min in increasing concentrations of IL2 or construct. For experiments to assess NK cell interaction with spleen stroma, DX 5-positively selected NK cells (for identification after fixation and permeabilization) were labeled with CFSE and recombined with NK-depleted stromal cells. For some studies, NKG2D was used, as described in the article^-/-NK cells were combined with wild-type splenocyte stromal cells for other experiments NK-depleted splenocytes from wild-type B6 mice were treated with saturating concentrations of either anti-IL2 α blocking antibody (clone 3C 7) or isotype control (both from Biolegend) prior to recombination with NK cells for such competitive STAT5 phosphorylation experiments, 100,000 cells were resuspended in 2 μ l of complete medium containing 1,000 IU/ml wtIL2, mutIL2, or OMCP-mut-IL2 (freshly prepared and pre-warmed), then the cells were incubated for 15 minutes at 37 ℃.

Flow cytometry all antibodies were anti-mouse and purchased from BD Bioscience or eBioscience and consisted of anti-CD 4 (clone GK1.5 or RM 4-5), anti-CD 8 (clone 53-6.7), anti-CD 278 (ICOS) (clone: 7 E.17G9), anti-CD 25 (clone PC 61), anti-KLRG 1 (clone 2F 1), CD49b (integrin α 2) (clone DX 5), anti-CD 3e (clone DX 5), all flow cytometry analyses were performed at 4 ℃ in a FACS buffer consisting of PBS containing 2% FBS and 0.4 EDTA using saturating concentrations of fluorochrome-conjugated antibodies1452C 11), anti-CD 45 (clone 30-F11), anti-CD 69 PE (clone H1.2F3), anti-GITR (clone DTA-1), anti-Foxp 3 (clone: FJK-16 s) and anti-Stat 5 (clone 47/Stat 5; pY 694). The antibody is conjugated with FITC, PE, PerCP-CyTM5.5, PE-Cyanine7, APC-eFluor 780, eFluor 450 or AlexaFluor 647.

phospho-Stat 5 evaluation was performed by paraformaldehyde fixation, methanol permeabilization and staining with AlexaFluor 488-conjugated anti-Stat 5 (pY694) (BD Pharmingen; clone 612599). To accomplish this, isolated NK cells or a combination of NK cells and NK-depleted splenocyte stromal cells were fixed in 2% Paraformaldehyde (PFA) for 10 min at 37 ℃ after 15 min stimulation with IL2. Cells were then washed once with ice-cold PBS and permeabilized by adding 0.5 ml/tube of 90% methanol on ice for 1 hour. Cells were washed once with ice-cold PBS (to remove methanol) and stained with anti-Stat 5 (pY694) antibody for 1 hour at room temperature, then washed once in PBS/0.5% fetal calf serum.

In vitro cytotoxicity: ⁵¹by contacting the target cells with 100 mCi sodium chromate⁵¹(Perkinelmer) was incubated for 1 hour for chromium release. A large number of splenocytes were used as effector cells and were plated in round bottom 96-well plates at 37 ℃ with defined effectors: target ratio and target were incubated for 4 hours. Specific cleavage is expressed as (experimental release-spontaneous release)/(maximum release-spontaneous release) X100%, with 0% specific cleavage as the lowest expression value.

In vivo cytokine injection: for the selection experiment, mice received intraperitoneal injections of a volume of 200 μ l of cytokine administered as ten equal doses given twice a day over a 5 day period. As described above, all cytokines were normalized to IUe on a molar basis. For selection experiments, mice were then sacrificed on day 6 and organs were fixed in 10% buffered formalin for histological analysis. For other experiments, spleen and lung lymphocyte populations were analyzed by flow cytometry. For all in vivo cytokine treatment experiments, animals were weighed(daily or every other day) and expressed as% change from cytokine treatment initiation.

To assess serum concentrations, wtIL2, mutIL2 or OMCP-mutIL2 were labeled with Alexa Fluor 647 (Life technologies Inc.) according to the manufacturer's instructions. Serum was collected at the indicated times and the cytokine concentrations were determined by fluoroscopy according to a standard curve.

In vivo tumor study: lewis Lung Carcinoma (LLC) cells in 100. mu.l sterile saline at 1X10 per mouse⁵One cell was injected subcutaneously into B6 or B6NKG2D^-/-In mice. Once visible tumors became evident, on day 5 post injection, a 5 day cytokine therapy course was initiated as described above. Measurement of the cross-sectional tumor diameter was performed using calipers and tumor volume was estimated to be 4/3 π r³. Mice were sacrificed at day 24 post injection or once they reached a maximum tumor diameter of 20 mm. For NK cell depletion, control with anti-NK 1.1 antibody (clone PK 136) or mouse IgG isotype (both from BioXcell) at 500 μ g on day-2 and 250 on day-1µMice were treated at 250 μ g per week for the duration of the experiment. For lymphoma clearance experiments, A/J mice were treated with 10 doses of cytokine over a 5 day period and at 5x10 on day 6 as described above⁶Cells/mice were injected intravenously with CFSE-labeled YAC-1 cells. Mice were sacrificed 4 hours later, lungs were digested, and passed through CFSE⁺Forward and side scatter analysis of cells determined the viability of YAC-1.

Statistics of : comparison of splenic and pulmonary resident lymphocytes between various cytokine treatment conditions was performed by unpaired T-test and Welch's correction to account for unequal variance or unequal sample size. Tumor growth was compared between different cytokine conditions by multiple unpaired T-tests performed between the various conditions at different time points using Sidak-Bonferroni corrections. Fold-change in STAT5 phosphorylation was assessed in a similar manner by unpaired T-test and Welch's correction.

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Introduction of examples 7 to 10

Intracellular surveillance mediated by MHC class I (MHC I) is a critical host immune function, and therefore MHC I molecules are often targeted for destruction or intracellular retention by viruses [1 ]. Many herpesviruses encode at least one protein that prevents cell surface expression of MHCI [1,2 ]. However, this immune evasion strategy sensitizes infected cells to NK cell-mediated lysis by the loss of inhibitory signals [3 ]. Viral infection also results in cell surface display of NKG2D ligand (NKG2 DL), recognized by the activation receptor NKG2D, further rendering infected cells susceptible to NK cell-mediated lysis. Thus, viruses that target mhc i expression also disrupt NKG 2D-mediated cellular responses by targeting NKG2DL on infected cells [4-7 ].

NKG2DL is not normally expressed on the cell surface, but the specific trigger that can induce expression of [8 ]. NKG2DL by cell stress is not clear, but NKG2DL is upregulated in response to several viral infections [9-12 ]. NKG2DL contains a large set of proteins, both recognized by NKG2D despite low sequence identity, the redundancy of NKG2DL includes the MIC (a and B) and ULBP (1-6) families in humans and the MULT1 and RAE-1 (α -e) and H60 (a-c) families [13 ]. NKG2 s in mice is likely due to a combination of ligand tissue-specific expression patterns and the need for NKG2D escape strategies [14] many viruses have evolved a mechanism to inhibit cell surface expression of NKG2DL as a means to interfere with viral infection NKG2D in beta-virus and gamma-herpesvirus-11-dna-binding strategies [14] as a strategy to prevent the expression of NKG 2-dna-RNA.

Like several herpesviruses, vaccinia virus (CPXV) also disrupts MHCI expression. CPXV expresses CPXV012 and CPXV203, respectively preventing TAP-mediated peptide transport and MHCI transport to two proteins on the cell surface [27-34 ]. Murine poxvirus, a related orthopoxvirus, induces NKG2DL expression, and NKG2D is critical for controlling murine poxvirus pathogenesis [35 ]. Infection with another orthopoxvirus, monkeypox virus, results in a dramatic expansion of NK cells, but impaired NK cell function [36 ]. Taken together, this suggests that CPXV-infected cells are sensitive to NK cell-mediated lysis.

Unlike herpes viruses, CPXV does not target NKG2 DL. In contrast, this virus is directly targeted to NKG2D [37,38] by a competitive inhibitor encoding NKG2DL, the orthopoxvirus MHC class I-like protein (OMCP). OMCP is a 152-residue protein that is secreted by infected cells and antagonizes NKG 2D-mediated killing of NKG2 DL-expressing target cells [37 ]. OMCP also plays an important role in vivo, and OMCP-empty CPXV is attenuated in mouse infection models (m. Sun et al, personal communication). OMCP binds murine NKG2D with an affinity equal to or greater than all murine NKG2DL tested and human NKG2D with an affinity 5,000 times that of NKG2DL [37-40 ].

despite their differences in sequence identity, all known host NKG2DL share common structural features [41,42 ]. NKG2DL contains an MHCI-like platform domain, which is composed of an eight-chain β -sheet with two helices [43-47 ]. the platform domain is subdivided into α 1 and α 2 domains, and each domain contains four β -chains and one α -helix unlike MHCI, the groove between the helices of the NKG2DL platform domain is closed, so NKG2DL does not bind peptides.

like host NKG2DL, OMCP also employs an MHCI-like platform domain [38 ]. however, the platform domain of OMCP has been tailored to have only a six-stranded β folds with shorter flanking helices we have termed the helix of the α 1 domain as H1 and the discontinuous helices of the α 2 domain as H2a and H2b the H2a and H2b helices of OMCP are also rearranged to be flatter and spaced apart from each other relative to the β folds.

here we report a 2.0-resolution structure of human NKG2D that binds OMCP from Brighton Red strains of vaccinia virus, which reveals significant reorientation of OMCP in the NKG2D binding groove relative to host NKG2DL the interface of OMCP and NKG2D is highly complementary, has a significantly larger surface area than the host NKG2DL buried, and remains continuous throughout the NKG2D binding groove this new binding adaptability and high affinity enables OMCP to compete with a high local concentration of membrane-bound host NKG2DL we further suggest that the mechanism of NKG2D antagonism requires secretion of OMCP in order not to cause NKG2D signaling.

Example 7 structural determination of OMCP-NKG 2D.

We have previously resolved the structure of OMCP alone, and shown to be similar to host NKG2DL, OMCP employs an MHCI-like platform domain [38 ]. Despite the overall similarity of the domain structure of OMCP to host NKG2DL, OMCP has several significant deviations in the putative NKG2D binding site that is hypothesized to be important for high affinity binding of OMCP to NKG2D. To further understand the exceptionally high affinity of OMCP for NKG2D, we crystallized and resolved the structure of OMCP binding to human NKG2D.

initial crystallization experiments with OMCP and NKG2D yielded 30 different crystallization conditions, subsequent data collection and molecular replacement of multiple low resolution crystal forms all yielded similar partial solutions, with alternating sheets of OMCP-NKG2D complex separated by an undefined density in the original structure of OMCP alone, the β -sheet package formed a trimer, and the α helix was oriented away from the center [38 ]. the same OMCP trimer was formed in OMCP-NKG2D partial solution, and NKG2D now bound to the outward helix (data not shown). to attempt to change lattice packing, we introduced mutations in the β -sheet of OMCP designed to break the trimer interface, these mutations were located opposite from the NKG2D binding site to avoid disrupting OMCP-NKG2D binding, the mutant form of OMCP (Y23D, F95D) crystallized with NKG2 in a new space group, and crystals from NKG2D to NKG 2.0 (table 24A) (fig. 24A).

this residue is located in the center of the OMCP largest ring, nor is there Å clear density of this residue in the OMCP structure alone [38 ]. the OMCP structure binding to NKG2D has no significant difference from our previous OMCP structure alone, and the RMSD of all atoms is 0.8 Å. likewise, NKG2D is also similar to the previous NKG2D structure, and the RMSD range is 0.5-0.9 Å. the β 3- β 4 ring of NKG2D is the region in OMCP or NKG2D that shows the only above average B factor, this ring is considered flexible, and has on average the above B factor [48 ]. in all previous NKG2D structures, the other cis-g 2D conformations between S193-S194 in our NKG2D structure (figure 29).

Example 8 interface between OMCP and NKG2D.

OMCP is assumed to bind to the same surface of NKG2D used by host NKG2DL because mutations in the NKG2DL binding pocket of (i) OMCP competes with host NKG2DL for NKG2D and (ii) NKG2D alter OMCP binding affinity [38 ]. OMCP does bind to NKG2D using the same concave binding pocket as host NKG2DL (fig. 24A). OMCP binds primarily with discontinuous helices H2a and H2B of its α 2 domain the positions of the H2a and H2B helices are such that the exposed side chains of each surface of the two helices within the binding site directly contact NKG2D (fig. 24B). only two contacts are found outside H2a and H2B, Ile49 and arg66. both residues are located within the α 1 domain but outside the H1 helix.

Twelve OMCP residues were contacted with eighteen NKG2D residues to form a mixture of bond types (table 2). The three residues in each NKG2D half-site are called core binding residues because they are in contact with all known host NKG2 DL. NKG2D subunit A (NKG 2D)^A) The core residues of (Tyr 152, Tyr199, Met 184) form two hydrogen bonds and form extensive hydrophobic contacts with OMCP residues. NKG2D^AThe core residue of (a) contacts the four OMCP residues, and the most critical of these residues is Phe 122. Phe122 and all three NKG2D^AThe core residues form multiple hydrophobic contacts, including pi stacking with Tyr 152. Phe122 also forms a backbone-side chain hydrogen bond with Tyr 152. Interestingly, OMCP is the first to not utilizeAll six NKG2D cores bind to the NKG2D ligand of residues, and only NKG2D subunit B (NKG 2D)^B) Met184 and Tyr152 in contact with OMCP. NKG2D^BMet184 and Tyr152 each form a single hydrogen bond and hydrophobic contact with OMCP residues. Two OMCP residues Trp127 and Asp132 are in contact with two NKG2D promoters. OMCP Trp127 and NKG2D^ALys50 of (a) forms hydrogen bonds and binds to NKG2D^BLeu148, NKG2D^ALys150 and Ser151 form several hydrophobic contacts. OMCP Asp132 and NKG2D^BTyr152 of (a) forms a hydrogen bond and binds to NKG2D^ALys150 of (A) forms a salt bridge (FIG. 25A).

Due to the high affinity of the OMCP-NKG2D interaction, we used a high-throughput in vitro selection method to find null mutants that bind NKG2D (table 3). Screening results identified D132 as an important residue to disrupt NKG2D binding. We then generated mutation D132R in an attempt to completely eliminate NKG2D binding. Surprisingly, the D132R mutant alone was not able to exceed K_DA 35-fold concentration bound NKG2D (fig. 25B), but did not affect OMCP binding to cells expressing FcRL5 (fig. 25C). This mutation may cause significant spatial conflict, as well as disruption of Asp132 and NKG2D^ALys150 and NKG2D^BInteraction of Tyr152 (fig. 25A).

previously, OMCP had 14-fold higher affinity for human NKG2D relative to mouse NKG2D, and three amino acid substitutions [38] located in the β 5' - β 5 loop (abbreviated L2) of NKG2D]. In addition to the substitutions themselves (I182V, M184I and Q185P), the position of the loop differs between NKG2D orthologs. L2 in human NKG2D curves toward the center of the concave binding cavity, compared to L2 of murine NKG2D. The overlay of murine NKG2D onto the human NKG2D-OMCP structure revealed, NKG2D^BMedium OMCP and Met184 (mNKG 2D residue I200) and NKG2D^Athe contact between Met184 (I200) and Glu185 (P201) in (1) will change due to different positions of the murine β 5' - β 5 loop (fig. 26A-B). this change will disrupt contact with three residues in OMCP H2a, three residues in H2B, and Arg66 within the α 1 domain hi the contact residue of L2 Met184 produces the most significant contact in two NKG2D (table 2) (fig. 26C)Of the 58 available NKG2D sequences, 54 preserved Met184 and Glu185 found in high affinity human NKG2D (fig. 26D).

18 OMCP variants between different CPXV and MPXV strains have been described [51]. In this study, we crystallized OMCP from Brighton Red strain from CPXV, which was in common with 17 other OMCP variants (collectively referred to as OMCP)_mpx) Has a highly conserved sequence of>60% sequence identity. Of the 12 OMCP contact residues observed, 9 were found to be in contact with OMCP_mpxThe same is true. In the remaining contacts, all three were conservative hydrophobic substitutions (I49L, T118I and M135I) (fig. 27). OMCP_mpxBinds to NKG2D and substitutions of NKG2D contact residues are less likely to severely affect OMCP_mpxAffinity for NKG2D [37]。

Example 9 novel NKG2D binding adaptation.

Host NKG2DL has low sequence identity but overall similar structure, and MHCI-like platform domains bind diagonally across the symmetric binding groove produced by NKG2D homodimers [13,41,52 ]. The host ligand contacts one NKG2D half-site with the H1 and S1-S2 loops and a second NKG2D half-site with H2 b. Despite having similar MHCI-like folds, OMCP binds the NKG2D binding groove in a new orientation, rotated-45 ° relative to host NKG2DL (fig. 27). Instead of using H1 and S1-S2 cyclic host ligands, OMCP replaces these contacts with H2 a. This rotation causes the helix of OMCP to be perpendicular to the NKG2D binding groove rather than placed diagonally across it.

the α helix of OMCP and host NKG2DL is discontinuous, and the two shorter helices are hinged relative to each other for the host ligand, the angle between H2a and H2b is-90 °, locating H2a away from the NKG2D interfaceclose complementarity (fig. 24B) tight coordination of α 2 helix with NKG2D is manifested by complementarity in high shape (0.77) and buried surface area (2,612 Å)²) in contrast, host NKG2DL has shape complementarity in the range of 0.63-0.72 and a buried surface area in the range of 1,700-2,180 Å²[43,44,46]。

the second unique feature of the α 2 helix is the separation of H2a and H2b from each other, this region also contains the translation of H2a and H2b completely into two distinct helices, this translation is critical for NKG2D binding as it allows each helix to be centered directly on the core binding site of each NKG2D monomer (fig. 27). this creates a symmetric binding site on OMCP that recognizes the symmetric binding groove produced by the NKG2D dimer, the symmetry between OMCP and NKG2D binding is in stark contrast to the typical binding of asymmetric host ligands to the symmetric NKG2D binding groove [52 ]. however, there is still an asymmetric element in the OMCP-NKG2D interaction as each NKG2D half-site recognizes a different N-to C-terminal oriented OMCP helix, again demonstrating the flexibility of rigid adaptation of NKG2D to recognized NKG 41, 53.

The contact site between NKG2D and host NKG2DL consists of two patches (batch) centered on the NKG2D and H1/S1-S2 loops and the core binding site of H2b of NKG2DL [41 ]. As a result, the NKG2D and NKG2DL interface was discontinuous, particularly in the center of the NKG2D binding groove (fig. 27). Due to the unique orientation of OMCP, H2a and H2b form continuous contacts along the entire NKG2D binding groove (fig. 27). The side chains of OMCP Lys126, Trp127, Glu131, and Asp132 contact residues in the center of the NKG2D binding groove and bridge the core binding site on each NKG2D monomer (fig. 24B). Specifically, OMCP Trp127 faces the center of the NKG2D dimer and makes hydrophobic contact with residues on both NKG2D monomers, effectively blocking any voids in the binding interface.

Example 10 signaling of NKG2D following ligand binding.

CPXV and MPXV infected cells secrete OMCP, which acts as an NKG2D antagonist [37 ]. This immune evasion strategy reminds one of the cancer-induced shedding of NKG2 DL. Some cancer cells use Matrix Metalloproteinases (MMPs) to proteolytically cleave NKG2DL from the cell surface while preventing the targeting of NKG 2D-bearing lymphocytes to cancer cells, and produce soluble NKG2DL to inhibit NKG2D in trans. Cell-associated NKG2DL triggered NKG2D effector function (fig. 28A), while cancer-induced soluble NKG2DL blocked NKG2D function (fig. 28B). Like detached NKG2DL, OMCP is soluble and blocks NKG2D function in trans [37] (fig. 28C). Unlike host NKG2DL, OMCP binds NKG2D in a novel orientation. Therefore, we asked whether OMCP can act as NKG2D agonist in the context of the cell membrane, similar to the host NKG2D ligand. Since OMCP is a secreted protein, artificial cell-associated OMCP [37] was constructed by using heterologous transmembrane domains from Thy1.1 (FIG. 28D). To measure NKG 2D-mediated cell killing, we stably transduced Ba/F3 cells with retroviral vectors expressing OMCP-thy1.1 constructs or host NKG2 DL. Target cells expressing OMCP-thy1.1 were killed equally to target cells transduced by host NKG2DL, suggesting that cell-associated OMCP is able to activate NKG2D signaling despite altered binding orientation (fig. 28E). Therefore, OMCP must be secreted in order to avoid its active NKG2D effector to act on its own, although efficacy may be lost due to diffusion.

Discussion of examples 7-10.

While many viruses employ the general mechanism of NKG2D destruction by attempting to retain multiple host-encoded NKG2D ligands within the infected cell, CPXV and MPXV employ very different approaches to directly target NKG2D. Since NKG2D is monomorphic, this mechanism has the significant advantage that a single protein is required to prevent NKG2D recognition of infected cells. The large number of sequence divergences host NKG2DL and its associated polymorphisms are thought to be driven by the selection of pathogen-encoded NKG2DL antagonists [14 ]. Likewise, viral NKG2L antagonists are in continuous cycles of adaptation under the selective pressure of different host NKG2 DL. NKG2D has a limited mutation space to accommodate because of the need to identify multiple NKG2 DLs. The limited ability of NKG2D mutations is another advantage of OMCP targeting directly to NKG2D rather than NKG2 DL.

Similar to OMCP, some cancer cells shed host NKG2DL to produce their own soluble NKG2D antagonists. However, this strategy has the additional benefit of removing host NKG2DL from the surface of cancer cells. In contrast, CPXV and MPXV lack known mechanisms for blocking surface expression of host NKG2 DL. Secreted OMCP must then be able to effectively compete for high local concentrations of NKG2DL in various hosts on infected cells, as well as spread out from infected cells. One possible approach to increase the ability of OMCP to compete with host ligands is to increase the affinity of OMCP by having multiple NKG2D binding domains. However, multimeric OMCP can cross-link NKG2D and possibly trigger NKG 2D-mediated killing. Therefore, secreted OMCP must be monomeric to prevent aberrant NKG2D signaling. Therefore, to compensate for these deficiencies, OMCP must have the highest affinity to compete effectively with the cell-associated host NKG2DL [37,38 ]. The half-life of the ligand-receptor interaction is closely related to physiological competitiveness [55 ]. OMCP binds human and murine NKG2D with half-lives of 348 and 54 seconds, respectively, compared to 1.5-18 seconds for most NKG2DL [38,44,56 ]. Indeed, the extended half-life of NKG2D enables OMCP to effectively antagonize NKG 2D-mediated immunity in a murine infection model (m.sun et al, personal communication).

To understand the molecular basis of OMCP for the long half-life of NKG2D, we have previously determined the structure of OMCP alone, where we report the structure of OMCP bound to NKG2D. The OMCP alone structure is very similar to that of the host NKG2D ligand, containing an atypical MHC I-like platform domain. The host NKG2D ligand binds to a helix of its platform domain oriented diagonally within the symmetric binding groove of NKG2D. Thus, OMCP is expected to be a viral mimetic of host NKG2D ligand, and similarly interacts with NKG2D.

the structure of OMCP-NKG2D instead reveals a new orientation of NKG2D ligand in the NKG2D binding groove the change in the α 2 domain helix allows OMCP to arrange its helix vertically within the binding groove this reorientation brings the H2a and H2b helices directly into contact with the core binding site of NKG2D and also forms the largest and most continuous binding interface with NKG2D because the forces mediating protein-protein interactions (hydrogen bonds, van der waals forces, hydrophobic interactions) are each weak, so the large continuous interface with high shape complementarity allows strong interactions to accumulate between proteins.

Since host NKG2DL and OMCP have similar MHC I-like platforms, it is reasonable to suspect why no host ligand has evolved a high affinity interaction similar to NKG2D. One possible reason is that the host's immune response must be carefully calibrated to balance the need to protect against autoimmune threats. Since expression of NKG2DL on the cell surface signals effector functions, even small amounts of high affinity host ligands on the cell surface may elicit an immune response, and the resulting tissue damage may be harmful to the host. Indeed, aberrant expression of NKG 2D-expressing cells and/or host NKG2DL has been associated with diabetes, celiac disease and rheumatoid arthritis [57-60 ]. Viruses are not limited by autoimmune selection pressure. Thus, CPXV and MPXV can freely evolve the virus NKG2DL with as high affinity as possible to maximize immune evasion potential.

Interestingly, OMCP triggers NKG2D signaling upon attachment to the target cell membrane, despite the new orientation of OMCP relative to the host NKG2 DL. The interaction of host NKG2DL with dimer NKG2D has a wide structural similarity to the interaction between MHC molecules and their cognate T Cell Receptors (TCRs). In both cases, NKG2DL/MHC is located diagonally on the surface generated by the dimer NKG 2D/TCR. However, there are several examples of MHC-TCR complexes which, like OMCP-NKG2D, interact in non-canonical directions [61-65 ]]. Several of these complexes involve autoimmune MHC-TCR complexes that tilt or rotate outside the normal range of MHC-TCR complexes [61,65]. Although these receptors may be inInduction of TCR signaling at high MHC concentrations, but their failure to assemble characteristic immune synapses [66]. In vitro peptide library-MHC-TCR (H2-L)^d-42F3) screening to generate p3A1-H2-L^d42F3 complex, a significant example of unconventional binding was found, the interface of which was relative to the other H2-L^drotation of the-42F 3 complex by-40 DEG this rotation makes the TCR nearly parallel to the MHC peptide binding groove and shifts the center of the interface nearly completely over one of the MHC α helices-a direction surprisingly similar to the OMCP-NKG2D interface [65 ]]. Interestingly, p3A1-H2-L^dthe-42F 3 complex is unable to induce TCR signaling [65]. Thus, unlike OMCP/NKG2D, the orientation of MHC relative to the TCR is an important factor in signaling.

OMCP-NKG2D and p3A1-H2-L^d42F3 has opposite signaling results, although with very similar orientation TCR signaling requires co-receptors to bind to the α 2/β 2 or α 3 domains of MHCII or MHCI, respectively P3A1-H2-L^dFailure of 42F3 to signal and other unconventional MHC-TCR complexes to form true immunological synapses may be due to failure of co-receptors to form the correct signaling quaternary structure [64, 65,67 ]]it is not known that co-receptor stimulation is required for signaling through NKG2D, and most NKG2DL lack the co-receptor binding α 2/β 2 or α 3 domains of authentic MHC molecules this difference in co-receptor dependence might explain why OMCP (when attached through a transmembrane) is still able to stimulate NKG 2D-signaling compared to MHC-TCR complexes with unconventional binding orientation, furthermore, it suggests that NKG2D aggregation at the cell surface is a major determinant of NKG 2D-mediated activation.

The method of examples 7-10.

A null mutant D132R was identified that binds NKG2D. High throughput in vitro selection methods based on combinatorial cell surface display were used to identify null mutants that bind NKG2D. The OMCP sequence was globally mutagenized using error-prone PCR, and the mutated amplicons were spliced by overlap extension PCR into signal-free thy1.1 cDNA. Mutations to be fused to unmutated Thy1.1This library of variant OMCPs was cloned into pMXs-IRES-EGFP retroviral transfer vector (Toshio Kitamura, University of Tokyo friendship) to generate a library of molecules for transfer into Ba/F3 cells. The transducers were then sorted for green fluorescence and anti-thy 1.1 expression to generate a library of cells, the members of which all had surface expression of OMCP, and mutations that produced frame shifts, early stop codons, and unfolded OMCP were filtered out. The OMCP library was sorted for NKG2D binding using NKG 2D-tetramer. Sorted cells were cloned by limiting dilution and analyzed. Retroviral cassettes of cells lacking or having reduced NKG2D binding activity were amplified and sequenced. Using this approach, we identified Asp132 as the key residue for NKG2D binding.

Protein expression and purification. The OMCP is described previously_BRAnd human NKG2D expression construct [38]。(D132R) OMCP_BRProtein and WT OMCP_BRThe same preparation was carried out. (23D/95D) OMCP-NKG2D Complex reconstituted from purified inclusion bodies by oxidative co-refolding as described previously [38]. The refolded protein was slowly diluted 10-fold with water and captured on a 5 ml HiTrap Q HP column (GE Healthcare) using a Profinia instrument (Bio-Rad). The captured proteins were washed with 50mM Tris, pH8.5, 20mM NaCl and bulk eluted with 50mM Tris, pH8.5, 250mM NaCl. The eluted protein was then concentrated and further purified by gel filtration chromatography on a Superdex S75 column (16/60; Amersham Biosciences). Fractions containing monodisperse OMCP-NKG2D complex (-50 KDa) were pooled and buffer exchanged into 25mM ammonium acetate pH 7.4.

Crystallization, data collection and processing. Seeded by streaking onto 0.2M MgCl containing 15% PEG 3350 by hanging drop vapor diffusion at 20 deg.C₂the crystals were freeze-protected directly prior to rapid freezing in a liquid nitrogen bath.diffraction data were collected at an advanced light Source synchrotron (beam line 4.2.2.) native (23D/95D) OMCP-hNKG2D crystal diffraction data were collected at 100K and at a wavelength of 1.00004A. Additional diffraction data are statistically summarized in table 1. Using HKL2000[68 ]]The data processing shows that the crystal belongs to the original monoclinic space group P2₁(space group # 4). The asymmetric unit of the crystal contains two copies of the (23D/95D) OMCP-hNKG2D complex.

And (5) constructing and refining the model. Human NKG2D (1 MPU) [48]]And OMCP (4 FFE) [38]The structure of (2) was used as a search model via Phenix molecular replacement [69 ]]. Using Phenix and Coot [70 ]]Repeated refinement and manual reconstruction were performed separately. Both the 2Fo-Fc and Fo-Fc plots were used for manual construction and placement of solvent molecules. R generated by the final model_{Work by}16.6% of R_{Free form}At 21.4%, 4% of all reflections were set for free R-factor cross validation. Also using a MOLPROBITY web server [71 ]]The progress of refinement was measured. The final Ramachandran statistic for this model has a 98% preference and an outlier of 0%. Table 1 summarizes other refined statistics. Application program PyMol [72 ]]A structural image is generated.

Structural analysis . Using procedure Ligplot + [73]、PISA[74]And SC [75]The OMCP-NKG2D interface was analyzed for contact residues, buried surface area, and shape complementarity. The architecture plan is compiled by the SBgrid alliance [76]. Use of Consurf Server [77-80 ]]NKG2D conservation analysis was performed. GenBank accession numbers for the species used for Consurf analysis are: human (30749494), northern chimpanzee (Borean orangutan) (21902299), chimpanzee (57113989), gibbon (332232684), macaque (355785888), green monkey (635063485), common marmoset (380848799), mouse (148667521), limo (149049263), guinea pig (348569092), squirrel (532114387), deer mouse (589967905), naked mole mouse (512868733), prairie voles (532053033), European shrew (505834608), starry-nosed mole (507978716), Chinese hamster (537136230), and cat (410963826).

Atomic coordinates . The atomic coordinates (accession number 4 PDC) have been stored in Protein Data Bank, research laboratory for Structural Bioinformatics (Rutgers University, N.Brunswick, N.J.).

In vitro NK cell killing assay . Splenocytes from C57BL/6 mice were preactivated with 200U/ml IL2 for 24 hours and used as cytotoxic effectors against the stably transduced Ba/F3 cell line in a standard killing assay. Target cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and contacted with activated splenocytes at 37 ℃, 5% CO2 at 10: 1. 20: 1 and 40: 1, effector of: target ratio was co-incubated for 4 hours. Percent killing was determined by incorporation of the dead cell exclusion dye 7-amino-actinomycin D (7 AAD) in the CFSE + target population as assessed by flow cytometry. Using the formula [ (experimental death% -background death%)/(maximum release death% -background death%)]X100 calculate the percent specific lysis. C57BL/6 mice were obtained from the National Cancer Institute (Charles River, MA). Mice were maintained under specific pathogen free conditions and used between 8 and 12 weeks of age. Generation of single cell suspensions of splenocytes for killing assays using standard protocols [81 ]]。

References to examples 7-10.

Example 11 individuals with dysfunctional natural killer cells are more likely to develop malignancies.

Fig. 14A shows AJ and 129 are lung cancer susceptible strains of mice, and B6 and C3H are lung cancer resistant strains of mice based on the larger tumor burden found in AJ and 129 mice. Figure 14B shows that when NK cells from various mouse strains were incubated with LM2 lung cancer cells at different ratios, freshly isolated NK cells from B6 and C3H mice (lung cancer resistant strains) resulted in significantly more lysis of LM2 lung cancer cells than freshly isolated NK cells from AJ and 129 mice (lung cancer susceptible strains). Taken together, these data indicate that mouse strains resistant to lung cancer have NK cells that lyse lung cancer cells more efficiently. In addition, susceptible lines have poorly functioning NK cells.

figure 15 shows that a greater percentage of NK cells appear to produce TNF α in "resistant" patients compared to "susceptible" patients, furthermore, tumors have been shown to down-regulate the lytic capacity of NK cells, even though they were previously highly functional.⁵³Thus, even individuals with highly functional NK cells may benefit from treatment that enhances NK cell function.

Notably, ex vivo cytokine activation can reverse natural killer cell dysfunction. Figure 16 shows that IL 2-activated NK cells from resistant (B6 and C3H) and susceptible (AJ and 129) mouse strains lyse LM2 lung cancer cells. Thus, mouse NK cells that did not show significant lysis of cancer cells (NK cells from 129 and AJ strains) were much more potent in lysis when treated with IL2. NK cells from anti-cancer lines also showed an increase in the percentage of specific lysis.

Example 12 OMCP-mutIL2 mediates immunotherapy in vivo.

Immunomodulation of malignant tumors involves complex interactions of various cellular components. CD4 has been shown in multiple models⁺Foxp3⁺T_regsPromote tumor specific tolerance and promote tumor growth^4,16,17. NK cells and CD8⁺CTLs contribute to the immunomodulation of a variety of tumors, such as melanoma^12,18. Other tumors, such as lung cancer, are almost entirely composed ofNK cell control with little contribution from the adaptive immune system^19,20(and unpublished data AS. Krupnick). To test OMCP-mutIL 2-mediated immunotherapy, we will rely on B16 melanoma expressing the model tumor antigen ovalbumin (MO 5 tumor cell line)²¹. Several studies have demonstrated NK cells and CD8⁺CTL's role in controlling melanoma growth^22-24. Thus, the melanoma model provides experimental advantages in studying OMCP-mutIL2, which can activate both types of cells (fig. 1E-F). An agent specific for the tumor, for example, MHC class I restricted CD8 specific for melanoma tumor associated antigen tyrosinase related protein 2 peptide SVYDFFVWL (SEQ ID NO:3)⁺T cell receptor tetramers, readily commercially available (Proimmune, Sarasota, Fl.). The use of an ovalbumin-expressing cell line also provides the advantage of studying the immune response to the highly immunogenic peptide SIINFEKL (SEQ ID NO:4) and to naturally occurring tumor-associated antigens such as tyrosinase-associated protein 2, which typically expands T-cells with low avidity^25,26。

For the study, B6 mice were injected subcutaneously with 1x10⁶MO5 melanoma cells. One week after tumor injection, mice were divided into 4 groups (10 mice per group) and treated with two injections per day as follows: wild type IL2 (group # 1); mutIL2 (group # 2); OMCP-mutIL2 (group # 3) or; saline (group # 4) (FIG. 12). The diameter is measured daily for 4 weeks after tumor growth, or until one of the groups yields a diameter>2cm of tumor. At this point, mice in all groups were sacrificed for analysis. In addition to tumor growth, tumors and lymphocyte infiltration draining inguinal lymph nodes will also be assessed by flow cytometry. We will quantify CD4⁺Foxp3⁺Total number of Tregs and activation status (assessed by ICOS and GITR upregulation). We will also assess NK cell number and activation as measured by IFN- γ production and up-regulation of CD 69. By quantifying CD8 primarily responsible for tumor clearance⁺T cells and CD8⁺CD44^hiCD62L^{Is low in}Effector Cells (EC) to assess antigen-specific CTL production^22,27,28. By identificationCD8 with T cell receptor specific for ovalbumin peptide SIINFEKL (SEQ ID NO:4) or melanoma specific tyrosinase related protein 2 peptide SVYDFFVWL (SEQ ID NO:3), both tetramers, from Proimmune, Sarasota, Fl⁺CTL to determine antigen specificity. Tumor cell apoptosis was quantified by TUNEL staining.

Based on our in vitro tumor data and in vivo phenotypic analysis, we suspected that the OMCP-mutIL2 group would show a reduction in tumor growth with a large number of NK cells, antigen-specific CTLs, in particular CD8⁺EC, and less CD4⁺Foxp3⁺T_regs. If this is demonstrated, we will determine CD8 through depletion experiments⁺Or the relative role of NK CTL. Even if CTL increases, it may not change the growth of MO 5. If this is proved to be the case, we will look in more detail at CD4⁺Foxp3⁺T_regsOr presence and activation of myeloid derived suppressor cells in OMCP-mutIL2 treated mice. Based on melanoma data, additional tumors will be tested using a similar method.

Example 13 CD8 after treatment with OMCP-mutIL2 fusion construct⁺Memory T cell production.

Naive CD8 upon activation by its T cell receptor⁺Differentiation of T cells predominantly into short-lived CD44 with cytolytic potential^hiCD62L^{Is low in}Effector Cells (EC). However, some of the activated cells differentiated into long-lived CD44^hiCD62L^hiCentral memory T cell (CD 8)⁺CMs)^29-31。CD8⁺CM serves as an antigen-specific reservoir for cytoprotection and, upon restimulation, differentiates into CD8 with cytolytic function⁺EC。CD8⁺The durability of CM makes it an ideal target for ex vivo generation and adoptive transfer for long-term protection³¹. The possibility of generating such cell populations in vivo offers a number of advantages over ex vivo systems, including the establishment of polyclonal populations reactive against a variety of tumor-associated antigens and the avoidance of donor singlesThe costs associated with ex vivo amplification. Tumor antigen specific CD8⁺In vivo expansion of CM may also eliminate the need for frequent apheresis and cell re-administration.

High dose IL2 therapy results in CD4⁺Foxp3⁺T_regsAnd CD8⁺Activation of T cells, but its specificity for tumor-associated antigens CD8⁺The effect of CM is unknown. Some have demonstrated that use of antibody depletion, CD4⁺Foxp3⁺T_regsInterfering with tumor-specific CD8⁺CM generation^17,32While others use a different model to prove CD4⁺Foxp3⁺T_regDepletion damage CD8⁺Memory formation^33,34. OMCP-mutIL2 produces a unique immune environment in which CD4 is maintained⁺Foxp3⁺T_regsBut not actively amplified (FIG. 3). CD8 although NKG2D is not at rest⁺Expression on T cells, but it is induced on this population after activation³⁵. Thus, unlike mutIL2, OMCP-mutIL2 results in CD8⁺The level of T cell proliferation was comparable to wild-type IL2 in NKG2D replete mice (FIGS. 1E-F). However, OMCP-mutII2 vs CD8⁺The impact of T cell memory formation is unknown, but is crucial for deciphering the long-term tumor-specific immunity conferred upon this cell population.

To test long-term memory formation following cytokine stimulation in vivo, we will use models of irradiated tumor cell seeding and cytokine therapy. To achieve this, we injected subcutaneously 1x10⁷Lethally irradiated (10 Gy) MO5 melanoma cells into C57BI/6 mice. Recipient mice were then treated with conventional IL2 (group # 1), mutIL2 (group # 2), OMCP-mutIL2 (group # 3) or saline (group # 4) twice daily over the course of 5 days (FIG. 13). Mice were sacrificed at different time points from one to three months post infection (fig. 13). CD8 resident through spleen, peripheral lymph nodes, lung and liver⁺CD44^hiCD62L^hiPhenotypic analysis of CMs to assess antigen-specific CD8⁺CM is formed. By adding ovalbumin peptide, SIINFEKL (SEQ ID)NO:4) or melanoma specific tyrosinase-related protein 2 peptide SVYDFFVWL (SEQ ID NO:3) (both from Proimmune, Sarasota, Fl.) MHC class I staining to determine antigen specificity.

To test the functional protection of this vaccination protocol in a single set of experiments, mice from the above four groups will not be sacrificed for phenotypic analysis, and live MO5 melanoma (subcutaneous 1 × 10) will be re-injected⁶Cell/mouse). The growth of melanoma will be assessed by continuous measurement of tumor diameter. CD8⁺The contribution of T cells to any immune protection will be assessed by CD8 specific antibody depletion in a subset of mice (clone YTS 169.4, BioXcell inc., West Lebanon, NH).

Example 14 mechanism of CTL activation by OMCP-mutIL2 fusion construct.

Understanding of the mechanism of enhanced activation of effector cell function by OMCP-mutIL2 chimeras is crucial to optimizing this therapeutic agent. The interaction of the fusion protein with IL2R and NKG2D may depend on several factors, including the length of the linker peptide (fig. 1E-F). Therefore, it is crucial to understand the mechanism of OMCP-mutIL2 chimera-mediated CTL activation to allow for optimization of the constructs and design of future immunotherapeutic protocols. The two-domain chimeric protein may potentially increase activation of NKG 2D-expressing cells through three non-mutually exclusive mechanisms. First and foremost, the OMCP-mutIL2 construct increases the binding of mutIL2 to target cellsAffinity force. This may result in an increased number of occupied receptors and an increased intensity of signaling compared to mutIL 2. In addition, dual binding to NKG2D and IL2R may be reducedReceptor internalizationAnd increase the duration of IL2 signaling. The OMCP-mutIL2 construct may also alter the signaling profile by the target cell by activating the IL2 and NKG2D stimulatory pathways. These three non-mutually exclusive effects may explain our increase in activation of our CTL constructs in a NKG 2D-mediated manner.

There are several ways to do this, either directly (radiolabelling, fluorescence) or indirectly (antigen exclusion)) Determination of the affinity of proteins for cells^38,39. We propose to use KinExA⁴⁰Determination of the wild-type IL2, mutIL2 or OMCP-mutIL2 vs CD4⁺Foxp3⁺T_regsNK cells and CD8⁺Avidity of T lymphocytes. To this end, we will use a magnetic bead isolation kit (Miltenyi Biotech, San Diego, Ca.) from wild-type C57BI/6 or NKG2D on a C57BI/6 background^-/-The cells were isolated from splenocytes from mice. Target cells were incubated in a medium containing 0.05% NaN₃In 11 falcon tubes in medium at 2-fold serial dilution. Tube 12 will contain only medium. OMCP-mutIL2 or mutIL2 alone was then added to wild-type or NKG2D^-/-Cells were placed in each tube and the cells were spun with cytokines at 4 ℃ for 36 hours. At the end of 36 hours, cells were centrifuged at 2400rpm for 4 minutes and the free construct present in the supernatant was measured by anti-IL2 ELISA. The equilibrium dissociation constant (Kd) is then calculated⁴¹. The advantage of this method is that the affinity of cell surface molecules at physiological density can be measured and no labeling is required, which can artificially reduce the affinity of an antibody for its antigen^42,43。

The double-domain structure of the fusion protein can be obviously increased at NKG2D⁺And IL2R⁺to address receptor internalization, we will incubate each construct with the cell types described above for a range of times, and monitor changes in cell surface expression of IL2R β γ and NKG2D using flow cytometry as previously described⁴⁴. Of most interest will be for each constructSignal transduction Profile. IL2-IL2R signals via the JAK-STAT pathway, while NKG2D signals via the DAP10/12 pathway. Although monomeric, soluble OMCP does not induce NKG2D signaling, OMCP can signal when locally concentrated on the cell surface⁴⁵. Therefore, it is crucial to determine whether the chimera is capable of inducing dual signaling through IL2R and NKG2D. IL 2-mediated signalling will be through fresh partitioning of in vitro incubations with constructsIsolated CD4+ Foxp3+ T_regsNK cells or CD8⁺Western blot of phosphorylated JAK1 and JAK3 in T cells to evaluate^46,47. NKG 2D-mediated signaling will be assessed by immunoprecipitation of DAP10 or DAP12 followed by Western blotting of phosphotyrosines as described previously^48,49。

Both IL2 and OMCP interact with their cognate receptors with high affinity; it is expected that fusion of the two proteins will greatly enhance the avidity of the chimeric construct for cells expressing IL2R and NKG2D. Thus, tethering of the construct to two cell surface receptors may result in reduced internalization and increased duration of signaling. Combining these two phenomena represents the most likely mechanism for increasing NK cell proliferation in vivo. Signaling through NKG2D dependent on receptor clustering⁴⁵. Since the construct is soluble, the chimera could cluster NKG2D and induce DAP10/12 signaling, although unlikely. However, if DAP10/12 signaling was detected, we will use cells derived from Vav1 knockout mice to investigate the importance of this signaling in expanding NK cells. Vav1 is a signal mediator downstream of DAP10⁵⁰. Compared to DAP knock-out, the use of Vav1 knock-out has the advantage that NKG2D expression remains intact⁵⁰. This would remove the NKG2D signaling component while leaving NKG 2D-dependent targeting intact. A clearer understanding of the mechanism of action of OMCP-IL2 chimera-dependent amplification would be critical to further improvement of therapeutics. Understanding these parameters will allow testing of different construct designs, primarily on the linker length between OMCP and IL2, to calibrate the effect of the chimera.

Example 15 in vivo immunotherapy with IL2, R38A/F42K IL2 or OMCP-targeted IL2 constructs.

To determine whether our constructs play a role in immune regulation of malignancies as well as viral infections, we will rely on in vivo models of B16 melanoma and Mouse Cytomegalovirus (MCMV). In one set of experiments, B6 mice were injected subcutaneously with 1x10⁶B16 of poor immunogenicityCells of a melanoma cell line. One week after tumor injection, mice were divided into 13 groups (5 mice per group) and treated with 5 injections per day of IL2, R38A/F42K IL2, OMCP fusion construct, or saline, as described in figure 18 and table 4. The diameters were measured daily for 4 weeks or until one group yielded diameters after tumor growth>2cm of tumor. At this point, mice in all groups were sacrificed for analysis. In addition to tumor growth, tumors and lymphocyte infiltration of draining inguinal lymph nodes will be assessed by flow cytometry. We will quantify CD4⁺Foxp3⁺Total number and activation status of Tregs (expressed as% of tumor infiltrating lymphocytes and% ICOS)⁺). We will also evaluate NK cell number and activation as measured by IFN- γ production and up-regulation of CD 69. Tumor cell apoptosis was assessed by TUNEL staining.

To assess the therapeutic potential of IL2 in models of infectious disease, B6 mice will be treated with sub-lethal doses of MCMV (5x 10) as described previously⁴) Particle Forming Unit (PFU) infection²⁹. On day 1 post-infection, mice were divided into 13 groups (5 mice per group) and treated with 5 injections per day of IL2, R38A/F42K IL2, OMCP fusion construct, or saline, as described in figure 18 and table 4. On day 6 post-infection, mice were sacrificed and splenic and pulmonary viral loads were determined by standard plaque assay.

Potential results include the discovery that treatment with pure IL2 had little effect on tumor growth or viral load, as we expect to see T_regPreferential activation over CTLs. We suspect that administration of IL2 in the form of mutant R38A/F42K will result in lower tumor and viral loads compared to wild-type IL2, due to CD4⁺Foxp3⁺Tregs are less activated. Nevertheless, despite T_regThe level of activation was lower, but the tumor burden between IL2 and R38A/F42K IL2 was likely the same, since the mutated form of IL2 could also reduce NK activation. Potential results include the discovery of pure cytokinesThe OMCP IL2 construct treated mice had a lower tumor burden and it was predicted that OMCP-R38A/F42K IL2 would show the best efficacy for immunotherapy with the most favorable side effect profile.

If we did not see the effect of the IL2 construct expressing OMCP, we will closely assess our confounding data, such as excessive CTL death due to extreme stimuli and possible CTL isolation in systemic organs such as liver and lung. If our hypothesis is supported and NK cells are activated and tumor growth is reduced after OMCP construct treatment, we will deplete both NK (using anti-NK 1.1 clone PK136, mouse anti-mouse depleting antibody) and CD8 (clone YTS169, rat anti-mouse CD 8)⁺T cell depleting antibodies) (both from BioXcell, West Lebanon, NH) were repeated. Based on these results, future work will focus on immunotherapy in primary carcinogenic models.

Example 16 Effect of IL2, R38A/F42K IL2 or OMCP targeting IL2 constructs on immunosuppression following radiation exposure.

Sublethal radiation exposure is a continuing risk to those involved in combat missions. In addition to the direct carcinogenic effects of radiation-induced DNA damage, sublethal radiation can also cause immune damage by selective death of a subpopulation of lymphocytes. CD8⁺T cells and CD44^loNaive T cells are specifically sensitive to radiation-induced death, while NK cell function is significantly reduced after irradiation. However, CD4⁺25⁺T cells and CD44^hiMemory-like T cells have a survival advantage after irradiation. CD4⁺25⁺T cells and CD8⁺CD44^hiT cells can down-regulate the immune response, explaining why even limited radiation exposure leads to significant immunosuppression. Pharmacological intervention to restore the immune system may reduce the morbidity and mortality of radiation poisoning. Surprisingly, the role of IL2 in mitigating radiation-induced changes has never been investigated. Low affinity IL2 receptor for bone marrow resident hematopoietic stem cells and commitmentIs expressed on NK progenitor cells. In turn, NK cells can secrete granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine that can help with hematopoietic recovery, when stimulated. Based on these data for this goal, we planned to test the hypothesis that IL2 or OMCP-IL2 constructs could help hematopoietic recovery after sublethal and lethal irradiation.

Based on the previously described radiation-induced hematopoietic damage and recovery model, we will irradiate B6 mice with sublethal 4.5 or lethal 7.5Gy from cesium sources. Within one hour of exposure, mice at both radiation doses were randomly divided into 13 groups as described in table 4 and treated with low, medium or high doses of IL2, R38A/F42K IL2 or an IL2 construct expressing OMPC for 5 days (figure 18). A portion of the mice were injected with saline after irradiation (group 13) (table 4), and untreated B6 mice that were not irradiated will also be included as controls (group 14). On day 6, hematopoietic recovery was monitored by flow cytometric analysis of peripheral blood obtained by superficial mandibular vein sampling. The total number of NK cells, T cells, B cells, granulocytes and monocytes and macrophages per ml of blood in the sample will be analyzed. Since 90% of untreated mice died 15-25 days after exposure to 7.5Gy, mice will be followed daily and survival curves in each treatment group will be compared by Kaplan-Meier analysis. Moribund mice in the 7.5Gy group will be carefully analyzed for cause of death, and bone marrow, spleen and peripheral organ infections and hematopoietic failure will be assessed by flow cytometry and tissue culture. Since mice in the sublethal 4.5Gy group were expected to survive long term, they were sacrificed one month after exposure and hematopoietic recovery of peripheral lymphoid organs as well as bone marrow was assessed by flow cytometry analysis.

Radiation-related DNA damage leads to malignant transformation. Hematopoietic malignancies are particularly prominent after radiation exposure. To assess the ability of the IL2 or OMCP-linked IL2 constructs to promote clearance of hematopoietic malignancies following radiation exposure, we will treat B6 mice with 4.5Gy of sublethal exposure from cesium sources. Two days after irradiation, mice were injected i.p. with 10³RMA-S lymphoma cells and three days later, low and mediumEqual or high doses of IL2, R38A/F42K IL2 or OMCP expressing IL2 construct were treated for 5 days of treatment (table 4, fig. 19). Unirradiated B6 mice were also included as controls (group 14). The survival of the mice will be followed.

We expect that wild-type IL2 alone has negligible effect on immune recovery as it is likely to result in preferential amplification of CD4⁺Foxp3⁺T_regsWhich has been preserved after irradiation. However, we suspect that R38A/F42K IL2, as well as the IL2 construct expressing OMCP, will amplify the NK fraction in peripheral blood and will contribute to extensive hematopoietic recovery, albeit indirectly through secretion of steady state cytokines such as GM-CSF. If we detected no difference in hematopoietic recovery between IL2 and saline treated groups, we will examine other confounding factors such as changes in the immune system induced by steady state proliferation and the effect of IL2 or the OMCP expressing IL2 construct on this proliferation. Although administration of 200,000 IU of IL2 to B6 mice per day was non-lethal, we realized that mice may be more debilitating in the face of irradiation. Thus, dosage adjustment may be required. For the "functional" part of the experiment, we plan to specifically utilize the well established RMA-S lymphoma challenge model because of the role of NK cells in the control of hematological malignancies. This established assay would enable us to obtain rapid experimental data to advance this goal. Based on these data, we will also expand this goal in the future using primary oncogenic models.

Example 17 OMCP targeted delivery of IL15 enhanced CD25 upregulation.

IL15 (IL 15) is a cytokine with structural similarity to IL2 like IL2, IL15 binds to and signals through a complex consisting of the IL2/IL15 receptor β chain (CD 122) and the common gamma chain (gamma-C, CD 132). IL15 is secreted by mononuclear phagocytes (and some other cells) following viral infection. IL15 regulates the activation and proliferation of T and Natural Killer (NK) cells.IL 15 provides a survival signal that maintains memory T cells in the absence of antigen.

OMCP was linked to the cytokine IL15 and its ability to activate NK cells was examined compared to IL15 alone. NK cell activation was measured by CD25 up-regulation. As shown in figure 21, higher levels of CD25 were evident when IL15 was delivered by OMCP at an equimolar dose compared to naked cytokine alone.

Example 18 OMCP targeted delivery of IL18 enhances NK cell activation.

OMCP was linked to WT human IL18, WT murine IL18 or mutant human IL18, which inhibited its interaction with IL18BP, and examined for its ability to activate NK cells (fig. 32). Peripheral blood lymphocytes were cultured in 4.4. mu.M wild-type IL18 (blue), OMCP-IL18 (red) or saline (black) for 48 hours. Activation of CD56+ CD 3-natural killer cells was superior to OMCP-IL18 compared to wild-type IL18, as measured by surface CD69 expression (fig. 33). This data indicates that linking OMCP to IL18 also enhances NK cell activation relative to IL18 without OMCP.

Example 19D 132R mutation in OMCP significantly reduced its NKG2D binding.

To further test the necessity of NKG2D binding in targeted delivery of IL2, we tested NK amplification and activation in the presence of mutIL2, OMCP-mutIL2 and (D132R) OMCP-mutIL 2. The D132R mutation reduced the advantage of natural killer cell activation over cytokine alone (fig. 22). Thus, high affinity NKG2D binding is critical for targeted delivery and lymphocyte activation by IL2.

Example 20 OMCP-IL2 is effective in treating infections caused by West Nile Virus (WNV).

The ability of various constructs of the invention to treat infections caused by West Nile Virus (WNV) was evaluated. Mice were given OMCP-IL2, a binding null mutant of OMCP, OMCP (D132R) -IL2, IL2 alone, IL2 alone (38R/42A), and PBS. All mice died from infection at approximately day 11 after treatment with OMCP (D132R) -IL2 and PBS. Approximately 20% of mice survived to day 21 after treatment with IL2 alone. However, treatment with IL2 (38R/42A) and OMCP-IL2 resulted in approximately 40% of mice surviving for more than 21 days (FIG. 30A). These results are consistently repeatable, as shown in fig. 30B.

References to examples 11-20.

Example 21 anti-NKG 2D antibody-mediated delivery of R38A/F42K mutant IL-2.

To compare antibody-mediated delivery of mutlL-2 with OMCP-mediated delivery of mutlL-2, 4 anti-human NKG2D single-chain variable fragment domains were engineered based on the sequences of the described KYK1 and KYK2 antibodies (r) ((r))J Mol Biol2008,384(5), 1143-1156). 1HL2 and 1LH2 from kyk1 antibody and 2HL2 and 2LH2 from kyk2 antibody. The binding coefficients of OMCP, KYK1 and KYK2 were 0.1nM, 27nM and 6nM, respectively.

Antibodies linked to OMCP-mutant IL-2, IgG-mutant IL-2, wild-type IL-2 and PBS controls were compared to 2.5X 10⁶Individual peripheral blood lymphocytes were co-cultured in 500 μ l medium at final concentrations of either 10U/ml or 100U/ml cytokine or construct. After 48 hours, NK activation was assessed as the relative median fluorescence intensity of intracellular perforin compared to PBS control. Will CD4⁺CD45RA^-Foxp3⁺Activation was assessed as the relative median fluorescence intensity of surface CD25 compared to the PBS control.

At 10U/ml, OMCP-mutant IL-2 showed a trend of increased perforin levels over antibody-mediated delivery, but it did not reach statistical significance (fig. 39). At 100U/ml, NK cells treated with 2HL2 and 2LH2 antibodies synthesized as much perforin as OMCP-mutIL-2 treated cells, but lower perforin levels were evident in 1HL2 and 1LH2 treated NK cells. Higher levels of CD25 were evident in wild type IL-2 treated cultures for all constructs. Thus, the results demonstrate that mutant IL-2 linked to the NKG2D antibody and OMCP linked to mutant IL-2 perform comparably.

Example 22 combination therapy with OMCP-IL2 and a PD-1 inhibitor.

This example describes the in vivo testing of combination therapy of OMCP-IL2 in combination with PD-1 antibodies.

A total of 16C 57BI/6 mice 6-9 weeks old were inoculated with 100,000 Lewis lung cancer cells per mouse by tail vein injection to induce tumor cell inoculation into the lungs. Mice were then randomized into four groups to receive the following treatments, which started 5 days after cell inoculation:

group 1-antibody isotype control,

group 2-anti-PD-1 antibody treatment,

group 3-antibody isotype control plus OMCP-IL2,

group 4-anti-PD-1 antibody plus OMCP-IL 2.

Group 1-mice were administered intraperitoneally (i.p.) 250 μ g of an isotype control antibody (Bioxcell clone No. 2A3, catalog No. BP0089) twice weekly for two weeks for a total of 4 doses (1000 μ g total) of antibody.

Group 2-mice were i.p. administered 250 μ g of anti-PD-1 antibody (Bioxcell clone number RMP1-14, catalog number BP0146) twice weekly for two weeks for a total of 4 doses (1000 μ g total) of antibody.

Group 3-mice were administered i.p. (i) 75,000IUe OMCP-IL2 fusion protein twice daily for five days, totaling 10 doses (750,000 IUe) of OMCP-IL2 fusion protein; and (ii) 250 μ g of isotype control antibody (Bioxcell clone No. 2a3, catalog No. BP0089) twice weekly for two weeks for a total of 4 doses (1000 μ g total) of antibody.

Group 4-mice were i.p. administered (i) 75,000IUe OMCP-IL2 fusion protein twice daily for five days, totaling 10 doses (750,000 IUe) of OMCP-IL2 fusion protein; and (ii) 250 μ g of anti-PD-1 antibody (Bioxcell clone No. RMP1-14, catalog No. BP0146) twice weekly for two weeks for a total of 4 doses (1000 μ g total) of antibody.

Mice were left for three weeks after completion of the respective treatments, at which time they were euthanized.

Since tumor burden measurably increases lung weight, lung weight is used as a primary measure of treatment efficacy. Fig. 34 depicts photographs of the lungs of the group 1-4 mice and fig. 35 depicts lung weights measured from the lungs of the group 1-4 mice. As shown in fig. 34 and 35, the combination of anti-PD-1 antibody and OMCP-IL2 (group 4) was found to virtually eliminate tumor growth in the lung and synergistically reduce tumor burden compared to OMCP-IL2 treatment alone (group 3) or anti-PD-1 antibody treatment alone (group 2). Thus, combination therapy showed surprisingly greater efficacy than each component administered alone.

Example 23 targeted delivery of the IL2 mutant by PD1 preferentially activates cytotoxic lymphocytes in vitro.

This example describes the in vitro testing of PD1 ligand therapy. In particular, this example will demonstrate improved immune cell activation of PDL1-mutIL2 and PDL2-mutIL2 fusion proteins relative to purified cytokines.

A total of 4 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. Splenocytes were cultured in triplicate for 36 hours according to the following group: group 1-saline control, group 2-100 IUe/mL wt IL2, group 3-100 IUe/mL mutIL2, group 4-100 IUe/mL PDL1, group 5-100 IUe/mL PDL2, group 6-100 IUe/mLPDL1-mutIL2, group 7-100 IUe/mL PDL2-mutIL 2. After a 36 hour incubation period, cells were stained for flow cytometry according to standard protocols and cell activation was assessed.

The cell population will be defined by the following gating strategy: Tregs-CD45+ CD3+ CD4+ Foxp3+, NK cells-CD 45+ CD3-CD49b + CD335+, Teff-CD45+ CD3+ CD8 +. Cell activation was further defined by assessing whether the following markers were upregulated: Tregs-ICOS, NK cells-CD 69 and KLRG1, Teff-CD 69.

Potential results include the discovery of significant activation of NK cells by treatment with wtIL2, PDL1-mutIL2 and PDL2-mutIL 2. Teff cells can also be activated by treatment with wtIL2, PDL1-mutIL2 and PDL2-mutIL 2. This is in contrast to Tregs, which should be activated by treatment with wtIL2 instead of PDL1-mutIL2 or PDL2-mutIL 2.

These results indicate that targeting IL2 therapy to PD1 cells via PD1 ligand fusion proteins significantly enhances the efficacy of IL2 therapy in anti-tumor cell populations such as NK cells and Teff cells, while avoiding activation of immune-tolerant populations such as Treg cells.

Example 24 Targeted delivery of IL2 mutant to PD1 induces proliferation of cytotoxic lymphocytes in vitro

This example describes the in vitro testing of PD1 ligand therapy. In particular, this example will demonstrate improved cytotoxic immune cell expansion by PDL1-mutIL2 and PDL2-mutIL2 fusion proteins relative to purified cytokines.

A total of 4 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. Splenocytes were stained with CFSE prior to culture. CFSE permanently binds DNA and provides an indication of cell proliferation through the subsequent decrease in fluorescence of cell division. Stained splenocytes will then be cultured for 6 days in triplicate according to the following set: group 1-saline control, group 2-1000 IUe/mL wt IL2, group 3-1000 IUe/mL mutIL2, group 4-1000 IUe/mL PDL1, group 5-1000 IUe/mL PDL2, group 6-1000 IUe/mL PDL1-mutIL2, group 7-1000 IUe/mLPDL2-mutIL 2. After a 6 day culture period, cells were stained for flow cytometry according to standard protocols and cell proliferation was assessed.

The cell population will be defined by the following gating strategy: Tregs-CD45+ CD3+ CD4+ Foxp3+, NK cells-CD 45+ CD3-CD49b + CD335+, Teff-CD45+ CD3+ CD8 +.

Potential results include the finding that wtIL2, PDL1-mutIL2 and PDL2-mutIL2 will induce significant proliferation in the NK cell population. We also found that Teff cell proliferation was induced by these same treatment groups. However, Treg cells will induce proliferation by wtIL2 treatment only, and will remain relatively quiescent with PDL1-mutIL2 and PDL2-mutIL2 treatment. Thus, PDL1-mutIL2 and PDL2-mutIL2 treatments would significantly enhance the NK cell to Treg cell ratio relative to wtIL2 treatment alone, which is a marker of prognosis of immune cell activation and cancer treatment response.

These results indicate that targeting IL2 therapy to PD1 cells via PD1 ligand fusion proteins significantly enhances the proliferative capacity of anti-tumor cell populations such as NK cells and Teff cells, while avoiding activation of immune-tolerant populations such as Treg cells.

Example 25 Targeted delivery of PD1 mutant IL2 enhances lymphocyte cytotoxicity

This example describes the in vitro testing of PD1 ligand therapy. In particular, this example will demonstrate improved cytotoxic immune cell response relative to purified cytokines after treatment with PDL1-mutIL2 and PDL2-mutIL2 fusion proteins.

A total of 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. A large number of splenocytes were cultured for 6 days according to the following group: group 1-saline control, group 2-1000 IUe/mL wt IL2, group 3-1000 IUe/mL mutIL2, group 4-1000 IUe/mL PDL1, group 5-1000 IUe/mL PDL2, group 6-1000 IUe/mL PDL1-mutIL2, group 7-1000 IUe/mL PDL2-mutIL 2. After a 6 day incubation period, cells were prepared using the Kit for 7-AAD/CFSE Cytotoxicity assays against K562 cells according to the manufacturer's protocol (Cayman Chemical, 7-AAD/CFSE Cell-media Cytotoxicity Assay Kit, product No. 600120). Splenocytes from each group will be seeded in triplicate with target K562 cells at the following ratio: target-free cells, 15.6: 1. 31.25: 1. 62.5: 1. 125: 1. 250: 1. 500: 1. after 4 hours, the ratio of live target cells to dead target cells will be assessed by flow cytometry.

Possible results include the finding that splenocytes incubated with wtIL2 will have enhanced cytotoxic function against target cells relative to saline controls. We further expect to find that PDL1-muIL2 and PDL2-mutIL2 treatment will further enhance splenocyte cytotoxicity.

These results indicate that the PD1 ligand IL2 fusion protein increases the cytotoxic activity of splenocytes relative to wtIL2 therapy. This may be a function of reduced Treg activation in the splenocyte population. This may further be the function of the mutIL2 portion of the fusion protein to enhance binding and signaling through IL2 receptors on T cells and NK cells.

Example 26 tumor growth and survival following in vivo treatment with PD1 Targeted therapy

This example describes in vivo proof of concept that PD1 ligand therapy inhibits tumor or cancer progression. In particular, this example will demonstrate improved tumor growth and overall survival indicators after in vivo treatment with PDL1-mutIL2 and PDL2-mutIL2 fusion proteins compared to purified cytokines.

A total of 50C 57BI/6 mice 6-9 weeks old will be used. Mice will be injected subcutaneously in the flank using Lewis Lung Carcinoma, 1 × 105 cells per mouse. Treatment will begin after 5 days when the tumor has grown sufficiently to become visible and measurable. Initial tumor size and mouse weight will be taken and the mice randomly divided into groups of 10 mice such that the initial tumor size and mouse weight are similar between the groups. The treatment groups were as follows: group 1-saline control, group 2-wt IL2, group 3-mut IL2, group 4-PDL 1-mutIL2, group 5-PDL 2-mutIL 2.

All mice will be treated twice daily for 5 days at 12 hour intervals for a total of 10 doses according to their group. Group 1-for all treatments, mice were administered intraperitoneally (i.p.) with 200 μ L saline as a negative control. Group 2-for each dose, mice were i.p. administered 75,000IUe wt IL2 for a total of 750,000 IUe wt IL2 after treatment. Group 3-for each dose, mice were i.p. administered 75,000IUe mut IL2 for a total of 750,000 IUe mut IL2 post treatment. Group 4-for each dose, mice were i.p. administered 75,000IUe PDL1-mutIL2, for a total of 750,000 IUe PDL1-mutIL2 after treatment. Group 5-for each dose, mice were i.p. administered 75,000IUe PDL2-mutIL2, for a total of 750,000 IUe PDL2-mutIL2 after treatment.

All tumors will be measured by caliper measurement and mouse weight will be measured daily during the treatment period. After the treatment process is complete, mouse weight and tumor will be measured three times per week. Mice will be monitored throughout the study for signs of distress or other effects of therapeutic treatment. All mice will be euthanized at a maximum tumor diameter of 20mm and the tumors will be retained for subsequent analysis. Any mice that died prematurely for a known or unknown reason will be subject to final measurements and tissues collected as soon as possible.

Possible results include the finding that mice treated with wt IL2 will exhibit considerable physiological distress compared to saline controls, and may even die prematurely from the treatment itself due to Vascular Leak Syndrome (VLS). Those mice that survived treatment may have some reduced tumor growth and increased survival compared to the saline control. Possible results further include the finding that mice treated with mutIL2 do not have VLS and associated physiological stress, but have little or no attenuation of tumor growth compared to saline control mice. In contrast, possible results may include the finding that treatment with PDL1-mutIL2 and PDL2-mutIL2 significantly reduced tumor growth and increased survival compared to the saline control and wtIL2 groups.

We will further analyze the lymphocyte infiltration of residual tumors by immunohistochemistry. In particular, we will assess intratumoral infiltration of CD8+ Teff cells and NK cells. Furthermore, we will assess the level of Apoptosis by the TUNEL assay (Millipore ApopTag Peroxidase In Situ Apoptosis Detection Kit, Cat. No. S7100). Possible results include the finding that treatment with PDL1-mutIL2 and PDL2-mutIL2 significantly increased intratumoral infiltration of CD8+ Teff and NK cells compared to saline control mice or wtIL2 treated mice.

These results suggest that PD1 ligand IL2 fusion proteins, particularly PDL1-mutIL2 and PDL2-mutIL2, have increased therapeutic benefit compared to wt IL2 or mutIL2 cytokine treatment alone. By targeting IL2 treatment to cells expressing PD1, unexpected toxicity and side effects will be reduced compared to wt IL2 treatment. In addition, the PD1 ligand IL2 fusion protein enhanced intratumoral infiltration of cytotoxic lymphocytes suggesting that targeted activation of these cell populations increases the ability of these cells to overcome tumor cell immunosuppression.

Example 27. targeted delivery of NKG2D OX40L preferentially activates cytotoxic lymphocytes in vitro.

This example describes an in vitro test for targeted delivery of NKG2D to OX40L therapy. In particular, this example will demonstrate improved immune cell activation of OMCP-OX40L relative to purified cytokines. This example will further demonstrate inhibition of OX40L signaling by OMCP-OX40L mut1 and OMCP-OX40L mut 2.

A total of 4 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. Splenocytes were cultured in triplicate for 36 hours according to the following group: group 1-saline control, group 2-100 IUe/mL OX40L, group 3-100 IUe/mL OX40L mut1, group 4-100 IUe/mL OX40L mut2, group 5-100 IUe/mL OMCP-OX40L, group 6-100 IUe/mL OMCP-OX40L mut1, group 7-100 IUe/mL OMCP-OX40L mut 2. After a 36 hour incubation period, cells were stained for flow cytometry according to standard protocols and cell activation was assessed.

Possible results include the finding that treatment by OX40L and OMCP-OX40L significantly activates NK cells. Teff cells can also be activated by treatment with OX40L and OMCP-OX 40L. This is in contrast to OX40L mut1, OX40L mut2, OMCP-OX40L mut1, and OMCP-OX40L mut2, which should inhibit NK cell activation. In addition, Teff cells, which should inhibit NK cell activation, can also be inhibited by treatment with OX40Lmut1, OX40L mut2, OMCP-OX40L mut1 and OMCP-OX40L mut 2.

These results suggest that targeting OX40L therapy to NKG 2D-expressing cells via OMCP ligand fusion proteins significantly enhances the efficacy of OX40L treatment in anti-tumor cell populations, such as NK cells and Teff cells.

Example 28 Targeted delivery of NKG2D OX40L induces proliferation of cytotoxic lymphocytes in vitro

This example describes an in vitro test for targeted delivery of NKG2D to OX40L therapy. In particular, this example will demonstrate the improved cytotoxic immune cell expansion of OMCP-OX40L relative to purified cytokines.

A total of 4 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. Splenocytes were stained with CFSE prior to culture. CFSE permanently binds DNA and provides an indication of cell proliferation through reduced fluorescence following subsequent cell division. Stained splenocytes will then be cultured in triplicate for 6 days according to the following set: group 1-saline control, group 2-1000 IUe/mL OX40L, group 3-1000 IUe/mL OX40L mut1, group 4-1000 IUe/mL OX40Lmut2, group 5-1000 IUe/mL OMCP-OX40L, group 6-1000 IUe/mL OMCP-OX40L mut1, group 7-1000 IUe/mL OMCP-OX40L mut 2. After a 6 day culture period, cells were stained for flow cytometry according to standard protocols and cell proliferation was assessed.

Possible results include the finding that OX40L and OMCP-OX40L will induce significant proliferation in NK cell populations. We also found that Teff cell proliferation was induced by these same treatment groups. However, possible results may include the finding that treatment with OX40L mut1, OX40L mut2, OMCP-OX40L mut1, and OMCP-OX40L mut2 did not induce NK or Teff cell expansion. OMCP-OX40L treatment will significantly enhance the NK cell to Treg cell ratio, which is a marker of immune cell activation and prognosis of cancer treatment response, over OX40L treatment alone.

These results suggest that targeting OX40L therapy to NKG 2D-expressing cells via NKG2D ligand fusion proteins significantly enhances the proliferative capacity of anti-tumor cell populations, such as NK cells and Teff cells.

Example 29 Targeted delivery of NKG2D OX40L enhances lymphocyte cytotoxicity

This example describes an in vitro test for targeted delivery of NKG2D to OX40L therapy. In particular, this example will demonstrate an improved cytotoxic immune cell response after treatment with OMCP-OX40L over the purified cytokine treatment.

A total of 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. A large number of splenocytes were cultured for 6 days according to the following group: group 1-saline control, group 2-1000 IUe/mL OX40L, group 3-1000 IUe/mLOX40L mut1, group 4-1000 IUe/mL OX40L mut2, group 5-1000 IUe/mL OMCP-OX40L, group 6-1000 IUe/mL OMCP-OX40L mut1, group 7-1000 IUe/mL OMCP-OX40L mut 2. After a 6 day incubation period, cells were prepared using the kit for 7-AAD/CFSE Cytotoxicity assays against K562 cells according to the manufacturer's protocol (Cayman Chemical, 7-AAD/CFSE Cell-media Cytotoxicity assay, product number 600120). Splenocytes from each group will be seeded in triplicate with target K562 cells at the following ratio: no target cells, 15.6: 1. 31.25: 1. 62.5: 1. 125: 1. 250: 1. 500: 1. after 4 hours, the ratio of live target cells to dead target cells will be assessed by flow cytometry.

Possible results include the finding that splenocytes incubated with OX40L have enhanced cytotoxic function against target cells compared to saline, OX40L mut1, and OX40L mut2 controls. Possible results also include the finding that OMCP-OX40L treatment will further enhance splenocyte cytotoxicity.

These results suggest that NKG2D ligand OX40L fusion protein increases splenic cytotoxic activity over OX40L therapy. This may be a function of enhancing binding and signaling of the OX40L portion of the fusion protein by OX40 receptors on T cells and NK cells.

Example 30 tumor growth and survival following in vivo treatment with OMCP-OX40L Targeted therapy

This example describes in vivo conceptual evidence that OMCP-OX40L therapy inhibits tumor or cancer progression. In particular, this example will demonstrate improved tumor growth and overall survival indicators following treatment with OMCP-OX40L fusion protein in vivo as compared to treatment with purified cytokines.

A total of 70C 57BI/6 mice 6-9 weeks old will be used. Mice will be injected subcutaneously in the flank using Lewis Lung Carcinoma, 1 × 105 cells per mouse. Treatment will begin after 5 days when the tumor has grown sufficiently to become visible and measurable. Initial tumor size and mouse weight will be taken and the mice randomly divided into groups of 10 mice such that the initial tumor size and mouse weight are similar between the groups. The treatment groups were as follows: group 1-saline control, group 2-OX 40L, group 3-OX 40L mut1, group 4-OX 40L mut2, group 5-OMCP-OX 40L, group 6-OMCP-OX 40L mut1, group 7-OMCP-OX 40L mut 2.

All mice will be treated twice daily for 5 days at 12 hour intervals for a total of 10 doses according to their group. Group 1-for all treatments, mice were administered intraperitoneally (i.p.) with 200 μ L saline as a negative control. Group 2-for each dose, mice were i.p. administered 75,000IUe OX40L for a total of 750,000 IUe OX40L after treatment. Group 3-for each dose, mice were i.p. administered 75,000IUe OX40L mut1 for a total of 750,000 IUe OX40L mut1 post treatment. Group 4-for each dose, mice were i.p. administered 75,000IUe OX40L mut2, for a total of 750,000 IUe OX40L mut2 post treatment. Group 5-for each dose, mice were i.p. administered 75,000IUe OMCP-OX40L for a total of 750,000 IUe OMCP-OX40L after treatment. Group 6-for each dose, mice were i.p. administered 75,000IUe OMCP-OX40Lmut1 for a total of 750,000 IUe OMCP-OX40L mut1 after treatment. For each dose, mice were administered i.p. 75,000IUe OMCP-OX40L mut2, for a total of 750,000 IUe OMCP-OX40L mut2 after treatment.

Possible results include the finding that mice treated with OX40L may have some reduced tumor growth and increased survival compared to saline controls. Possible results further include the finding that mice treated with OX40L mut1 or OX40L mut2 had little or no reduction in tumor growth compared to saline control mice. In contrast, possible results might include the finding that treatment with OMCP-OX40L significantly reduced tumor growth and increased survival compared to saline, OMCP-OX40L mut1, and OMCP-OX40 Lmut2 controls, and OX40L groups.

We will further analyze the lymphocyte infiltration of residual tumors by immunohistochemistry. In particular, we will assess intratumoral infiltration of CD8+ Teff cells and NK cells. Furthermore, we will assess the level of Apoptosis by the TUNEL assay (Millipore ApopTag Peroxidase In Situ Apoptosis Detection Kit, Cat. No. S7100). Possible results include the finding that treatment with OMCP-OX40L significantly increased intratumoral infiltration of CD8+ Teff and NK cells compared to saline control mice or OX40L treated mice.

These results suggest that NKG2D ligand OX40L fusion proteins, particularly OMCP-OX40L, have increased therapeutic benefit compared to OX40L treatment alone. In addition, NKG2D ligand OX40L fusion protein should enhance intratumoral infiltration of cytotoxic lymphocytes, suggesting that targeted activation of these cell populations increases the ability of these cells to overcome tumor cell immunosuppression.

Example 31 targeted delivery of 4-1BBL by NKG2D preferentially activates cytotoxic lymphocytes in vitro.

This example describes an in vitro test for NKG2D targeted delivery of 4-1BBL therapy. In particular, this example will demonstrate improved immune cell activation of OMCP-4-1BBL relative to purified cytokines.

A total of 4 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. Splenocytes were cultured in triplicate for 36 hours according to the following group: group 1-saline control, group 2-100 IUe/mL4-1BBL, group 3-100 IUe/mL OMCP-4-1 BBL. After a 36 hour incubation period, cells were stained for flow cytometry according to standard protocols and cell activation was assessed.

Cell populations will be defined by the following gating strategy as Tregs-CD45+ CD3+ CD4+ Foxp3+, NK cells-CD 45+ CD3-CD49b + CD335+, Teff-CD45+ CD3+ CD8 +. Cell activation was further defined by assessing whether the following markers were upregulated: Tregs-ICOS, NK cells-CD 69 and KLRG1, Teff-CD 69.

Possible results include the finding that NK cells are significantly activated by treatment with 4-1BBL and OMCP-4-1 BBL. Teff cells can also be activated by treatment with-1 BBL and OMCP-4-1 BBL. Possible results further include the finding that OMCP-4-1BBL will exhibit greater NK activation and possibly Teff cell activation than the 4-1BBL ligand alone.

These results suggest that targeting 4-1BBL therapy to NKG 2D-expressing cells via OMCP ligand fusion proteins significantly enhances the efficacy of 4-BBL therapy in anti-tumor cell populations, such as NK cells and Teff cells.

Example 32 NKG2D Targeted delivery of 4-1BBL induces proliferation of cytotoxic lymphocytes in vitro

This example describes an in vitro test for NKG2D targeted delivery of 4-1BBL therapy. In particular, this example will demonstrate improved cytotoxic immune cell expansion of OMCP-4-1BBL relative to purified cytokines.

A total of 4 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. Splenocytes were stained with CFSE prior to culture. CFSE permanently binds DNA and provides an indication of cell proliferation through reduced fluorescence following subsequent cell division. Stained splenocytes will then be cultured in triplicate for 6 days according to the following set: group 1-saline control, group 2-1000 IUe/mL4-1BBL, group 3-1000 IUe/mL OMCP-4-BBL. After a 6 day culture period, cells were stained for flow cytometry according to standard protocols and cell proliferation was assessed.

Cell populations were defined by the following gating strategy: Tregs-CD45+ CD3+ CD4+ Foxp3+, NK cells-CD 45+ CD3-CD49b + CD335+, Teff-CD45+ CD3+ CD8 +.

Possible results include the finding that 4-1BBL and OMCP-4-1BBL will induce significant proliferation in NK cell populations. We also found that Teff cell proliferation was induced by these same treatment groups. Possible results further include the finding that OMCP-4-1BBL treatment significantly enhances the NK cell to Treg cell ratio, which is a marker of immune cell activation and prognosis of cancer treatment response, compared to 4-1BBL treatment alone.

These results suggest that targeting 4-1BBL therapy to NKG 2D-expressing cells via NKG2D ligand fusion proteins significantly enhances the proliferative capacity of anti-tumor cell populations, such as NK cells and Teff cells.

Example 33 Targeted delivery of NKG2D 4-1BBL enhances lymphocyte cytotoxicity

This example describes an in vitro test for NKG2D targeted delivery of 4-1BBL therapy. In particular, this example will demonstrate an improved cytotoxic immune cell response following treatment with OMCP-4-1BBL than after treatment with purified cytokines.

A total of 6-9 week old C57BI/6 mice will be used to prepare fresh spleen cell cultures. A large number of splenocytes were cultured for 6 days according to the following group: group 1-saline control, group 2-1000 IUe/mL4-1BBL, group 3-1000 IUe/mLOMCP-4-1 BBL. After a 6 day incubation period, cells were prepared using the Kit for 7-AAD/CFSE Cytotoxicity assays against K562 cells according to the manufacturer's protocol (Cayman Chemical, 7-AAD/CFSE Cell-media Cytotoxicity Assay Kit, product number 600120). Splenocytes from each group will be seeded in triplicate with target K562 cells at the following ratio: target-free cells, 15.6: 1. 31.25: 1. 62.5: 1. 125: 1. 250: 1. 500: 1. after 4 hours, the ratio of live target cells to dead target cells will be assessed by flow cytometry.

Possible results include the finding that splenocytes incubated with 4-1BBL will have enhanced cytotoxic function against target cells compared to saline controls. Possible results also include the finding that OMCP-4-1BBL treatment will further enhance splenocyte cytotoxicity.

These results suggest that the NKG2D ligand 4-1BBL fusion protein increases the cytotoxic activity of splenocytes over 4-1BBL therapy. This may be a function of enhanced binding and signaling of the 4-1BBL portion of the fusion protein by the 4-1BB receptor on T cells and NK cells.

Example 34 tumor growth and survival following in vivo treatment with OMCP-4-1BBL Targeted therapy

This example describes in vivo proof of concept that OMCP-4-1BBL therapy inhibits tumor or cancer progression. In particular, this example demonstrates improved tumor growth and overall survival indices following treatment with OMCP-4-1BBL fusion protein in vivo as compared to treatment with purified cytokines.

A total of 30C 57BI/6 mice 6-9 weeks old will be used. Mice will be injected subcutaneously in the flank using Lewis Lung Carcinoma, 1 × 105 cells per mouse. Treatment will begin after 5 days when the tumor has grown sufficiently to become visible and measurable. Initial tumor size and mouse weight will be taken and the mice randomly divided into groups of 10 mice such that the initial tumor size and mouse weight are similar between the groups. The treatment groups were as follows: group 1-saline control, group 2-4-1 BBL, group 3-OMCP-4-1 BBL.

All mice will be treated twice daily for 5 days at 12 hour intervals for a total of 10 doses according to their group. Group 1-for all treatments, mice were administered intraperitoneally (i.p.) with 200 μ L saline as a negative control. Group 2-for each dose, mice were i.p. administered 75,000IUe 4-1BBL, for a total of 750,000 IUe 4-1BBL after treatment. Group 3-for each dose, mice were i.p. administered 75,000IUe OMCP-4-1BBL for a total of 750,000 IUe OMCP-4-1BBL after treatment.

Possible results include the finding that mice treated with 4-1BBL may have some reduced tumor growth and increased survival compared to saline controls. In contrast, possible results further include the finding that treatment with OMCP-4-1BBL significantly reduced tumor growth and increased survival compared to saline controls and the 4-1BBL group.

We will further analyze the lymphocyte infiltration of residual tumors by immunohistochemistry. In particular, we will assess intratumoral infiltration of CD8+ Teff cells and NK cells. Furthermore, we will assess the level of Apoptosis by the TUNEL assay (Millipore ApopTag Peroxidase In Situ Apoptosis Detection Kit, Cat. No. S7100). Possible results include the finding that treatment with OMCP-4-1BBL significantly increased intratumoral infiltration of CD8+ Teff and NK cells over saline control mice or 4-1 BBL-treated mice.

These results suggest that NKG2D ligand 4-1BBL fusion proteins, particularly OMCP-4-1BBL, have increased therapeutic benefit compared to 4-1BBL treatment alone. In addition, NKG2D ligand 4-1BBL fusion protein should enhance intratumoral infiltration of cytotoxic lymphocytes, suggesting that targeted activation of these cell populations increases the ability of these cells to overcome tumor cell immunosuppression.

Example 35 OMCP-IL2 was used to expand ex vivo cell therapy cultures.

Experiments were performed to evaluate the utility of targeted cytokine delivery on cytotoxic lymphocyte expansion in vitro. The disclosed chimeric peptides, in particular OMCP-IL2, can be used to expand T cells, such as CAR-T cells or Tumor Infiltrating Lymphocytes (TILs). These therapies are typically cultured ex vivo in the presence of IL2 to facilitate their expansion. Experiments were performed to determine if OMCP-IL2 expanded ex vivo cultured lymphocytes better than IL2 alone.

In this study, 2.5X 10 cultures were grown at 1000IUe/ml in the presence of plate-bound anti-CD 3 and wild-type IL-2 or OMCP-mutIL-2⁶And C57BL/6 spleen cells. CD3 stimulation was removed after 72 hours and medium containing cytokines was replenished every other day to avoid medium depletion. Counting of CD3 by flow cytometry over the course of 2 weeks⁺T cells and NK1.1⁺CD3^-Total number and proliferation of NK cells (KI 67 expression), viability (staining by exclusion of the viability dye L34959) and depletion (surface PD1 expression) phenotypic markers. At the completion of the experiment (day 13), cells were evaluated for other depletion markers, such as lang 3 and Tim 3.

The results demonstrate that the increased number of T cells and NK cells in cultures expanded with OMCP-mutIL-2 compared to wild-type IL-2 is evident (FIG. 37, FIG. 38). Similar proliferation levels were evident between the two cultures, but OMCP-mutLL-2 the viability of the treated cells was higher, which probably explains the increase in cell number. PD-1 levels at NK and CD3⁺T cells were all increased, but were significantly decreased at days 6-9 when cultured in OMCP-mutIL-2 treated cells but not wild-type IL-2 treated cells. Other depletion markers such as Tim-2 and Lag-3 were also increased in wild-type IL-2 treated cultures. Thus, these results demonstrate that OMCP-mutIL2 is more effective than wild-type IL2 in ex vivo expansion of lymphocytes. This is of great interest for therapies such as adoptive cellular immunotherapy. Adoptive cellular immunotherapy is a T cell-based immunotherapy in which T cells are taken from a subject and stimulated and/or genetically manipulated in vitro and then transferred back to the patient to fight the tumor or infection.

Claims

1. A chimeric peptide comprising a cytokine linked to an immune cell surface protein targeting ligand.

2. The chimeric peptide of claim 1, wherein the cytokine is part of the IL2 subfamily.

3. The chimeric peptide of claim 1, wherein the cytokine is selected from the group consisting of IL2, IL7, IL15, IL18, IL21, and mutants thereof.

4. The chimeric peptide of claim 1, wherein the cytokine is interleukin-2 (IL 2), mutant IL2, or a variant thereof.

5. The chimeric peptide of claim 4, wherein the mutated IL2 comprises at least one mutation selected from the group consisting of R38A, F42K, and C125S.

6. The chimeric peptide of claim 4, wherein the IL2 comprises the amino acid sequence set forth in SEQ ID NO 5 or SEQ ID NO 6.

7. The chimeric peptide of claim 1, wherein the cytokine is part of the IL1 family.

8. The chimeric peptide of claim 1, wherein the cytokine is IL8 or a mutant thereof.

9. The chimeric peptide of any one of the preceding claims, wherein the immune cell surface protein targeting ligand is an NKG2D ligand.

10. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody.

11. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody having high affinity for the NKG2D receptor.

12. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody, wherein the antibody is KYK-1.

13. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody, wherein the antibody is an scFv of KYK-1.

14. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody, wherein the antibody is KYK-2.

15. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody, wherein the antibody is an scFv of KYK-2.

16. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody, wherein said antibody comprises the amino acid sequence set forth in SEQ ID No. 35.

17. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody, wherein said antibody comprises the amino acid sequence set forth in SEQ ID NO 36.

18. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody, wherein said antibody comprises the amino acid sequence set forth in SEQ ID NO 37.

19. The chimeric peptide of claim 9, wherein the NKG2D ligand is an antibody, wherein said antibody comprises the amino acid sequence set forth in SEQ ID No. 38.

20. The chimeric peptide of claim 12, wherein the NKG2D ligand is an antibody, wherein said antibody comprises the amino acid sequences set forth in SEQ ID NO 35 and SEQ ID NO 36.

21. The chimeric peptide of claim 14, wherein the NKG2D ligand is an antibody, wherein said antibody comprises the amino acid sequences set forth in SEQ ID NO 37 and SEQ ID NO 38.

22. The chimeric peptide of claim 13, wherein the NKG2D ligand is an antibody, wherein said antibody comprises the amino acid sequence set forth in SEQ ID No. 39 or SEQ ID No. 40.

23. The chimeric peptide of claim 15, wherein the NKG2D ligand is an antibody, wherein the antibody comprises the amino acid sequence set forth in SEQ ID NO 41 or SEQ ID NO 42.

24. The chimeric peptide of claim 9, wherein the NKG2D ligand is an orthopoxvirus major histocompatibility complex class I-like protein (OMCP) or a portion thereof.

25. The chimeric peptide of claim 24, wherein the OMCP comprises the amino acid sequence set forth in SEQ ID NO 7, SEQ ID NO 13, or SEQ ID NO 14, or a portion thereof.

26. The chimeric peptide of claim 24, wherein the portion of OMCP comprises an activating portion of OMCP.

27. The chimeric peptide of claim 26, wherein the activating portion of OMCP comprises the H2B helical portion of OMCP.

28. The chimeric peptide of any of claims 24 to 27, wherein the cytokine is a member of the Tumor Necrosis Factor Superfamily (TNFSF) or a mutant thereof.

29. the chimeric peptide of claim 28, wherein the cytokine is selected from the group consisting of TNF- β, OX40L, 4-1BBL, CD40L, receptor activator of nuclear factor kappa-B ligand (RANKL), lymphotoxin- β (LTA), lymphotoxin-beta (LTB), Fas ligand (FASL), and CD 27L.

30. The chimeric peptide of claim 29, wherein the OX40L comprises the amino acid sequence set forth in SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 59, SEQ ID NO 60, or SEQ ID NO 61.

31. The chimeric peptide of claim 29, wherein the 4-1BBL comprises the amino acid sequence set forth in SEQ ID No. 65 or SEQ ID No. 66.

32. The chimeric peptide of any one of claims 1 to 8, wherein the immune cell surface protein targeting ligand is a PD1 ligand.

33. The chimeric peptide of claim 32, wherein the PD1 ligand is an antibody.

34. The chimeric peptide of claim 32, wherein the PD1 ligand is PDL1 or a portion thereof.

35. The chimeric peptide of claim 32, wherein the PD1 ligand is PDL2 or a portion thereof.

36. The chimeric peptide of claim 32, wherein the PD1 ligand comprises the amino acid sequence set forth in SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, or SEQ ID NO 55.

37. A chimeric peptide comprising an orthopoxvirus major histocompatibility complex class I-like protein (OMCP) or a portion thereof linked to a targeting molecule.

38. The chimeric peptide of claim 37, wherein the targeting molecule binds to a target molecule expressed on a cell.

39. The chimeric peptide of claim 38, wherein the target molecule is a protein expressed on the surface of a cell.

40. The chimeric peptide of claim 39, wherein the protein is a receptor.

41. The chimeric peptide of claim 38 or 39, wherein the cell is selected from the group consisting of a cancer cell, a vascular endothelial cell, a tumor-associated fibroblast, and a monocyte.

42. The chimeric peptide of claim 37, wherein the targeting molecule is an antibody.

43. The chimeric peptide of claim 42, wherein the antibody binds to an antigen present in the stroma of the tumor.

44. The chimeric peptide of claim 42, wherein the antibody binds mesothelin.

45. The chimeric peptide of claim 42, wherein the antibody binds carcinoembryonic antigen (CEA).

46. The chimeric peptide of claim 37, wherein the targeting molecule is a receptor-targeted agonist antibody.

47. The chimeric peptide of claim 37, wherein the targeting molecule is a receptor-targeted antagonist antibody.

48. The chimeric peptide of claim 37, wherein the targeting molecule is a targeting molecule that induces TNF-related apoptosis.

49. The chimeric peptide of claim 48, wherein the targeting molecule is a 4-1BB agonist.

50. The chimeric peptide of any one of claims 37 to 49, wherein the OMCP comprises the amino acid sequence set forth in SEQ ID NO 7, 13, or 14, or a portion thereof.

51. The chimeric peptide of any one of claims 37 to 49, wherein the portion of OMCP comprises an activated portion of OMCP.

52. The chimeric peptide of claim 51, wherein the activating portion of OMCP comprises the H2B helix of OMCP.

53. The chimeric peptide of any one of the preceding claims, wherein the linker is a peptide linker and comprises from about 20 to about 30 amino acids.

54. The chimeric peptide of any one of claims 1 to 52, wherein the linker comprises a linker selected from the group consisting of (AAS)_n、(AAAL)_n(SEQ IDNO:68)、(G_nS)_nOr (G)₂S)_nWherein A is alanine, S is serine, L is leucine, and G is glycine, and wherein n is an integer from 1 to 20, or from 1 to 10, or from 3 to 10.

55. The chimeric peptide of any one of claims 1 to 52, wherein the linker comprises at least one tag selected from a FLAG tag and a His tag.

56. The chimeric peptide of any one of claims 1 to 52, wherein the linker comprises the amino acid sequence set forth in SEQ ID NO 8.

57. A chimeric peptide comprising the amino acid sequence set forth in SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, or SEQ ID NO 46.

58. A chimeric peptide comprising the amino acid sequence set forth in SEQ ID NO 47, 48, 49 or 50.

59. A chimeric peptide comprising the amino acid sequence shown in SEQ ID NO 62, 63, 64 or 67.

60. A nucleic acid molecule comprising a sequence encoding the chimeric peptide of any one of claims 1-59.

61. A pharmaceutical composition comprising the chimeric peptide of any one of claims 1-59.

62. A combination therapy comprising the pharmaceutical composition of claim 61 and a PD-1 inhibitor and/or a PD-L1 inhibitor.

63. The combination therapy of claim 62, wherein the PD-1 inhibitor is a PD-1 antibody.

64. The combination therapy of claim 63, wherein the PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, REGN2810, PDR001, and MEDI 0680.

65. The combination therapy of claim 62, wherein the PD-1 inhibitor is a PD-L1 antibody.

66. The combination therapy of claim 65, wherein the PD-L1 antibody is selected from the group consisting of dolvacizumab, avizumab, altuzumab, or BMS-936559, or STI-A1010, STI-A1011, STI-A1012, STI-A1013, STI-A1014, and STI-A1015.

67. A method of treating a subject diagnosed with cancer comprising administering to the subject the pharmaceutical composition of claim 61.

68. The method of claim 67, wherein the cancer is selected from the group consisting of melanoma, renal cell carcinoma, lung cancer, and blood cancer.

69. The method of claim 67, further comprising administering the combination therapy of claim 62.

70. The method of any one of claims 67-69, wherein said composition comprises a chimeric peptide comprising: (i) OMCP or a part thereof linked to IL2 or a mutant thereof, (ii) an anti-NKG 2D antibody linked to IL2 or a mutant thereof, (iii) PDL1 or PDL2 linked to IL2 or a mutant thereof, (iv) OMCP or a part thereof linked to OX40L or a mutant thereof, or (v) OMCP or a part thereof linked to 4-1BBL or a mutant thereof.

71. A method of delivering a cytokine to a target cell, the method comprising contacting the target cell with the chimeric peptide of any one of claims 1-59.

72. The method of claim 71, wherein said chimeric peptide comprises OMCP or a portion thereof linked to IL2 or a mutant thereof.

73. The method of claim 71 or claim 72, wherein the target cell is an NK cell or a CD8+ CTL.

74. A method of activating an immune cell, the method comprising contacting an immune cell with the chimeric peptide of any one of claims 1-59.

75. The method of claim 74, wherein said chimeric peptide comprises: (i) OMCP or a part thereof linked to IL2 or a mutant thereof, (ii) an anti-NKG 2D antibody linked to IL2 or a mutant thereof, (iii) PDL1 or PDL2 linked to IL2 or a mutant thereof, (iv) OMCP or a part thereof linked to OX40L or a mutant thereof, or (v) OMCP or a part thereof linked to 4-1BBL or a mutant thereof.

76. The method of claim 74 or claim 75, wherein the immune cells are NK cells and CD8+ CTLs.

77. The method of claim 76, wherein the activated NK cells lyse tumor cells.

78. The method of claim 74, wherein the chimeric peptide comprises OMCP or a portion thereof linked to IL15, IL18, or a mutant thereof.

79. The method of claim 78, wherein said immune cell is an NK cell.

80. A method of treating a viral infection, the method comprising administering to a subject the pharmaceutical composition of claim 61.

81. The method of claim 80, wherein said viral infection is caused by a flavivirus.

82. The method of claim 81, wherein said flavivirus is West Nile Virus (WNV).

83. The method of any one of claims 80-82, wherein the composition comprises a chimeric peptide comprising OMCP or a portion thereof linked to IL2 or a mutant thereof.

84. A method of ex vivo expansion of lymphocytes, the method comprising culturing lymphocytes in the presence of the chimeric peptide of any one of claims 1-59.

85. The method of claim 84, wherein said chimeric peptide comprises OMCP or a portion thereof linked to IL2 or a mutant thereof.

86. A method of improving adoptive cellular immunotherapy in a subject, the method comprising administering to the subject a therapeutic composition comprising lymphocytes that have been cultured in the presence of the chimeric peptide of any one of claims 1-59.

87. The method of claim 86, wherein said chimeric peptide comprises OMCP or a portion thereof linked to IL2 or a mutant thereof.

88. A method of treating lung cancer in a subject, the method comprising administering to the subject an effective amount of a combination therapy, wherein the combination therapy comprises (i) a chimeric peptide comprising OMCP or a portion thereof and IL2 or a mutant thereof, and (ii) an anti-PD-1 antibody.