Modulation of pglyrp1 for treatment of cancer and autoimmunity

Abstract

The technology described herein relates to methods of inhibiting Peptidoglycan Recognition Protein 1 (Pglyrp1) in order to improve immune-checkpoint inhibitor treatments for cancer and autoimmune diseases.

Description

MODULATION OF PGLYRP1 FOR TREATMENT OF CANCER AND AUTOIMMUNITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 533,499 filed August 18, 2023, the contents of which are incorporated herein by reference in their entireties.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Number AI073748, Grant Number AI039671, Grant Number AI056299, and Grant Number Al 144166, awarded by the National Institutes of Health. The Government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 22, 2024, is named 043214-190070WOPT_SL.xml and is 32,024 bytes in size.TECHNICAL FIELD

[0004] The disclosure relates generally to compositions and methods relating to treatment of cancer and autoimmunity.BACKGROUND

[0005] Immune checkpoint inhibition is an important method for enhancing the immune system’s response against cancer cells. Immune checkpoint inhibition aims at removing inhibitory co-stimulatory signals from T cells, mainly tumor-specific cytotoxic CD8+cells, via blockade of inhibitory receptors such as cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and / or programmed death protein- 1 (PD-l) / PD-ligand 1 (PD-L1), among others.

[0006] The physiological role of immune checkpoint molecules is to restrain the immune system from mounting inappropriate T cell responses, particularly autoimmune responses. This homeostatic mechanism may be exploited by malignant cells to escape immunological surveillance (Fritz and Lenardo, 2019). It follows that administration of monoclonal antibodies that target CTLA-4, PD-1 and PD-L1 restores the cytotoxic function of lymphocytes and induces effective antineoplastic responses (Wilky, 2019).

[0007] Unfortunately, treatment with immune checkpoint inhibitors, while improving the immune response against cancer cells, can also induce autoimmunity to other tissues in the subject being treated.SUMMARY

[0008] The methods and compositions provided herein are based, in part, on the discovery that inhibiting Peptidoglycan Recognition Protein (Pglyrpl) can augment the anti -cancer effect of an immune checkpoint inhibitor while also reducing autoimmunity produced by the immune checkpoint inhibitor. That is, inhibitors of Pglyrpl can help reduce treatment-limiting side effects of immune checkpoint inhibitors while also providing improved anti-tumor efficacy.

[0009] Accordingly, provided herein in one aspect is a method of treating cancer, the method comprising administering to a subject in need thereof an immune checkpoint inhibitor and an inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl), wherein the inhibitor of Pglyrpl potentiates antitumor immune activity of the immune checkpoint inhibitor and suppresses immune checkpoint inhibitor-mediated autoimmunity.

[0010] In one embodiment of this aspect and all other aspects provided herein, the inhibitor of Pglyrp 1 comprises an antibody or antigen-binding fragment thereof, or a nucleic acid molecule (e.g., an RNAi molecule, shRNA, miRNA, among others) or an RNA-targeting system (e.g., a CRISPR-Casl3 system (see, e.g., Kushawa et al., Developmental Cell 54: 805-817 (2020) and references therein)).

[0011] In another embodiment of this aspect and all other aspects provided herein, the antibody inhibitor of Pglyrpl comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof. Such antibodies or fragments thereof may also be defined in terms of their complementaritydetermining regions (CDRs). These antibodies are commercially available and can be found at LS Bio (Cat. No. LS-B4940-50, Seattle, WA), Abeam (Cat. No. abl3903, Cambridge, UK), and Creative Biolabs (Cat. No. MOB-1405CT, Shirley, NY) respectively. The term “complementarity-determining region” or “hypervariable region”, when used herein, refers to the regions of an antibody in which amino acid residues involved in antigen binding are situated. The CDRs are generally comprised of amino acid residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31- 35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; (Kabat et al. (1991)

[0012] In another embodiment of this aspect and all other aspects provided herein, the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4). SEQ ID Nos 1 and 2 are complementary and together form a functional RNAi molecule; similarly, SEQ ID Nos 3 and 4 are complementary and together fom a functional RNAi molecule. SEQ ID NO. 1 targets residues 541-565 in the Mus musculus Pglyrpl transcript. SEQ ID NO. 2 targets residues 565-539 in the Mus musculus Pglyrpl transcript. SEQ ID NO. 3 targets residues 433-457 in the Mus musculus Pglyrpl transcript. SEQ ID NO. 4 targets residues457-431 in the ATAs musculus Pglyrpl transcript. As used herein, “r” is used to indicate a ribonucleoside (e.g., rA, rC, rG, rU). Residues without the “r” notation are deoxyribonucleotides (e.g., A, C, G, T).

[0013] In another embodiment of this aspect and all other aspects provided herein, the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.

[0014] In another embodiment of this aspect and all other aspects provided herein, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of: CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.

[0015] In some embodiments, the PD-1 inhibitor is an anti -PD-1 antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and a light chain variable region (LCVR) comprising three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein: HCDR1 has an amino acid sequence of SEQ ID NO: 5; HCDR2 has an amino acid sequence of SEQ ID NO: 6; HCDR3 has an amino acid sequence of SEQ ID NO: 7; LCDR1 has an amino acid sequence of SEQ ID NO: 8; LCDR2 has the amino acid sequence of AAS; and LCDR3 has an amino acid sequence of SEQ ID NO: 10. In some embodiments, the HCVR comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the LCVR comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 13 and 14. In some embodiments, the anti- PD-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain, wherein the light chain has an amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence of SEQ ID NO: 11 and the light chain has an amino acid sequence of SEQ ID NO: 12. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof comprising a HCVR with 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 13. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof comprising a LCVR with 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 14. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof comprising a HCVR with 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 13, and a LCVR with 90%, 95%, 97%, or 98% sequence identity to SEQ ID NO: 14. In some embodiments, the PD-1 inhibitor is cemiplimab or a bioequivalent thereof.

[0016] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from the group consisting of cemiplimab, nivolumab, pembrolizumab, pidilizumab, MEDI0608 (AstraZeneca, Cambridge, UK), ezabenlimab (BI 754091), PF-06801591 (Pfizer, New York, NY), spartalizumab, camrelizumab, JNJ-63723283 (Janssen, Beerse, Belgium), and MCLA-134 (Merus, Cambridge, MA). In other embodiments, the PD-1 inhibitor is an anti-PD-Ll antibody selected from the group consisting of REGN3504 (Kelly, M.P. et al. 2021. Preclinical PET imaging with the novel human antibody 89Zr-DFO-REGN3504 sensitively detects PD-L1 expression in tumors and normal tissues. 9(1), the contents of which are incorporated herein by reference in its entirety), avelumab, atezolizumab, durvalumab, MDX-1105 (Bristol-Myers Squibb, New York, NY), LY3300054 (Eh Lilly and Company, Indianapolis, IN), FAZ053 (Novartis, Cambridge, MA), socazolimab (STI-1014), CX-072 (CytomX, San Francisco, CA), KN035 (Alphamab, Suzhou, China), and Cosibelimab (CK-301). In some embodiments, the PD-1 inhibitor is administered at a dose of 5 mg to 1500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of 200 mg, 250 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 750 mg, 800 mg, 1000 mg, 1050 mg, or 1200 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of 1 mg / kg to 20 mg / kg of the patient's body weight. In some embodiments, the PD-1 inhibitor is administered at a dose of 1 mg / kg, 3 mg / kg or 10 mg / kg of the patient's body weight. In some embodiments, the PD-1 inhibitor is administered as one or more doses, wherein each dose is administered every week, two weeks, three weeks, four weeks, five weeks or six weeks. In certain embodiments, the PD-1 inhibitor is administered intravenously, subcutaneously, or intraperitoneally.

[0017] In another embodiment of this aspect and all other aspects provided herein, the immune checkpoint inhibitor is an inhibitor of PD-1 selected from the group consisting of: pembrolizumab, nivolumab, and cemiplimab. Exemplary complementarity determining regions (CDRs) for these and other anti-PDl antibodies can be found in e.g., US Patent Application Nos: US20210403567, US20220064034, and US Patent Nos.: 8,008,449, 8,354,509 and 8,900,587, the contents of each of which are incorporated herein by reference in their entirety.

[0018] In another embodiment of this aspect and all other aspects provided herein, the immune checkpoint inhibitor is an inhibitor of PD-L1 selected from the group consisting of: atezolizumab, avelumab, and durvalumab.

[0019] In another embodiment of this aspect and all other aspects provided herein, the immune checkpoint inhibitor is an inhibitor of CTLA4 comprising ipilimumab.

[0020] In another embodiment of this aspect and all other aspects provided herein, the checkpoint inhibitor and the inhibitor of Pglyrpl are administered concurrently.

[0021] In another embodiment of this aspect and all other aspects provided herein, the Pglyrp 1 inhibitor is initially administered before the checkpoint inhibitor.

[0022] In another embodiment of this aspect and all other aspects provided herein, the checkpoint inhibitor is initially administered before the inhibitor of Pglyrp 1.

[0023] Another aspect provided herein relates to a method of suppressing autoimmune side effects of checkpoint inhibitor cancer immunotherapy, the method comprising: administering a Pglyrpl inhibitor to a subject who is being or has been treated with an immune checkpoint inhibitor cancer immunotherapy.

[0024] In one embodiment of this aspect and all other aspects provided herein, the inhibitor of Pglyrp 1 comprises an antibody or antigen-binding fragment thereof, or a nucleic acid or RNA-targeting system as described herein or known in the art.

[0025] In another embodiment of this aspect and all other aspects provided herein, the antibody inhibitor of Pglyrpl comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof. Exemplary complementarity determining regions (CDRs) for Pglyrpl antibodies can be found, for example, in US Patent No.: 9,663,568, the contents of which are incorporated herein by reference in their entirety.

[0026] In another embodiment of this aspect and all other aspects provided herein, the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).

[0027] In another embodiment of this aspect and all other aspects provided herein, the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.

[0028] In another embodiment of this aspect and all other aspects provided herein, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of: CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.

[0029] In another embodiment of this aspect and all other aspects provided herein, the side effect is selected from: colitis, endocrinopathy, hepatitis, thyroiditis, myasthenia gravis, autoimmune autonomic ganglionpathy, nephritis, liver toxicity, skin rash or pruritus, meningitis, headaches, encephalopathy, fatigue, diarrhea, and pneumonitis.

[0030] Also provided herein, in another aspect, is a method of treating cancer in a subject in need thereof, the method comprising: (i) detecting or receiving results of an assay detecting the expression of Pglyrpl by cells of the subject’s cancer; and (ii) administering an inhibitor of Pglyrpl to the subject, wherein the inhibitor promotes an anti-cancer immune response while suppressing autoimmune dysfunction.

[0031] In one embodiment of this aspect and all other aspects provided herein, the detecting comprises detecting Pglyrpl protein and / or RNA encoding Pglyrpl protein.

[0032] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof, or an RNAi molecule.

[0033] In another embodiment of this aspect and all other aspects provided herein, the antibody inhibitor of Pglyrpl comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.

[0034] In another embodiment of this aspect and all other aspects provided herein, the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrUrCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).

[0035] Another aspect provided herein relates to a method of treating autoimmune disease, the method comprising administering to a subject in need thereof an inhibitor of Pglyrpl.

[0036] In one embodiment of this aspect and all other aspects provided herein, the inhibitor of Pglyrp 1 comprises an antibody or antigen-binding fragment thereof, or an RNAi molecule.

[0037] In another embodiment of this aspect and all other aspects provided herein, the autoimmune disease affects mucosal tissues or tissues of the gastrointestinal tract.

[0038] In another embodiment of this aspect and all other aspects provided herein, the autoimmune disease is selected from pneumonitis, colitis, enteritis, and gastritis.

[0039] In another aspect, provided herein is a composition comprising an immune checkpoint inhibitor and an inhibitor of Pglyrp 1.

[0040] In one embodiment of this aspect and all other aspects provided herein, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of: CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.

[0041] In another embodiment of this aspect and all other aspects provided herein, the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof, or an RNAi molecule.

[0042] In another embodiment of this aspect and all other aspects provided herein, the antibody inhibitor of Pglyrpl comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.

[0043] In another embodiment of this aspect and all other aspects provided herein, the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).

[0044] In another embodiment of this aspect and all other aspects provided herein, the composition promotes an anti-cancer immune response and suppresses immune checkpoint inhibitor-mediated autoimmunity when administered to a subject having cancer.

[0045] Another aspect provided herein relates to a method of treating cancer via immunotherapy while limiting cancer immunotherapy-related autoimmune activity, the method comprising administering to a subject in need thereof an immune checkpoint inhibitor and an inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl), wherein the inhibitor of Pglyrpl potentiates anti-tumor immune activity of the immune checkpoint inhibitor and suppresses immune checkpoint inhibitor-mediated autoimmunity.BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIGs. 1A-1H show co-expression of Pglyrpl with co-inhibitory receptors in T cells. FIGs.1A-1C examine how Pglyrpl is co-expressed with co-inhibitory receptors in B16 tumor-infdtratingsingle CD8+T cells. FIG. 1A shows the correlation analysis of genes positively correlated with co- inhibitory genes (Spearman correlation coefficient > 0, FDR < 0.05, marked in red) and genes positively correlated with stem-like genes (Spearman correlation coefficient > 0, FDR < 0.05, marked in blue) in single CD8+T cells. FIG.1B depicts a heatmap displaying the expression of Pglyrpl and selected co- inhibitory genes (top cluster, rows) and selected stem -like genes (bottom cluster, rows) in CD8+T single cells (columns). Color indicates the scaled expression level (cell-wise z-normalized log2(CPM+l)). Genes and cells were ordered by hierarchical clustering using Euclidean distance and Ward’s clustering criterion. FIG. 1C depicts a UMAP plot showing the expression of the CD8+T cell dysfunction signature and Pglyrpl, Pdcdl . Havcr2. Lag3, and Tigit in tumor-infiltrating single CD8+T cells. FIG. ID shows the relative expression (RE) of Pglyrpl in CD8+T cells isolated from MC38-OVA tumors by qPCR (n=3-4). FIG. IE shows the relative expression (RE) of Pglyrpl in CD4+(left) and CD8+(right) T cells in vitro differentiated + / - IL-27 by qPCR (n=3). FIG. IF shows the elative expression (RE) of Pglyrpl in CD4+T cells from wildtype (WT) or Maf'Prdml'1' mice in vitro differentiated + / - IL-27 by qPCR (n=3). FIGs. 1G and 1H examine the influence of IL-27-signaling on the expression of Pglyrpl in vivo. Expression of Pglyrpl (normalized TPM) in CD8+T cells isolated from B16 melanoma tumors of wildtype (WT) and Il27rcd~ mice (FIG. 1G) (n=4-5) or Maf'Prdml1' mice (FIG. 1H) (n=3-7) by bulk RNA-seq (24). In FIGs. 1D-1H, data are presented as the mean with ± SEM. Unpaired two-tailed t-tests were performed. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001.

[0047] FIGs. 2A-2K examine how Pglyrpl-doii cient mice show enhanced anti-tumor immunity. FIGs. 2A-2C show how MC38-OVA tumors were implanted into WT and Pglyrpl'1' mice (n=9). (FIG. 2A) Mean tumor growth, (FIG. 2B) tumor sizes and (FIG. 2C) tumor weights on day 15 are shown. FIGs. 2D-2G examine how TILs were harvested for flow cytometry from mice bearing MC38-OVA tumors. FIG. 2D shows representative flow cytometry data and summary plots of the frequency of Tregcells (CD45+TCRp+CD4+FOXP3+) (n=7-10). FIG. 2E shows representative flow cytometry data and summary plots of the frequency of indicated cytokines in CD8+T cells (n=8-10). FIG. 2F-2G show representative flow cytometry data and summary plots of the frequency of PD and TIM3+(FIG. 2F) and PDU TIM3+double -positive (FIG. 2G) CD8+T cells (n=8-10). FIG. 2H-2K depict how CD8+T cells were isolated from WT and Pglyrpl1' mice bearing MC38-OVA tumors for population RNA-seq. FIG. 2H shows a volcano plot of differentially expressed genes comparing WT vs. Pglyrpl1' CD8+T cells (n=4). Differentially expressed genes were determined as FDR < 0.15, fold change > 2. Positive log2 fold change corresponds to up-regulation in Pglyrpl1' CD8+T cells and vice versa. FIG. 21 examines a heatmap of selected differentially expressed genes (FDR < 0.15, fold change > 2) comparing WT vs. Pglyrpl1' CD8+T cells (n=4). FIG. 2J-2K examines gene set enrichment analysis of selected Gene Ontology (GO) terms (FIG. 2J) and published CD8+T cell signatures (FIG. 2K) enriched in Pglyrpl1' vs. WT CD8+T cells. Wherry exhaustion; effector vs. memory, exhausted vs. memory; effector vs. memory LCMV, effector vs. exhausted; effector memory vs. naive; human exhaustion. InFIGs. 2A, 2D-2G, data are presented as the mean with ± SEM. In FIGs. 2B, 2C, data are presented as the mean. Unpaired two-tailed t-tests were performed. *, P <0.05; **, P <0.01.

[0048] FIGs 3A-3I examine scRNA-seq of tumor-infiltrating T cells in Pglyrpl-defi cient mice. FIG. 3A shows a UMAP of tumor-infiltrating T single cells (1,820 cells). TILs were isolated from MC38-OVA tumors grown in Pglyrpl1' and WT mice and sorted for viable CD45+CD3+cells for scRNA-seq. Cells are colored by cluster assignment. FIG. 3B examines a violin plot and dot plot depicting the expression of Pglyrpl in the tumor-infiltrating T cell clusters defined in FIG. 3A. Only WT cells were included in the analysis. P-values were computed using Wilcoxon rank-sum tests. FIG. 3C shows a UMAP of tumor-infiltrating T single cells as in FIG. 3A. Cells are colored by genotype. FIG. 3D depicts bar plots comparing the number of differentially expressed genes (FDR < 0.05 and |log2 fold change] > 0.25) (top) and the distance (bottom) between Pglyrpl1' and WT cells in clusters defined in FIG. 3A. Distance between Pglyrpl1' and WT cells was computed based on the nonoverlapping of cells’ nearest neighbors in the principal component space. FIG. 3E depicts bar plots depicting the genotype composition of the tumor-infiltrating T cell clusters defined in FIG. 3A. Relative frequency (left) and log2 odds ratio of chi-square test (right) are shown. FIG. 3F shows a volcano plot of differentially expressed genes comparing WT vs. Pglyrpl1' cells in the CD8+T cell-1 cluster. Differential genes were determined as FDR < 0.05 and |log2 fold change] > 0.25. Positive log2 fold change corresponds to up-regulation in Pglyrpl1' cells and vice versa. FIG. 3G examines a gene set enrichment analysis of selected Gene Ontology (GO) terms and KEGG and Reactome pathways (left) and published CD8+T cell signatures (right) enriched in WT vs. Pglyrpl1' cells in the CD8+T cell-1 cluster. IL-27 signature; exhaustion; terminally exhausted; exhaustion CD8 T cells; transitory vs. stemlike, self-renewing; progenitor exhausted; RNA processing. FIG. 3H-3I shows how MC38-OVA tumors were implanted into WT and E8iCKPglyrplaiamice (n=7). FIG. 3H shows mean tumor growth. FIG. 31 examines how TILs were harvested for flow cytometry. Summary plots of the frequency of CD8+T cells, PDU, and TIGIT CD8+T cells (n=6-7). In FIGs. 3H-3I, data are presented as the mean with ± SEM. Unpaired two-tailed t-tests were performed. *, P <0.05.

[0049] FIGs. 4A-4G shows / ’^ / )!r / -deficiency results in protection from EAE. FIG. 4A-4C examines how EAE was induced by CFA / MOG in WT and Pglyrpl1' mice (n=13-14). The mean clinical score (FIG. 4A), the disease course summary table (FIG. 4B), and the mean clinical score (n=8- 14) (FIG. 4C) are shown. Data are merged from two independent experiments. FIGs. 4D-4E shows on day 24 after immunization, histology was performed of the CNS parenchyma and meninges (FIG. 4D) and the optic nerve (FIG. 4E) (n=8-9). WM: white matter, GM: grey matter.

[0050] FIG. 4F-4G shows at day 18 after immunization, CNS-infiltrating lymphocytes were extracted and were analyzed for the number of different immune populations (FIG. 4F) and for the expression of intracellular cytokines (FIG. 4G) by flow cytometry. In FIGs. 4A, 4C, 4F, and 4G, data are presentedas the mean with ± SEM. Unpaired two-tailed t-tests were performed. *, P <0.05; **, P <0.01; ****, P <0.0001.

[0051] FIGs. 5A-5I examines how Pglyrpl expression in myeloid cells contributes to EAE disease. FIGs. 5A-5C shows how EAE was induced by CFA / MOG. The mean clinical score of EAE in E8iCTePglyrpl™ (n=10) and WT littermate controls (n=15) (FIG. 5A), in Cd4CiePglyrpl™ (n=8) and WT littermate controls (n=10) (FIG. 5B), and in I.ysMr' Pglyrpl™ (n=15) and WT littermate controls (n=16) (FIG. 5C) are shown. FIG. 5D depticts a bar plot depicting the mean clinical score at day 23 of EAE in LysM^ Pglyrpl™ and WT littermate controls (n=15). FIG. 5E shows the concentration of IL- 6 in the blood of EAE mice at peak of disease (day 15) by legendplex (n=4). FIG. 5F examines the MHC2 expression on myeloid cell populations in the CNS in EAE mice at disease onset (day 10) by flow cytometry (n=7-9). FIG. 5G and FIG. 5H show phenotypic characterization of T cells in the dLN in EAE mice at disease onset (day 10) by flow cytometry (n=7-8). FIG. 51 examines cytokine concentration in the culture medium during recall assay, determined by legendplex (n=7-9). In FIG. 5A-5I, data are presented as the mean with ± SEM. Unpaired two-tailed t-tests were performed. *, P <0.05; **, P <0.01.

[0052] FIGs. 6A-6F examines scRNA-seq reveals changes in monocytes and neutrophils in gZyr f-deficient mice during EAE. FIG. 6A examines the expression of Pglyrpl in immune populations in the dLNs at EAE onset (day 10) by qPCR. FIG. 6B shows a UMAP of CNS -infiltrating myeloid cells (3,698 cells). EAE was induced in Pglyrpl' ' and WT littermate controls and CNS- infiltrating myeloid cells were sorted (CD45+CD3 CD 19 ) for scRNA-seq at disease onset (day 10). Cells are colored by cluster assignment. FIG. 6C shows a heatmap representing cluster-specific upregulated genes (FDR <0.05, fold change >1.5). Cluster assignments as in FIG. 6A. If more than 20 genes met the cut-off criteria, plotted the top 20 genes (ranked by fold change; each gene is only shown once if ranked top 20 in multiple clusters). FIG. 6D depicts bar plots comparing the distance between Pglyrpl'1' and WT cells in clusters defined in FIG. 6B. Distance between Pglyrpl'1' and WT cells was computed based on the non-overlapping of cells’ nearest neighbors in the principal component space. FIG. 6E shows a volcano plot of differentially expressed genes comparing WT vs. Pglyrpl'1' cells in the mono / MACS and neutrophil clusters as defined in FIG. 6B. Differential genes were determined as FDR < 0.05, fold change > 1.19. Positive log2 fold change corresponds to up-regulation in Pglyrpl'1' cells and vice versa. Log2 Fold changes and -logic p-values were capped within [-2.5, 2.5] and [0, 30] for visualization purpose. FIG. 6F examines a gene set enrichment analysis of selected Gene Ontology (GO) terms and KEGG / Reactome pathways in WT vs. Pglyrpl'1' cells in the mono / MACS (left) and neutrophils (right).

[0053] FIG. 7 depicts Pglyrpl is expressed on exhausted T cells in human cancer. Expression of Pglyrpl and co-inhibitory receptors in immune cells of human melanoma samples (27). Plotted are the percent of cells with UMI count > 1.

[0054] FIG. 8 shows Pglyrpl is co-expressed with co-inhibitory receptors in Tregcells. Heatmap displaying the normalized relative expression of Pglyrpl and co-inhibitory receptors in conventional CD4+T cells (Tconv, CD4+TCRp+Foxp3QV?~) and Treg cells (Treg, CD4+TCRp+oxp3GFP+) from 'oxpF '"’-reporter mice in different tissues by qPCR. Gene expression is displayed relative to the spleen Tconv sample, n=3.

[0055] FIGs. 9A-9E shows immune-profiling of tumors in Pglyrpl- deficient mice. FIG. 9A shows Pglyrpl1' mouse validation. Relative expression of Pglyrpl transcript in WT, Pglyrp . and Pglyrp ' CD8+T cells from the spleen by qPCR (n=2-3). FIG. 9B-9C shows how B16-OVA tumors were implanted into WT and Pglyrpl'1' mice (n=7). FIG. 9B shows mean tumor growth and FIG. 9C shows tumor sizes on day 16. FIG. 9D depicts relative expression of Pglyrpl transcript in different immune populations isolated from MC38-OVA tumors grown in WT mice by qPCR (n=4). FIG. 9E shows the frequency of different immune populations in MC38-OVA tumors grown in WT and Pglyrpl1' mice (n=9) by flow cytometry.

[0056] FIGs. 10A-10D examines immune populations in the dLN and spleen of tumor-bearing Pglyrpl-deii cient mice. FIG. 10A-10D shows how MC38-OVA tumors were implanted into WT and Pglyrpl1' mice (n=8-10) and TILs were harvested for flow cytometry. FIG. 10A shows summary plots of the frequency of Tregcells (CD45+TCRp+CD4+FOXP3+) in the dLN and spleen. FIG. 10B depicts summary plots of the frequency of indicated cytokines in CD8+T cells in the dLN and spleen. FIG. 10C shows summary plots of the frequency of PD1- and TIM3-expressing CD8+T cells in the dLN and spleen. FIG. 10D shows summary plots of the frequency of PD1- and TIM3-expressing CD4+T cells in the tumor.

[0057] FIG. 11 examines the association between the expression levels of Pglyrpl and survival rate in different human cancers. Overall survival of patients with breast cancer (BRCA) (n=l, 100), HER2- positive BRCA (BRCA-Her2) (n=82), colon adenocarcinoma (COAD) (n=458), and lung squamous cell carcinoma (LUSC) (n=501) grouped by PGLRYP1 expression through TIMER2 database. Split expression percentage of patients: 20%. Hazard ratio (HR) and p-value are provided

[0058] FIG. 12A-12D shows the characterization of tumor-infiltrating T cells in Pg / )’r / -deficient mice. FIG. 12A shows a heatmap representing cluster-specific upregulated genes (FDR <0.05, fold change > 1.5). FIG. 12B examines a gene set enrichment analysis of selected Gene Ontology (GO) terms and KEGG and Reactome pathways (left) and published CD8+T cell signatures (right) enriched in CD8+T cell-1 cluster vs. CD8+T cell-2 cluster. Only WT cells were included in the analysis. Naive CD8-1; naive CD8-2; terminally exhausted-1; transitory vs. stem-like; terminally exhausted-2; transitory vs. exhausted; exhausted T cells; effector-like. FIG. 12C depicts RNA velocity analysis was performed on the CD8+T cell clusters -1 and -2 (FIG. 3A) using scVelo. The velocity vector field displayed as streamlines (left) and the velocity vector field at single-cell level (right) with each arrow showing the direction and speed of movement of an individual cell. FIG. 12D shows a volcano plot ofdifferentially expressed genes comparing WT vs. Pglyrpl1' cells in the Treg cluster defined in FIG. 12A. Differential genes were determined as FDR < 0.05 and |log2 fold change] > 0.25. Positive log2 fold change corresponds to up-regulation in Pglyrpl1' cells and vice versa.

[0059] FIGs. 13A-13B examines the analysis of the tumor phenotype in Pglyrplninmice. FIG. 13A shows the relative expression (RE) of Pglyrpl in CD8+T cells, CD4+T cells, CD1 lb+cells, B cells and neutrophils by qPCR (n=5). MC38-OVA tumors were implanted into WT and E8iCKPglyrplaiamice. FIG. 13B shows the mean tumor growth of MC38-OVA tumors implanted into WT and LysM^Pglyrpl mice (n=8-9). In FIGs. 13A-13B, data are presented as the mean with ± SEM. Unpaired two-tailed t-tests were performed. Ns: not significant; ***, P <0.001.

[0060] FIGs. 14A-14B shows the antigen-presentation defect of LysM^ Pglyrpla'asplenocytes. FIG. 14A shows an antigen-presentation assay with splenocytes from LysM^ Pglyrpl'' " or WT littermate controls with 2D2 naive CD4+T cells with or without MOG peptide (n=4). Cell proliferation was measured by CellTrace Violet (CTV) staining. Quantification (left) and representative plots (right). FIG. 14B shows cytokine concentration in the culture medium during antigen-presentation assay as in A, determined by legendplex (n=4). In FIGs. 14A-14B, data are presented as the mean with ± SEM. Unpaired two-tailed t-tests were performed. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001.

[0061] FIGs. 15A-15C depicts scRNA-seq of CNS-infiltrated myeloid cells in Pglyrpl1' mice during EAE. FIG. 15A shows a UMAP of CNS-infiltrating myeloid cells (3,698 cells). EAE was induced in Pglyrpl'1' and WT littermate controls and CNS -infiltrating myeloid cells were sorted (CD45+CD3 CD 19 ) for scRNA-seq at disease onset (day 10). Cells are colored by genotype.

[0062] FIG. 15B depicts bar plots depicting the genotype composition of the CNS-infiltrating myeloid cell clusters defined in FIG. 6B. Relative frequency (left) and log2 odds ratios and p-values of chi- square test (right) are shown. Postive log2 odds ratio corresponds to a higher proportion in Pglyrpl'1' cells and vice versa. FIG. 15C shows a Venn diagram depicting the overlap in up-regulated genes (FDR < 0.05, fold change > 1.19) in WT cells (left) and Pglyrpl'1' cells (right) both in mono / MACS and neutrophil clusters as defined in FIG. 15B.DETAILED DESCRIPTION

[0063] Provided herein are methods and compositions related to combination treatment of cancer with an immune checkpoint inhibitor and a Pglyrpl inhibitor. This combination improves cancer treatment by enhancing the efficacy of the immune checkpoint inhibitor while also reducing autoimmunity associated with immune checkpoint inhibitors. In addition, the methods and compositions described herein can be applied to the treatment of autoimmune disease.Definitions

[0064] For convenience, certain terms employed in the specification, examples, and appended claims, are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0065] As used herein, the term "inhibitor" refers to any compound, natural or synthetic, which can antagonize or reduce the activity of a target protein or signaling pathway. An inhibitor can be, for example, a peptide, a protein, an antibody, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound. An inhibitor may attenuate or prevent the activity of a target protein either directly or indirectly.

[0066] Direct inhibition can be obtained, for instance, by binding to a protein and preventing the protein from interacting with an endogenous molecule (i.e. ligand), such as an enzyme, an ion, a hormone, a growth factor, a cytokine, a substrate, or other binding partner, thereby diminishing the activity of the protein. For instance, an inhibitor may bind an enzyme active site and sterically preclude binding of an endogenous substrate at this location, thus decreasing the enzymatic activity of the protein. Direct inhibitors can also act directly on the corresponding gene and / or mRNA of a named target protein. In some embodiments of any of the aspects, an inhibitor of a named target described herein is a direct inhibitor of that named target.

[0067] As used herein, the term “Pglyrp 1 inhibitor” refers to an agent that can reduce and / or inhibit Pglyrpl expression or activity. Non-limiting examples of an appropriate assay for Pglyrp 1 activation can include, but not be limited to, stimulating in vitro macrophages with PGLYRP 1+peptidogly can in the presence or absence of an inhibitor and detecting the levels of proinflammatory cytokines (e.g., IL- 1[3 and TNFa).

[0068] In some embodiments of any of the aspects, the Pglyrp 1 inhibitor is a direct Pglyrp 1 inhibitor. In one embodiment, a Pglyrpl inhibitor binds to Pglyrpl polypeptide and inhibits Pglyrpl activity. In another embodiment, the Pglyrpl inhibitor can interfere with Pglyrpl function or expression intracellularly, e.g., via interference with translation of mRNA encoding Pglyrpl. As a non-limiting example, such interference can involve targeted cleavage of Pglyrpl transcripts. In another embodiment, the Pglyrp 1 inhibitor can induce degradation of Pglyrp 1. Exemplary agents include, but are not limited to an antibody or antigen-binding fragment thereof, a small molecule, a peptide, polypeptide, nucleic acid, an RNA interference (RNAi) molecule (including but not limited to a short interfering RNA (siRNA), a short hairpin RNA (shRNA) or a micro-RNA (miRNA)), antisense, an aptamer or a guide RNA for CRISPR-mediated inhibition.

[0069] A “Pglyrp 1 inhibitor” refers to an agent that reduces Pglyrp 1 expression level or activity in cells by at least 20% when compared to Pglyrp 1 expression level or activity in such cells in the absence of the inhibitor and can include, for example, a decrease by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more.

[0070] As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” refers to 100% inhibition (i.e. expression or activity that is below detectable limits using a standard assay measuring Pglyrpl expression or activity in cells (e.g., tumor cells)).

[0071] As used herein, a "cell-surface marker" refers to any molecule that is expressed on the surface of a cell, such as a cancer or tumor cell. Cell-surface expression usually requires that a molecule possesses a transmembrane domain. Many naturally occurring cell-surface markers are characterized among the "CD" or "cluster of differentiation" molecules. Cell-surface markers vary depending upon cell type, and often provide antigenic determinants to which antibodies can bind, e.g., for targeting of an agent to a desired cell or tissue. Exemplary tumor cell markers include, but are not limited to, alpha fetoprotein, CA15-3, CA27.29, CA19.9, CA-125, calcitonin, calretinin, carcinoembryonic antigen, CD34, CD99, CD 117, chromogranin, cytokeratin, desmin, epithelial membrane antigen, CD31, glial fibrillary acidic protein, hPG80, HMB-45, MART-1, prostate-specific antigen, S100, synaptophysin, thymidine kinase, and vimentin.

[0072] As used herein, the term “intracellular marker” refers to any molecule that is expressed inside a given tumor or cancer cell; such markers can be associated with one or more intracellular compartments such as the mitochondria, peroxisomes, endoplasmic reticulum, nucleus, cytoplasm, or the intracellular side of the cytoplasmic membrane, etc.

[0073] As used herein, “small molecule inhibitors” include, but are not limited to, small peptides or peptide-like molecules, soluble peptides, and non-peptidyl organic or inorganic compounds with inhibitory activity to a given target or function. A small molecule inhibitor or antagonist can have a molecular weight of about 100 to about 20,000 Daltons (Da), for example about 500 to about 15,000 Da, or about 1000 to about 10,000 Da.

[0074] As used herein, the terms “antibody reagent,” “antibodies” or “antigen-binding fragments thereof’ include monoclonal, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, and / or antigen-binding fragments of any of the above. Antibodies can also refer to immunoglobulin molecules and immunologically active portions that contain antigen or target binding sites or “antigen-binding fragments.” The immunoglobulin molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule, as is understood by one of skill in the art.

[0075] As used herein, an antibody reagent (e.g., an antibody or antigen-binding domain thereof) specifically binds to Pglyrpl as an inhibitor or to a cell marker present either on the cell-surface or in some cases intracellularly, with a KDof IO-5M (10000 nM) or less, e.g., IO-6M or less, 10-7M or less,IO-8M or less, IO-9M or less, IO-10M or less, 1011M or less, or IO-12M or less and binds to that target at least lOOx, or lOOOx, or 10,000x and preferably more strongly than it binds to an off-target protein or distinct cell-surface or intracellular marker. The person of ordinary skill in the art can determine appropriate conditions under which the polypeptide agents described herein selectively bind a given target using any suitable methods, such as titration of the antibody reagent in a suitable cell binding assay.

[0076] As used herein, an "antigen-binding fragment” refers to that portion of an antibody that is necessary and sufficient for binding to a given antigen. At a minimum, an antigen binding fragment of a conventional antibody will comprise six complementarity determining regions (CDRs) derived from the heavy and light chain polypeptides of an antibody arranged on a scaffold that permits them to selectively bind the antigen. A commonly used antigen-binding fragment includes the VH and VL domains of an antibody, which can be joined either via part of the constant domains of the heavy and light chains of an antibody, or, alternatively, by a linker, such as a peptide linker. Non-conventional antibodies, such as camelid antibodies have only 2 heavy chain sequences, denoted, for example VHH. These can be used in a manner analogous to Vi i / Vi -containing antigen-binding fragments. Non-limiting examples of antibody fragments encompassed by the term antigen-binding fragment include: (i) a Fab fragment, having VL, CL, VH and CHI domains; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) an Fd fragment having VH and CHI domains; (iv) a Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) an Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment (Ward etal., Nature 341, 544-546 (1989)), also referred to as a singledomain antibody, which consists of a VH domain and has only 3 CDRs; (vii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (viii) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988); and Huston etal., PNAS (USA) 85:5879-5883 (1988)); (ix) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and (x) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8(10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870).

[0077] An "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to Pglyrpl is substantially free of antibodies that specifically bind antigens other than Pglyrpl). An isolated antibody that specifically binds to Pglyrpl may, however, have cross-reactivity to other antigens, such as to Pglyrpl molecules from other species. Moreover, an isolated antibody can be substantially free of other cellular material and / or chemicals.

[0078] As used herein, the term “specificity” refers to the number of different types of antigens or antigenic determinants to which an antibody or antibody fragment thereof as described herein can bind. The specificity of an antibody or antibody fragment thereof can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation (KD) of an antigen with an antigen-binding protein, is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein, such as an antibody or antigen-binding fragment thereof: the less the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD). Accordingly, an antibody or antigen-binding fragment thereof as described herein is said to be "specific for" a first target or antigen compared to a second target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed, for example as a KDvalue) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to another target or polypeptide. Antibody affinities can be determined, for example, by a surface plasmon resonance based assay (such as the BIACORE assay described in PCT Application Publication No. W02005 / 012359); Forte Bio Octet™ analysis, enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA’s), for example.

[0079] As used herein, “avidity” is a measure of the strength of binding between an antigen-binding molecule (such as an antibody or antibody fragment thereof described herein) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule, and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins (such as an antibody or portion of an antibody as described herein) will bind to their cognate or specific antigen with a dissociation constant (KD) of 10’5to 1012moles / liter or less, such as 10-7to 1012moles / liter or less, or 10-8to 1012moles / liter (i.e., with an association constant (KA) of 105to 1012liter / moles or more, such as 107to 1012liter / moles or 108to 1012liter / moles). Any KD value greater than 10'4mol / liter (or any KAvalue lower than 104M1) is generally considered to indicate non-specific binding. The KDfor biological interactions which are considered meaningful (e.g., specific) are typically in the range of l010M (0.1 nM) to 10'5M (10000 nM). The stronger an interaction, the lower is its KD. For example, a binding site on an antibody or portion thereof described herein will bind to the desired antigen with an affinity less than 500 nM, such as less than 200 nM, or less than 10 nM, such as less than 500 pM. Specific binding of an antigenbinding protein to an antigen or antigenic determinant can be determined in any suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art; as well as other techniques as known in the art and / or mentioned herein.

[0080] Accordingly, as used herein, “selectively binds” or “specifically binds” refers to the ability of an antibody or antigen-binding fragment thereof as described herein to bind to a target, such as Pglyrp 1 or a given cell marker, with a KD of IO-5M (10000 nM) or less, e.g., IO-6M, 10-7M, 10-8M, IO-9M, 10’10M, 10-11M, IO-12M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which polypeptide agents as described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay.

[0081] As used herein, the term “selectively inhibits” means that an agent inhibits, as that term is used herein, the association of a first ligand-receptor pair but does not substantially inhibit the association of a relevant second ligand-receptor pair.

[0082] As used herein, the term “bispecific polypeptide agent” refers to a polypeptide that comprises a first polypeptide domain which has a binding site that has binding specificity, e.g., via an antigen-binding domain of an antibody, for a first target, and a second polypeptide domain which has a binding site that has specificity, e.g., via an antigen-binding domain of an antibody, for a second target, i.e., the agent has specificity or is specific for two targets. The first target and second target are not the same, but are both present in an in vivo situation, such that one bispecific agent can encounter and simultaneously bind both targets. Due to the avidity effect of having two closely apposed binding domains, a bispecific agent, including a bispecific polypeptide agent, will bind to targets, including antigen epitopes that are, themselves, closely apposed, more strongly (i.e., with greater avidity) than the bispecific agent will bind either target or antigen when the targets or antigens are not in close apposition to the other. Such difference in avidity thereby provides a preference or selectivity of the bispecific agent, such as a bispecific polypeptide agent, that can be exploited for therapy.

[0083] As used herein, the term "multispecific polypeptide agent" refers to a polypeptide that comprises at least a first polypeptide domain having a binding site that has binding specificity for a first target, and a second polypeptide domain having a binding site that has binding specificity for a second target. A multispecific polypeptide agent can include further, e.g., third, fourth, etc. binding sites for additional targets. The various targets are not the same (i.e., are different targets (e.g., proteins)), but are each present in an in vivo situation, such that one bispecific agent can potentially encounter and potentially bind simultaneously to each of the targets. In one embodiment, the third, fourth or further binding site comprises a site that targets the multispecific agent to a desired location, e.g., via binding specificity for a cell- or tissue-specific marker. For the avoidance of doubt, a bispecific polypeptide agent is a type of multispecific polypeptide agent.

[0084] As used herein, the term "target" refers to a biological molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate, etc.) to which a polypeptide domain which has a binding site can selectively bind. The target can be, for example, an intracellular target (e.g., an intracellular protein target) or a cell surface target (e.g., a membrane protein, a receptor protein). Exemplary “target” biological moleculesfor the purposes of the methods and compositions described herein include Pglyrpl, an immune checkpoint molecule or a cell marker as described herein.

[0085] The term "universal framework" refers to a single antibody framework sequence corresponding to the regions of an antibody conserved in sequence as defined by Kabat ("Sequences of Proteins of Immunological Interest", US Department of Health and Human Services) or corresponding to the human germline immunoglobulin repertoire or structure as defined by Chothiaand Lesk, J. Mol. Biol. 196:910-917 (1987). The Kabat database is now also maintained on the world wide web. The compositions and methods described herein provide for the use of a single framework, or a set of such frameworks, which have been found to permit the derivation of virtually any binding specificity though variation in the hypervariable regions alone. The universal framework can be a VL framework (V% or VK), such as a framework that comprises the framework amino acid sequences encoded by the human germline DPK1, DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPKIO, DPK12, DPK13, DPK15, DPK16, DPK18, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24, DPK25, DPK26 or DPK 28 immunoglobulin gene segment. If desired, the VL framework can further comprise the framework amino acid sequence encoded by the human germline JK1, JK2, JK3, JK4, or JK5 immunoglobulin gene segments. In other embodiments the universal framework can be a VH framework, such as a framework that comprises the framework amino acid sequences encoded by the human germline DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP38, DP45, DP46, DP47, DP49, DP50, DP51, DP53, DP54, DP65, DP66, DP67, DP68 or DP69 immunoglobulin gene segments. In some embodiments, the VH framework can further comprise the framework amino acid sequence encoded by the human germline JHI, JH2, JH3, JH4, Jn4b, JH5 or JH6 immunoglobulin gene segments.

[0086] An "Fv" fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in a single-chain Fv or scFv (see below). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

[0087] As used herein, "antibody variable domain" refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of Complementarity Determining Regions (CDRs; i.e., CDR1, CDR2, and CDR3), and Framework Regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. For the methods and compositions described herein, the amino acid positions assigned to CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987 and 1991)). Amino acid numbering of antibodies or antigen binding fragments is also according to that of Kabat.

[0088] A "Fab" or “Fab fragment” contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

[0089] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which permits the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0090] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0091] The expression "linear antibodies" refers to the antibodies described in Zapata et al., Protein Eng., 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH- CHI-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0092] An "affinity matured" antibody is one with one or more alterations in one or more CDRs thereof which result in improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169: 147155 (1995); Yelton et al. J. Immunol. 155: 1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0093] As used herein in relation to antibody domains, "complementary" refers to when two immunoglobulin domains belong to families of structures which form cognate pairs or groups or are derived from such families and retain this feature. For example, a VH domain and a VL domain of a natural antibody are complementary; two VH domains are not complementary, and two VL domains are not complementary. Complementary domains can be found in other members of the immunoglobulinsuperfamily, such as the Vaand Vp (or y and 5) domains of the T cell receptor. Domains which are artificial, such as domains based on protein scaffolds which do not bind epitopes unless engineered to do so, are non-complementary. Likewise, two domains based on, for example, an immunoglobulin domain and a fibronectin domain are not complementary.

[0094] The process of designing, selecting and / or preparing a bispecific of multispecific polypeptide agent as described herein is also referred to herein as "formatting" the amino acid sequence, and an amino acid sequence that is made part of a bispecific or multispecific polypeptide agent described herein is said to be "formatted" or to be in the format of that bispecific or multispecific polypeptide agent. Examples of ways in which an amino acid sequence can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted amino acid sequences form a further aspect of the bispecific or multispecific polypeptide agents described herein.

[0095] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will ideally comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol 2:593-596 (1992)). The constant region, can if desired, include one or more modifications that modify or disrupt interaction of the human or humanized antibody with an Fc receptor, as described herein. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-3'27 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.

[0096] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0097] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of autoimmune disease, cancer, or allergy. A subject can be male or female.

[0098] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for cancer, or the one or more complications related to cancer. Alternatively, a subject can also be one who has not been previously diagnosed as having cancer or one or more complications related thereto. For example, a subject can be one who exhibits one or more risk such diseases or disorders.

[0099] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0100] The term "therapeutically effective amount" refers to an amount of an inhibitor effective to promote or potentiate an anti-tumor immune response. A therapeutically effective amount of a Pglyrpl inhibitor as described herein will potentiate an anti-tumor immune response promoted by an immunotherapeutic agent such as a checkpoint inhibitor while mitigating off-target or autoimmune effects thereof (e.g., multiple sclerosis). Amounts will vary depending on the specific disease or disorder, its state of progression, and the age, weight and gender of a subject, among other variables. Thus, it is not possible to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. When used in reference to treatment of an autoimmune disease, e.g., comprising administration of a Pglyrp 1 inhibitor, a “therapeutically effective amount” of a Pglyrp 1 inhibitor is an amount effective to reduce at least one symptom or indicator of the auoimmune disease.

[0101] As used herein, the term “anti-tumor immune activity” refers to an increase of at least 20% in immune -mediated markers within the tumor or tumor microenvironment. Exemplary immune- mediated markers can include one or more of: cytokines, macrophage infiltration, death of cancer cells, and the like. In some embodiments, the anti-tumor immune activity is increased by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold or higher than the anti -tumor immune activity in the tumor prior to treatment as described herein or in a substantially similar tumor in a subject that is not being treated as described herein.

[0102] As used herein, the term “suppresses immune checkpoint inhibitor-mediated autoimmunity” refers to a decrease of at least 20% in at least one symptom or measure of autoimmunity or autoimmune dysfunction in a subject being treated with an immune checkpoint inhibitor and administered a Pglyrpl inhibitor, as compared to the degree of autoimmune activity in the subject prior to administration of the Pglyrpl treatment or as compared to a reference sample, subject or population that is treated with an immune checkpoint inhibitor without co-treatment with a Pglyrpl inhibitor. Exemplary symptoms or clinical measures of autoimmunity can include, but are not limited to, level of autoimmune antibodies, joint pain, joint swelling, abdominal pain, digestive issues, fever, swollen glands, colitis,endocrinopathy, hepatitis, thyroiditis, myasthenia gravis, autoimmune autonomic ganglionpathy, nephritis, liver toxicity, skin rash or pruritus, meningitis, headaches, encephalopathy, fatigue, diarrhea, and pneumonitis. In other embodiments, administration of Pglyrpl in combination with an immune checkpoint inhibitor reduces at least one symptom or clinical measure of autoimmunity in a subject by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or even 100% (i.e., absence of a given symptom or below detectable levels of a clinical measure of autoimmunity).

[0103] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of at least one symptom of atargeted disease or disorder, e.g., cancer (e.g., tumor growth rate, tumor size, degree of angiogenesis in tumor, metastasis etc.) or an autoimmune disease (e.g., inflammation). The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a disease or disorder. Treatment is generally “effective" if one or more symptoms or clinical markers of the cancer or autoimmune disease being treated is reduced. Alternatively, treatment is “effective" if the progression of cancer or the autimmune disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0104] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to autoimmune side effects or autoimmune dysfunction. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[0105] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, anact of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0106] The terms “decrease”, “reduce”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction", “decrease" or “inhibit” means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level.

[0107] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0108] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alphaamino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0109] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it canbe a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA.

[0110] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0111] In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is / are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is / are global. In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is systemic.

[0112] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0113] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one expression product. As used herein, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.

[0114] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0115] The term “effective amount” as used herein is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. The effective amount of a compound of the invention may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0116] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0117] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0118] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0119] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0120] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0121] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0122] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Bruce Alberts et al., Molecular Biology of the Cell, published by W.W. Norton & Company, 2022 (ISBN 0393884821, 978-0393884821); John M. Lackie eat al.(eds.), The Dictionary of Cell and Molecular Biology, 5thEdition, published by Academic Press, 2013 (ISBN 0123849314, 978-0123849311); Nalini Chandar et al., Lippincott Illustrated Reviews: Cell and Molecular Biology, 3rdEdition, published by LWW, 2023 (ISBN 1975180895, 978-1975180898);Teresa Atwood et al., Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); Johnathan Law et al., (eds.), A Dictionary of Chemistry, 8thEdition, published by Oxford University Press, 2020 (ISBN 9780198841227, 9780191876783); Robert C. King et al. (eds.), A Dictionary of Genetics, 8thEdition, published by Oxford University Press, 2013(ISBN 9780199766444, 9780199376865); Richard Cammack et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006 (ISBN 9780198529170, 9780191727641); John Lackie et al. (eds.), A Dictionary of Biomedicine, 2ndEdition, published by Oxford University Press, 2019 (ISBN 9780191829116); Lodish et al., Molecular Cell Biology, 8thEdition, published by W.H. Freeman, 2016 (ISBN 1464183392, 978- 1464183393); Abul K. Abbas et al., Cellular and Molecular Immunology, 10thEdition, published by Elsevier, 2021 (ISBN 0323757480, 978-0323757485); Kenneth M. Murphy et al., Janeway's Immunobiology, 10thEdition, published by W. W. Norton & Company, 2022 (ISBN 0393884899, 978-0393884890); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 0444569464); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, 1987-2010 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0123] In all embodiments where a sample is obtained or has been obtained or provided, the sample can be sample taken, obtained, or provided via minimally invasive methods and / or involves only a minor intervention. In some embodiments of any of the aspects, a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and / or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc), fecal sampling, semen / seminal fluid sampling, or clippings (e.g., of hair or nails). In some emodiments of any of the aspects, the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and / or hair or nail clippings.

[0124] Other terms are defined herein within the description of the various aspects of the invention.

[0125] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0126] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.Pglyrpl

[0127] Peptidoglycan recognition protein 1 (otherwise known as PGLYRP1, PGRP-S, TNFSF3L, PGRP, TAG7 and PGRPS) is a protein involved in the innate immune response and has both antibacterial and anti-fungal properties. Increased expression of Pglyrpl has been associated with anumber of inflammatory diseases including, but not limited to, rheumatoid arthritis, type I diabetes, and cardiovascular disease. Pglyrpl is highly expressed in bone marrow, neutrophils, and eosinophils, and is released from neutrophils upon their activation.

[0128] As used herein, the term “PGLYRP1” includes any naturally occurring form of PGLYRP1, which may be derived from any suitable organism, such as an invertebrate species or a vertebrate species. In some embodiments, PGLYRP1 is ahighly conserved, 196 amino acid long protein consisting of a signal peptide and a peptidoglycan binding domain. PGLYRP1 as described herein can be vertebrate PGLYRP1, such as mammalian PGLYRP1, such as PGLYRP1 from a primate (such as a human, a chimpanzee, a cynomolgus monkey or a rhesus monkey); a rodent (such as a mouse or a rat), a lagomorph (such as a rabbit), or an artiodactyl (such a cow, sheep, pig or camel), among others. Preferably, the PGLYRP1 is human PGLYRP1. Exemplary protein and nucleic acid sequences for Pglyrpl are as follows.

[0129] Human Pglyrpl protein sequence (SEQ ID NO: 28) msrrsmllaw alpsllrlga aqetedpacc spivprnewk alasecaqhl slplrywvs htagsscntp ascqqqarnv qhyhmktlgw cdvgynflig edglvyegrg wnftgahsghIwnpmsigis fmgnymdrvp tpqairaaqg llacgvaqga Irsnyvl kgh rdvqrtlspg nqlyhliqnw phyrsp

[0130] Human Pglyrp 1 mRNA sequence (SEQ ID NO: 29) agcggtctcc cggaccctgc cgccctgcca ctatgtcccg ccgctctatg ctgcttgcct gggctctccc cagcctcctt cgactcggag cggctcagga gacagaagac ccggcctgct gcagccccat agtgccccgg aacgagtgga aggccctggc atcagagtgc gcccagcacc tgagcctgcc cttacgctat gtggtggtat cgcacacggc gggcagcagc tgcaacaccc ccgcctcgtg ccagcagcag gcccggaatg tgcagcacta ccacatgaag acactgggct ggtgcgacgt gggctacaac ttcctgattg gagaagacgg gctcgtatac gagggccgtg gctggaactt cacgggtgcc cactcaggtc acttatggaa ccccatgtcc attggcatca gcttcatggg caactacatg gatcgggtgc ccacacccca ggccatccgg gcagcccagg gtctactggc ctgcggtgtg gctcagggag ccctgaggtc caactatgtg ctcaaaggac accgggatgt gcagcgtaca ctctctccag gcaaccagct ctaccacctc atccagaatt ggccacacta ccgctccccc tgaggccctg ctgatccgca ccccattcct cccctcccat ggccaaaaac cccactgtct ccttctccaa taaagatgta gctcaaaa

[0131] In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 is an antibody or antigenbinding fragment thereof. In one embodiment, an anti -Pglyrp 1 antibody or antigen-binding fragment thereof binds to a Pglyrp 1 protein (e.g., SEQ ID NO: 28) and inhibits one or more activities of Pglyrp 1. Exemplary acitivies of Pglyrp 1 that can be inhibited include pro-inflammatory activities or activation of downstream effectors (e.g., TREM1).

[0132] Exemplary Pglyrp 1 antibodies can be also be found in e.g., US Patent No. 9,663,568, the content of which is incorporated by reference herein in its entirety. Exemplary Mus musculus Pglyrp 1 variable heavy chain sequences include SEQ ID Nos: 15-20 and Mus musculus exemplary Pglyrp 1 variable light chains include SEQ ID Nos: 21-26, which can be used to generate a Pglyrp 1 antibody comprising any combination of SEQ ID Nos: 15-26. Humanized forms of SEQ ID Nos: 15-26 are specifically contemplated herein.

[0133] In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 comprises a nucleic acid molecule. In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 comprises a nucleic acid molecule comprising an RNAi molecule, shRNA, miRNA, or encoding an RNA targeting CRISPR-Cas system. In one embodiment, an anti-Pglyrpl RNAi molecule binds to a Pglyrp 1 mRNA sequence (e.g., SEQ ID NO: 29) and inhibits expression of the Pglyrp 1 protein. Exemplary RNAi sequences that target the Pglyrp 1 mRNA include, but are not limited to, rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).Antibody inhibitors

[0134] As discussed above, in some embodiments, an inhibitory agent can comprise an antibody or antigen-binding fragment thereof that binds Pglyrpl and inhibits Pglyrpl activity. As used herein, an “antibody reagent" is a polypeptide that includes at least one antigen-binding immunoglobulin variable domain sequence, and which specifically binds a given antigen (e.g., Pglyrpl).

[0135] In one embodiment, an inhibitory antibody or antigen binding fragment thereof binds the Pglyrpl and inhibits its function. The mechanism of inhibition can include, for example, interference with Pglyrpl interaction with its known peptidoglycan ligand, as well as interference with Pglyrpl interaction with other (co)regulatory molecules; the key is that binding of the antibody reagent inhibits Pglyrpl -mediated signals and this can be verified in an appropriate cell culture assay. Non-limiting examples of an appropriate cell culture assay can include, but not be limited to in vitro macrophages stimulated with PGLYRPl+peptidoglycan in the presence or absence of the inhibitor and analyzed for levels of proinflammatory cytokines in the supernatant such as interleukin- 1 beta (IL1[3) and tumor necrosis factor alpha (TNFa). An in vivo example of an appropriate assay can include, but not be limited to, performing a tumor experiment and an EAE experiment in the presence and absence of inhibitor and analyzing if the inhibitor leads to the substantially the same phenotype as observed in the PGLYRP1- deficient mice.

[0136] A variety of suitable antibody reagent formats are known in the art, such as complete antibodies, e.g., an IgG, or modified forms or fragments of such antibodies, including, as non-limiting examples, single chain antibodies, heterodimers of antibody heavy chains and / or light chains, an Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, a Fab' fragment, a F(ab')2 fragment), a single variable domain (e.g., VH, VL, VHH), a dAb, and modified versions of any of the foregoing (e.g., modified by the covalent attachment of polyalkylene glycol (e.g., polyethylene glycol, polypropylene glycol, polybutylene glycol) or other suitable polymer). Antibody reagents or constructs can, if desired, be linked to an antibody Fc region, comprising one or both of CH2 and CH3 domains, and optionally, a hinge region. Such linkage can provide benefits such as increased serum half-life or promotion of effector fiinction(s). Alternatively, antibody reagents or constructs can be fused to a carrier such as serum albumin to promote increased serum half-life. As another alternative, fusion with serum albumin can also extend serum half-life. The benefits of serum albumin binding can also be realized with an antigen-binding domain that binds serum albumin. For example, a multispecific polypeptide agent can comprise a first antigen-binding domain specific for Pglyrpl, that is fused to an immune checkpoint specific antibody binding domain and an antigen-binding domain that binds serum albumin.

[0137] In some embodiments, a polypeptide agent, including an antibody reagent, can be formatted as a bispecific polypeptide agent as described herein, and in US 2010 / 0081796 and US 2010 / 0021473, the contents of which are herein incorporated in their entireties by reference. Bispecific agents can include, for example, agents including separate binding sites specific for Pglyrpl and an immune checkpoint inhibitor molecule. In other embodiments, a polypeptide agent, including an antibody reagent, can beformated as a multispecific polypeptide agent, for example as described in WO 03 / 002609, the entire teachings of which are incorporated herein by reference.

[0138] Antibodies suitable for practicing the methods described herein are preferably monoclonal, and can include, but are not limited to, human, humanized or chimeric antibodies, including single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, and / or antigen-binding fragments of any of the above. Antibody reagents also include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain at least one, at least two, at least three or more antigen binding sites that specifically bind Pglyrpl. Such immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule, as understood by one of skill in the art.

[0139] Additional types of antibodies include, but are not limited to, chimeric, humanized, and human antibodies. For application in man, it is often desirable to reduce immunogenicity of antibodies originally derived from other species, like mouse. This can be done by construction of chimeric antibodies, or by a process called "humanization". In this context, a "chimeric antibody" is understood to be an antibody comprising a domain (e.g. a variable domain) derived from one species (e.g. mouse) fused to a domain (e.g. the constant domains) derived from a different species (e.g. human).

[0140] The term "monoclonal antibody" as used herein refers to a population of antibodies that comprise an identical antigen-binding domain. In some embodiments, a monoclonal antibody can be produced by a single B cell clone, B cell hybrodima or its equivalent. Such a cell produces only one antibody, such that all antibodies produced by such a clone have the same antigen-binding domain. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes) on a given target antigen, each monoclonal antibody is directed against a single determinant on the antigen. The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. It is to be understood that the term “monoclonal antibody” refers to an antibody that is derived from a single isolated clone, including any eukaryotic, prokaryotic or phage clone, and not the method by which the antibody is produced. For example, the monoclonal antibodies to be used in accordance with the methods and compositions described herein can be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597 (1991), for example. A wide variety of methods for producing constructs with the antigen-binding domain of monoclonal antibodies are known to those of ordinary skill in the art.

[0141] Bispecific and multispecific polypeptide agents can comprise immunoglobulin variable domains that have different binding specificities. Such bispecific and multispecific polypeptide agents can comprise combinations of heavy and light chain domains. For example, a VH domain and a VL domain,which can be linked together in the form of an scFv (e.g., using a suitable linker such as (Gly4Ser)n (where n = 1-8, e.g., 2, 3, 4, 5, 6, or 7) (SEQ ID NO: 27) can provide one antigen-binding domain that binds one or each target of a multispecific polypeptide agent e.g. Pglyrpl and an immune checkpoint molecule. A construct that includes, e.g., an scFv that binds Pglyrpl and an scFv that binds an immune checkpoint molecule, is said to be bispecific for Pglyrpl and the immune checkpoint inhibitor. Similar arrangements can be applied in the context of, e.g., a bispecific F(ab')2 construct.

[0142] Single domain antibody constructs are also contemplated for the development of bispecific or multispecific reagents described herein. In some embodiments of the aspects described herein, the bispecific and multispecific polypeptide agents do not comprise complementary VH / VL pairs which form an antigen-binding site that binds to a single antigen or epitope co-operatively as found in conventional two chain antibodies. Instead, in some embodiments, the bispecific and multispecific polypeptide agents can comprise a domain, wherein the V domains each have different binding specificities, such that two different epitopes or antigens are specifically bound. Camelid antibodies for example comprise only VH domains, and can be used to generate bispecific constructs when modified to a humanized scaffold.

[0143] In addition, in some embodiments, bispecific and multispecific polypeptide agents comprise one or more CH or CL domains. A hinge region domain can also be included in some embodiments. Such combinations of domains can, for example, mimic natural antibodies, such as IgG or IgM, or fragments thereof, such as Fv, scFv, Fab or F(ab') 2 molecules. Other structures, such as a single arm of an IgG molecule comprising VH, VL, CHI and CL domains, are also encompassed within the embodiments described herein. Alternatively, in another embodiment, a plurality of bispecific polypeptide agents is combined to form a multimer. An Fc domain that binds human FcRn can extend circulating half-life by directing internalized antibodies into the FcRn-mediated recycling / secretory pathway. As another alternative, fusion with serum albumin can also extend serum half-life.

[0144] It will be appreciated by one skilled in the art that the light and heavy variable regions of a monospecific, bispecific or multispecific polypeptide agent produced according to the methods described herein can be on the same polypeptide chain, or alternatively, on different polypeptide chains. In the case where the variable regions are on different polypeptide chains, then they can be linked via a linker, generally a flexible linker (such as a polypeptide chain), a chemical linking group, or any other method known in the art.

[0145] In another aspect, bispecific antibodies having an IgG-like format are provided. Such formats have the conventional four chain structure of an IgG molecule (2 heavy chains and two light chains), in which one antigen-binding region (comprised of a VH and a VL domain) specifically binds Pglyrp 1 and a second antigen-binding region (also comprised of a VH and a VL domain) binds an immune checkpoint molecule. In some embodiments, each of the variable regions (2 VH regions and 2 VL regions) is replaced with a dAb or single chain variable domain. The dAb(s) or single chain variable domain(s) that are included in an IgG-like format can have the same specificity or different specificities.In some embodiments, the IgG-like format is tetravalent and can have two, three or four specificities. For example, the IgG-like format can be bispecific and comprise 3 dAbs that have the same specificity and another dAb that has a different specificity; bispecific and comprise two dAbs that have the same specificity and two dAbs that have a common but different specificity; trispecific and comprise first and second dAbs that have the same specificity, a third dAb with a different specificity and a fourth dAb with a different specificity from the first, second and third dAbs; or tetraspecific and comprise four dAbs that each have a different specificity. Antigen-binding fragments of IgG-like formats (e.g., Fab, F(ab')2, Fab', Fv, scFv) can be prepared as is known to one of skill in the art, and as described herein.

[0146] In some embodiments of the aspects described herein, antigen-binding fragments of antibodies can be combined and / or formatted into non-antibody multispecific polypeptide structures to form multivalent complexes, which bind target molecules having the same epitope, thereby providing superior avidity. For example, natural bacterial receptors such as SpA can be used as scaffolds for the grafting of CDRs to generate ligands which bind specifically to one or more epitopes. Details of this procedure are described in U.S. Pat. No. 5,831,012, the contents of which are herein incorporated by reference in their entirety. Other suitable scaffolds include those based on fibronectin and affibodies. Details of suitable procedures are described in WO 98 / 58965, herein incorporated by reference in its entirety. Exemplary scaffolds are described in van den Beuken et al., J. Mol. Biol. 310:591-601 (2001), and in WO 00 / 69907 (Medical Research Council), the contents of each of which are herein incorporated by reference in their entireties. As but one example, a scaffold can be based on the ring structure of bacterial GroEL or other chaperone polypeptides. In some embodiments, protein scaffolds can be combined.

[0147] Generally, the orientation of the polypeptide domains that have a binding site with binding specificity for a target, and whether a bispecific or multispecific polypeptide agent comprises a linker, are a matter of design choice. However, some orientations, with or without linkers, can provide better binding characteristics than other orientations. All orientations are encompassed by the aspects and embodiments described herein, and bispecific or multispecific polypeptide agents that contain an orientation that provides desired binding characteristics can be easily identified by screening.

[0148] Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually done by affinity chromatography steps, but the product yields are low. Similar procedures are disclosed in WO 93 / 08829, and in Traunecker et al, EMBO J., 10:3655-3659 (1991), herein incorporated by reference in their entireties.

[0149] According to another approach, described, for example in WO96 / 27011, herein incorporated by reference in its entirety, the interface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered from recombinant cell culture. Such interfaces can comprise at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0150] In one aspect, the bispecific antibodies described herein include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies can be made using any convenient crosslinking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. In one embodiment, the bispecific antibodies do not comprise a heteroconjugate.

[0151] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. For example, Brennan et al., Science, 229: 81 (1985) describe aprocedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. A bispecific antibody produced using this method can be used in any of the compositions and methods described herein.

[0152] Another option for production of bispecific or multispecific antibody reagents uses the dual variable domain immunoglobulin, or DvDIg approach. See, e.g., DiGiammarino et al., Meth. Mol. Biol. 899: 145-156 (2012). In this approach, each arm of the immunoglobulin molecule has two or more antigen-binding domains, which can be different, linked in tandem. The design has the benefit of providing bi- or multi-specificity without the problems generated by random assortment of differing light chains with differing heavy chains.

[0153] In some embodiments, bispecific antibodies for use in the compositions and methods described herein can be produced using any of the methods described in U.S. Patent Application No.: 20100233173; U.S. Patent Application No.: 20100105873; U.S. Patent Application No.: 20090155275; U.S. Patent Application No.: 20080071063; and U.S. Patent Application No.: 20060121042, the contents of each of which are herein incorporated in their entireties by reference. In some embodiments,a bispecific antibody specific for Pglyrp 1 and an immune checkpoint inhibitor can be produced using any ofthe methods described in U.S. Patent Application No.: 20090175867 and U.S. Patent Application No.: 20110033483, the contents of which are herein incorporated in their entireties by reference.

[0154] In some embodiments, bispecific antibodies can be made by the direct recovery of Fab'-SH fragments recombinantly expressed, e.g., in E. coli. and chemically coupled to form bispecific antibodies. Chemical conjugation is based on the use of homo- and heterobifunctional reagents with E- amino groups or hinge region thiol groups. Homobifimctional reagents such as 5,5'-Dithiobis(2- nitrobenzoic acid) (DNTB) generate disulfide bonds between the two Fabs, and 0- phenylenedimaleimide (O-PDM) generate thioether bonds between the two Fabs (Brenner et al., 1985, Glennie et al., 1987). Heterobifiinctional reagents such as N-succinimidyl-3-(2-pyridylditio) propionate (SPDP) combine exposed amino groups of antibodies and Fab fragments, regardless of class or isotype (Van Dijk et al., 1989). For example, Shalaby et al., J Exp. Med, 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0155] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described, and can be used in the generation of bispecific antibodies. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol, 148(5): 1547-1553 (1992)). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VH and VL domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994). Alternatively, the antibodies can be "linear antibodies" as described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-C Hl) which form a pair of antigen binding regions. Linear antibodies can be bispecific or multispecific.

[0156] Antibodies useful in the present methods can be described or specified in terms of the particular CDRs they comprise. The compositions and methods described herein encompass the use of an antibodyor derivative thereof comprising a heavy or light chain variable domain, where the variable domain comprises (a) a set of three CDRs, and (b) a set of four framework regions, and in which the antibody or antibody derivative thereof specifically binds Pglyrpl, and an immune checkpoint inhibitor.

[0157] Also provided herein are chimeric antibody derivatives of the bispecific and mutispecific polypeptide agents, i.e., antibody molecules in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is / are identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibody molecules can include, for example, one or more antigen binding domains from an antibody of a mouse, rat, or other species, with human constant regions. A variety of approaches for making chimeric antibodies have been described and can be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes the selected antigens, on the surface of differentiated cells or tumor-specific cells. See, for example, Takeda et al., 1985, Nature 314:452; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al.; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom patent GB 2177096B).

[0158] The bispecific and multispecific polypeptide agents described herein can also include humanized antibody derivatives. Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non- human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0159] In some embodiments, antibodies described herein include derivatives that are modified, i.e., by the covalent attachment of another type of molecule to the antibody that does not prevent the antibodyfrom binding to its target. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can contain one or more non- classical amino acids.

[0160] Antibodies can be produced in bacteria, yeast, fungi, protozoa, insect cells, plants, or mammalian cells (see e.g., Frenzel et al. (2013) Front Immunol. 4: 217). A mammalian expression system is generally preferred for manufacturing most therapeutic proteins, such as antibodies, as they require post-translational modifications. A variety of mammalian cell expression systems are now available for expression of antibodies, including but not limited to immortalized Chinese hamster ovary (CHO) cells, mouse myeloma (NSO), mouse L-cells, myeloma cell lines like J558L and Sp2 / 0, baby hamster kidney (BHK), or human embryo kidney (HEK-293).

[0161] As used herein, the term “Complementarity Determining Regions” (CDRs, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for specific antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region can comprise amino acid residues from a “complementarity determining region” as defined by Kabat (i.e., about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95- 102 (H3) in the heavy chain variable domain). Likewise, “frameworks” (FWs) comprise amino acids 1-23 (FW1), 35-49 (FW2), 57-88 (FW3), and 98-107 (FW4) in the light chain variable domain and 1- 30 (FW1), 36-49 (FW2), 66-94 (FW3), and 103-113 (FW4) in the heavy chain variable domain taking into account the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1987, 1991)).

[0162] The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. Methods and computer programs for determining sequence similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12: 387, 1984), BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990), and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm may also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at ncbi.nlm.nih.gov / blast / ). Incomparing sequences, these methods account for various substitutions, deletions, and other modifications.

[0163] Anti-Pglyrpl antibodies are known in the art and commercial available. For example, known anti-Pglyrpl antibodies known in the art include:

[0164] In some embodiments of any of the aspects, the inhibitor of Pglryp 1 is an antibody or antigenbinding fragment thereof selected from LS-B4940, 188C424, 299D535, 1040002, and 1040023. In some embodiments of any of the aspects, the inhibitor of Pglryp 1 is an antibody or antigen-binding fragment thereof comprising the six CDRs of one of LS-B4940, 188C424, 299D535, 1040002, and 1040023.

[0165] In some embodiments of any of the aspects, the inhibitor of Pglryp 1 is an antibody or antigenbinding fragment thereof selected from LS-B4940, 188C424, and 299D535. In some embodiments of any of the aspects, the inhibitor of Pglryp 1 is an antibody or antigen -binding fragment thereof comprising the six CDRs of one of LS-B4940, 188C424, and 299D535.RNA interference (RNAi)

[0166] Other approaches for inhibiting Pglyrpl expression and / or activity include the use of RNA interference (RNAi), aptamers and / or the use of small molecules. The design and testing of RNAi molecules that inhibit the expression of Pglyrp 1 are known to those skilled in the art. For example, RNAi molecules that inhibit Pglyrpl can be obtained from commercial sources such as ThermoFisher, Creative Biolabs, and Dharmacon, among others.

[0167] In some embodiments of any of the aspects, the inhibitor of Pglyrpl comprises a nucleic acid molecule. In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 comprises an inhibitory nucleic acid molecule. In some embodiments of any of the aspects, the inhibitor of Pglyrpl comprises an inhibitory RNA molecule.

[0168] As used herein, “inhibitory nucleic acid” refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double -stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), and the like. In some embodiments of any of the aspects, the inhibitory nucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA). Inhibitory nucleic acids can also include guide sequence molecules (e.g., a guide RNA) that function, e.g., in combination with anenzyme, to induce insertions, deletions, indels, and / or mutations of a target, thereby inhibiting the expression of the target.

[0169] In some embodiments of any of the aspects, an iNA comprises a sequence that is complementary to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 15 nucleotides in length that is complementary to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 20 nucleotides in length that is complementary to at least a portion of a target sequence described herein.

[0170] In some embodiments of any of the aspects, an iNA comprises a sequence that is the reverse complement to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 15 nucleotides in length that is the reverse complement to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 20 nucleotides in length that is the reverse complement to at least a portion of a target sequence described herein.

[0171] In some embodiments of any of the aspects, an iNA comprises a sequence that can specifically hybridize to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 15 nucleotides in length that can specifically hybridize to at least a portion of a target sequence described herein. In some embodiments of any of the aspects, an iNA comprises a sequence at least 20 nucleotides in length that can specifically hybridize to at least a portion of a target sequence described herein.

[0172] As used herein, the term “iRNA” refers to an agent that contains RNA (or modified nucleic acids as described below herein) and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In some embodiments of any of the aspects, an iRNA as described herein effects inhibition of the expression and / or activity of a target, e.g.Pglyrpl. In some embodiments of any of the aspects, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA. In some embodiments of any of the aspects, administering an inhibitor (e.g. an iRNA) to a subject results in a decrease in the target mRNA level in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the subject without the presence of the iRNA.

[0173] In some embodiments of any of the aspects, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, toa target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target, e.g., it can span one or more intron boundaries. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length nucleotides in length, inclusive. In some embodiments of any of the aspects, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

[0174] Exemplary embodiments of types of inhibitory nucleic acids can include, e.g,. siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art. One skilled in the art would be able to design further siRNA, shRNA, or miRNA to target the nucleic acid sequence of Pglyrpl (e.g., SEQ ID NO: 29), e.g., using publically available design tools. siRNA, shRNA, or miRNA is commonly made using companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).

[0175] In some embodiments of any of the aspects, the inhibitor of Pglyrpl is an RNAi molecule, shRNA, or miRNA.

[0176] The RNAi molecule can be siRNA, shRNA, endogenous microRNA (miRNA), or artificial miRNA. In one embodiment, an RNAi molecule as described herein effects inhibition of the expression and / or activity of a target, e.g. Pglyrp 1. In some embodiments of any of the aspects, the RNAi molecule is an siRNA that inhibits Pglyrpl activity and / or expression.

[0177] One skilled in the art can design siRNA, shRNA, or miRNA to target Pglyrpl, e.g., using publically available design tools. siRNA, shRNA, or miRNA can be synthetically made or expressed from a vector. Commercial sources include companies such as Dharmacon (Lafayette, CO) and Sigma Aldrich (St. Louis, MO), among others.

[0178] In some embodiments, the RNAi molecule can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions

[0179] The nucleobases in the RNAi molecule can be chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the methods and compositions described herein can be synthesized and / or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.

[0180] In one embodiment, the Pglyrpl inhibitor comprises an miRNA. microRNAs or miRNAs are small non-coding RNAs with an average length of 22 nucleotides. These molecules act by binding to complementary sequences within mRNA molecules, usually in the 3' untranslated (3'UTR) region, thereby promoting target mRNA degradation or inhibited mRNA translation. The interaction between microRNA and mRNAs is mediated by what is known as the “seed sequence”, a 6-8-nucleotide region of the microRNA that directs sequence-specific binding to the mRNA through imperfect Watson-Crick base pairing. More than 900 microRNAs are known to be expressed in mammals. Many of these can be grouped into families on the basis of their seed sequence, thereby identifying a “cluster” of similar microRNAs. A miRNA can be encoded by a nucleic acid that is expressed in the cell, e.g., from naked DNA, or can be encoded by a nucleic acid that is contained within a vector.

[0181] The agent may result in gene silencing of the target gene (e.g., Pglyrpl), such as with an RNAi molecule (e.g. siRNA or miRNA). This entails a decrease in the Pglyrpl mRNA level in a cell by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or even about 100% (i.e., below detectable limits by standard mRNA assay detection methods). One skilled in the art will be able to readily assess whether the siRNA, shRNA, or miRNA effectively targets e.g., Pglyrpl, for downregulation, for example by transfecting the siRNA, shRNA, or miRNA into cultured cells and detecting the levels of a gene product (e.g., Pglyrpl) found within the cell via western-blotting.

[0182] In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 comprises one or more sequences selected from: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4). In some embodiments of any of the aspects, the inhibitor of Pglyrpl comprises the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 comprises the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the inhibitor of Pglyrpl comprises the sequence ofSEQ ID NO: 3. In some embodiments of any of the aspects, the inhibitor of Pglyrpl comprises the sequence of SEQ ID NO: 4.

[0183] In some embodiments of any of the aspects, the inhibitor of Pglyrpl consists essentially of one or more sequences selected from: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4). In some embodiments of any of the aspects, the inhibitor of Pglyrpl consists essentially of the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the inhibitor of Pglyrpl consists essentially of the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the inhibitor of Pglyrpl consists essentially of the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the inhibitor of Pglyrpl consists essentially of the sequence of SEQ ID NO: 4.

[0184] In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 consists of one or more sequences selected from: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4). In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 consists of the sequence of SEQ ID NO: 1. In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 consists of the sequence of SEQ ID NO: 2. In some embodiments of any of the aspects, the inhibitor of Pglyrpl consists of the sequence of SEQ ID NO: 3. In some embodiments of any of the aspects, the inhibitor of Pglyrpl consists of the sequence of SEQ ID NO: 4.

[0185] Expression Vectors: The RNAi agent can be contained in or expressed by a desired vector. Many such vectors useful for transferring exogenous genes into target mammalian cells are available. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc.

[0186] An expression vector can direct expression of an RNA or polypeptide (e.g., a Pglyrp 1 inhibitor) from nucleic acid sequences contained therein linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector can comprise additional elements, for example, the expression vector can have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. Expression refers to the cellular processes involved in producing RNA and / or proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from agene and processing derivatives thereof, such as siRNA, shRNA, miRNA, etc., and polypeptides obtained by translation of mRNA transcribed from a gene or gene construct.

[0187] Vectors can be episomal, e.g. plasmids, virus-derived vectors such as cytomegalovirus, adenovirus, etc., or can be integrated into the target cell genome, through homologous recombination or random integration, e.g. for retrovirus-derived vectors such as MMLV, HIV-1, ALV, etc. In some embodiments, combinations of retroviruses and an appropriate packaging cell line may also find use, where the capsid proteins will be functional for infecting the target cells. Commonly used retroviral vectors are "defective", i.e. unable to produce viral proteins required for productive infection. Replication of the vector requires growth in the packaging cell line.

[0188] Integrating vectors, such as retroviral vectors, lentiviral vectors, hybrid adenoviral vectors, and herpes simplex viral vector are specifically contemplated for use in the methods described herein. Alternatively, non-integrative vectors (e.g., non-integrative viral vectors) can be used and can eliminate the risks posed by integrative retroviruses, as they do not incorporate their genome into the host DNA. Non-limiting examples of non-integrating viral vectors include Epstein Barr oriP / Nuclear Antigen- 1 (“EBNA1”) vector, RNA Sendai viral vector, or an F-deficient Sendai virus vector. Another example of a non-integrative vector is a minicircle vector. Minicircle vectors are circularized vectors in which the plasmid backbone has been released leaving only the eukaryotic promoter and cDNA(s) that are to be expressed.

[0189] In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 is one or more nucleic acids comprising an RNA targeting CRISPR-Cas system, e .g, the inhibitor is a guide RNA of a RNA targeting CRISPR-Cas system. As used herein, the terms “guide nucleic acid,” “guide sequence,” “crRNA,” “guide RNA,” “single guide RNA,” “gRNA” or “CRISPR guide sequence” refer to a nucleic acid comprising a sequence that determines the specificity of an enzyme, e.g., the Cas DNA binding protein of a CRISPR / Cas system, to a polynucleotide target. The gNA can comprise a polynucleotide sequence with at least partial complementarity with a target nucleic acid sequence, sufficient to hybridize with the target nucleic acid sequence and to direct sequence-specific binding of an enzyme, e.g, a nuclease, to the target nucleic acid sequence.

[0190] In some embodiments, the enzyme directed by the gNA is a gene-editing protein, e.g., any nuclease that induces a nick or double-strand break into a desired recognition site. Such enzymes can be native or engineered. These breaks can then be repaired by the cell in one of two ways: non- homologous end joining and homology-directed repair (homologous recombination). In non- homologous end joining (NHEJ), the double-strand breaks are repaired by direct ligation of the break ends to one another. As such, no new nucleic acid material is inserted into the site, although some nucleic acid material may be lost, resulting in a deletion. In homology-directed repair, a donor polynucleotide with homology to the cleaved target DNA sequence can be used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the target DNA. Therefore, new nucleic acid material may be inserted / copied into thesite. The modifications of the target DNA due to NHEJ and / or homology-directed repair can be used for gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.

[0191] In one embodiment, the gene-editing protein is a CRISPR-associated nuclease. The native prokaryotic CRISPR-associated nuclease system comprises an array of short repeats with intervening variable sequences of constant length (i.e., clusters of regularly interspaced short palindromic repeats), and CRISPR-associated ("Cas") nuclease proteins. The RNA of the transcribed CRISPR array is processed by a subset of the Cas proteins into small guide RNAs, which generally have two components as discussed below. There are at least three different systems: Type I, Type II and Type III. The enzymes involved in the processing of the RNA into mature crRNA are different in the 3 systems. In the native prokaryotic system, the guide RNA ("gRNA") comprises two short, non-coding RNA species referred to as CRISPR RNA ("crRNA") and trans-acting RNA ("tracrRNA"). In an exemplary system, the gRNA forms a complex with a nuclease, for example, a Cas nuclease. The gRNA: nuclease complex binds a target polynucleotide sequence having a protospacer adjacent motif ("PAM") and a protospacer, which is a sequence complementary to a portion of the gRNA. The recognition and binding of the target polynucleotide by the gRNA: nuclease complex induces cleavage of the target.

[0192] Any CRISPR-associated nuclease can be used in the system and methods of the invention. CRISPR nuclease systems are known to those of skill in the art, e.g. Cas9, Cas 12, Cas 12a, or the like, see Patents / applications 8,993,233, US 2015 / 0291965, US 2016 / 0175462, US 2015 / 0020223, US 2014 / 0179770, 8,697,359; 8,771,945; 8, 795,965; WO 2015 / 191693; US 8,889,418; WO 2015 / 089351; WO 2015 / 089486; WO 2016 / 028682; WO 2016 / 049258; WO 2016 / 094867; WO 2016 / 094872; WO 2016 / 094874; WO 2016 / 112242; US 2016 / 0153004; US 2015 / 0056705; US 2016 / 0090607; US 2016 / 0029604; 8,865,406; 8,871,445; each of which are incorporated by reference in their entirety. The nuclease can also be a phage Cas nuclease, e.g., Casfb (e.g., Pausch et al. Science 369:333-7 (2020); which is incorporated by reference herein in its entirety).

[0193] The full-length guide nucleic acid strand can be any length. For example, the guide nucleic acid strand can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments of the various aspects described herein, a nucleic acid strand is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. For example, the guide nucleic acid sequence is 10-30 nucleotides long.

[0194] In addition to a sequence that is complementary to a target nucleic acid, in some embodiments, the gNA also comprises a scaffold sequence. Expression of a gNA encoding both a sequence complementary to a target nucleic acid and scaffold sequence has the dual function of both binding (hybridizing) to the target nucleic acid and recruiting the endonuclease to the target nucleic acid, which may result in site-specific CRISPR activity. In some embodiments, such a chimeric gNA may be referred to as a single guide RNA (sgRNA).

[0195] In some embodiments of the various aspects described herein, the guide nucleic acid is designed using a guide design tool (e.g., Benchling™; Broad Institute GPP™; CasOFFinder™; CHOPCHOP™; CRISPOR™; Deskgen™; E-CRISP™; Geneious™; GenHub™; GUIDES™ (e.g., for library design); Horizon Discovery™; IDT™; Off-Spotter™; and Synthego™; which are available on the world wide web).Immune checkpoint inhibition

[0196] The immune system has multiple inhibitory pathways that are critical for maintaining selftolerance and modulating immune responses. In T-cells, the amplitude and quality of response is initiated through antigen recognition by the T-cell receptor and is regulated by immune checkpoint proteins that balance co-stimulatory and inhibitory signals. Exemplary immune checkpoint proteins can include PD-1 (e g., NCBI Gene ID: 5133); PD-L1 (e g., NCBI Gene ID: 29126); PD-L2 (e.g., NCBI Gene ID: 80380); TIM-3 (e.g., NCBI Gene ID: 84868); CTLA4 (e.g., NCBI Gene ID: 1493); TIGIT (e.g., NCBI Gene ID: 201633); KIR (e g., NCBI Gene ID: 3811); LAG3 (e.g., NCBI Gene ID: 3902); DDl-a (e g., NCBI Gene ID: 64115); A2AR (e g., NCBI Gene ID: 135); B7-H3 (e g., NCBI Gene ID: 80381); B7-H4 (e g., NCBI Gene ID: 79679); BTLA (e.g., NCBI Gene ID: 151888); IDO (e.g., NCBI Gene ID: 3620); TDO (e.g., NCBI Gene ID: 6999); HVEM (e.g., NCBI Gene ID: 8764); GAL9 (e.g., NCBI Gene ID: 3965); 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, y5, and memory CD8+ (a[3) T cells) (e.g., NCBI Gene ID: 51744); CD160 (also referred to as BY55) (e.g., NCBI Gene ID: 11126); and various B-7 family ligands. B7 family ligands include, but are not limited to, B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7.

[0001] The term “immune checkpoint inhibitor”, as used herein, refers to a factor or agent that blocks the activity of one or more molecules involved in attenuating the immune response.

[0197] Examples of immune checkpoint inhibitors include, but are not limited to inhibitors of CTLA- 4, PD-1, LAG-3, B7-H3, 67-H4, TIM3, A2AR, and IDO.

[0198] In some embodiments, the one or more immune checkpoint inhibitors are selected from an inhibitor of: Programmed Death-Ligand 1 (PD-L1, also known as B7-HI, CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CDI37, CDI60, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, ID02, ICOS (inducible T cell costimulator), KIR, LAIRI, LIGHT, MARCO (macrophage receptor with collagenous structure), PS (phosphatidylserine), OX-40, SLAM, TIGIT, VISTA, and VTCNI. In some embodiments of any of the aspects, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of: CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.

[0199] In some embodiments of any of the aspects, the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.

[0200] In some aspects of this and other embodiments the immune checkpoint inhibitor is selected from a group consisting of an anti-PD-1 antibody, an anti PD-L1 antibody, an anti-CTLA-4 antibody, and combinations thereof. Preferably, the immune checkpoint inhibitor is selected from a group consisting of nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GlaxoSmithKline / Amplimmune), MPDL3280A (Roche), MDX-1105 (Medarex, Inc. / Bristol Myer Squibb), MEDI-4736 (Medimmune / AstraZeneca), arelumab (Merck Serono), ipilimumab (YERVOY, (Bristol-Myers Squibb), tremelimumab (Pfizer), pidilizumab (CureTech, Ltd.), IMP321 (Immutep S.A.), MGA271 (Macrogenics), BMS-986016 (Bristol-Meyers Squibb), lirilumab (Bristol-Myers Squibb), urelumab (Bristol-Meyers Squibb), PF-05082566 (Pfizer), IPH2101 (Innate Pharma / Bristol- Myers Squibb), MEDI-6469 (Medlmmune / AZ), CP-870,893 (Genentech), Mogamulizumab (Kyowa Hakko Kirin), Varlilumab (CellDex Therapeutics), Avelumab (EMD Serono), Galiximab (Biogen Idee), AMP -514 (Amplimmune / AZ), AUNP 12 (Aurigene and Pierre Fabre), Indoximod (NewLink Genetics), NLG-919 (NewLink Genetics), INCB024360 (Incyte) and combinations thereof. Exemplary complementarity determining regions (CDRs) for certain anti-PDl antibodies can be found in e. g. US Patent Application Nos: US20210403567, US20220064034, US2020 / 031933, US 2019 / 010232, and US Patent Nos.: 8,008,449, 8,354,509 and 8,900,587, the contents of each of which are incorporated herein by reference in their entirety.

[0201] Non-limiting examples of immune checkpoint inhibitors (with checkpoint targets and manufacturers noted in parantheses) can include :MGA271 (B7-H3: MacroGenics); ipilimumab (CTLA-4; Bristol Meyers Squibb); pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb) ; atezolizumab (PD-L1; Genentech); galiximab (B7.1; Biogen); IMP321 (LAG3: Immuntep); BMS-986016 (LAG3; Bristol Meyers Squibb); SMB-663513 (CD137; Bristol-Meyers Squibb); PF- 05082566 (CD137; Pfizer); IPH2101 (KIR; Innate Pharma); KW-0761 (CCR4; Kyowa Kirin); CDX- 1127 (CD27; CellDex); MEDI-6769 (0x40; Medlmmune); CP-870,893 (CD40; Genentech); tremelimumab (CTLA-4; Medimmune); pidilizumab (PD-1; Medivation); MPDL3280A (PD-L1; Roche); MEDI4736 (PD-L1; AstraZeneca); MSB0010718C (PD-L1; EMD Serono); AUNP12 (PD-1; Aurigene); avelumab (PD-L1; Merck); durvalumab (PD-L1; Medimmune); IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211); the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834); TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94); anti-CTLA-4 antibodies described in US Patent Nos: 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238; tremelimumab, (ticilimumab, CP-675,206); ipilimumab (also known as 10D1, MDX-D010); PD-1 and PD-L1 blockers described in US Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT Published Patent Application Nos: W003042402, WO2008156712, W02010089411, W02010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699; nivolumab (MDX 1106, BMS 936558, ONO 4538); lambrolizumab (MK-3475 or SCH 900475); CT-Oi l; AMP -224; and BMS-936559 (MDX- 1105-01). The foregoing references are incorporated by reference herein in their entireties.

[0202] Exemplary anti-CTLA4 antibodies and their sequences (including CDRs) are described for example at U.S. Pat. Nos. 6,682,736; 7,109,003; 7,123,281; 7,411,057; 7,824,679; 8,143,379; 7,807,797; and 8,491,895 (Tremelimumab is 11.2.1, therein), the contents of which are herein incorporated by reference in their entirety.

[0203] Exemplary anti-CCR4 antibodies and their sequences (including CDRs) can be found in e.g., US Patent Nos.: 8,980,263; 9,458,238; 7,138,117; 6,989,145; 9,789,182; and 8,574,574, and US Patent Application No.: US2011 / 0262431, the contents of each of which are incorporated herein by reference in their entirety.

[0204] Exemplary anti-CD27 antibodies and their sequences (including CDRs) are described, for example, in US Patent Nos: 9,169,325; 10,301,392; 9,023,999; and 9,403,914, the contents of each of which are incorporated herein by reference in their entirety.

[0205] Exemplary anti-CD80 antibodies and their sequences are described, for example, in US Patent Nos.: 7,417,128; or 7,192,585, the contents of which are incorporated herein by reference in their entirety.

[0206] Exemplary anti-KIR antibodies and their sequences (including CDRs) are described, for example, in US Patent Application No.: US2017 / 0334993; and in US Patent No.: 9,789,182, the contents of each of which are incorporated herein by reference in their entirety.

[0207] Exemplary anti-CD137 antibodies and their sequences (including CDRs) are described, for example, in US Patent Application Nos.: US2019 / 0382491, the contents of which are incorporated herein by reference in its entirety.

[0208] Further examples of checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, PDL2, B7-H3, B7-H4, BTLA, HVEM, GAL9, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, y5, and memory CD8+ (a[3) T cells), CD 160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, TIGIT, DD1, TIM-3, Lag-3, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7.

[0209] In some embodiments, the one or more immune checkpoint inhibitors are selected from an inhibitor of: PD-L1, PD-1, CTLA-4, PD-L2, LAG3, TIM3, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, ID02, ICOS, KIR, LAIRI, LIGHT, MARCO, PS, OX-40, SLAM, TIGIT, VISTA, VTCNI, IDO, CXCR4, CISH, CBL-B, B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6,B7-H7, CGEN-15049, CHK1, CHK2, A2aR, and DDE

[0210] In some embodiments of any of the aspects, the immune checkpoint inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab. In some embodiments of any of the aspects, the immune checkpoint inhibitor is selected frompembrolizumab, nivolumab, and cemiplimab. In some embodiments of any of the aspects, the immune checkpoint inhibitor is selected from atezolizumab, avelumab, and durvalumab. In some embodiments of any of the aspects, the immune checkpoint inhibitor is ipilimumab.Compositions, Combinations, and Kits

[0211] In some embodiments of any of the aspects, a method, composition, or kit described herein relates to at least one inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl).

[0212] In some embodiments of any of the aspects, a method, composition, combination, or kit described herein relates to at least one immune checkpoint inhibitor and at least one inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl).

[0213] In one aspect of any of the embodiments, described herein is a combination of at least one immune checkpoint inhibitor and at least one inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl). As used herein “combination” refers to a group of two or more substances or elements for use together, e.g., for use in treating cancer. The two or more substances can be present in the same formulation in any molecular or physical arrangement, e.g., in an admixture, in a solution, in a mixture, in a suspension, in a colloid, in an emulsion. The formulation can be a homogeneous or heterogenous mixture. In some embodiments of any of the aspects, the two or more substances can be comprised by the same or different superstructures, e.g., nanoparticles, liposomes, vectors, cells, scaffolds, or the like, and said superstructure is in solution, mixture, admixture, suspension with a solvent, carrier, or some of the two or more substances. Alternatively, the two or more substances can be present in two or more separate formulations, e.g., in a kit or package comprising multiple formulations in separate containers, to be mixed or brought into contact with each other when treatment is to be performed.

[0214] In some embodiments, the elements of a combination are provided in the same formulation. In some embodiments, the elements of a combination are provided in separate formulations or containers.

[0215] In one aspect of any of the embodiments, described herein is a kit comprising at least one inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl). In one aspect of any of the embodiments, described herein is a kit comprising at least one immune checkpoint inhibitor and at least one inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl).

[0216] A kit is an assemblage of materials or components, including at least one element or reagent described herein. The exact nature of the components configured in the kit depends on its intended purpose. In some embodiments of any of the aspects, a kit includes instructions for use. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit, e.g., to administer to a patient. Still in accordance with the present invention, “instructions for use” may include a tangible expression describing the preparation of at least oneelement described herein, such as dilution, mixing, or dosing instructions, and the like, typically for an intended purpose. Optionally, the kit also contains other useful components, such as, measuring tools, diluents, buffers, syringes, pharmaceutically acceptable carriers, or other useful paraphernalia as will be readily recognized by those of skill in the art.

[0217] The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging may also preferably provide an environment that protects from light, humidity, and oxygen. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, polyester (such as polyethylene terephthalate, or Mylar) and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of a composition containing a volume of at least one reagent described herein. The packaging material generally has an external label which indicates the contents and / or purpose of the kit and / or its components.

[0218] In some embodiments of any of the aspects, a composition, combination, or kit described herein comprises a bispecific antibody, which specific for Pglyrpl and an immune checkpoint protein.

[0219] In some embodiments of any of the aspects, a kit or combination described herein is for use in treating cancer. In some embodiments of any of the aspects, a kit or combination described herein is for use in treating an autoimmune disease.Cancer

[0220] The methods and compositions described herein provide an improved method of treating cancer in a subject, with increased efficacy and reduced autoimmune side effects. In some embodiments of these aspects and all such aspects described herein, the subject in need thereof has or has been diagnosed with cancer. In some embodiments of these aspects and all other aspects described herein, the subject is being treated or will be treated with a modulator of an immune checkpoint molecule.

[0221] In one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising administering: at least one immune checkpoint inhibitor; and at least one inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl); to the subject. In one aspect of any of the embodiments, described herein is a composition, combination, or kit comprising at least one immune checkpoint inhibitor; and at least one inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl) for use in a method of treating cancer in a subject in need thereof.

[0222] In some embodiments of any of the aspects, the inhibitor of Pglyrp 1 potentiates anti -tumor immune activity of the immune checkpoint inhibitor and suppresses immune checkpoint inhibitor- mediated autoimmunity. Anti-tumor immune activity can be measured as a reduction in tumor size, a reduction in tumor size increase, an increase in one or more immune cells, an increase in T cells, an increase in the activity or activation of one or more immune cells, an increase in T cell activation, an increase in cytokine release, and / or a decrease in the level or activity of one or more checkpoint inhibitors.

[0223] In some embodiments of any of the aspects, cancer immunotherapy-related autoimmune activity and / or autoimmune dysfunction is reduced or limited as compared to treatment without the inhibitor of Pglyrp 1. Cancer immunotherapy-related autoimmune activity and / or autoimmune dysfunction can be measured as an the damage in a non-tumor tissue, the rate of damage in a non-tumor tissue, inflammation, inflammation in a non-tumor tissue, the number of one or more immune cells in non- tumor tissue, the number of T cells in non-tumor tissue, the activity or activation of one or more immune cells in non-tumor tissue, T cell activation in non-tumor tissue, cytokine levels in non-tumor tissue, or an autoimmune side effect. Specific autoimmune side effects of checkpoint inhibitor cancer immunotherapy and / or autoimmune dysfunction include colitis, endocrinopathy, hepatitis, thyroiditis, myasthenia gravis, autoimmune autonomic ganglionpathy, nephritis, liver toxicity, skin rash or pruritus, meningitis, headaches, encephalopathy, fatigue, diarrhea, and pneumonitis.

[0224] In one aspect of any of the embodiments, described herein is a method of suppressing one or more autoimmune side effects of checkpoint inhibitor cancer immunotherapy in a subject who is being or has been treated with an immune checkpoint inhibitor cancer immunotherapy, the method comprising administering at least one Pglyrp 1 inhibitor to the subject.

[0225] In one aspect of any of the embodiments, described herein is a method of treating cancer, the method comprising administering an inhibitor of Pglyrp 1 to a subject in need of treatment for cancer and determined to have an increased level of expression of Pglyrp 1 as compared to a healthy reference level.

[0226] As described herein, levels of Pglyrp 1 can be increased in subjects with autoimmune dysfunction or autoimmune side effects. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising administering at least one Pglyrp 1 inhibitor to a subject determined to have a level of Pglyrp 1 that is incresaed relative to a reference. In one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising: a) determining the level of Pglyrp 1 in a sample obtained from a subject; and b) administering at least one Pglyrp 1 inhibitor to the subject if the level of Pglyrp 1 is increased relative to a reference. In one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising: a) determining the level of Pglyrp 1 in a sample obtained from a subject; b) administering at least one Pglyrp 1 inhibitor to the subject if the level of Pglyrp 1 is increased relative to a reference; and c) administering at least oneimmune checkpoint inhibitor but not a Pglyrpl inhibitor if the level of Pglyrpl is not increased relative to the reference.

[0227] In some embodiments of any of the aspects, the method comprises administering at least one Pglyrp 1 inhibitor to a subject previously determined to have a level of Pglyrp 1 that is increased relative to a reference. In some embodiments of any of the aspects, described herein is a method of treating cancer in a subject in need thereof, the method comprising: a) first determining the level of Pglyrpl in a sample obtained from a subject; and b) then administering at least one Pglyrpl inhibitor to the subject if the level of Pglyrpl is increased relative to a reference.

[0228] In one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising: a) determining if the subject has an increased level of Pglyrpl; and b) administering at least one Pglyrpl inhibitor to the subject if the level of Pglyrpl is increased relative to a reference. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of Pglyrpl can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of Pglyrpl in the subject. In some embodiments of any of the aspects, the step of determining if the subj ect has an increased level of Pglyrp 1 can comprise performing or having performed an assay on a sample obtained from the subject to determine / measure the level of Pglyrpl in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of Pglyrpl can comprise ordering or requesting an assay on a sample obtained from the subject to determine / measure the level of Pglyrpl in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of Pglyrpl can comprise receiving the results of an assay on a sample obtained from the subject to determine / measure the level of Pglyrpl in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of Pglyrpl can comprise receiving a report, results, or other means of identifying the subject as a subject with an incresed level of Pglyrpl.

[0229] In one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising: a) determining if the subject has an increased level of Pglyrpl; and b) instructing or directing that the subject be administered at least one Pglyrpl inhibitor if the level of Pglyrpl is increased relative to a reference. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of Pglyrpl can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of Pglyrpl in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of Pglyrpl can comprise performing or having performed an assay on a sample obtained from the subject to determine / measure the level of Pglyrpl in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of Pglyrpl can comprise ordering or requesting an assay on a sample obtained from the subject to determine / measure the level of Pglyrpl inthe subject. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and / or treatment recommendations in view of the assay results.

[0230] In one aspect of any of the embodiments, described herein is a method of determining if a subject has autoimmune dysfunction or autoimmune side effects or is in need of treatment for autoimmune dysfunction or autoimmune side effects, the method comprising: determining the level of Pglyrpl in a sample obtained from the subject, wherein a level of Pglyrpl which is increased relative to a reference indicates the subject has autoimmune dysfunction or autoimmune side effects or is in need of treatment for autoimmune dysfunction or autoimmune side effects.

[0231] In some embodiments of any of the aspects, measurement of the level of a target and / or detection of the level or presence of a target, e.g. of an expression product (nucleic acid or polypeptide of one of the genes described herein) or a mutation can comprise a transformation. As used herein, the term “transforming” or “transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance. The transformation can be physical, biological or chemical. Exemplary physical transformation includes, but is not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation. A biological / chemical transformation can involve the action of at least one enzyme and / or a chemical reagent in a reaction. For example, a DNA sample can be digested into fragments by one or more restriction enzymes, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase. In some embodiments of any of the aspects, a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).

[0232] Transformation, measurement, and / or detection of a target molecule, e.g. a Pglyrpl mRNA or polypeptide can comprise contacting a sample obtained from a subject with a reagent (e.g. a detection reagent) which is specific for the target, e.g., a target-specific reagent. In some embodiments of any of the aspects, the target-specific reagent is detectably labeled. In some embodiments of any of the aspects, the target-specific reagent is capable of generating a detectable signal. In some embodiments of any of the aspects, the target-specific reagent generates a detectable signal when the target molecule is present.

[0233] Methods to measure gene expression products are known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent. For example, the antibody can be labeled with a detectable marker whose presence and location in the subject is detected by standard imaging techniques.

[0234] For example, antibodies for the various targets described herein are commercially available and can be used for the purposes of the invention to measure protein expression levels. Alternatively, since the amino acid sequences for the targets described herein are known and publically available at the NCBI website, one of skill in the art can raise their own antibodies against these polypeptides of interest for the purpose of the methods described herein.

[0235] The amino acid sequences of the polypeptides described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. In particular, the NCBI accession numbers for the amino acid sequence of human Pglyrpl is included herein, e.g. SEQ ID NO: 28.

[0236] In some embodiments of any of the aspects, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change of color, upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.

[0237] In some embodiments of any of the aspects, the assay can be a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two- dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.

[0238] Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence -linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), and protein A immunoassays. Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available. In some embodiments of any of the aspects, the immunoassay can be a quantitative or a semi-quantitative immunoassay.

[0239] An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as blood or serum, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. For the methods and assays described herein, specific binding of the target polypeptides with respective proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein / peptide complex. The complex is then detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody.

[0240] Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.

[0241] In one embodiment, an ELISA involving at least one antibody with specificity for the particular desired antigen (e.g., Pglyrpl) can also be performed. A known amount of sample and / or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ Anorogenic substrates with much higher sensitivity.

[0242] In another embodiment, a competitive ELISA is used. Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface. A second batch of purified antibodies that are not conjugated on any solid support is also needed. These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal. A sample (e.g., a blood sample) from a subject is mixed with a known amount of desired antigen (e.g., a known volume or concentration of a sample comprising a target polypeptide) together with the horseradish peroxidase labeled antibodies and the mixture is then are added to coated wells to form competitive combination. After incubation, if the polypeptide level is high in the sample, a complex of labeled antibody reagentantigen will form. This complex is free in solution and can be washed away. Washing the wells will remove the complex. Then the wells are incubated with TMB (3, 3', 5, 5'-tetramethylbenzidene) color development substrate for localization of horseradish peroxidase -conjugated antibodies in the wells. There will be no color change or little color change if the target polypeptide level is high in the sample.If there is little or no target polypeptide present in the sample, a different complex in formed, the complex of solid support bound antibody reagents-target polypeptide. This complex is immobilized on the plate and is not washed away in the wash step. Subsequent incubation with TMB will produce significant color change. Such a competitive ELSA test is specific, sensitive, reproducible and easy to operate.

[0243] There are other different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904. These references are hereby incorporated by reference in their entirety.

[0244] In one embodiment, the levels of a polypeptide in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample . There are currently many LFIA tests used for medical diagnostics, either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen. Depending upon the level of target polypeptides present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and / or homogenized tissue samples etc. Strip tests are also known as dip stick tests, the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments of any of the aspects, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target / its analogue. Unlabelled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.

[0245] The use of "dip sticks" or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U. S. patent applications Ser. No. 10 / 278,676; U.S. Ser. No. 09 / 579,673 and U.S. Ser. No. 10 / 717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Examples of patents that describe the use of "dip stick" technology to detect soluble antigens via immunochemical assays include, but are not limited to US Patent Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a "dip stick" which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the "dip stick," prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teachings of this "dip stick" technology for the detection of polypeptides using antibody reagents as described herein.

[0246] Other techniques can be used to detect the level of a polypeptide in a sample . One such technique is the dot blot, an adaptation of Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)). In a Western blot, the polypeptide or fragment thereof can be dissociated with detergents and heat, and separated on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose or PVDF membrane. The membrane is incubated with an antibody reagent specific for the target polypeptide or a fragment thereof. The membrane is then washed to remove unbound proteins and proteins with non-specific binding. Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested. A dot blot immobilizes a protein sample on a defined region of a support, which is thenprobed with antibody and labelled secondary antibody as in Western blotting. The intensity of the signal from the detectable label in either format corresponds to the amount of enzyme present, and therefore the amount of polypeptide. Levels can be quantified, for example by densitometry.

[0247] In some embodiments of any of the aspects, the level of a target can be measured, by way of non-limiting example, by Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy and / or immunoelectrophoresis assay.

[0248] In certain embodiments, the gene expression products as described herein can be instead determined by determining the level of messenger RNA (mRNA) expression of the genes described herein. Such molecules can be isolated, derived, or amplified from a biological sample, such as a blood sample. Techniques for the detection of mRNA expression is known by persons skilled in the art, and can include but not limited to, PCR procedures, RT-PCR, quantitative RT-PCR Northern blot analysis, differential gene expression, RNAse protection assay, microarray based analysis, next-generation sequencing; hybridization methods, etc.

[0249] In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.

[0250] In some embodiments of any of the aspects, the level of an mRNA can be measured by a quantitative sequencing technology, e.g. a quantitative next-generation sequence technology. Methods of sequencing a nucleic acid sequence are well known in the art. Briefly, a sample obtained from a subject can be contacted with one or more primers which specifically hybridize to a single-strand nucleic acid sequence flanking the target gene sequence and a complementary strand is synthesized. In some next-generation technologies, an adaptor (double or single-stranded) is ligated to nucleic acid molecules in the sample and synthesis proceeds from the adaptor or adaptor compatible primers. In some third-generation technologies, the sequence can be determined, e.g. by determining the location and pattern of the hybridization of probes, or measuring one or more characteristics of a single molecule as it passes through a sensor (e.g. the modulation of an electrical field as a nucleic acid molecule passes through a nanopore). Exemplary methods of sequencing include, but are not limited to, Sanger sequencing, dideoxy chain termination, high-throughput sequencing, next generation sequencing, 454 sequencing, SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrent sequencing, sequencing by hybridization, nanopore sequencing, Helioscope sequencing, single molecule real time sequencing, RNAP sequencing, long read sequencing, and the like. Methods and protocols for performing these sequencing methods are known in the art, see, e.g. “Next Generation Genome Sequencing” Ed. Michal Janitz, Wiley-VCH; “High-Throughput Next Generation Sequencing” Eds. Kwon and Rieke, Humanna Press, 2011; and Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); which are incorporated by reference herein in their entireties.

[0251] The nucleic acid sequences of the genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. For example, the human Pglyrpl mRNA (e.g. SEQ ID NO: 29) is known. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.

[0252] Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw andalkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).

[0253] In some embodiments of any of the aspects, one or more of the reagents (e.g. an antibody reagent and / or nucleic acid probe) described herein can comprise a detectable label and / or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.

[0254] In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, biolumine scent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

[0255] In other embodiments, the detection reagent is label with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments of any of the aspects, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3TM, Cy5TM, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyesTM, 6-carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy- 2',4',7',4,7-hexachlorofiuorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfmorescein (JOE or J), N,N,N',N'-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazinedyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments of any of the aspects, a detectable label can be a radiolabel including, but not limited to 3H, 1251, 35S, 14C, 32P, and 33P. In some embodiments of any of the aspects, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments of any of the aspects, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments of any of the aspects, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0256] In some embodiments of any of the aspects, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA. A reagent can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0257] A level which is less than a reference level can be a level which is less by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, or less relative to the reference level. In some embodiments of any of the aspects, a level which is less than a reference level can be a level which is statistically significantly less than the reference level.

[0258] A level which is more than a reference level can be a level which is greater by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or more than the reference level. In some embodiments of any of the aspects, a level which is more than a reference level can be a level which is statistically significantly greater than the reference level.

[0259] In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and / or do not exhibit signs or symptoms of cancer. In some embodiments of any of the aspects, the reference can be a level of thetarget molecule in a population of subjects who do not have or are not diagnosed as having, and / or do not exhibit signs or symptoms of autoimmune dysfunction. In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and / or do not exhibit signs or symptoms of autoimmune side effects. In some embodiments of any of the aspects, the reference can also be a level of expression of the target molecule in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same. In some embodiments of any of the aspects, the reference can be the level of a target molecule in a sample obtained from the same subject at an earlier point in time, e.g., the methods described herein can be used to determine if a subject’s sensitivity or response to a given therapy is changing overtime.

[0260] In some embodiments of any of the aspects, the level of expression products of no more than 200 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 100 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 20 other genes is determined. In some embodiments of any of the aspects, the level of expression products of no more than 10 other genes is determined.

[0261] In some embodiments of the foregoing aspects, the expression level of a given gene can be normalized relative to the expression level of one or more reference genes or reference proteins.

[0262] In some embodiments, the reference level can be the level in a sample of similar cell type, sample type, sample processing, and / or obtained from a subject of similar age, sex and other demographic parameters as the sample / subject for which the level of Pglyrpl is to be determined. In some embodiments, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g. the same number and type of cells.

[0263] The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or plasma sample from a subject. In some embodiments of any of the aspects, the present invention encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and / or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, atest sample can comprise cells from a subject. In some embodiments of any ofthe aspects, the test sample can be a biopsy sample, or sample of cancer cells.

[0264] The test sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person).

[0265] In some embodiments of any of the aspects, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and / or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, fdtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any of the aspects, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments of any of the aspects, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments of any of the aspects, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments of any of the aspects, the test sample can be treated with a chemical and / or biological reagent. Chemical and / or biological reagents can be employed to protect and / or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.

[0266] In some embodiments of any of the aspects, the methods, assays, and systems described herein can further comprise a step of obtaining or having obtained a test sample from a subject. In some embodiments of any of the aspects, the subject can be a human subject. In some embodiments of any of the aspects, the subject can be a subject in need of treatment for (e.g. having or diagnosed as having) cancer or a subject at risk of or at increased risk of developing cancer as described elsewhere herein.

[0267] In some embodiments of any of the aspects, the sample obtained from a subject can be a biopsy sample. In some embodiments of any of the aspects, the sample obtained from a subject can be a sample comprising cancer cells. In some embodiments of any of the aspects, the sample obtained from a subject can be a sample consisting of cancer cells. In some embodiments of any of the aspects, the sample obtained from a subject can be a blood or serum sample.

[0268] In certain embodiments, the cancer is metastatic or has the potential to be metastatic. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.

[0269] Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.

[0270] In addition, cancers that can be treated using a Pglyrp 1 inhibitor in combination with an immune checkpoint inhibitor can be an angiogenic tumor.

[0271] Examples of cancer that can be treated with the methods and compositions provided herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; cholangiocarcinoma; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small -cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; teratocarcinoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), tumors of primitive origins and Meigs' syndrome.

[0272] In some embodiments of any of the aspects, the cancer is melanoma. In some embodiments of any of the aspects, the cancer is colorectal cancer. In some embodiments of any of the aspects, the cancer is melanoma or colorectal cancer.

[0273] The data present in the Examples herein demonstrates that a Pglyrp 1 inhibitor potentiates the response to an immune checkpoint inhibitor in diverse cancers. Accordingly, there is rational basis to conclude that the use of a Pglyrp 1 inhibitor and an immune checkpoint inhibitor will be therapeutic in any cancer where immune checkpoint inhibitors have been demonstrated to be efficacious as a monotherapy. Such cancers include melanoma, hepatocellular carcinoma, lung cancer, gastric cancer, and esophageal cancer. In some embodiments of any of the aspects, the cancer is melanoma,hepatocellular carcinoma, lung cancer, gastric cancer, or esophageal cancer. In some embodiments of any of the aspects, the cancer is colorectal cancer, melanoma, hepatocellular carcinoma, lung cancer, gastric cancer, or esophageal cancer.

[0274] The use of a Pglyrp 1 inhibitor with an immune checkpoint inhibitor is considered a combination therapy. However, in some embodiments, a Pglyrp 1 inhibitor / immune checkpoint inhibitor as described herein is used in combination with at least one additional anti-cancer therapy, such as an anti-cancer agent or chemotherapeutic, X-rays, gamma rays or other sources of radiation to destroy cancer stem cells and / or cancer cells.

[0275] In some embodiments described herein, the methods further comprise administering an anticancer therapy or agent to a subject in addition to the inhibitor of Pglyrp 1 activity or expression. In some embodiments, inhibitors of Pglyrp 1 are administered simultaneously, in the same or in separate compositions, or sequentially with the at least one additional anti-cancer therapy. For sequential administration, the Pglyrp 1 inhibitor described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed. The Pglyrp 1 inhibitor and / or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The agent can be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder. In one embodiment, the Pglyrp 1 inhibitor is administered prior to the onset of metastasis to prevent or reduce the degree of metastasis. In another embodiment, the Pglyrp 1 inhibitor is administered following the detection of metastasis.

[0276] The term "anti -cancer therapy" refers to a therapy useful in treating cancer other than the Pglyrp or immune checkpoint inhibitors disclosed herein. Examples of anti-cancer therapies include, but are not limited to, e.g., surgery, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, such as anti-HER2 antibodies (e.g., HERCEPTIN®), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1 / EGFR inhibitor (e.g., erlotinib (TARCEVA®)), platelet derived growth factor inhibitors (e.g., GLEEVEC™ (Imatinib Mesylate)), a COX2 inhibitor (e.g., celecoxib), interferons, cytokines, and other bioactive and organic chemical agents, etc. In some embodiments, an anti-cancer therapy can be one or more antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: TIM-1, CEACAM1 or any CEACAM family member, ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptor(s), or TRAIL / Apo2. Combinations thereof are also specifically contemplated for the methods described herein.

[0277] An anti -cancer therapy can include a cytotoxic agent, such as a radioactive isotope (e.g. At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32or a radioactive isotope of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including active fragments and / or variants thereof.

[0278] Non-limiting examples of chemotherapeutic agents can include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophy cin 8); dolastatin; duocarmycin (including the synthetic analogues, KW2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g, calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g. , Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T2 toxin, verracurin A, roridin A and anguidine);urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB.); inhibitors of PKC -alpha, Raf, H-Ras, EGFR (e.g., erlotinib (TARCEVA®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation or radiation therapy.

[0279] Chemotherapeutic agents as used herein encompass both chemical and biological agents. These agents function, for example, to inhibit a cellular activity upon which the cancer cell depends for continued survival. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most if not all of these agents are directly toxic to cancer cells and do not require immune stimulation. In one embodiment, a chemotherapeutic agent is an agent of use in treating neoplasms such as solid tumors. In one embodiment, a chemotherapeutic agent is a radioactive molecule. One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff s Clinical Oncology, 2013 Elsevier; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003)).

[0280] By "reduce” or “inhibit" in terms of the cancer treatment methods described herein refers to a reduction in at least one parameter or symptom of a cancer by at least 20% at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60 %, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, or at least 95% or greater. Parameters or symptoms of cancer that can be reduced or inhibited with the methods described herein include, but are not limited to, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or the size of the dormant tumor, tumor growth rate, pain, degree of angiogenesis in the tumor, etc. A patient or subjectwho is being treated for a cancer or tumor is one who a medical practitioner has diagnosed as having such a condition. Diagnosis can be by any suitable means.Autoimmune Disease

[0281] In one aspect of any of the embodiments, described herein is method of treating autoimmune disease in a subject in need thereof, the method comprising administering an inhibitor of Pglyrpl to the subject. In one aspect of any of the embodiments, described herein an inhibitor of Pglyrpl for use in a method of treating autoimmune disease. As used herein, the term "autoimmune disease", “autoimmune condition”, or "autoimmune disease or disorder” refers to a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom.

[0282] Auto-immune related diseases and disorders arise from an overactive and / or abnormal immune response of the body against substances (autoantigens) and tissues normally present in the body, otherwise known as self or autologous substance. This dysregulated inflammatory reaction causes an exaggerated response by macrophages, granulocytes, lymphocytes, and / or T-lymphocytes leading to abnormal tissue damage and cell death. Subsequent loss of function is associated with inflammatory tissue damage.

[0283] In one embodiment of any one of the methods described, the autoimmune disorder is selected from the group consisting of thyroiditis, type 1 diabetes mellitus, Hashimoto's thyroiditis, Graves' disease, celiac disease, multiple sclerosis, Guillain-Barre syndrome, Addison's disease, and Raynaud's phenomenon, Goodpasture's disease, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis (e.g., post treatment Lyme disease syndrome), proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), palindromic arthritis, inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatorybowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infdtration of T cells and chronic inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic large-artery disorder, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and giant-cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa / periarteritisnodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA- associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia pemiciosa), Addison's disease, pure red cell anemia or aplasia (PRC A), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus -membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, and autoimmune or immune -mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, lupoid hepatitis, giant-cell hepatitis, autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such asautoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies including channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressier's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post- cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, Sampler's syndrome, Caplan's syndrome, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, SCID, sepsis, endotoxemia, post-vaccination syndromes, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenesis, autoimmune hemolysis, Boeck's disease, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, ileitis regionalis, leucopenia, transverse myelitis, primary idiopathic myxedema, ophthalmia symphatica, polyradiculitis acuta, pyoderma gangrenosum, acquired spenic atrophy, vitiligo, toxic-shock syndrome, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmuneatrophic gastritis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), myocarditis, nephrotic syndrome, primary sclerosing cholangitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infdtration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, granulomas containing eosinophils, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, allergic hypersensitivity disorders, glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, and autoimmune uveoretinitis (AUR). In some embodiments, the autoimmune disease or condition or T cell mediated inflammation can be neurodegeneration, e.g, Alzheimer’s or Parksinson disease.

[0284] In some embodiments of any of the aspects, the autoimmune disease affects mucosal tissues or tissues of the gastrointestinal tract. In some embodiments of any of the aspects, the autoimmune disease is selected from pneumonitis, colitis, enteritis, and gastritis. In some embodiments of any of the aspects, the autoimmune disease is multiple sclerosis.Dosage and Administration

[0285] The methods and compositions provided herein enhance anti-cancer activity of an immune checkpoint inhibitor and / or treat cancer in a subject by administering a therapeutically effective amount of a Pglyrpl inhibitor as an adjunct to immune checkpoint inhibition. In one embodiment, the subject can be a mammal. In another embodiment, the mammal can be a human, although the approach is effective with respect to all mammals.

[0286] The appropriate dosage range for a Pglyrp 1 inhibitor or a given anti -cancer agent depends upon the potency of the agent, and includes amounts large enough to produce the desired effect, e.g., treatment of one or more symptoms associated with cancer (e.g., tumor size, tumor growth, pain, etc). Although adverse side effects are often associated with anti-cancer agents, the dosage should not be so large as to cause unacceptable or life-threatening adverse side effects. Generally, the dosage will vary with the type of inhibitor, and with the age, condition, and sex of the patient. The dosage can be determined by one of skill in the art and can also be adjusted by the individual physician in the event of any complication. It is contemplated herein that the dose of an immune checkpoint inhibitor can be higher when administered in combination with a Pglyrp 1 inhibitor than the immune checkpoint inhibitor can be safely administered as a monotherapy. This is because Pglyrpl reduces autoimmune side effects associated with immune checkpoint inhibition. Alternatively, the dose of the immune checkpointinhibitor can be lower than the dose of the immune checkpoint inhibitor when administered as a monotherapy as the Pglyrpl inhibitor can potentiate or enhance the anti -cancer effects of the immune checkpoint inhibitor.

[0287] The effective amount may be based upon, among other things, the size of the compound, the biodegradability of the compound, the bioactivity of the compound and the bioavailability of the compound. For example, if the compound does not degrade quickly, is bioavailable and highly active, a smaller amount will be required to be effective. One of skill in the art can perform routine empirical activity tests for a compound to determine the bioactivity in bioassays and thus determine the effective amount.

[0288] The efficacy of a given treatment for a cancer can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if any one or all of the signs or symptoms of the cancer is / are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved, or ameliorated, e.g., by at least 10% following treatment with an anti-cancer agent or combination thereof described herein. Efficacy can also be measured by failure of an individual to worsen as assessed by stabilization of the disease, or the need for medical interventions (i.e., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing progression of the cancer; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of the disease, or preventing secondary diseases / disorders associated with the cancer (e.g., cancer metastasis).

[0289] Typically, the dosage ranges for a Pglyrpl inhibitor, an immune checkpoint inhibitor (or an accompanying combination anti-cancer therapy) are in the range of O.OOlmg / kg body weight to 5 g / kg body weight. In some embodiments, the dosage range is from 0.001 mg / kg body weight to Ig / kg body weight, from 0.001 mg / kg body weight to 0.5 g / kg body weight, from 0.001 mg / kg body weight to 0.1 g / kg body weight, from 0.001 mg / kg body weight to 50 mg / kg body weight, from 0.001 mg / kg body weight to 25 mg / kg body weight, from 0.001 mg / kg body weight to 10 mg / kg body weight, from 0.001 mg / kg body weight to 5 mg / kg body weight, from 0.001 mg / kg body weight to 1 mg / kg body weight, from 0.001 mg / kg body weight to 0.1 mg / kg body weight, from 0.001 mg / kg body weight to 0.005 mg / kg body weight. Alternatively, in some embodiments the dosage range is from 0. 1 g / kg body weight to 5 g / kg body weight, from 0.5 g / kg body weight to 5 g / kg body weight, from 1 g / kg body weight to 5 g / kg body weight, from 1.5 g / kg body weight to 5 g / kg body weight, from 2 g / kg body weight to 5 g / kg body weight, from 2.5 g / kg body weight to 5 g / kg body weight, from 3 g / kg body weight to 5 g / kg body weight, from 3.5 g / kg body weight to 5 g / kg body weight, from 4 g / kg body weight to 5 g / kg body weight, from 4.5 g / kg body weight to 5 g / kg body weight, from 4.8 g / kg body weight to 5 g / kg bodyweight. In one embodiment, the dose range is from 5pg / kg body weight to 30pg / kg body weight. Alternatively, the dose range will be titrated to maintain serum levels between 5pg / mL and 30pg / mL.

[0290] In certain embodiments, a therapeutically effective amount of a Pglyrpl inhibitor and / or an immune checkpoint inhibitor can be from about 0.05 mg to about 1500 mg, from about 1 mg to about 800 mg, from about 5 mg to about 600 mg, from about 10 mg to about 550 mg, from about 50 mg to about 400 mg, from about 75 mg to about 350 mg, or from about 100 mg to about 300 mg of the antibody. For example, in various embodiments, the amount of the Pglyrpl inhibitor and / or an immune checkpoint inhibitor is about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, about 1000 mg, about 1010 mg, about 1020 mg, about 1030 mg, about 1040 mg, about 1050 mg, about 1060 mg, about 1070 mg, about 1080 mg, about 1090 mg, about 1200 mg, about 1210 mg, about 1220 mg, about 1230 mg, about 1240 mg, about 1250 mg, about 1260 mg, about 1270 mg, about 1280 mg, about 1290 mg, about 1300 mg, about 1310 mg, about 1320 mg, about 1330 mg, about 1340 mg, about 1350 mg, about 1360 mg, about 1370 mg, about 1380 mg, about 1390 mg, about 1400 mg, about 1410 mg, about 1420 mg, about 1430 mg, about 1440 mg, about 1450 mg, about 1460 mg, about 1470 mg, about 1480 mg, about 1490 mg, or about 1500 mg.

[0291] As for when the compound, composition and / or agent is to be administered, one skilled in the art can determine when to administer such a compound and / or agent. The administration may be constant for a certain period of time or periodic and at specific intervals. The compound may be delivered hourly, daily, weekly, monthly, yearly (e.g. in a time release form) or as a one-time delivery. The delivery may be continuous delivery for a period of time, e.g. intravenous delivery. In one embodiment of the methods described herein, the agent is administered at least once per day. In one embodiment of the methods described herein, the agent is administered daily. In one embodiment ofthe methods described herein, the agent is administered every other day. In one embodiment of the methods described herein, the agent is administered every 6 to 8 days. In one embodiment of the methods described herein, the agent is administered weekly.

[0292] As one of skill in the art will appreciate, the dosage of a Pglyrpl inhibitor (or an immune checkpoint molecule) can vary depending upon the dosage form employed and the route of administration utilized. Compositions, methods, and uses that exhibit large therapeutic indices (i.e., the dose ratio between toxic and therapeutic effects) are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50, which achieves a half-maximal inhibition of measured function or activity as determined in cell culture, or in an appropriate animal model. The effects of any particular dosage can be monitored by a suitable bioassay. A therapeutically effective amount is an amount of an agent that is sufficient to produce a statistically significant, measurable change of a given symptom of a cancer (see “Efficacy " below). Such effective amounts can also be gauged in clinical trials as well as animal studies for a given agent.

[0293] An appropriate therapeutic amount or dose for treating a human subject can be informed by data collected in cell cultures or animal models. In some embodiments, the therapeutic efficacy can be estimated by the ED50 in an animal model (the dose therapeutically effective in 50% of the population) or in a cell cytotoxicity assay (where at least 50% of the cancer cells are killed).

[0294] Therapeutic compositions can be conventionally administered in a unit dose. The term "unit dose" when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of an anti-cancer agent calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle.

[0295] Administration of the doses recited above or as employed by a skilled clinician can be repeated for a limited and defined period of time. In some embodiments, the doses are given once a day, or multiple times a day (e.g., at least two times a day, at least three times a day etc). In a preferred embodiment, the doses recited above are administered daily for several weeks or months. The duration of treatment depends upon the subject’s clinical progress and continued responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.

[0296] The agents described herein can be administered to a subject in need thereof by any appropriate route which results in an effective treatment in the subject. For example, agents useful in the methods and compositions described herein can be administered topically, intravenously (by bolus or continuous infusion), orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and can be delivered by peristaltic means, if desired, or by other means known by those skilled in the art. The agent can be administered systemically, if so desired.

[0297] In some embodiments, the Pglyrpl inhibitor described herein can be administered to a subject by any mode of administration that delivers the agent systemically or locally to a desired surface ortarget, and can include, but is not limited to, injection, infusion, instillation, and inhalation administration. To the extent that polypeptide agents can be protected from inactivation in the gut, oral administration forms are also contemplated. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.

[0298] The phrases “parenteral administration" and “administered parenterally” as used herein, refer to modes of administration other than enteral and topical administration, usually by injection. The phrases “systemic administration," “administered systemically", “peripheral administration" and “administered peripherally" as used herein refer to the administration of the agents described herein, other than directly into a target site, tissue, or organ, such that it enters the subject’s circulatory system and, thus, is subject to metabolism and other like processes.

[0299] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. An agent can be targeted by means of a targeting moiety, such as e.g., an antibody or targeted liposome technology.

[0300] Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

[0301] Combination Therapies:

[0302] Combination therapy using a Pglyrpl inhibitor and an immune checkpoint inhibitor, with or without an additional anti -cancer treatment, can comprise administration of the therapeutics to a subject concurrently. The term “concurrently” is not limited to the administration of the cancer therapeutics at exactly the same time, but rather, it is meant that they are administered to a subject in a sequence and within a time interval such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise). For example, the combination therapies can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic effect, preferably in a synergistic fashion. The combination cancer therapeutics (including a Pglyrpl inhibitor) can be administered separately, in any appropriate form and by any suitable route. When the combination therapies are not administered in the same pharmaceutical composition, it is understood that they can be administered in any order to a subject in need thereof. For example, a firstprophylactically and / or therapeutically effective regimen can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second cancer therapeutic, to a subject in need thereof.

[0303] When administered in combination with a Pglyrpl inhibitor / immune checkpoint molecule, the anti -cancer agent or drug can be administered in an amount or dose that is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) or the same as the amount or dosage of the agent used individually, e.g., as a monotherapy.

[0304] In some embodiments of any of the aspects, the Pglyrp 1 inhibitor is initially administered before the checkpoint inhibitor. In some embodiments of any of the aspects, the checkpoint inhibitor is initially administered before the inhibitor of Pglyrp 1.

[0305] Currently available anti-cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physician's Desk Reference (60th ed., 2017).Compositions, Formulations and Packaging

[0306] Also provided herein are compositions, including pharmaceutical compositions, comprising a Pglyrpl inhibitor (optionally including an immune checkpoint inhibitor) as described herein. In one embodiment, the compositions are pharmaceutical compositions. Pharmaceutical compositions for use with the methods described herein can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates can be formulated for administration by, for example, aerosol, intravenous, oral or topical route. The compositions can be formulated for intralesional, intratumoral, intraperitoneal, subcutaneous, intramuscular or intravenous injection; infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, per rectum, intrabronchial, nasal, transmucosal, intestinal, oral, ocular or otic delivery.

[0307] In embodiments where the Pglyrp 1 inhibitor comprises an anti -Pglyrp 1 RNAi agent, the RNAi agent can be mixed with a delivery system, such as a liposome system, and optionally can include an acceptable excipient. In a preferred embodiment, the composition is formulated for injection.

[0308] Techniques and formulations generally may be found in Remmington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank’ssolution or Ringer’s solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

[0309] For oral administration, the pharmaceutical composition can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fdlers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., pharmaceutically acceptable oils, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[0310] Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use as described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0311] The anti-Pglyrpl inhibitors can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with or without an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0312] The Pglyrpl inhibitor can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0313] In addition to the formulations described previously, the Pglyrpl inhibitors can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation(for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0314] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories.

[0315] The compositions can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

[0316] For therapies involving the administration of nucleic acids, such Pglyrpl inhibitors can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, such as injection, RNAi comprising Pglyrpl inhibitors can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank’s solution or Ringer’s solution. In addition, the oligomers can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

[0317] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0318] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A method of treating cancer, the method comprising administering to a subject in need thereof an immune checkpoint inhibitor and an inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl), wherein the inhibitor of Pglyrpl potentiates anti -tumor immune activity of the immune checkpoint inhibitor and suppresses immune checkpoint inhibitor-mediated autoimmunity.2. The method of paragraph 1 , wherein the inhibitor of Pglyrp 1 comprises an antibody or antigenbinding fragment thereof, or a nucleic acid molecule.3. The method of paragraph 2, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA or encodes an RNA targeting CRISPR-Cas system.4. The method of paragraph 1 or 2, wherein the antibody inhibitor of Pglyrpl comprises LS- B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.5. The method of paragraphs 1-4, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).6. The method of any one of paragraphs 1-5 wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.7. The method of paragraph 6, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of: CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.8. The method of paragraph 7, wherein the immune checkpoint inhibitor is an inhibitor of PD1 selected from the group consisting of: pembrolizumab, nivolumab, and cemiplimab.9. The method of paragraph 7, wherein the immune checkpoint inhibitor is an inhibitor of PD- L1 selected from the group consisting of: atezolizumab, avelumab, and durvalumab.10. The method of paragraph 7, wherein the immune checkpoint inhibitor is an inhibitor of CTLA4 comprising ipilimumab.11. The method of any one of paragraphs 1-10, wherein the checkpoint inhibitor and the inhibitor of Pglyrpl are administered concurrently.12. The method of any one of paragraphs 1-10, wherein the Pglyrpl inhibitor is initially administered before the checkpoint inhibitor.13. The method of any one of paragraphs 1-10, wherein the checkpoint inhibitor is initially administered before the inhibitor of Pglyrp 1.14. A method of suppressing autoimmune side effects of checkpoint inhibitor cancer immunotherapy, the method comprising: administering a Pglyrpl inhibitor to a subject who is being or has been treated with an immune checkpoint inhibitor cancer immunotherapy.15. The method of paragraph 14, wherein the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof, or an RNAi molecule.16. The method of paragraph 14 or 15, wherein the antibody inhibitor of Pglyrpl comprises LS- B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.17. The method of paragraph 14 or 15, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAAT (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).18. The method of any one of paragraphs 14-17, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.19. The method of any one of paragraphs 14-17, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of: CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.20. The method of any one of paragraphs 14-19, wherein the side effect is selected from: colitis, endocrinopathy, hepatitis, thyroiditis, myasthenia gravis, autoimmune autonomic ganglionpathy, nephritis, liver toxicity, skin rash or pruritus, meningitis, headaches, encephalopathy, fatigue, diarrhea, and pneumonitis.21. A method of treating cancer in a subject in need thereof, the method comprising: detecting or receiving results of an assay detecting the expression of Pglyrpl by cells of the subject’s cancer; and administering an inhibitor of Pglyrpl to the subject, wherein the inhibitor promotes an anticancer immune response while suppressing autoimmune dysfunction.22. The method of paragraph 21, wherein the detecting comprises detecting Pglyrpl protein and / or RNA encoding Pglyrpl protein.23. The method of paragraph 21 or 22, wherein the inhibitor of Pglyrp 1 comprises an antibody or antigen-binding fragment thereof, or an RNAi molecule.24. The method of paragraph 23, wherein the antibody inhibitor of Pglyrp 1 comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.25. The method of paragraph 23, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).26. A method of treating autoimmune disease, the method comprising administering to a subject in need thereof an inhibitor of Pglyrp 1.27. The method of paragraph 26, wherein the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof, or an RNAi molecule.28. The method of paragraph 26 or 27, wherein the autoimmune disease affects mucosal tissues or tissues of the gastrointestinal tract.29. The method of paragraph 28, wherein the autoimmune disease is selected from pneumonitis, colitis, enteritis, and gastritis.30. A composition comprising an immune checkpoint inhibitor and an inhibitor of Pglyrp 1.31. The composition of paragraph 30, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of: CTLA4, TIM-3, LAG-3, PD- 1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.32. The composition of paragraph 30 or 31, wherein the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof, or an RNAi molecule.33. The method of paragraph 32, wherein the antibody inhibitor of Pglyrp 1 comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.34. The method of paragraph 32, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).35. The composition of any one of paragraphs 30-34, wherein the composition promotes an anticancer immune response and suppresses immune checkpoint inhibitor-mediated autoimmunity when administered to a subject having cancer.36. A method of treating cancer via immunotherapy while limiting cancer immunotherapy-related autoimmune activity, the method comprising administering to a subject in need thereof an immune checkpoint inhibitor and an inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl), wherein the inhibitor of Pglyrpl potentiates anti -tumor immune activity of the immune checkpoint inhibitor and suppresses immune checkpoint inhibitor-mediated autoimmunity.

[0319] In some embodiments, the present technology may be defined in any of the following numbered paragraphs:1. A method of treating cancer in a subject in need thereof, the method comprising administering: an immune checkpoint inhibitor; and an inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl); to the subject.2. The method ofparagraph 1, wherein the inhibitor of Pglyrpl potentiates anti -tumor immune activity of the immune checkpoint inhibitor and suppresses immune checkpoint inhibitor-mediated autoimmunity.3. The method of paragraph 1 or 2, whereby cancer immunotherapy-related autoimmune activity is reduced or limited as compared to treatment without the inhibitor of Pglyrp 1.4. The method of any one of the preceding paragraphs, wherein the inhibitor of Pglyrp 1 comprises an antibody or antigen-binding fragment thereof.5. The method of paragraph 4, wherein the antibody or antigen-binding fragment thereof comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.The method of any one of the preceding paragraphs, wherein the inhibitor of Pglyrpl comprises a nucleic acid molecule. The method of paragraph 6, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system. The method of any one of paragraphs 6-7, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4). The method of any one of the preceding paragraphs, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof. The method of any one of the preceding paragraphs, wherein the immune checkpoint inhibitor is selected from an inhibitor of:PD-L1, PD-1, CTLA-4, PD-L2, LAG3, TIM3, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, ID02, ICOS, KIR, LAIRI, LIGHT, MARCO, PS, OX- 40, SLAM, TIGIT, VISTA, VTCNI, IDO, CXCR4, CISH, CBL-B, B7- 1, B7-2, B7-DC, B7- Hl, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6,B7-H7, CGEN-15049, CHK1, CHK2, A2aR, and DDE The method of any one of the preceding paragraphs, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of:CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B. The method of paragraph 11, wherein the immune checkpoint inhibitor is an inhibitor of PD1 selected from the group consisting of: pembrolizumab, nivolumab, and cemiplimab. The method of paragraph 11, wherein the immune checkpoint inhibitor is an inhibitor of PD-L1 selected from the group consisting of: atezolizumab, avelumab, and durvalumab. The method of paragraph 11, wherein the immune checkpoint inhibitor is an inhibitor of CTLA4 comprising ipilimumab. The method of any one of the preceding paragraphs, wherein the checkpoint inhibitor and the inhibitor of Pglyrpl are administered concurrently. The method of any one of the preceding paragraphs, wherein the Pglyrpl inhibitor is initially administered before the checkpoint inhibitor. The method of any one of the preceding paragraphs, wherein the checkpoint inhibitor is initially administered before the inhibitor of Pglyrp 1.A method of suppressing one or more autoimmune side effects of checkpoint inhibitor cancer immunotherapy in a subject who is being or has been treated with an immune checkpoint inhibitor cancer immunotherapy, the method comprising administering a Pglyrpl inhibitor to the subject. The method of paragraph 18, wherein the inhibitor of Pglyrpl comprises an antibody or antigenbinding fragment thereof. The method of paragraph 18, wherein the antibody or antigen-binding fragment thereof comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof. The method of any one of paragraphs 18-20, wherein the inhibitor of Pglyrp 1 comprises a nucleic acid molecule. The method of paragraph 21, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system. The method of any one of paragraphs 21-22, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4). The method of any one of paragraphs 18-23, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof. The method of any one of paragraphs 18-24, wherein the immune checkpoint inhibitor is selected from an inhibitor of:PD-L1, PD-1, CTLA-4, PD-L2, LAG3, TIM3, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, ID02, ICOS, KIR, LAIRI, LIGHT, MARCO, PS, OX- 40, SLAM, TIGIT, VISTA, VTCNI, IDO, CXCR4, CISH, CBL-B, B7- 1, B7-2, B7-DC, B7- Hl, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6,B7-H7, CGEN-15049, CHK1, CHK2, A2aR, and DDE The method of any one of paragraphs 18-25, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of:CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B. The method of paragraph 26, wherein the immune checkpoint inhibitor is an inhibitor of PD1 selected from the group consisting of: pembrolizumab, nivolumab, and cemiplimab. The method of paragraph 26, wherein the immune checkpoint inhibitor is an inhibitor of PD-L1 selected from the group consisting of: atezolizumab, avelumab, and durvalumab.The method of paragraph 26, wherein the immune checkpoint inhibitor is an inhibitor of CTLA4 comprising ipilimumab. The method of any one of paragraphs 18-29, wherein the one or more autoimmune side effects of checkpoint inhibitor cancer immunotherapy comprise one or more of: colitis, endocrinopathy, hepatitis, thyroiditis, myasthenia gravis, autoimmune autonomic ganglionpathy, nephritis, liver toxicity, skin rash or pruritus, meningitis, headaches, encephalopathy, fatigue, diarrhea, and pneumonitis. A method of treating cancer, the method comprising administering an inhibitor of Pglyrpl to a subject in need of treatment for cancer and determined to have an increased level of expression of Pglyrpl as compared to a healthy reference level. The method of paragraph 31, further comprising a first step of detecting the expression of Pglyrpl in cells of the subject’s cancer. A method of treating cancer in a subject in need thereof, the method comprising: detecting or receiving results of an assay detecting the expression of Pglyrpl by cells of the subject’s cancer; and administering an inhibitor of Pglyrpl to the subject. The method of any one of paragraphs 31-33, wherein the inhibitor promotes an anti-cancer immune response while suppressing autoimmune dysfunction. The method of any one of paragraphs 31-34, wherein the determining or detecting comprises detecting Pglyrpl protein and / or RNA encoding Pglyrpl protein. The method of any one of paragraphs 31-35, wherein the inhibitor of Pglyrp 1 comprises an antibody or antigen-binding fragment thereof. The method of paragraph 36, wherein the antibody or antigen-binding fragment thereof comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof. The method of any one of paragraphs 31-37, wherein the inhibitor of Pglyrpl comprises a nucleic acid molecule. The method of paragraph 38, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system. The method of any one of paragraphs 38-39, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4). A method of treating autoimmune disease in a subject in need thereof, the method comprising administering an inhibitor of Pglyrpl to the subject.The method of paragraph 41, wherein the inhibitor of Pglyrpl comprises an antibody or antigenbinding fragment thereof. The method of paragraph 42, wherein the antibody or antigen-binding fragment thereof comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof. The method of any one of paragraphs 41-43, wherein the inhibitor of Pglyrpl comprises a nucleic acid molecule. The method of paragraph 44, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system. The method of any one of paragraphs 44-45, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4). The method of any one of paragraphs 41-46, wherein the autoimmune disease affects mucosal tissues or tissues of the gastrointestinal tract. The method of any one of paragraphs 41-47, wherein the autoimmune disease is selected from pneumonitis, colitis, enteritis, and gastritis. A composition, combination, or kit comprising: an immune checkpoint inhibitor; and an inhibitor of Pglyrp 1. The composition, combination, or kit of paragraph 49, wherein the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof. The composition, combination, or kit of paragraph 50, wherein the antibody or antigen-binding fragment thereof comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof. The composition, combination, or kit of any one of paragraphs 49-51, wherein the inhibitor of Pglyrpl comprises a nucleic acid molecule. The composition, combination, or kit of paragraph 52, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system. The composition, combination, or kit of any one of paragraphs 52-53, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).55. The composition, combination, or kit of any one of paragraphs 49-54, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.56. The composition, combination, or kit of any one of paragraphs 49-55, wherein the immune checkpoint inhibitor is selected from an inhibitor of:PD-L1, PD-1, CTLA-4, PD-L2, LAG3, TIM3, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, ID02, ICOS, KIR, LAIRI, LIGHT, MARCO, PS, OX- 40, SLAM, TIGIT, VISTA, VTCNI, IDO, CXCR4, CISH, CBL-B, B7- 1, B7-2, B7-DC, B7- Hl, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6,B7-H7, CGEN-15049, CHK1, CHK2, A2aR, and DDL57. The composition, combination, or kit of any one of paragraphs 49-56, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of:CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.58. The composition, combination, or kit of paragraph 57, wherein the immune checkpoint inhibitor is an inhibitor of PD1 selected from the group consisting of: pembrolizumab, nivolumab, and cemiplimab.59. The composition, combination, or kit of paragraph 57, wherein the immune checkpoint inhibitor is an inhibitor of PD-L1 selected from the group consisting of: atezolizumab, avelumab, and durvalumab.60. The composition, combination, or kit of paragraph 57, wherein the immune checkpoint inhibitor is an inhibitor of CTLA4 comprising ipilimumab.61. The composition, combination, or kit of any one of paragraphs 49-60, for use in the treatment of cancer.62. The composition, combination, or kit of any one of paragraphs 49-60, for use in the treatment of an autoimmune disease.63. The composition, combination, or kit of paragraph 62, wherein the autoimmune disease affects mucosal tissues or tissues of the gastrointestinal tract.64. The composition, combination, or kit of any one of paragraphs 62-63, wherein the autoimmune disease is selected from pneumonitis, colitis, enteritis, and gastritis.

[0320] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1:

[0321] Introduction

[0322] The use of immune checkpoint blockade for the treatment of cancer has recently revolutionized oncology. Immune checkpoint blockade increases antitumor immunity by blocking inhibitory receptors on immune cells. Antibodies that block checkpoint inhibitory receptors have shown efficacy in a significant subset of cancer patients. Anti-CTLA4 (Ipilimumab) was the first immune checkpoint blockade antibody approved by the Food and Drug Administration (FDA), followed by antagonistic antibodies against the PD1 / PD-L1 axis. While additional agents are currently being tested for efficacy in clinical trials, to date only the checkpoint molecules CTLA4, PD1, PD-L1 and LAG-3 are targets by FDA-approved drugs. However, current immune checkpoint blockade is only effective in a subset of patients with certain cancer types and ineffective in many others. Hence, there is a strong need to identify new checkpoint molecules as immunotherapy targets.

[0323] A major obstacle of current immunotherapy approaches are immune-related adverse events (irAEs). IrAEs arise due to the important homeostatic function of immune checkpoint molecules in regulating immune responses thereby preventing pathological immune overreactions. Hence, blockade of checkpoint molecules can lead to autoimmune-like tissue inflammation, especially at sites with high exposure to foreign antigens such as mucosal surfaces. The frequency and severity of irAEs is mainly dependent on the patient characteristics and the treatment agent used. Even though most organs can be affected, irAEs are most frequently observed at the gastrointestinal tract, skin, liver, and endocrine glands. The underlying immunological mechanisms leading to irAEs remain unknown. While therapy- induced irAEs correlate with anti-tumor immunity, it is unclear whether the same or different mechanisms drive anti-tumor immunity and the autoimmune-like tissue inflammation. Novel therapy approaches and targets that induce anti -tumor immunity but do not induce autoimmune-like toxicity are needed and would significantly enhance the benefit and applicability of immune checkpoint blockade in the clinic.

[0324] PGLYRP1 belongs to the family of peptidoglycan recognition proteins (PGRPs) that is highly conserved from insects to mammals. Mammalian PGLYRP1 is -200 amino acids long with a molecular mass of -18-20 kDa, and has a signal peptide. Structural analysis of human PGLYRP1 revealed the PGLYRP1 molecule to contain at the front a conserved peptidoglycan-binding groove and on the back a PGLYRP1 -specific groove that might constitute a binding site for specific effector and signaling molecules. PGLYRP1 is expressed along the digestive tract, including the throat, oesophagus jejunum, ileum and caecum. Concerning the cell type, PGLYRP1 has mainly been described to be expressed in the granules of neutrophils and eosinophils and to a lesser extent in non-immune cells including epithelial cells, fibroblasts, and intestinal M-cells. PGLYRP1 is directly bactericidal against both grampositive and gram -negative bacteria and acts likely by direct binding to cell wall peptidoglycan (PGN). PGLYRP1 -deficient mice have an increased susceptibility to infection with lowly pathogenic grampositive bacteria and PGLYRP1 -deficient neutrophils have defects in intracellular killing and digestion of bacteria. In the tumor setting, PGLYRP1 has been reported to form a cytotoxic complex with the heat shock protein 70 (HSP70) that induces apoptotic cell death in tumor cells. The proinflammatoryreceptor TREM-1 has been suggested as receptor of PGLYRP1 on myeloid cells. Binding of the complex of PGLYRP1 with PGN to TREM-1 was described to induce proinflammatory cytokine expression in neutrophils and macrophages. While PGLYRP1 has been found to be up-regulated in tumor-associated Treg cells, until today, no function of PGLYRP1 in T cells has been described.

[0325] Here, Pglyrpl is identified to be highly co-expressed with co-inhibitory receptors in T cells. The IL-27-PRDM1 / MAF axis was found to regulate the expression of Pglyrpl in T cells. Genetic deletion of PGLYRP1 in mice led to decreased tumor growth accompanied with an enhanced activation / effector phenotype in CD8+T cells. Interestingly, Pglyrpl -deficiency at the same time resulted in strong protection against EAE with defects in antigen-presentation in myeloid cells and major transcriptional alterations in macrophages / monocytes and neutrophils. The findings highlight PGLYRP1 as a promising novel target in immunotherapy, the targeting of which can induce a potent anti-tumor immune response while not triggering autoimmune -like side effects.

[0326] Results

[0327] Pglyrpl is co-expressed with co-inhibitory molecules in T cells

[0328] Previous work showed that co-inhibitory molecules are expressed in a shared gene module and are highly co-expressed in T cells. Hence, the identification of genes highly co-expressed with co- inhibitory molecules in CD8+T cells in the tumor could yield in the discovery of novel T-cell co- inhibitory molecules. The previously published scRNA-seq data of CD8+tumor-infiltrating lymphocytes (TILs) from B16F10 melanoma was used in order to perform an unbiased correlation analysis of all genes with co-inhibitory genes (Pdcdl, Tigit, Ctla4, Havcr2. Lag3) in comparison to stem-like genes (Ccr7, Cxcr5. Tcf7, Sell) (FIG. 1A). This analysis identified multiple molecules with known functions in T cells, including Cxcr6. Nkg7, Gzmb. Ifng. Prfl, IrJ8, and Bhlhe40. The analysis also identified the gene Pglyrpl with no known function in T cells. The expression of Pglyrpl was positively correlated with co-inhibitory molecules (Spearman correlation coefficient = 0.47, p=1.2e-21) and negatively correlated with stem-like genes (Spearman correlation coefficient = -0.27, p=1.8e-7). Clustering analysis of the scRNA-seq CD8+TIL data revealed that Pglyrpl is expressed in the same cluster as the co-inhibitory molecules (Pdcdl , Tigit, Ctla4, Havcr2. Lag3) and the dysfunction signature (26), but in a separate cluster than the stem-like genes (Slamf6, Ccr7. Cxcr5, Tcf7. Sell) (FIGs. IB and 1C). An up-regulation of Pglyrpl expression was validated ex vivo in exhausted CD8+T cells (PD1+TIM3+) from MC38-OVA tumors by qPCR (FIG. ID).

[0329] To analyze the expression of Pglyrpl in human tumors, published scRNA-seq data of immune cells was utilized from human melanoma tumors. Pglyrpl was found to be expressed the highest in the exhausted CD8+T cell cluster, similar to the co-inhibitory receptors (FIG. 9), suggesting a coexpression of Pglyrpl and the co-inhibitory receptors also in humans.

[0330] Co-inhibitory molecules are highly expressed on regulatory T cells (Tregs) and play an important role for Treg function. Hence, Fox / GGFP-reporter mice were obtained and the expression of Pglyrpl and co-inhibitory molecules measured on conventional T cells (Tconv, CD4+TCRp+Foxp3GV?~) and Tregs (CD4+TCRp+Foxp3GFP+) in different tissues by qPCR (FIG. 10). Indeed, Pglyrpl was found to be up-regulated with co-inhibitory molecules on Tregs.

[0331] Together, it was found, in both mice and humans, that Pglyrpl is highly co-expressed with co- inhibitory molecules in T cells.

[0332] The expression of Pglyrpl in T cells is regulated by the IL-27-PRDMl / c-MAF axis

[0333] It was previously demonstrated that the cytokine IL-27 induces a co-inhibitory module in T cells that is shared across different states of T cell non -responsiveness. Notably, Pglyrpl was identified as part of this IL-27-induced co-inhibitory module. To validate the induction of Pglyrpl expression by IL- 27 in T cells, naive CD4+and naive CD8+T cells were cultured in vitro with or without IL-27 cytokine and confirmed a significant up-regulation of Pglyrpl expression in CD4+(~27 fold) and CD8+(~2.5 fold) T cells upon IL-27- signaling (FIG. IE). The transcription factors PRDM1 and c-MAF were identified as cooperative regulators of the IL-27- induced co-inhibitory module. To test whether PRDM1 and c-MAF contribute to the IL-27-mediated induction of Pglyrpl expression in T cells, PRDM1 / c-MAF -deficient mice were obtained Maf1' Prdml1') and naive CD4+T cells were cultured in vitro with or without IL-27 cytokine (FIG. IF). The induction of Pglyrpl expression by IL-27 was significantly reduced in Maf1’ PrdmlG~ T cells. Together, these data suggest that the IL-27-PRDMl / c- MAF axis induces Pglyrpl expression in T cells in vitro. To analyze whether IL-27 and PRDMl / c- MAF play a role for Pglyrpl expression in T cells in vivo, previously published bulk RNA-seq data was analyzed from CD8+T cells isolated from B16 melanoma tumors ofWT and IL-27 receptor-deficient (Il27rcrl~) mice or MafPrdmr1' mice (FIGs. 1G and 1H). In both cases, a reduced expression of Pglyrpl expression was found in comparison to the respective WT controls. Hence, the results suggest that the IL-27-PRDM1 / MAF axis regulates the expression of Pglyrpl in T cells in vitro as well as in vivo.

[0334] Decreased tumor growth in P / yr iJ-deficient mice

[0335] To analyze whether PGLYRP1 plays a functional role in anti -tumor immunity, Pglyrpl- deficient mice Pglyrpl1) were obtained (FIG. 9A) and MC38-OVA colon carcinoma cells were injected into the g / yr / »7 / ’ mice and matching wildtype (WT) littermate controls (FIGs. 2A-2K). Decreased tumor sizes and weights were found in Pglyrpl1' mice (FIGs. 2A-2C), suggesting PGLYRP1 as a potential negative regulator of anti -tumor immunity. Decreased tumor growth was also found in Pglyrpl1' mice challenged with B16-OVA melanoma cells (FIGs. 9B and 9C), highlighting that the effect of Pglyrpl -deficiency on tumor growth is conserved across tumor types.

[0336] To understand in which immune cell type PGLYRP1 might play a role in the tumor microenvironment, the expression of Pglyrpl was assessed in different immune cell populations isolated from MC38-OVA tumors by qPCR (FIG. 9D). Consistent with the previous analysis (FIGs. 1A-1H, FIG. 7 and FIG. 8), the highest expression of Pglyrpl was found in exhausted CD8+T cells and Tregs. In the myeloid compartment, NK cells and neutrophils were found to express the highest levels of Pglyrpl in the tumor. General immune profiling of immune cell population frequencies inMC38-OVA tumors grown in PglyrpP1' mice revealed no major differences compared to tumors grown in WT mice (FIG. 9E) except for Tregs that were significantly decreased in tumors of PglyrpP1' mice but unchanged in the draining lymph node (dLN) and spleen (FIG. 2D, FIG. 10A). Due to the coexpression oiPglyrpl with co-inhibitory molecules in CD8+T cells, a focused analysis was performed of the CD8+T cell compartment. Interestingly, the CD8+T cells in the tumor and dLN in PglyrpP1' mice were found to express enhanced frequencies of proinflammatory cytokines, including IFNy, TNFa and GM-CSF, suggesting an increased effector function (FIG. 2E, FIG. 10B). TIM3 and PD1 are co- inhibitory molecules up-regulated during T cell activation that can also be used as markers for exhausted T cells. An increased expression of TIM3 and PD1 was found on CD8+T cells and an increased frequency of TIM3+PD1+CD8+T cells in the tumor, dLN, and spleen m ' PglyrpP1' mice (FIGs. 2F and 2G, FIG. IOC). Together, these data suggest an enhanced activation and effector phenotype of CD8+TILs in PglyrpP1' mice.

[0337] To obtain an unbiased understanding of the CD8+T cell phenotype in Pglyrpl / _mice, bulk RNA- seq of CD8+TILs was performed from PglyrpP1' mice and matching WT littermate controls. 330 up- regulated genes and 35 down-regulated genes were found in PglyrpP1' CD8+T cells when compared to WT cells (FDR < 0.15, fold change > 2) (FIG. 2H). In accordance with the flow analysis, the up- regulated genes in PglyrpP1' CD8+T cells included genes involved in T cell activation, effector functions, and exhaustion (Nr4a2, Prfl , Lag3, IrJ8, Atf3, Ifrig, Havcr2. Tigii. Maf, Ccl4) (FIGs. 2H and 21). Pathway enrichment analysis using the Gene Ontology (GO) resource and published signatures identified T cell activation and effector signatures to be significantly enriched in PglyrpP1' CD8+T cells (FIGs. 2J and 2K), further supporting an enhanced activation and effector phenotype of CD8+TILs in PglyrpP1' mice.

[0338] Survival analysis of the association between the expression levels of PGLYRP1 and survival rate in different human cancers revealed a significantly worse outcome in tumors with high PGLYRP1 expression (FIG. 11), suggesting that PGLYRP1 might also act as negative regulator of anti -tumor immunity in human cancers.

[0339] ScRNA-seq reveals a shift of stem-like / progenitor cells to activated and effector cells in the PglyrpP’ CD8+TIL compartment

[0340] To obtain a more granular understanding of the changes in the CD8+T cell compartment in PglyrpP1' mice, scRNA-seq of tumor-infiltrating T cells (CD45+CD3+cells) was performed from PglyrpP1' mice and matching WT littermate controls (FIGs. 3A-3I). Clustering analysis divided 1,820 T cell single cells into four subsets (FIG. 3A), each expressing distinct signatures (FIG. 12A). The four clusters were annotated by differential gene signatures as Tregs, CD4+Tconv, CD8+T cells- 1, and CD8+T cells-2. Pathway enrichment analysis revealed significant enrichment of stem-like / progenitor-like pathways in the CD8+T cells- 1 cluster including signatures related to WNT and TCF signaling and stem- and naive-like cells (FIG. 12B). In contrast, the CD8+T cells-2 cluster was enriched for signatures involved in T cell activation, effector functions, and exhaustion, indicating CD8+T cells- 1 as the stem-like / progenitor cluster and CD8+T cells-2 as the effector / exhausted cluster. RNA velocity analysis using scVelo suggested a trajectory from stem-like / progenitor cluster to the effector / exhausted cluster (FIG. 12C), further suggesting CD8+T cells-1 as the stem-like / progenitor cells and CD8+T cells-2 cluster as the effector / exhausted cluster. In accordance with the previous analysis identifying high expression of Pglyrpl in exhausted CD8+T cells and Tregs (FIG. 1A-1H, FIG. 8), the expression of Pglyrpl was the highest in the effector / exhausted cluster and Treg cluster (FIG. 3B).

[0341] While cells in the Tregs and CD4+Tconv clusters from WT and Pglyrpl1' mice were well-mixed and had only few differentially expressed genes (Tregs (CD4+Tconv) 8 genes up-regulated, 4 genes down-regulated; FDR <0.05, |log2 fold change] >0.25), the CD8+T cells were more distinguishable between the two conditions and had a larger number of differentially expressed genes (239 up- and 226 down-regulated; two CD8+T cell clusters combined) (FIGs. 3C and 3D). Interestingly, of the CD8+T cell clusters especially the stem-like / progenitor cluster had a large number of differentially expressed genes (137 up- and 124 down-regulated) (FIG. 3D). Analysis of the genotype composition ofthe tumorinfiltrating T cell clusters identified a significant increase in the effector / exhausted cluster (FIG. 3E), consistent with the previous flow analysis (FIGs. 2D-2G). These data suggest major transcriptional changes in the stem-like / progenitor cluster and an increase in the effector / exhausted cluster, potentially suggesting a shift of stem-like / progenitor cells to activated / exhausted cells in PglyrpP1’ mice.

[0342] Focused differential gene expression analysis and pathway enrichment analysis of the stem- like / progenitor cluster from WT and PglyrpP1' mice revealed an up-regulated expression of genes (Statl, Cd69. Jak2. Tbx21, Irfl, PrfP) and pathways (interferon signaling, leukocyte-mediated cytotoxicity, exhaustion) involved in T cell activation and effector functions in PglyrpP1' cells (FIGs. 3F and 3G). These data further suggest a shift of stem-like / progenitor cells to activated / exhausted cells in the CD8+TIL compartment in PglyrpP1' mice.

[0343] In accordance with the previous flow analysis (FIG. 2D), a significant decrease in the Treg cluster was identified in tumors in PglyrpP1' mice (FIG. 3E). Of the few differentially expressed genes in the Treg cluster between WT and PglyrpP1' mice, Itgae (encoding CD 103) was the top down- regulated gene (p = 1.2e-5, fold change = 2.73) m ' Pglyrpl1' mice (FIG. 12D). CD103 is usually highly expressed on tumor-infiltrating Tregs and marks a particularly suppressive subset of Tregs (32, 33). Hence, a dysregulated expression of CD103 on PglyrpP1' Tregs might be involved in their low representation in the tumors.

[0344] To examine whether PGLYRP1 has a cell-intrinsic function in CD8+T cells, Pglyrpl- conditional knockout mice (Pglyrpl™) were obtained and bred to E8r " mice to generate mice with a specific deletion of Pglyrpl in CD8+T cells (E8iCiePglyrpPn) (FIG. 13A). MC38-OVA tumors were implanted into WT and E8i ~' Pglyrpl™ mice and found E8i ~' Pglyrpl™ mice to exhibit improved tumor growth control with increased frequencies of CD8+TILs that expressed higher levels of the activation and exhaustion markers PD1 and TIGIT (FIGs. 3H and 31). Importantly, mice that lacked Pglyrpl expression specifically in myeloid cells (LysPf-^ Pglyrpl™) showed no alteration in tumorgrowth, suggesting that the expression of Pglyrpl on myeloid cells is dispensable for anti-tumor immunity (FIG. 13B).

[0345] In summary, the data suggests that the deletion of Pglyrpl in CD8+TILs leads to a shift of stem- like / progenitor cells to activated / exhausted cells indicating that PGLYRP1 is potentially a novel inhibitory molecule in CD8+T cells.

[0346] Pglyrplcient mice are protected from EAE

[0347] A major obstacle of blocking co-inhibitory molecules for cancer therapy are irAEs that arise due to the important homeostatic function of immune inhibitory molecules in regulating immune responses. Hence, the blockade and deletion of T-cell inhibitory molecules, such as CTLA4, PD1, TIM3, and TIGIT, leads to exacerbated autoimmune disease in murine mouse models, including experimental autoimmune encephalomyelitis (EAE). To examine whether the deficiency of PGLYRP1, similar to other inhibitory molecules, exacerbates autoimmune disease, EAE was induced in Pglyrpl1' mice and matching wildtype (WT) littermate controls. Pglyrpl ' mice were highly protected against EAE with a decreased incidence rate Pglyrpl1': 62%, WT: 100%), maximal clinical EAE score (Pglyrpl1' 2.3, WT: 2.9), and mean clinical EAE score Pglyrpl1' 1.6, WT: 2.3) (FIGs. 4A-4C). Histological analysis further revealed reduced CNS lesions and optic neuritis in Pglyrpl1' mice (FIG. 4D and 4E). In the CNS of Pglyrpl1' mice, a general reduction was found of hematopoietic infiltrates (CD45+) and CD4+T cell-, CD8+T cell-, Treg-, and B cell- infiltrates, cell types known to drive EAE disease (FIG. 4F). The CD4+T cells infiltrated into the CNS of Pglyrpl1' mice expressed significantly lower levels of the inflammatory cytokines IL-17A, IFNy, and GM-CSF, and the anti-inflammatory cytokine IL- 10 (FIG. 4G), indicating a potential defect in T-cell priming during EAE without PGLYRP1.

[0348] In summary, in contrast to known T-cell inhibitory molecules, Pglyrpl1' mice are protected against EAE and PGLYRP1 seems to contribute to EAE disease pathology.

[0349] PGLYRP1 contributes to EAE disease by acting as a pro-inflammatory molecule in myeloid cells

[0350] To reveal the cell type responsible for the reduced EAE phenotype in Pglyrpl- global knockout mice (Pglyrpl1), the Pglyrpl -conditional knockout mouse (Pglyrpl™) was utilized and crossed to E8i™ mice to generate mice with a specific deletion of Pglyrpl in CD8+T cells E8i™ Pglyrpl™) and to Cd4CTemice to generate mice with a specific deletion of Pglyrpl in CD4+and CD8+T cells (Cd4™ Pglyrpl™). In both mouse models no alteration was observed in the EAE disease, suggesting that the expression ofPGLYRPl on T cells is not responsible for the reduced EAE phenotype in Pglyrpl1' mice (FIGs. 5A-5B)

[0351] Based on the defect in T-cell priming in Pglyrpl'1' mice during EAE (FIGs. 4F-4G) and the important function of myeloid cells in priming autoreactive T cells during EAE, it was hypothesized that the expression of PGLYRP1 on myeloid cells could contribute to the reduced EAE phenotype in Pglyrpl'1' mice. Hence, the Pglyrpl™ mice were crossed to LysM™ mice generating mice with a specific deletion of Pglyrpl in myeloid cells, including monocytes, mature macrophages, granulocytes,and some dendritic cells (DCs) (PysM '' Pglyrpl ). LysM '' Pglyrpl™ mice were protected against EAE (FIGs. 5C-5D), with lower systemic levels of the proinflammatory cytokine IL-6 (FIG. 5E). The CNS-infdtrated myeloid cells were less activated, as measured by the expression of MHC2 (FIG. 5F). In accordance with reduced myeloid cell activation, CD4+T cells in the dLN appeared less primed, with reduced expression of the activation marker CD44 and the proinflammatory cytokines GM-CSF, IL-2, and TNFa (FIG. 5G), suggesting a defect in T-cell priming in LysM™ Pglyrpl™ mice during EAE. In addition, lower levels of pathogenic Th 17 cells (IL-17A+IFNy+) were detected in the CNS (FIG. 5H). Together, these data indicate that PGLYRP1 expression in myeloid cells is required for sufficient antigen-presentation and priming of T cells during EAE.

[0352] To directly test a role of PGLYRP 1 expression in myeloid cells for antigen-presentation to CD4+T cells, an antigen-presentation assay was performed. Splenocytes were cocultured from LysM™ Pglyrpl™ mice with naive CD4+T cells from 2D2 T-cell receptor (TCR) transgenic mice that express a TCR specific for the myelin oligodendrocyte glycoprotein (MOG) (44) in culture medium with or without MOG peptide (FIG. 14A-14B). In the culture wells with LysM™ Pglyrpl'' " splenocytes, the 2D2 cells showed lower proliferation rates (FIG. 14A) and lower levels of the proinflammatory cytokines IL- la, IL-6, MCP-1, and TNFa were secreted into the media (FIG. 14B), suggesting a role of PGLYRP1 expression in myeloid cells for antigen-presentation and priming of CD4+T cells.

[0353] To analyze whether PGLYRP 1 expression in myeloid cells plays a role for T-cell priming during EAE, we performed a recall assay and induced EAE in LysM™ Pglyrpl™ mice and WT littermate controls. At day 10 post immunization, the spleens and cultured splenocytes were harvested with or without MOG peptide. In the culture wells with LysM™ Pglyrpl™ splenocytes, lower concentrations of proinflammatory cytokines (IFNp, IL-6, MCP-1, TNFa) were detected in the media (FIG. 51), suggesting a role of PGLYRP 1 expression in myeloid cells for priming of CD4+T cells during EAE.

[0354] Together, these data suggest PGLYRP 1 is needed by myeloid cells for antigen-presentation and priming of CD4+T cells during EAE.

[0355] Substantial transcriptional changes in monocytes and neutrophils in Pglyrpl1' mice during EAE

[0356] To reveal which myeloid populations are affected by Pglyrpl -deficiency during EAE, the expression of Pglyrpl in immune populations was analyzed in the dLNs during EAE onset (FIG. 6A). The highest expression was detected in monocytes and neutrophils. This high Pglyrpl -expression was induced by EAE, as monocytes and neutrophils derived from naive mice showed low Pglyrpl- expression.

[0357] To obtain an unbiased view on the effect of Pglyrpl -deficiency on the myeloid cell compartment, scRNA-seq was performed of CNS-infiltrating myeloid cells (CD45+CD3 CD 19 ) derived from Pglyrpl1' mice and matching WT littermate controls during EAE onset. Clustering analysis divided 3,698 myeloid single cells into 14 subsets (FIG. 6B, FIG. 15A) with distinct expression signatures (FIG. 6C). The 14 clusters were annotated by using cell type markers anddifferential gene signatures as monocytes / macrophages (mono / MACS), neutrophils, microglia, B cells, DC1 / DC2, pDC, DC3, NK cells, macrophages, and monocytes (FIG. 6C). Distance analysis between PglyrpP1' and WT cells in each cluster revealed the greatest distance in mono / MACS and neutrophil clusters, suggesting substantial transcriptional changes in monocytes / macrophages and neutrophils in PglyrpP1' mice during EAE (FIG. 6D).

[0358] Focused differential gene expression analysis revealed substantial expression changes in mono / MACS (44 genes upregulated, 119 genes downregulated, FDR<0.05 and fold change > 1.19) and neutrophils (166 genes upregulated, 54 genes downregulated, FDR<0.05 and fold change > 1.19) in PglyrpP1' mice (FIG. 6E). Genes down-regulated in mono / MACS included genes encoding members of the complement system (C / &, Clqb) and the pro -inflammatory chemokine CXCL10 (CxcllO). Cd74 encoding the HLA-DR antigens-associated invariant chain (CD74) was down-regulated in PglyrpP1’ neutrophils. Nine genes were downregulated in both PglyrpP1' mono / MACS and neutrophils including Apoe and LgalsS (FIG. 15C). Apoe was especially highly downregulated in both PglyrpP1' mono / MACS (rank 1 by fold change) and neutrophils (rank 3 by fold change). Apoe encodes the apolipoprotein E (APOE) that has recently been shown to have a critical function in myeloid cell antigen presentation and T-cell priming, in accordance with our findings of defects in antigen presentation and T-cell priming in PglyrpP1' mice during EAE (Figure 4G, 51 and Figure S8). Lgals3 encodes GALECTIN-3 that has been shown to regulate the migration of monocytes / macrophages. Genes upregulated in both PglyrpP1' mono / MACS and neutrophils include Jun and Lilra6, both involved in myeloid cell activation. Multiple genes upregulated in PglyrpP1' neutrophils belong to interferon- stimulated genes (Ifit3, Ifit3b, Ifit Ifi204. Ifi27l2a, Oasl2. Irf7), indicating an increased IFN-signaling. Pathway enrichment analysis of the differentially expressed genes revealed WT mono / MACS to be enriched for pathways involved in the complement cascade, inflammatory response, and antigen presentation, while PglyrpP1' mono / MACS were enriched in pathways associated with DNA repair and cell division (FIG. 6F). WT neutrophils were enriched for pathways involved in cell killing, neutrophil degranulation, and antigen presentation, while pathways associated with interferon signaling were enriched in PglyrpP1' neutrophils. Together, these analyses suggest a downregulation of pro- inflammatory pathways, cell activation, and antigen presentation in both PglyrpP1' mono / MACS and neutrophils.

[0359] In summary, a high expression of Pglyrpl was found in monocytes and neutrophils and substantial transcriptional changes in these cell populations in PglyrpP1' mice during EAE. Hence, PGLYRP1 expression in the myeloid populations monocytes and neutrophils might contribute to EAE pathogenesis. Deficiencies in antigen presentation by mono / MACS and neutrophils might be responsible for the attenuated EAE disease in PglyrpP1' and in LysPP^ PglyrpP " mice.

[0360] Discussion

[0361] In this study PGLYRP1 was identified as a potential novel target for immunotherapy. Pglyrpl was found to be highly co-expressed with co-inhibitory molecules in T cells and hypothesized thatPGLYRP1 could play an important role in anti -tumor immunity. Indeed, reduced tumor growth in Pglyrpl1' mice was found with an enhanced activation and effector phenotype in CD8+T cells, but at the same time found Pglyrpl1' mice to be strongly protected from EAE with a potential function of PGLYRP1 in activation and antigen presentation in mono / MACS and neutrophils.

[0362] The Pglyrpl -expression was found in T cells to be induced by the cytokine IL-27. IL-27 is an immunoregulatory cytokine that suppresses T cell responses by multiple mechanisms, including inhibition of T cell effector differentiation and induction of a “co-inhibitory” module in T cells. In addition, IL-27 induces IL- 10 in a wide range of cell types and can differentiate naive T cells to type 1 regulatory T (Tri) cells. In accordance with these functions, Il27r(P' T cells have defects in producing IL- 10. Based on the findings that PGLYRP1 plays an inhibitory function in T cells, it makes sense that it is induced by the immunoregulatory cytokine IL-27, as part of a general anti-inflammatory module together with IL-10 and co-inhibitory molecules.

[0363] A major obstacle of current immunotherapy approaches are irAEs. In fact, the severity of irAEs is often the limiting factor for the applicability of immunotherapy in the clinic. IrAEs are observed in a high frequency of p...

Claims

CLAIMSWhat is claimed herein is:

1. A method of treating cancer in a subject in need thereof, the method comprising administering: an immune checkpoint inhibitor; and an inhibitor of Peptidoglycan Recognition Protein 1 (Pglyrpl); to the subject.

2. The method of claim 1, wherein the inhibitor of Pglyrpl potentiates anti-tumor immune activity of the immune checkpoint inhibitor and suppresses immune checkpoint inhibitor-mediated autoimmunity.

3. The method of claim 1 or 2, whereby cancer immunotherapy-related autoimmune activity is reduced or limited as compared to treatment without the inhibitor of Pglyrp 1.

4. The method of any one of the preceding claims, wherein the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof.

5. The method of claim 4, wherein the antibody or antigen-binding fragment thereof comprises LS- B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.

6. The method of any one of the preceding claims, wherein the inhibitor of Pglyrpl comprises a nucleic acid molecule.

7. The method of claim 6, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system.

8. The method of any one of claims 6-7, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).

9. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.

10. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is selected from an inhibitor of:PD-L1, PD-1, CTLA-4, PD-L2, LAG3, TIM3, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, ID02, ICOS, KIR, LAIRI, LIGHT, MARCO, PS, OX- 40, SLAM, TIGIT, VISTA, VTCNI, IDO, CXCR4, CISH, CBL-B, B7- 1, B7-2, B7-DC, B7- Hl, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6,B7-H7, CGEN-15049, CHK1, CHK2, A2aR, and DDE11. The method of any one of the preceding claims, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of:CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.

12. The method of claim 11, wherein the immune checkpoint inhibitor is an inhibitor of PD1 selected from the group consisting of: pembrolizumab, nivolumab, and cemiplimab.

13. The method of claim 11 , wherein the immune checkpoint inhibitor is an inhibitor of PD-L 1 selected from the group consisting of: atezolizumab, avelumab, and durvalumab.

14. The method of claim 11, wherein the immune checkpoint inhibitor is an inhibitor of CTLA4 comprising ipilimumab.

15. The method of any one of the preceding claims, wherein the checkpoint inhibitor and the inhibitor of Pglyrpl are administered concurrently.

16. The method of any one of the preceding claims, wherein the Pglyrpl inhibitor is initially administered before the checkpoint inhibitor.

17. The method of any one of the preceding claims, wherein the checkpoint inhibitor is initially administered before the inhibitor of Pglyrp 1.

18. A method of suppressing one or more autoimmune side effects of checkpoint inhibitor cancer immunotherapy in a subject who is being or has been treated with an immune checkpoint inhibitor cancer immunotherapy, the method comprising administering a Pglyrpl inhibitor to the subject.

19. The method of claim 18, wherein the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof.

20. The method of claim 18, wherein the antibody or antigen-binding fragment thereof comprises LS- B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.

21. The method of any one of claims 18-20, wherein the inhibitor of Pglyrpl comprises a nucleic acid molecule.

22. The method of claim 21, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system.

23. The method of any one of claims 21-22, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).

24. The method of any one of claims 18-23, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.

25. The method of any one of claims 18-24, wherein the immune checkpoint inhibitor is selected from an inhibitor of:PD-L1, PD-1, CTLA-4, PD-L2, LAG3, TIM3, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, ID01, ID02, ICOS, KIR, LAIRI, LIGHT, MARCO, PS, OX- 40, SLAM, TIGIT, VISTA, VTCNI, IDO, CXCR4, CISH, CBL-B, B7- 1, B7-2, B7-DC, B7- Hl, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6,B7-H7, CGEN-15049, CHK1, CHK2, A2aR, and DDL26. The method of any one of claims 18-25, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of:CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.

27. The method of claim 26, wherein the immune checkpoint inhibitor is an inhibitor of PD1 selected from the group consisting of: pembrolizumab, nivolumab, and cemiplimab.

28. The method of claim 26, wherein the immune checkpoint inhibitor is an inhibitor of PD-L1 selected from the group consisting of: atezolizumab, avelumab, and durvalumab.

29. The method of claim 26, wherein the immune checkpoint inhibitor is an inhibitor of CTLA4 comprising ipilimumab.

30. The method of any one of claims 18-29, wherein the one or more autoimmune side effects of checkpoint inhibitor cancer immunotherapy comprise one or more of: colitis, endocrinopathy, hepatitis, thyroiditis, myasthenia gravis, autoimmune autonomic ganglionpathy, nephritis, liver toxicity, skin rash or pruritus, meningitis, headaches, encephalopathy, fatigue, diarrhea, and pneumonitis.

31. A method of treating cancer, the method comprising administering an inhibitor of Pglyrpl to a subject in need of treatment for cancer and determined to have an increased level of expression of Pglyrpl as compared to a healthy reference level.

32. The method of claim 31, further comprising a first step of detecting the expression of Pglyrpl in cells of the subject’s cancer.

33. A method of treating cancer in a subject in need thereof, the method comprising: detecting or receiving results of an assay detecting the expression of Pglyrpl by cells of the subject’s cancer; and administering an inhibitor of Pglyrpl to the subject.

34. The method of any one of claims 31-33, wherein the inhibitor promotes an anti-cancer immune response while suppressing autoimmune dysfunction.

35. The method of any one of claims 31-34, wherein the determining or detecting comprises detecting Pglyrpl protein and / or RNA encoding Pglyrpl protein.

36. The method of any one of claims 31-35, wherein the inhibitor of Pglyrp 1 comprises an antibody or antigen-binding fragment thereof.

37. The method of claim 36, wherein the antibody or antigen-binding fragment thereof comprises LS- B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.

38. The method of any one of claims 31-37, wherein the inhibitor of Pglyrp 1 comprises a nucleic acid molecule.

39. The method of claim 38, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system.

40. The method of any one of claims 38-39, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).

41. A method of treating autoimmune disease in a subject in need thereof, the method comprising administering an inhibitor of Pglyrp 1 to the subject.

42. The method of claim 41, wherein the inhibitor of Pglyrp 1 comprises an antibody or antigen-binding fragment thereof.

43. The method of claim 42, wherein the antibody or antigen-binding fragment thereof comprises LS- B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.

44. The method of any one of claims 41-43, wherein the inhibitor of Pglyrp 1 comprises a nucleic acid molecule.

45. The method of claim 44, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system.

46. The method of any one of claims 44-45, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).

47. The method of any one of claims 41-46, wherein the autoimmune disease affects mucosal tissues or tissues of the gastrointestinal tract.

48. The method of any one of claims 41-47, wherein the autoimmune disease is selected from pneumonitis, colitis, enteritis, and gastritis.

49. A composition, combination, or kit comprising: an immune checkpoint inhibitor; and an inhibitor of Pglyrp 1.

50. The composition, combination, or kit of claim 49, wherein the inhibitor of Pglyrpl comprises an antibody or antigen-binding fragment thereof.

51. The composition, combination, or kit of claim 50, wherein the antibody or antigen-binding fragment thereof comprises LS-B4940, 188C424 or 299D535, or an antigen-binding fragment thereof.

52. The composition, combination, or kit of any one of claims 49-51, wherein the inhibitor of Pglyrpl comprises a nucleic acid molecule.

53. The composition, combination, or kit of claim 52, wherein the nucleic acid molecule comprises an RNAi molecule, shRNA, miRNA, or encodes an RNA targeting CRISPR-Cas system.

54. The composition, combination, or kit of any one of claims 52-53, wherein the RNAi molecule comprises one or more sequences selected from the group consisting of: rCrArA rCrUrC rUrArU rCrArG rGrUrC rArUrC rCrArA rArGC T (SEQ ID NO: 1), rArGrC rUrUrU rGrGrA rUrGrA rCrCrU rGrArU rArGrA rGrUrU rGrGrU (SEQ ID NO: 2), rCrGrU rGrCrU rGrCrC rCrUrA rArArU rCrUrU rCrUrG rGrAA T (SEQ ID NO: 3), and / or rArUrU rCrCrA rGrArA rGrArU rUrUrA rGrGrG rCrArG rCrArC rGrGrA (SEQ ID NO: 4).

55. The composition, combination, or kit of any one of claims 49-54, wherein the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.

56. The composition, combination, or kit of any one of claims 49-55, wherein the immune checkpoint inhibitor is selected from an inhibitor of:PD-L1, PD-1, CTLA-4, PD-L2, LAG3, TIM3, 2B4, A2aR, B7HI, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, ID02, ICOS, KIR, LAIRI, LIGHT, MARCO, PS, OX- 40, SLAM, TIGIT, VISTA, VTCNI, IDO, CXCR4, CISH, CBL-B, B7- 1, B7-2, B7-DC, B7- Hl, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6,B7-H7, CGEN-15049, CHK1, CHK2, A2aR, and DDE57. The composition, combination, or kit of any one of claims 49-56, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from the group consisting of:CTLA4, TIM-3, LAG-3, PD-1, PD-L1, TIGIT, IDO, CXCR4, CISH, and CBL-B.

58. The composition, combination, or kit of claim 57, wherein the immune checkpoint inhibitor is an inhibitor of PD 1 selected from the group consisting of: pembrolizumab, nivolumab, and cemiplimab.

59. The composition, combination, or kit of claim 57, wherein the immune checkpoint inhibitor is an inhibitor of PD-L1 selected from the group consisting of: atezolizumab, avelumab, and durvalumab.

60. The composition, combination, or kit of claim 57, wherein the immune checkpoint inhibitor is an inhibitor of CTLA4 comprising ipilimumab.

61. The composition, combination, or kit of any one of claims 49-60, for use in the treatment of cancer.

62. The composition, combination, or kit of any one of claims 49-60, for use in the treatment of an autoimmune disease.

63. The composition, combination, or kit of claim 62, wherein the autoimmune disease affects mucosal tissues or tissues of the gastrointestinal tract.

64. The composition, combination, or kit of any one of claims 62-63, wherein the autoimmune disease is selected from pneumonitis, colitis, enteritis, and gastritis.

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