Methods and compositions utilizing IDO1-dependent angiogenic cells for the treatment of pathological conditions involving neovascularization - Patents.com

Abstract

The present invention is a composition and method for treating retinopathy or inhibiting pathological neovascularization in a subject. [Means for solving the problem] In one embodiment, the method includes ablating or inhibiting IDO-dependent angiogenic cells (IDVCs) in the eye of a subject. Various methods for ablating or inhibiting IDVCs are described, including inhibiting IDO1 and the integrated response node.

Description

[Technical field]

[0001] The present invention relates to methods and compositions utilizing IDO1-dependent angiogenic cells for the treatment of pathological conditions involving neovascularization. (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 122,121, filed December 7, 2020, the entire disclosure of which is incorporated herein by reference. (Incorporation by reference of material submitted in electronic form) Incorporated herein by reference in its entirety is a sequence listing submitted via EFS-Web as a text file named SEQLIST.txt, created on December 7, 2021, and having a size of 4,212 bytes. [Background technology]

[0002] Angiogenesis, the tightly regulated process of new blood vessel formation, is developmentally required for organogenesis but is rare in normal adult tissues, except for the female reproductive cycle and wound healing. In contrast, pathological angiogenesis is characterized by dysregulated neovascularization and is involved in a variety of diseases, including the development of tumors that require a blood supply from the host to grow beyond a certain stage (1). Tumor angiogenesis is induced through interaction with the local inflammatory milieu, which subverts the physiological process of wound healing. (2) One hallmark of the inflammatory tumor microenvironment is the presence of a heterogeneous population of immature myeloid cells, collectively referred to as MDSCs (myeloid-derived suppressor cells) (3). In addition to their remarkable immunosuppressive capacity, the ability of MDSCs to promote angiogenesis is now considered a defining feature, and MDSCs are believed to be key drivers of tumor neovascularization (4).

[0003] IFNγ and IL6 are two important proinflammatory cytokines that appear to function antagonistically in suppressing or promoting angiogenesis, respectively (5, 6). Although the biological effects of these apparently antagonistic activities are unclear, recent genetic data implicate the tryptophan catabolic enzyme IDO1 (indoleamine 2,3-dioxygenase 1) as a key regulatory node that sustains neovascularization by responding to local IFNγ and counteracts its antiangiogenic activity by signaling for increased IL6 production (7). Although the ability of IFNγ to induce IDO1 has been extensively characterized ( 8 ), the regulatory relationship between IDO1 and IL6 is not well understood, complicated by apparently contradictory previously published findings in which IDO1 was found to either positively or negatively affect IL6 induction ( 9 – 12 ). Even with regard to IDO1, which is a positive driver of IL6 induction, at least two distinct signaling pathways are involved. Some studies have attributed the induction of IL6 by IDO1 to signaling through the integrated stress response (ISR) pathway following IDO1-mediated activation of the general control nonderepressible 2 (GCN2) serine kinase by tryptophan depletion (11), whereas others have implicated IDO1-initiated AHR (aryl hydrocarbon receptor) signaling in response to production of the endogenous AHR ligand kynurenine (13). One possible explanation for these discrepancies is the environment, i.e., the associated cell type, in which IDO1 is expressed. IDO1 expression has been reported in a variety of immune and non-immune cells, including MDSCs, DCs (dendritic cells), macrophages, NK (natural killer) cells, endothelial cells, mesenchymal stromal cells, and fibroblasts (14). Genetic evidence strongly implicates IL6 as a downstream effector required for IDO1 to promote neovascularization, but the cell types in which IDO1 is expressed in the neovascular environment have yet to be identified, and therefore the underlying signaling pathways involved in IDO1-IL6 binding remain to be elucidated. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides, in one aspect, a method of treating retinopathy or inhibiting pathological neovascularization in a subject. [Means for solving the problem]

[0005] The method involves removing or inhibiting IDO-dependent vascularizing cells (IDVCs) in the subject's eye. IDVCs are functionally characterized as having a role in establishing and maintaining the requirement for induction of IDO1 in IDVCs during neovascularization. In one embodiment, inhibition of IDVC activity is achieved by inhibition of IDO1. In another embodiment, IDVCs are inhibited or removed using antibodies or antibody drug conjugates (ADCs) directed against cell surface markers of IDVCs. In another embodiment, the IDVCs are inhibited or eliminated by blocking or inhibiting the integrated stress response (ISR) in the IDVCs.

[0006] In one embodiment, the method comprises: (a) blocking or inhibiting the expression, induction, activity or signaling of GCN2 (general control nonderepressible 2); and / or (b) blocking or inhibiting expression, induction, activity or signaling of activating transcription factor 4 (ATF4); and / or (c) Blocking or inhibiting the expression, induction, activity or signal transduction of CHOP (C / EBP homologous protein / Ddit3 DNA-damage inducible transcript 3).

[0007] In another aspect, the method involves blocking or inhibiting a signaling molecule downstream of the integrated stress response.

[0008] In one embodiment, the signaling molecule is a cytokine, hi another embodiment, the cytokine is IL-6.

[0009] In another embodiment of the methods described herein, the methods further comprise blocking or inhibiting any form of vascular endothelial growth factor (VEGF) expression, induction, activity, or signaling. In another embodiment, the method further comprises administering an inhibitor of indoleamine 2,3 dioxygenase-1 (IDO1) expression, induction, activity, or signaling.

[0010] Treatment compositions including treatments, including kits for administration, as well as other aspects and advantages, are described in the detailed description below. [Brief description of the drawings]

[0011] [Figure 1-1]Figures 1A-1D show GCN2 phenocopy loss of IDO1 in limiting 4T1 lung metastasis growth, neovascularization, and IL6 elevation. (Figure 1A) Lung staining with India Ink to visualize metastatic burden 5 weeks after orthotopic 4T1 breast tumor cell engraftment in WT, IdoT- / -, and Gcn2- / - mice. (Figure IB) Kaplan-Meier survival curves for cohorts of WT, IdoT- / -, and Gcn2- / - mice (N7 mice / group) after orthotopic implantation of IxlO44T1 cells. Significance was assessed by two-group log-rank test. [Figure 1-2] As above. (Fig. 1C) Confocal images of immunofluorescently stained blood vessels (anti-CAVl; Cy3) and nuclei (DAPI) within 4T1 lung metastases from WT, IdoT- / -, and Gcn2- / - mice at 5-6 weeks. Scale bar = 100 µm. (Fig. ID) Quantitative assessment of neovascular density within lung metastases from WT, IdoT- / -, and Gcn2- / - mice 5-6 weeks after orthotopic 4T1 breast tumor cell engraftment (N = 3 mice / group). Significance between the WT control group and the IdoT- / - and Gcn2- / - groups (shown along the straight bars) was determined by one-way ANOVA with Dunnett's multiple comparison test, and significance between the Ido1- / - and Gcn2- / - groups (shown in brackets) was graphed as means ± SEM and determined by one-way unpaired Student's t-test. [Figure 2-1] Figures 2A-2C demonstrate that inhibition of ISR but not AHR signaling blocks induction of IL6 expression as well as inhibition of IDO1 activity in vitro. (Figure 2A) To assess the effect of IDO1 inhibitor (Epacadostat; left column), ISR inhibitor (ISRIB; middle column) and AHR inhibitor (BAY-218; right column) treatment on IDO1 activity, kynurenine levels were measured spectrophotometrically in the supernatants of IFNγ+LPS-treated U937 and HL60 cells. Fold induction compared to controls without IFNγ+LPS induction is graphed as mean ± SEM (N = 3 trials / condition). [Figure 2-2] Ibid. (FIGS. 2B-2C) qPCR-based assessment of IFNγ+LPS-mediated induction (by row) of Il6, Atf4, Chop, and Cyp1a1 gene expression relative to untreated baseline levels in U937 and HL60 cells, and the effect of IDO1 inhibitor (Epacadostat; left column), ISR inhibitor (ISRIB; center column), and AHR inhibitor (BAY-218; right column) treatment on the induction of each of these genes. Fold induction compared to control without IFNγ+LPS induction is graphed as mean ± SEM (N = 3 trials / condition). Statistical significance of the effect of different inhibitor treatments was determined by two-tailed Student's t-test. Percent inhibition by inhibitor treatment is also noted at the top of each graph. [Figure 2-3] Same as above [Figure 3-1] Figures 3A-3G demonstrate that blockade of ISR signaling in oxygen-induced retinopathy limits angiogenesis and IL6 induction, but IDO1 remains elevated. (Figure 3A) Fluorescence microscopy of vasculature in retinal squamous epithelium after OIR and normoxia. (Figure 3B-D) Eyes from WT, Ido1- / - and Gcn2- / - OIR cohorts assessed at P17 for (Figure 3B) neovascular area across the whole retinal area (N ≥ 6 eyes / group). (Figure 3C) IL6 present in pooled vitreous humor (40 eyes / pool; N ≥ 2 pools / group), (Figure 3D) Kynurenine present in pooled vitreous humor (40 eyes / pool; N ≥ 2 pools / group). (Figure 3B-3D) Straight bars: ANOVA with Dunnett's test comparing WT to other groups. [Figure 3-2] As above (Fig. 3E) WT OIR cohorts injected intraocularly with siRNA targeting Gcn2, Atf4, Chop, Ahr, or non-targeting controls assessed by t-test for neovascular area across the total retinal area (N > eyes / group). (Fig. 3F, 3G) Confocal images of retinal flat mounts (Fig. 3B and 3E) stained for blood vessels (B4-Alexa 488-isolectin) and IDO1 (CY3). Scale bar = 50 μm. [Figure 4-1](FIGS. 4A-4E) Demonstrating the localization of IDO1 expression to Gr1-positive immune cell populations in 4T1 metastases and OIR. Confocal images stained for: (FIG. 4A) IDO1 (Cy3) and blood vessels (antiCAV1; FITC) from WT, IDO1- / -, and Gcn2- / - (scale bar = 20 μm); (FIG. 4B) IDO1 (Cy3) and CD45 (FITC) from WT and IDO1- / - mice compared to primary tumors and spleens from WT mice (scale bar = 50 μm); (FIG. 4C) IDO1 (Cy3) and Cr1 or CD11b (Alexa 488) from WT and IDO1- / - mice (scale bar = 50 μm). [Figure 4-2] As above (Fig. 4D) Retinal flat mounts stained for IDO1 (Cy3) and CD45 or Gr1 (FITC) from WT OIR-induced pups (scale bar = 10 μm). (Fig. 4E) Comparison of neovascular area over total retinal area between α-Gr1 and isotype control antibody-injected WT OIR-induced pups (N ≥ eyes / group) by t-test. [Figure 5-1] Figures 5A-5F demonstrate that the Gr1+ CD11blo subpopulation promotes neovascularization via expression of IDO1. (Figure 5A) Flow cytometry plots of CD45+ magnetic bead-selected, Gr1+ gated immune cells isolated from 4T1 metastasis-burdened lungs, showing gating for the separation of CD11blo (P3) and CD11bhi (P4) cells (see Figure 12 for full gating scheme). [Figure 5-2]Ibid. (FIG. 5B) Confocal images of isolated CD45+ Gr1+ CD11blo and CD45+ Gr1+ CD11bhi cells attached to slides and stained for IDO1 (Cy3), CD11b (Alexa 488) and nuclei (DAPI). Scale bar=50 μm. (FIG. 5C, FIG. 5E) Matrigel photos paired with confocal images stained for blood vessels (anti-CAV1; Cy3) and nuclei (DAPI). Matrigel plugs were incorporated with PBS alone or with 1.5×106 Gr1+ CD11blo or Gr1+ CD11bhi cells obtained from: (FIG. 5C) WT or Ido1− / − mice and transplanted into WT mice, or (FIG. 5E) WT mice and transplanted into WT mice that received vehicle or 50 mg / kg epacadostat bid for the last 3 days. Scale bar = 100 μm. (Figures 5D, 5F) Neovascular density quantification corresponding to images in Figures 5C and 5E (N ≧ 5 plugs / condition). Straight bars: ANOVA with Dunnett's test for PBS compared to other groups. Brackets: ANOVA with Sidak test for selected pairs. [Figure 5-3] Figures 5D-5E Same as above [Figure 5-4] Figure 5F Same as above [Figure 6-1]Figures 6A-6F show that Gr1+ CD11blo cells require both IL6 and GCN2 to promote neovascularization and counter IFNγ by inducing IDO1. (Figures 6A, 6E) Matrigel plug photographs paired with confocal images stained for blood vessels (anti-CAV1; Cy3) and nuclei (DAPI). Matrigel plugs were incorporated with PBS alone or with 1.5x106 Gr1+ CD11blo or GR1+ CD11bhi cells obtained from: (Figure 6A) Il6- / - or Gcn2- / - mice and transplanted into WT mice, or (Figure 6E) WT or Ido1- / - mice and transplanted into Ifng- / - mice. Scale=100 μm. (Figures 6B, 6F) Quantitative assessment of neovascular density (N≧3 plugs / condition) corresponding to the images in Figures 6A and 6E. Straight bars: ANOVA with Dunnett's test for PBS compared to other groups. Brackets: ANOVA with Sidak's test for selected pairs. (Fig. 6C, 6D) Confocal images obtained from WT and Ifng- / - mice: (Fig. 6C) retinal OIR flat mounts stained for blood vessels (B4-Alexa 488 isolectin) and IDO1 (Cy3), or (Fig. 6D) 4T1 lung metastases for blood vessels (anti-CAV1: FITC) and IDO1 (Cy3). Scale = (Fig. 6C) 50 μm or (Fig. 6D) 10 μm. [Figure 6-2] Figures 6B to 6D [Figure 6-3] Figures 6E-6F as above [Figure 7-1]Figures 7A-7E show that the autofluorescent subpopulation of Gr1+ CD11blo cells promoting neovascularization contains an IDO1-dependent subset characterized by high CD11c and asialo-GM1. (Figure 7A) Confocal images of autofluorescent high (AFhi) and low (AFlo) subsets of Gr1+ CD11blo cells from 4T1 lung metastases stained for IDO1 (Cy3) and nuclei (DAPI). Scale = 100 μm. (Figure 7B) Matrigel plug photographs paired with confocal images (scale = 100 μm) stained for blood vessels (anti-CAV1; Cy3) and DAPI. Matrigel plugs were isolated from WT mice and incorporated with 1.5x106 AFhi or AFlo cells implanted into WT mice. (Figure 7C) Plots following Gr1+ CD11blo gating: (left) gating on 488 / 530 intensity, and (right) subsequent gating on CD11c-PE (y-axis) and asialo-GM1-APC intensity (x-axis) intensities. (Figure 7D) CD1chi asialo-GM1hi and CD11clo asialo-GM1lo cells were isolated as shown in Figure 7C and stained with: (left panel) IDO1 (Cy3) and DAPI (scale = 50 μm) for confocal imaging, or (right panel) Giemsa (scale = 20 μm) for light microscopy. (FIG. 7E) Quantitative assessment of neovascular density in Matrigel plugs incorporated with PBS alone or with 5x104 CD11chi asialo-GM1hi (hi / hi) or CD11clo asialo-GM1lo (lo / lo) cells obtained and implanted from: (left) WT or IDO1- / - mice and implanted into WT mice, or (right) WT, IDO1- / -, Il61- / -, or Gcn2- / - mice and implanted into either WT or Ifng- / - mice (N ≥ plugs / condition). WT and IDO1- / - to WT groups are included in both panels for comparison purposes. Straight bars: ANOVA with Dunnett's test for comparing (left) PBS or (right) WT with the other groups. Brackets: ANOVA with Sidak's test for selected pairs. [Figure 7-2] Figures 7D ~7E Same as above [Figure 8] Figures 8A-8B show validation of transgenic mouse strains. (Figure 8A) Genotyping. Purified genomic DNA from each of the four transgenic mouse strains utilized in this study and wild-type controls was assayed by PCR using primer pairs designed to detect each of the genetic alterations that result in functional disruption of the target genes (see Tables 1-3). The primers used for each set of analyses are indicated at the top of each gel image, and the order in which the PCR products were loaded is listed on the side. Changes in the banding patterns of each transgenic staining are consistent with the predictions of each primer set. (Figure 8B) Cytokine analysis. Lung homogenates from (left) Il6- / - and (right) Ifng- / - strain mice and corresponding WT controls were assessed for IL6 and IFNγ levels, respectively, 24 hours after intranasal instillation of 25 μg LPS by a cytokine bead array immunoassay-based analysis (BD Biosciences) as previously described (Smith, 2012, ref. 9). Means ± SEM (N = 4 mice) are plotted on a logarithmic scale because of the large difference between values ​​obtained for WT and knockout animals. [Figure 9] Figure 9 shows that primary 4T1 tumors grow at comparable rates in WT, Ido1- / - and Gcn2- / - mice. Female WT, Ido1- / - and Gcn2- / - mice each received orthotopic implants of 1x104T1 cells (N = 5 tumors). Caliper measurements were taken biweekly and primary tumor volumes were calculated at day 11 post-engraftment, when palpable tumor masses were evident. Data are plotted as means ± SEM. [Figure 10]Figure 10 shows the assessment of cell viability in response to various inhibitor treatments. Viability of U937 and HL60 cells induced with IFNγ+LPS and treated with either the IDO1 inhibitor Epacadostat, the ISR inhibitor ISRIB or the AHR inhibitor BAY-218 (top to bottom) was assessed using the LDI Cell CountEZ TPS assay kit. Cell viability of the various treatment groups was determined by normalization to the corresponding set of untreated control cells and graphed as mean ± SEM (N = 3 wells / condition). An additional set of cells from each group was treated with the proteasome inhibitor Bortezomib (BTZ, 20 nM) as a positive control for pharmacologically induced cell death. [Figure 11] Figure 11 shows a comparison of retinal flat mount images to visualize the effect on neovascularization of Gcn2 loss compared to Ido1 loss in a mouse model of oxygen-induced retinopathy. As an example of normal retinal neovascularization, (top) representative images of blood vessels in retinal flat mounts obtained at P17 from WT neonates exposed to hyperoxia from P7-P12 to induce OIR or maintained under constant normoxia, (bottom) Ido1- / - and Gcn2- / - neonates exposed to hyperoxia from P7-P12 to induce OIR. [Figure 12] FIG. 12 shows the isolation of cells by flow cytometry for assessment of vascular promotion using surface markers Gr-1 and CD11b. Fluorescence-activated cell sorting was used to enrich the cell population identified as IDO1+ from 4T1 lung metastases for incorporation into Matrigel plugs. Plots of the sequential gating used are shown from left to right: (P1) gating on forward scatter (X-axis) and side scatter (Y-axis), (P2) gating on PerCP-Cy5 intensity to select Gr-1+ cells, (P3 and P4) gating on FITC intensity to select CD11blo and CD11bhi cells, respectively. The gap between the P3 and P4 gates was included to reduce the possibility of cross-contamination between the two sorted populations. [Figure 13-1] Figures 13A-13B show FACS-based identification and isolation of highly autofluorescent subpopulations of Gr1+ CD11blo cells. (Figure 13A) Plots of sequential gating used to isolate autofluorescence positive cells are shown from left to right: (P1) gating on forward scatter (X-axis) and side scatter (Y-axis), (P2) gating on PerCP-Cy5 intensity to select Gr1+ cells, (P3) gating on PE-Cy7 intensity to select CD11blo cells, and (P4,5) gating on 488 / 530 nM excitation / emission channels to select AFhi (autofluorescence-high, P4) and AFlo (autofluorescence-low, P5) cell populations. (Figure 13B) Detection of autofluorescence in different channels. Following sequential gating of Gr1+ CD11blo cells (plots 1-3 from left to right), the autofluorescence profiles at five different excitation / emission wavelengths are shown in plots 4-8. [Figure 13-2] Same as above [Figure 14-1]Figures 14A-14B show the identification of CD11c and asialo-GM1 as markers of Ido1-expressing cell populations. (Figure 14A) Evaluation of a panel of cell surface markers on the Gr1+ CD11blo AFhi population by flow cytometry. Antibodies against F480, B220, CD3ε, CD11c, asialo-GM1, Siglec-F, CD31, IFNGR1 (CD119), MHC-II and PDL1 (CD274) were used for detection in either the 633 / 660 (Alexa647) or 633 / 780 (APC-Cy7) channels. The blue lines on each graph show histograms representing each antibody-associated signal relative to the baseline indicated by the red line. (Figure 14B) Simultaneous evaluation of CD11c and asialo-GM1 surface markers in the Gr1+ CD11blo AFhi population. The sequential gating plots used are shown from left to right: (P1) gating on forward scatter (X-axis) and side scatter (Y-axis), (P2) gating on PerCP-Cy5 intensity to select for Gr1+ cells, (P3) gating on PE-Cy7 intensity to select for CD11blo cells, (P4) gating on 488 / 530 intensity to select for AFhi cells, (P5, 6) gating on CD11c-PE intensity (X-axis) and asialo-GM1-APC intensity (Y-axis) to select for CD11chi asialo-GM1hi (top right) and CD11clo asialo-GM1lo (bottom left) cells. [Figure 14-2] Same as above [Figure 15-1]15A-15D show a comparison of the neovascularization potential of CD11chi asialo-GM1hi and CD11clo asialo-GM1lo cells within the AFhi population. (Fig. 15A, 15C) Matrigel plug photographs paired with confocal images stained for blood vessels (anti-CAV1; Cy3) and nuclei (DAPI). Matrigel plugs were incorporated with PBS alone or with 5x104 CD11chi asialo-GM1hi or CD11clo asialo-GM1lo cells obtained and implanted from: (Fig. 15A) WT or Ido1- / - mice and implanted into WT mice, or (Fig. 15C) Il6- / - or Gcn2- / - mice and implanted into WT mice. Scale = 100 μm. (Fig. 15B) Photographic images of Matrigel plugs excised 9 days after subcutaneous implantation into mice. Plugs incorporated 3-fold serial dilutions (shown on the left) of CD11chi asialo-GM1hi and CD11clo asialo-GM1lo cells isolated from and transplanted into WT mice. (FIG. 15D) Quantitative assessment of angiogenesis density (N≧2 plugs / condition) corresponding to the images in FIG. 15C. Straight bars: ANOVA with Dunnett's test for PBS compared with other groups. Brackets: ANOVA with Sidak's test for selected pairs. [Figure 15-2] Same as above [Figure 15-3] Same as above [Figure 16-1]Figures 16A-16B. CD11chi asialo-GM1hi IDVCs from Ido1- / -, Il6- / - and Gcn2- / - mice show impaired ability to support host IFNγ-dependent neovascularization. (Figure 16A) Photographic images of Matrigel plugs incorporating CD11chi asialo-GM1hi cells (left) paired with confocal images (right) of sections cut from each Matrigel plug excised 9 days after subcutaneous implantation into mice and stained for blood vessels (anti-CAV1; Cy3) and nuclei (DAPI). From top to bottom, rows represent Matrigel plugs introduced into WT and Ifng- / - mice. From left to right, columns represent Matrigel plugs incorporating cells obtained from WT, Ido1- / -, Il6- / - and Gcn2- / - mice. Scale bar = 100 μm. These images are relevant for the quantitative assessment of neovascular density shown in Figure 7E. (Figure 16B) Detection of IDO1 in CD11chi asialo-GM1hi cells isolated from different transgenic mouse strains. Confocal images of isolated CD11chi asialo-GM1hi cells were attached to slides and stained with either a secondary Cy3-conjugated antibody (first slide) or anti-IDO1 (Cy3) and nuclei (DAPI). Scale bar = 50 μm. The transgenic mouse strains from which the cells were isolated (WT, Ido1- / -, Gcn2- / -, Il6- / -, Ifng- / -) are listed on top of each image. [Figure 16-2] Same as above [Figure 17-1]Figures 17A-17B show flow cytometry-based validation of the fluorescent signals obtained with the antibodies used to identify and isolate IDVCs. (Figure 17A) Fluorescence subtracts one confirmation of CD11c and asialo-GM1 detection for IDVCs from 4T1 lung metastases. Plots from the sequential gating strategy used to identify and isolate CD11chi asialo-GM1hi IDVCs are shown from left to right: (P1) gating on forward scatter (X-axis) and side scatter (Y-axis), (P2) gating on PerCP-Cy5 intensity (X-axis) to select the Gr1+ population, (P3) gating on PE-Cy7 intensity (X-axis) to select the CD11blo population, (P4) gating on 488 / 530 nm excitation / emission channels to select the AFhi population, and (P5, 6) gating on PE intensity (Y-axis) and Alexa647 intensity (X-axis) to separate the CD11chi asialo-GM1hi and CD11clo asialo-GM1lo populations. (Top) All antibodies present, (middle) no anti-CD11c antibody, (bottom) no anti-asialo-GM1 antibody. (FIG. 17B) Fluorescence minus one confirmation of CD45 detection on IDVCs from 4T1 lung metastases. Plots from the sequential gating strategy used to identify and isolate CD11chi asialoGM1hi IDVCs, modified as follows: (P1) gating on forward scatter (X-axis) and side scatter (Y-axis), (P2) gating on APC-Cy7 intensity (X-axis) to select the CD45+ population, (P3) gating on Pacific blue intensity (X-axis) to select the Gr-1+ population, (P4) gating on PE-Cy7 intensity (X-axis) to select the CD11blo population, (P5) gating on 488 / 530 nm excitation / emission channels to select the AFhi population, and (P6, 7) gating on PE intensity (Y-axis) and Alexa647 intensity (X-axis) to separate the CD11chi asialo-GM1hi and CD11clo asialo-GM1lo populations. (Top row) All antibodies present; (bottom row) anti-CD45 antibodies were not included. [Figure 17-2] Same as above [Figure 18-1] Figures 18A-18C show validation of the CD11blo status of IDVCs. (Figure 18A) Fluorescence minus one assessment of CD11b staining of CD11chi asialo-GM1hi AFhi IDVCs from 4T1 lung metastases. Plots from the modified sequential gating strategy used to identify CD11chi asialo-GM1hi CD1blo IDVCs are shown from left to right: (P1) gating on forward scatter (X-axis) and side scatter (Y-axis), (P2) gating on PE intensity (Y-axis) and Alexa647 intensity (X-axis) to select CD11chi asialo-GM1hi cells, (P3, 4) gating on 488 / 530 nm excitation / emission channels (X-axis) and PE-Cy7 intensity (Y-axis) to separate AFhi CD11chi and AFhi CD11blo cells. (Top) All antibodies present, (Bottom) anti-CD11b antibody was not included. The rightmost column shows an overlay from the previous column, demonstrating a shift (top) in PE-Cy7 intensity of both the P3 and P4 gated populations relative to the CD11b unstained population, and an overlap (bottom) in 488 / 530 autofluorescence between the P3 and P4 gated populations and the CD11b unstained population. (Figure 18B) FACS sorted AFhi CD11chi and AFhi CD11blo cells isolated as shown in Figure 18A were adhered to slides and stained for IDO1 (Cy3) and DAPI for confocal imaging (scale bar = 50 μm). (Figure 18C) Photographic images of excised Matrigel plugs 9 days after subcutaneous implantation into WT mice paired with confocal images (right) of frozen sections cut from each Matrigel plug and stained for blood vessels (anti-CAV1; Cy3) and nuclei (DAPI). The rows from top to bottom show examples of Matrigel plugs loaded with AFhi CD11chi or AFhi CD11blo cells. [Figure 18-2] Same as above [Figure 19-1]Figures 19A-19C. Flow cytometry-based assessment of Gr-1, Ly6C and Ly6G status of IDVCs compared to MDSCs. FIG. 19A is a representative example of a branched gating strategy used to identify both conventional MDSCs and IDVCs from 4T1 lung metastases as follows: (P1 top) forward scatter (X-axis) and side scatter (Y-axis), followed by (P2 left) gate on either Pacific Blue intensity (X-axis) and PE-Cy7 intensity (Y-axis) to select the CD45hi CD11b hi population (MDSCs) or (P2 right) gate on Pacific Blue intensity (X-axis) to select the CD45+ population, (P3) gate on PE intensity (X-axis) and Alexa647 intensity (Y-axis) to select the CD11c hi asialo-GM1 hi population, and (P4) gate on 488 / 530 nm excitation / emission channels (X-axis) and PE-Cy7 intensity (Y-axis) to select the AFhi CD11blo population. The bottom three columns show streptavidin-APC-Cy7 fluorescent staining intensity in MDSC (Figure 19B) and IDVC (Figure 19C) populations from three independent samples labeled with either biotin-conjugated anti-Gr-1, anti-Ly6C or anti-Ly6G antibodies (blue histograms) compared to streptavidin-APC-Cy7 alone (red histograms). [Figure 19-2] Same as above [Figure 19-3] Same as above [Figure 20-1]Figures 20A-20F show that IDO1 prevents IFNg-mediated neovascular regression by suppressing the induction of Nos2. (Figure 20A) The lung metastasis survival benefit resulting from loss of IDO1 is eliminated by concomitant loss of IFNγ. Kaplan-Meier survival curves of cohorts of WT, Ido1- / -, Ifng- / - and Ifng- / -Ido1- / - mice following orthotopic implantation of 1x104 4T1 cells (N>17 mice) (significance assessed by two-group log-rank test). (Figure 20B) The lung metastasis survival benefit resulting from loss of IDO1 is similarly eliminated by concomitant loss of NOS2. Kaplan-Meier survival curves for cohorts of WT, Ido1- / -, Nos2- / - and Nos2- / -Ido1- / - mice after orthotopic engraftment of 1x104 4T1 cells (N>9 mice) (significance assessed by two-group log-rank test). (Figure 20C) IDVCs lacking IDO1 restore the ability to induce neovascularization in Nos2- / - mice. Photographic images of matrigel plugs incorporating CD11chi asialo-GM1hi cells obtained from WT or Ido1- / - mice and excised 9 days after subcutaneous implantation into WT or Nos2- / - mice (left) shown paired with confocal images of sections cut from each matrigel plug and stained for blood vessels (anti-CAV1; Cy3) (right). Scale bar = 100 μM. (FIG. 20D) (Left) Quantitative assessment of neovascular density corresponding to images from Nos2− / − mice in C (N ≧ 2 plugs / condition) plotted as mean ± SEM and assessed for statistical significance by Student's t test. (Right) Quantitative assessment of neovascular density in Matrigel plugs from WT and Ifng− / − mice from FIG. 7 was included for comparison. ****, P < 0.0001; ns, not significant. (FIG. 20E) Loss of IDO1 leads to elevated NOS2 expression in lung metastases. Confocal images from 4T1 lung metastases stained for NOS2 (FITC, green) from WT, Ido1− / −, Nos2− / − and Ifng− / − mice. (FIG. 20F) IDO1 inhibition similarly induces elevated NOS2 in lung metastases.Confocal images from 4T1 lung metastases stained for NOS2 (FITC, green) and nuclei (DAPI, blue) from WT mice orally dosed with either vehicle or 50 mg / kg epacadostat bid for 3 days. The dotted white line in the top panel delineates the area of ​​enlargement shown in the corresponding bottom panel. [Figure 20-2] Same as above [Figure 20-3] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The compositions and methods described herein are based on the inventors' identification of IDVCs in the retina, where induction of IDO1 leads to integrated stress response (ISR) activation that is central to the ability of IDVCs to support the establishment and maintenance of neovascularization.

[0013] In addition to immunosuppression, it is generally accepted that MDSCs (myeloid-derived suppressor cells) also support tumor angiogenesis. Recently, it has been reported that the tryptophan catabolic enzyme IDO1 (indoleamine 2,3-dioxygenase) is involved in promoting neovascularization through its position as a key regulatory node between the inflammatory cytokines IFNγ (interferon-γ) and IL6 (interleukin 6). However, the cell type expressing IDO1 has not been reported so far. The ability of Gr-1+ MDSCs to induce neovascularization in vivo within their heterogeneous expansion is mainly due to the highly autofluorescent CD11b lo It is reported herein to be associated with a small subset of cells. IDO1 expression is further restricted to a distinct CD11c, asialo-GM1 double positive subpopulation of these cells, referred to here as IDVCs (IDO1-dependent vascularization cells), due to the key role that IDO1 activity in these cells plays in promoting neovascularization. Mechanistically, induction of IDO1 in IDVCs provides a negative feedback constraint on the anti-angiogenic effects of host IFNγ by signaling for the production of IL6 via GCN2-mediated activation of the integrated stress response in these cells. These findings reveal fundamental molecular and cellular insights into how IDO1 interacts with the inflammatory milieu to promote neovascularization.

[0014] I. Composition Components and Methods In describing the compositions and methods described herein, the various components can be defined by using technical and scientific terms that have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and by reference to published texts. Such texts provide one of ordinary skill in the art with a general guide to many of the terms used in the present application. The definitions contained herein are provided for clarity in describing the components and compositions herein and are not intended to limit the invention as claimed.

[0015] A. Eligibility "Patient" or "subject" or "individual" as used herein means mammals, including humans, domestic or agricultural animals, household animals or pets, and animals typically used in clinical trials. In one embodiment, the subject of these methods and compositions is a human. In one embodiment, the subject has an eye disease. In another embodiment, the subject has an eye disease and has not yet been treated with any therapy. In another embodiment, the subject has an ocular disease and is treated with conventional methodologies, e.g., intraocular administration of a vascular endothelial growth factor (VEGF) inhibitor, but does not respond optimally or in a sufficient manner to achieve sufficient therapeutic benefit. In another embodiment, the subject having the ocular disease has been administered a VEGF inhibitor or blocking agent, but has not achieved the desired maximal therapeutic response observed in other patients upon administration of the VEGF inhibitor or blocking agent monotherapy.

[0016] B. Eye diseases The term "ocular disorder" refers to a disorder or disease of the eye. In one embodiment, the eye disease is characterized by neovascularization, ie, new or abnormal blood vessel formation in a tissue or portion of the eye, or the excessive blood vessel formation is in a tissue or portion of the eye. In one embodiment, the eye disorder is retinopathy. In certain embodiments, the eye disease is characterized by abnormal / altered angiogenesis. In certain embodiments, the ocular disease is characterized by intraocular neovascularization, which may be, but is not limited to, neovascularization of the optic disc, iris, retina, choroid, cornea, and / or vitreous humor. Examples of ocular diseases include, but are not limited to, glaucoma, pannus (corneal vascular proliferation), pterygium, macular edema, macular degeneration (e.g., age-related macular degeneration), retinopathies (e.g., diabetic retinopathy, vascular retinopathy, retinopathy of prematurity), diabetic retinal ischemia, diabetic macular edema, retinal degeneration, retrolental fibroplasia, corneal graft neovascularization, central retinal vein occlusion, pathologic myopia, uveitis, ocular inflammatory diseases, and proliferative vitreoretinopathy. In certain embodiments, the ocular disease is selected from the group consisting of retinopathy (e.g., retinopathy of prematurity, diabetic retinopathy (e.g., proliferative diabetic retinopathy), and macular degeneration (e.g., dry or wet macular degeneration). In yet another embodiment, the ocular disease or disorder is a macular dystrophy (eg, Stargardt's disease, Viteriform macular dystrophy (VTM), North Carolina macular dystrophy, or Best's disease).

[0017] Age-related macular degeneration (AMD) is a degenerative disease of the macula that often results in progressive vision loss. It is diagnosed as either dry (non-neovascular) or wet (neovascular). The rate of disease progression can vary between individuals and is associated with multiple risk factors. Early AMD includes clinical signs such as drusen and abnormalities of the retinal pigment epithelium. Late stage AMD can be neovascular (also known as wet or exudative) or non-neovascular (known as atrophic, dry, or non-exudative). Late-stage non-neovascular AMD is characterized by the development of macular geographic atrophy (GA) and loss of central vision, leading to severe and permanent visual impairment and legal blindness (see, e.g., Mitchell, P. et al. (2018)). Age-related macular degeneration is discussed in Lancet, 392(10153), 1147-1159, which is incorporated herein by reference. Late stage neovascular AMD is characterized by the development of edema and retinal detachment with loss of central vision, leading to severe and permanent visual impairment and legal blindness.

[0018] C. IDO-dependent angiogenic cells (IDVCs) Described herein is a newly isolated cell type, termed IDO1-dependent angiogenic cells (IDVCs). This novel immune cell subtype was isolated in mice from within the heterogeneous Gr-1+ MDSC population and is functionally distinguishable from the majority of MDSCs by its ability to induce neovascularization and sustain these new blood vessels in the presence of IFNγ via ISR-driven production of IL6 activated by IDO1.

[0019] In addition to immunosuppression, it is generally accepted that MDSCs (myeloid-derived suppressor cells) also support tumor angiogenesis. Recently, it has been reported that the tryptophan catabolic enzyme IDO1 (indoleamine 2,3-dioxygenase) is involved in promoting neovascularization through its position as a key regulatory node between the inflammatory cytokines IFNγ (interferon-γ) and IL6 (interleukin 6). As described herein, the ability of Gr-1+ MDSCs to induce neovascularization in vivo within a heterogeneous population is primarily due to the expression of highly autofluorescent CD11b lo Associated with a small subset of cells. IDO1 expression is further restricted to a distinct CD11c, asialo-GM1 double positive subpopulation of these cells, referred to here as IDVCs, due to the key role that IDO1 activity in these cells plays in promoting neovascularization. Mechanistically, induction of IDO1 in IDVCs provides a negative feedback constraint on the anti-angiogenic effects of host IFNγ by signaling for the production of IL6 via GCN2-mediated activation of the integrated stress response in these cells. In one embodiment, the IDVCs are localized to the retina of the eye. In another embodiment, the IDVCs are localized to the choroid of the eye. In another embodiment, the IDVCs are localized to the retina and choroid of the eye.

[0020] Induction of IDO1 in IDVC cells leads to ISR (Integrated Stress Response) activation, which is central to the ability of IDVCs to support the establishment and maintenance of neovascularization. IDVCs can be isolated nearly homogeneously from mice by flow cytometry using antibodies to detect high levels of CD45, CD11c, asialo-GM1 and Gr-1, and low levels of CD11b on the cell surface, coupled with their relatively high levels of intrinsic autofluorescence.

[0021] IDVCs are leukocytes or other cell types that respond to cytokines, including the cytokine IFNγ (interferon-γ), and have been functionally characterized as having a role in neovascularization by inducing IDO1, the establishment and maintenance of which requires the induction of IDO1 in IDVCs. Characterization of the cells as IDVCs may involve isolating the cells and incorporating them into Matrigel plugs that are implanted under the skin of mice. After several days, the implantation site is evaluated for the presence of blood vessels.

[0022] In one embodiment, the IDVCs are characterized by the following phenotype: CD11b+CD14-CD15+ or CD11b+CD14-CD66b+. Alternatively, the CD33 myeloid marker can be used instead of CD11b, since very few CD15+ cells are CD11b-. In another embodiment, the IDVCs are characterized by the following phenotype: Lin-HLA-DR- / loCD33+ or Lin-HLA-DR- / loCD11b+CD14-CD15+CD33+. In one embodiment, one or more of the following cell surface markers are localized on IDVCs: CD11c, CD274 (PDL1), MHC class II, CD4, CD31 (PECAM-1), CD202B (TIE2), CD205 (DEC-205), Siglec 8, or EMR1.

[0023] D. Integrated Stress Response (ISR) Pathway Nodes The integrated stress response (ISR) is an inducible and ubiquitous signaling pathway in eukaryotic cells that is activated in response to a variety of physiological stimuli and pathological conditions. Such stimuli generally include cell-extrinsic factors and stressors, such as hypoxia, amino acid deprivation, glucose deprivation, and viral infection. In addition, intracellular stresses, such as endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins in the endoplasmic reticulum, can also activate the ISR. Furthermore, in the context of cancer biology, the ISR can be triggered by oncogene activation.

[0024] As described herein, the ISR is shown to be a biologically relevant signaling pathway through which IDO1 acts to promote pathological neovascularization in retinopathies. Inhibition of key ISR nodes such as GCN2, CHOP, and ATF4 has been shown to reduce neovascularization in an animal model of retinopathy, namely the oxygen-induced retinopathy (OIR) mouse model. As used herein, the term "ISR node" refers to any component along a signaling pathway initiated by stress-activated eIF2α kinase. In certain embodiments, the ISR node refers to GCN2, CHOP, and / or ATF4.

[0025] General control nonderepressible 2 (GCN2) is a master regulator kinase of amino acid homeostasis and is important for cancer survival in an amino acid-deprived tumor microenvironment. GCN2 is also designated in humans as eukaryotic translation initiation factor 2-alpha kinase 4 (EIF2AK4), eIF-2-alpha kinase GCN2, and GCN2-like protein, and is encoded by the EIF2AK4 gene. GCN2 is a metabolic stress-sensing protein kinase that phosphorylates the α subunit of eukaryotic translation initiation factor 2 (EIF2S1 / eIF-2-α) in response to low amino acid availability. The sequence of hGCN2 is known in the art and can be found, for example, in UniProtKB-Q9P2K8.

[0026] C / EBP homologous protein (CHOP), also known as growth arrest and DNA damage-inducible protein 153 (GADD153), belongs to the CCAAT / enhancer-binding protein (C / EBP) family. CHOP dimerizes with other C / EBP members, altering their DNA-binding and transactivation properties. It induces growth arrest and apoptosis after endoplasmic reticulum stress or DNA damage. The sequence of hCHOP is known in the art and can be found, for example, in UniProtKB-P35638.

[0027] Activating transcription factor 4 (ATF4) is a transcription factor that binds to the cAMP response element (CRE) (consensus: 5'-GTGACGT[AC][AG]-3') and acts both as a regulator of normal metabolic and redox processes and as a master transcription factor during the integrated stress response (ISR) (PubMed:1847461, PubMed:16682973, PubMed:31444471, PubMed:32132707). ATF4 is a core effector of the ISR and is required for adaptation to various stresses, such as endoplasmic reticulum (ER) stress, amino acid starvation, mitochondrial stress, or oxidative stress. The sequence of hATF4 is known in the art and can be found, for example, in UniProtKB-P18848.

[0028] E. IDO1 Indoleamine 2,3-dioxygenase (IDO1) is a tryptophan catabolic enzyme that catalyzes the first step in the conversion of tryptophan to kynurenine. IDO1 is an extrahepatic enzyme that catabolizes the essential amino acid tryptophan independently of tryptophan metabolic processing in the liver. IDO1 monotherapy for the treatment of eye disorders is described in International Publication No. WO 2016 / 100851, see also U.S. Patent No. 10,535,035, which are incorporated herein by reference. IDO1 may be referred to herein as IDO.

[0029] F. VEGF and VEGFR "VEGF" refers to vascular endothelial growth factor, which induces angiogenesis or the angiogenic process. As used herein, the term "VEGF" includes various subtypes or isoforms of VEGF (also known as vascular permeability factor (VPF) and VEGF-A) that are generated by alternative splicing of the VEGF-A / VPF gene, including, for example, VEGF121, VEGF165, and VEGF189. Additionally, as used herein, the term "VEGF" includes VEGF-B, VEGF-C, VEGF-D and VEGF-E, which act through their cognate VEGF receptors (i.e., VEGFRs) to induce angiogenesis or the angiogenic process. The term "VEGF" includes any member of a class of growth factors that bind to a VEGF receptor, such as VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), or VEGFR-3 (FLT-4). The term "VEGF" can be used to refer to a "VEGF" polypeptide or a gene or nucleic acid encoding "VEGF." See U.S. Patent Application Publication No. 2019 / 0380187, which is incorporated herein by reference.

[0030] G. Inhibitors, Blockers, and Antagonists The general terms "blocker," "inhibitor," or "antagonist" refer to an agent that partially or completely inhibits the activity or production of a target molecule or target cell, e.g., as used herein, IDVCs, ISR nodes, IDO1, or VEGF. In certain embodiments, these terms refer to compositions or compounds or agents capable of decreasing the gene expression levels, mRNA levels, protein levels or protein activity of a target molecule. Exemplary forms of antagonists include, for example, proteins, polypeptides, peptides (such as cyclic peptides), antibodies or antibody fragments, peptidomimetics, nucleic acid molecules, antisense molecules, ribozymes, aptamers, RNAi molecules, and small organic molecules. Illustrative non-limiting mechanisms of antagonist inhibition include inhibiting ligand synthesis and / or stability (e.g., using antisense, ribozyme or RNAi compositions targeted to the ligand gene / nucleic acid), blocking binding of the ligand to its cognate receptor (e.g., using anti-ligand aptamers, antibodies or soluble, decoy cognate receptors), inhibiting receptor synthesis and / or stability (e.g., using antisense, ribozyme or RNAi compositions targeted to the ligand receptor gene / nucleic acid), blocking binding of the receptor to its cognate receptor (e.g., using receptor antibodies), blocking activation of the receptor by its cognate ligand (e.g., using receptor tyrosine kinase inhibitors), blocking the active site of an enzyme as a result of the enzyme binding of an inhibitor molecule that prevents binding of a natural substrate, i.e., inhibiting the interaction of an inhibitor molecule that binds to either the activity or binding site of the protein, or via an allosteric site on the protein, in order to modulate the biological activity of the protein. Furthermore, a blocker or inhibitor may directly or indirectly inhibit a target molecule.

[0031] I. Salts The compositions described herein also include any salt of the specific IDVC, ISR node, IDO, or VEGF inhibitor compound described herein. As used herein, "salt" refers to derivatives of the disclosed compounds, where the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of salts include, but are not limited to, mineral acid (HCl, HBr, H2SO4, etc.) or organic acid (acetic acid, benzoic acid, trifluoroacetic acid, etc.) salts of basic residues such as amines; alkali (Li, Na, K, Mg, Ca, etc.) or organic (trialkylammonium, etc.) salts of acidic residues such as carboxylic acids; The salts of the compounds described or referenced herein can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile (ACN) are preferred.

[0032] The "pharmaceutically acceptable salts" of the compounds described herein or incorporated by reference include a subset of the "salts" described above, the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa, 1985, p. 1418, and Journal of Pharmaceutical Science, 66, 2(1977), each of which is incorporated herein by reference in its entirety.

[0033] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0034] ii. Prodrugs The term "prodrug" refers to a compound or molecule or drug that, after administration, is metabolized (i.e., converted in the body) to the original pharmacologically active molecule or compound, e.g., an active ingredient IDO inhibitor (see, e.g., WO 2019 / 051198), a VEGF or VEGFR inhibitor or antagonist, or an ISR node antagonist or inhibitor. A prodrug is typically, if not completely, a pharmacologically inactive form that is converted or metabolized within or inside the body, e.g., by the action of endogenous enzymes or other chemicals and / or conditions, to an active form (i.e., a drug). Instead of administering the active molecule directly, a corresponding prodrug is used to improve the way the composition / active molecule is absorbed, distributed, metabolized, and / or excreted. Prodrugs are often designed to improve the bioavailability or how the drug selectively interacts with cells or processes that are not its intended target, reducing harmful or unintended, unwanted or severe side effects of the active molecule or drug.

[0035] iii. Biosimilars A "biosimilar" is a biological product, generally a large, complex molecule. It is produced from a living organism and is guaranteed to be of consistent quality with a high degree of similarity to a reference product (e.g., a biological drug already approved by the FDA). FDA-approved biosimilars must not have clinically meaningful differences from the reference drug in terms of purity, safety, molecular structure and biological activity, or potency.

[0036] iv. Antibodies and Fragments The term "antibody" or "antibody molecule" is any peptide or protein, including antibodies and fragments thereof, that binds to a specific antigen. As used herein, antibody or antibody molecule contemplates intact immunoglobulin molecules, immunologically active portions of immunoglobulin molecules, and fusions of immunologically active portions of immunoglobulin molecules.

[0037] The antibody may be a naturally occurring antibody or a synthetic or modified antibody (e.g., a recombinantly produced antibody; a chimeric antibody; a bispecific antibody; a humanized antibody; a camelid antibody, etc.). The antibody may comprise at least one purification tag. In certain embodiments, the framework antibody is an antibody fragment. The term "antibody fragment" includes a portion of an antibody that is an antigen-binding fragment or a single chain thereof. Antibody fragments can be synthetic or genetically engineered polypeptides. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be produced using recombinant techniques as a single protein chain that allows the VL and VH regions to pair to form a monovalent molecule, known as a single chain Fv (scFv). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as whole antibodies. Antibody fragments include, but are not limited to, immunoglobulin fragments including single domains (Dab; e.g., single variable light or heavy chain domains), Fab, Fab', F(ab')2, and F(v); and fusions (e.g., via linkers) of these immunoglobulin fragments, including, but not limited to, scFv, scFv2, scFv-Fc, minibodies, diabodies, triabodies, and tetrabodies. The antibody may also be a protein (eg, a fusion protein) comprising at least one antibody or antibody fragment.

[0038] Antibodies useful in the methods of the present invention are preferably "immunologically specific," which refers to proteins / polypeptides, particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

[0039] The antibody of the present invention may be further modified. For example, the antibody may be humanized. In certain embodiments, the antibody (or a portion thereof) is inserted into the backbone of an antibody or antibody fragment construct. For example, the variable light chain domain and / or the variable heavy chain domain of the antibody of the present invention may be inserted into another antibody construct. Methods for recombinantly producing antibodies are well known in the art. Indeed, commercially available vectors for certain antibody and antibody fragment constructs are available.

[0040] The antibody of the present invention may also be conjugated / linked to other moieties. For example, the antibody may be operably linked (e.g., covalently linked, optionally via a linker) to at least one cell-penetrating peptide, detectable agent, imaging agent, or imaging agent. The antibody of the present invention may also include at least one purification tag (e.g., His tag). In certain embodiments, the antibody is conjugated to a cell-penetrating peptide.

[0041] v. Aptamers The term "aptamer" refers to a peptide or nucleic acid that has an inhibitory effect on a target. Inhibition of a target by an aptamer can occur by binding to the target, by catalytically altering the target, by reacting with the target in a manner that modifies the target or its functional activity, by binding ionically or covalently to the target as a suicide inhibitor, or by facilitating a reaction between the target and another molecule. Aptamers can be peptides, ribonucleotides, deoxyribonucleotides, other nucleic acids, or mixtures of different types of nucleic acids. Aptamers can contain one or more modified amino acids, bases, sugars, polyethylene glycol spacers, or phosphate backbone units, as described in more detail herein.

[0042] vi. RNA and DNA The terms "RNA interference," "RNAi," "miRNA," "shRNA," "siRNA," and "siRNA" refer to any method in which the expression of a gene or gene product is reduced by introducing into a target cell one or more double-stranded RNAs that are homologous to the gene of interest (particularly the messenger RNA of the gene of interest). Gene therapy involves the introduction of modified RNA or DNA into cells via a number of well-known investigational vectors, recombinant viruses, and CRISPR technology; gene therapy can also involve the manipulation of RNA or DNA using recombinant techniques and / or the treatment of disease. Effective inhibition of the IDO / TDO pathway and gene products, and the VEGF pathway and gene products, can be delivered via modified RNA or modified DNA to achieve the combination therapeutic results described herein. Such genetic manipulation can also employ gene editing techniques such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and TALEN (transcription activator-like effector genome modification), among others. See, for example, the textbook Engineering, and Medicine. 2017. Human Genome Editing: Science, Ethics, and Governance. Washington, DC: the National Academies Press. https: / / doi.org / 10.17226 / 24623. Details of such methods are incorporated herein by reference.

[0043] vii. Small molecules The term "small molecule" as applied to pharmaceuticals generally refers to non-biological, organic compounds that have a relatively low molecular weight, below about 900 daltons, that affect biological processes. Small molecule drugs have easily identifiable structures and can be synthetically replicated with high confidence. In one embodiment, small molecules have a molecular weight of less than 550 Daltons, increasing the likelihood that the molecule is compatible with the intracellular absorption capacity of the human digestive system. Small molecule drugs are often administered orally as a tablet. The term small molecule drugs is used to contrast them with biologic drugs, which are often relatively large molecules such as peptides, proteins, and recombinant protein fusions, and are often produced using living organisms.

[0044] viii. Specific inhibitors of IDO1 The phrase "compositions that block or inhibit IDO1 expression, induction, activity, and / or signaling" includes, but is not limited to, small molecule enzyme inhibitors that directly target IDO1, or salts, enantiomers or prodrugs thereof. Another embodiment of such compositions includes small molecules that block or inhibit one or more targets upstream or downstream of IDO1 (including ISR nodes such as GCN2, CHOP10, and / or ATF4) in their respective pathways to inhibit the action of IDO1 and other tryptophan catabolic enzymes, or salts, enantiomers or prodrugs thereof. In another embodiment, the IDO1 inhibitor composition comprises a molecule that mimics the presence of tryptophan. In yet another embodiment, the composition containing an IDO inhibitor comprises a nucleic acid molecule, such as an siRNA or shRNA, that inhibits the translation or transcription of IDO1. In another embodiment, the IDO1 inhibitor composition comprises a protein therapeutic that binds to and inhibits the activity of IDO1. Such a protein therapeutic can include an anti-IDO1 antibody or a binding fragment thereof.

[0045] More specifically, the composition that blocks or inhibits IDO1 expression, induction, activity, and / or signaling is the following compound or a prodrug, salt, and / or therapeutically effective formulation thereof: Indoximod (1-methyl-D-tryptophan, 1MT, NLG-8189), 1-methyl-L-tryptophan, Racemic mixture of 1-methyl-D-tryptophan and 1-methyl-L-tryptophan epacadostat (INCB024360; Incyte; Wilmington, DE; Liu et al. (2010) Blood 115(17):3520-3530; Koblish et al. (2010) Mol. Cancer Ther., 9(2):489-498), Navoximod (NLG-919, GDC-0919, RG6078; Lumos Pharma / NewLink Genetics / Genentech), indoximod prodrug NLG802, BMS-986205 / ONO-7701(F001287, Hunt et al., AACR 2017, Abstract 4964), PF-06840003 / EOS200271 (EOS, Wythes et al, SITC 2016, Abstract 253), A compound of the formula 1-RD-tryptophan or 1-RL-tryptophan, where R is C1-C12 alkyl; MK-7162 / IOM2983 (Merck), LY3381916 (Lily), KHK2455 (Kyowa Hakko Kirin), HTI-1090 / SHR9146 (Hengrui Therapeutics, Inc.), DN1406131 (De Novo Pharmatech), RG70099 (Roche / CuraDev), Roxyl-WL (Xu et al. J Enzyme Inhib Med Chem.2018; 33(1): 1089-94.), TPST-8844 (Tempest Therapeutics), Ethyl pyruvate, AMG-1 (Amgen), described in Smith JR et al., Novel indoleamine 2,3-dioxygenase-1 inhibitors from a multistep in silico screen, Bioorganic & Medicinal Chemistry, 20(3): 1354-13631 February 2012, incorporated herein by reference; Methylthiohydantoin-DL-tryptophan (MTH-Trp), β-(3β)-DL-alanine, β-(3-benzo(b)thienyl)-DL-alanine, 6-nitro-L-tryptophan, Indole 3-carbinol, 3,3'-diindolylmethane, Epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-DL-tryptophan, 5-bromoindoxyl diacetate, Naphthoquinones, S-allylbrassinin, S-benzylbrassinin, 5-bromobrassinine, Phenylimidazoles, 4-phenylimidazole, Exiguamine A NSC401366, β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, Flick et al. (2013) Int. J. Tryp. Res. 6:35-45) (ARQ 761; ArQule, now owned by Merck); DX-03-12 and other compounds described in Wen, H. et al. (Design and Synthesis of Indoleamine 2,3-Dioxygenase 1 Inhibitors and Evaluation of Their Use as Anti-Tumor Agents, Molecules 2019, 24, 2124; doi:10.3390 / molecules -24112124, incorporated by reference); Compounds described in WO2014 / 186035 and U.S. Patent Application Publication No. 2018 / 030026 (Curadev); and / or Compounds described in WO2014 / 081689 and U.S. Pat. No. 9,499,497 (Vertex Pharmaceuticals).

[0046] Further suitable inhibitors are described in Wang, XX et al. (Recent advances in discovery of indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, MedChemComm, and grouped as tryptophan derivatives, inhibitors with a imidazole, 1,2,3-triazole or tetrazole scaffold, inhibitors with quinone or iminoquinone, N-hydroxyamidines).

[0047] Further examples of small molecule IDO1 inhibitors include, but are not limited to, those disclosed in PCT / US2014 / 022680 (e.g., tricyclic compounds related to imidazoisoindoles), PCT / US2012 / 033245 (e.g., fused imidazole derivatives), PCT / US2010 / 054289 (e.g., imidazole derivatives), PCT / US2009 / 041609 (e.g., compounds of formula I-VIII), PCT / US2008 / 057032 (e.g., naphthoquinone derivatives), PCT / US2009 / 041609 (e.g., compounds of formula I-VIII), PCT / US2008 / 0416032 (e.g., naphthoquinone derivatives), PCT / US2009 / 0416036 (e.g., compounds of formula I-VIII), PCT / US2009 / 0416038 (e.g., compounds of formula I-VIII), PCT / US2009 / 0416039 (e.g., compounds of formula I-VIII), PCT / US2009 / 0416037 (e.g., compounds of formula I-VIII), PCT / US2009 / 0416039 ... US2008 / 085167 (e.g., compounds of formula I-XLIV), PCT / US2006 / 42137 (e.g., compounds of formula I), PCT / US2006 / 0179837 (e.g., compounds of formula I), PCT / US2004 / 005155 [e.g., phenyl-TH-DL-trp (3-(N-phenylthiohydantoin)-indole), propenyl-TH-DL-trp (3-(N-allylthiohydantoin)-indole), and methyl-TH-DL-trp (3-(N-methyl-thiohydantoin)-indole)], PCT / US2004 / 005154 (e.g., compounds of formula I or II), U.S. Pat. No. 7,705,022 (e.g., compounds of formula I), U.S. Pat. No. 8,008,281 (e.g., phenyl-TH-DL-trp (3-(N-phenylthiohydantoin)-indole), propenyl-TH-DL-trp (3-(N-allylthiohydantoin)-indole), and methyl-TH-DL-trp (3-(N-methylthiohydantoin)-indole), U.S. Pat. No. 7,714,139 (e.g., compounds of Formula I or II), U.S. Pat. Appl. Pub. No. 20140066625 (e.g., fused imidazole derivatives; compounds of Formula I or II), U.S. Pat. Appl. Pub. No. 20130177590 (e.g., N-hydroxyamidino heterocycles; compounds of Formula I-III), U.S. Pat. Appl. Pub. No. 20140023663 (e.g., 1,2,5-oxadiazoles; No. 20080146624 (e.g., amidines; compounds of formula I or II); U.S. Patent Application Publication No. 20080119491 (e.g., amidinoheterocycles;Compounds of formula I-IV), U.S. Patent Application Publication No. 20080182882 (e.g., N-hydroxyamidino heterocycles; compounds of formula I), U.S. Patent Application Publication No. 20080214546 (e.g., N-hydroxyamidino heterocycles; compounds of formula I), U.S. Patent Application Publication No. 20060258719 (compounds of formula I), Banerjee et al. (2008) Oncogene 27:2851-2857 (e.g., brassinin derivatives;), and Kumar et al. (2008) J. Med.Chem., 51:1706-1718 (e.g., phenylimidazole derivatives). In certain embodiments, the IDO1 inhibitor is a prodrug (see, e.g., U.S. Patent Application Publication No. 20170022157 and U.S. Provisional Application No. 62 / 555,726). All references are incorporated herein by reference, particularly with respect to the IDO1 inhibitors provided therein.

[0048] In certain embodiments, the IDO1 induction inhibitor is ethyl pyruvate (Muller, et al. (2010) Cancer Res. 70:1845-1853) or Gleevec (imatinib, see Balachandran et al. (2011) Nat. Med. 17: 1094-1100). In certain embodiments, the IDO1 pathway inhibitor (e.g., an inhibitor of a downstream signaling pathway) is 1-methyltryptophan, in particular 1-methyl D-tryptophan (indoximod, NLG-8189; 1-methyl-D-tryptophan; NewLink Genetics), salts and prodrugs thereof (US Patent Publication No. 20170022157) or racemic mixtures containing same.

[0049] Further inhibitors of IDO1 expression include, but are not limited to, the following inhibitors: JAK / STAT (e.g. JAK, STAT3, STAT1) [Du et al. (2000) J. Interferon Cytokine Res., 20:133-142, Muller et al. (2005) Nature Med., 11:312-319); Yu et al. (2014) J. Immunol., 193:2574-2586], NFκB (Muller et al. (2005) Nature Med., 11:312-319; Muller et al. (2010) Cancer Res., 70:1845-1853), KIT (Balachandran et al. (2011) Nature Med., 17:1094-1100), MET (Rutella et al. (2006) Blood 108:218-227; Giannoni et al. (2014) Haematologica 99:1078-1087), RAS / RAF / MEK (Liu(2010)Blood 115:3520-3530), Aryl hydrocarbon receptor (AHR) (Bessede et al. (2014) Nature 511:184-190; Litzenburger et al. (2014) Oncotarget 5:1038- 1051), or Vascular endothelial growth factor receptor (VEGFR) (Marti et al. (2014) Mem Inst Oswaldo Cruz 109:70-79). In certain embodiments, the inhibitor is not an inhibitor of VEGFR.

[0050] Still other IDO inhibitors, salts or prodrugs thereof, are described in the following U.S. patents, which are incorporated herein by reference: U.S. Patent No. 10,501,458: Substituted bicyclic fused ring compounds as indoleamine-2,3-dioxygenase inhibitors; U.S. Patent No. 10,494,360: Inhibitors of indoleamine 2,3-dioxygenase; U.S. Patent No. 10,472,336: Modulators of indoleamine 2,3-dioxygenase; U.S. Patent No. 10,436,785: Anti-indoleamine 2,3-dioxygenase 1 antibodies and diagnostic uses thereof; U.S. Patent No. 10,399,933: Inhibitors of indoleamine-2,3-dioxygenase for the treatment of cancer; U.S. Patent No. 10,399,932: Inhibitors of indoleamine-2,3-dioxygenase for the treatment of cancer; U.S. Patent No. 10,369,137: 1,2,5-oxadiazoles as inhibitors of indoleamine 2,3-dioxygenase; U.S. Patent No. 10,358,427: Modulators of indoleamine 2,3-dioxygenase; U.S. Patent No. 10,336,731: Substituted 1H-indole-2-carboxamide compounds as indoleamine-2,3-dioxygenase inhibitors; U.S. Patent No. 10,329,297: Compounds for the inhibition of indoleamine-2,3-dioxygenase; U.S. Patent No. 10,323,004: Inhibitors of indoleamine 2,3-dioxygenase and methods of their use; U.S. Patent No. 10,287,252: Inhibitors of tryptophan-2,3-dioxygenase or indoleamine-2,3-dioxygenase; U.S. Patent No. 10,280,163: 5- or 8-substituted imidazo[1,5-a]pyridines as indoleamine and / or tryptophan 2,3-dioxygenases; U.S. Patent No. 10,280,157: Methods for the synthesis of indoleamine 2,3-dioxygenase inhibitors; U.S. Patent No. 10,239,894: Inhibitors of indoleamine 2,3-dioxygenase; U.S. Patent No. 10,208,002: Modulators of indoleamine 2,3-dioxygenase and methods of use thereof; US Patent 10,034,864: 1,2,5-Oxadiazoles as inhibitors of indoleamine 2,3-dioxygenase; U.S. Patent No. 9,873,688: Methods for the synthesis of indoleamine 2,3-dioxygenase inhibitors; U.S. Patent No. 9,789,094: 1,2,5-Oxadiazoles as Indoleamine 2,3-Dioxygenase Inhibitors; U.S. Patent No. 9,771,370: Compounds for the inhibition of indoleamine-2,3-dioxygenase; U.S. Patent No. 9,675,571: Inhibitors of indoleamine 2,3-dioxygenase (IDO); U.S. Patent No. 9,499,497: Compounds useful as inhibitors of indoleamine 2,3-dioxygenase; US Patent No. 9,463,239: Use of inhibitors of indoleamine-2,3-dioxygenase in combination with other treatment modalities; U.S. Patent No. 9,433,666: Indoleamine 2,3-dioxygenase-based immunotherapy; U.S. Patent No. 9,321,755: Methods for the synthesis of indoleamine 2,3-dioxygenase inhibitors; U.S. Patent No. 9,320,732: 1,2,5-Oxadiazoles as inhibitors of indoleamine 2,3-dioxygenase; U.S. Patent No. 9,073,875: Compounds useful as inhibitors of indoleamine 2,3-dioxygenase; U.S. Patent No. 8,993,605: 1,2,5-Oxadiazoles as inhibitors of indoleamine 2,3-dioxygenase; U.S. Patent No. 8,951,536: N-Hydroxyamidinoheterocycles as modulators of indoleamine 2,3-dioxygenase; U.S. Patent No. 8,846,726: Modulators of indoleamine 2,3-dioxygenase and methods of use thereof; U.S. Patent Nos. 8,822,511 and 8,796,319: 1,2,5-oxadiazoles as inhibitors of indoleamine 2,3-dioxygenase; US Patent No. 8,580,844: Use of inhibitors of indoleamine-2,3-dioxygenase in combination with other treatment modalities U.S. Patent Nos. 8,507,541, 8,450,351 and 8,377,976: N-hydroxyamidino heterocycles as modulators of indoleamine 2,3-dioxygenase; U.S. Patent No. 8,436,151: Indoleamine 2,3-dioxygenase-2 antibodies; U.S. Patent No. 8,372,870: Modulators of indoleamine 2,3-dioxygenase and methods of use thereof for treating cancer; US Patent No. 8,088,803: 1,2,5-Oxadiazoles as inhibitors of indoleamine 2,3-dioxygenase; U.S. Patent No. 8,058,416: Nucleic acid molecules encoding indoleamine 2,3-dioxygenase-2; U.S. Patent No. 8,034,953: Modulators of indoleamine 2,3-dioxygenase and methods of use thereof; U.S. Patent No. 7,799,776: Indoleamine 2,3-dioxygenase (IDO) inhibitors; US Patent No. 7,598,287: Use of inhibitors of indoleamine-2,3-dioxygenase in combination with other treatment modalities. In yet another embodiment, the IDO inhibitor is an IDO-targeting peptide-based vaccine (see Iversen et al. (2014) Clin. Cancer Res., 20:221-32).

[0051] ix. Specific inhibitors of ISR nodes GCN2 inhibitors include, but are not limited to, the following: GCN2-IN-1(A-92)(Medchemexpress), GCN2iA (Nakamura et al, Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response, PNAS, epub July 30, 2018 115 (33) E7776-E7785), which are also described by Fujimoto et al. (Identification of Novel, Potent, and Orally Available GCN2 Inhibitors with Type I Half Binding Mode, ACS Med Chem Lett.2019 Oct 10; 10(10): 1498-1503, epub Sept 2019), GZD824 (Kato et al, GZD824 inhibits GCN2 and sensitizes cancer cells to amino acid starvation stress, Molecular Pharmacology, 98(6) (October 8, 2020)), Inhibitors based on the triazolo[4,5-d]pyrimidine scaffold; as described by Lough et al. (Triazolo[4,5-d]pyrimidines as Validated General Control Nonderepressible 2 (GCN2) Protein Kinase Inhibitors Reduce Growth of Leukemia Cells, Volume 16, September 2018, Pages 350-360), GCN2 inhibitors; being tested by RAPT therapeutics (e.g., GCN2i-282, GCN2i-490, FLX-GCN2i-A, FLX-GCN2i-B) (Marshall et al, Targeting the Stress Response Kinase GCN2 to Restore Immunity And Decrease Tumor Cell Survival SITC 2019). Other GCN2 inhibitors include those disclosed in U.S. Patent Application Publication No. 20190375753, including those having the following general formula (Rapt Therapeutics):

[0052] [ka] Each of these documents is incorporated herein by reference.

[0053] CHOP inhibitors include Klar et al. (Abstract 3275: LNAplus for improving cancer immunotherapy) TM Antisense oligonucleotides for inhibition of ER stress factor C / EBP homologous protein (Chop), DOI: 10.1158 / 1538-7445.AM2019-3275, published July 2019) and ISRIB, but are not limited to these.

[0054] ATF4 inhibitors include, but are not limited to, ursolic acid, tomatidine and its derivatives, Ebert et al. [Identification and Small Molecule Inhibition of an Activating Transcription Facto4 (ATF4)-Dependent Pathway Induction in Age-Related Skeletal Muscle Weakness and Atrophy, J Biol Chem. 2015 Oct 16; 290(42): 25497-25511], GSK2606414, and TRIB3. Other ATF4 inhibitors include those compounds disclosed in U.S. Patent Application Publication No. 9,034,299, entitled "ATF4 Inhibitors and Their Use for Neuroprotection, Repair, Regeneration, and Plasticity" by Ratan, U.S. Patent Application Publication No. 20160317526, entitled "Prolylhydroxylase / atf4 Inhibitors and Methods of Use for Treating Neuronal Cell Injury or Death" by Ratan and Karuppagounder, and WO2017 / 212423, entitled "Chemical Compounds" by Axten et al. Each of these documents is incorporated herein by reference.

[0055] ISR node inhibitors further include nucleic acid molecules that bind to and block or reduce the activity, expression, or translation of ISR node genes, gene products, or transcripts. Examples of ISR node inhibitors include siRNA, shRNA, and antisense oligonucleotides (ASOs).

[0056] x. Specific inhibitors of VEGF and VEGFR The phrase "a composition that blocks or inhibits the expression, induction, activity, and / or signaling of one or more of VEGF subtypes or isoforms or subforms of VEGF receptors (VEGFR)" refers to a composition including, but not limited to, any VEGF antagonist that can neutralize, block, inhibit, abrogate, reduce, or interfere with VEGF activity, including its binding to one or more VEGFRs. Among such inhibitors or antagonists are anti-VEGF antibodies or antibody fragments directed against epitopes of VEGF isotypes A-F or VEGF-binding fragments thereof, artificial single-chain antibody fragments, or molecules that mimic the VEGF receptor to bind to a VEGF isotype, or drugs that interfere with receptor signaling. For example, the VEGF antagonist can be, but is not limited to, an anti-VEGF antibody, a VEGF trap, an anti-VEGFR antibody, a VEGFR inhibitor, thalidomide, a DI 14-Notch inhibitor, an anti-tubulin vascular disrupting agent (VDA), an angiopoietin-Tie2 inhibitor, a nitric oxide synthase (NOS) inhibitor, or a cationic polyamino acid dendrimer. As used herein, the term "VEGF trap" refers to a decoy VEGF receptor that blocks the VEGF signaling pathway by preferentially binding to VEGF, thereby inhibiting its binding to its cognate receptor. In one embodiment, a VEGF trap is a recombinant fusion protein that includes one or more extracellular domains of a VEGF receptor, or a portion of such an extracellular domain, fused to a second protein, For example, a VEGF receptor extracellular domain can be fused to an Fc isoform, such as the Fc fragment of an immunoglobulin. In one embodiment, a recombinant fusion protein consisting of portions of the human VEGF receptor 1 and 2 extracellular domains, in which a VEGF trap is fused to the Fc portion of human IgG1, formulated as an isotonic solution for intravitreal administration, is available under the tradename EYLEA. (登録商標) The drug is Regeneron. In another embodiment, the VEGF trap is OPT-302 (Opthea; Dugel, Pravin U. et al., Phase 1 Study of OPT-302 Inhibition of Vascular Endothelial Growth Factors C and D for Neovascular Age-Related Macular Degeneration Ophthalmic Retinal, DOI: https: / / doi.org / 10.1016 / j.oret.2019.10.008), i.e., a fusion protein comprising immunoglobulin-like domains 1-3 of the extracellular domain of VEGFR-3 fused to the Fc fragment of human immunoglobulin G1 (IgG1). Still other traps include other receptor domains and other Fc isoforms.

[0057] In certain embodiments, specific examples of VEGF inhibitors include, but are not limited to, rapamycin, everolimus, temserolimus, low molecular weight heparin, SPARC (osteonectin) peptide, bevacizumab, ranibizumab, ramucirumab, aflibercept, interleukin 17 (IL-17), DC101, sunitinib, sorafenib, pazopanib, AMG706, cediranib, banetanib, nintedanib (vargatef), brivanib, XL-184, axitinib, tivozanib, thalidomide, ranalidomide, DMXAA, nadroparin, 2,5-dimethylcelecoxib, cyclophosphamide, HBC, and tasquinimod.

[0058] In certain embodiments, the VEGF inhibitor or antagonist comprises: Ranibizumab (Lucentis (登録商標) ), bevacizumab (Avastin (登録商標) ), aflibercept (Eylea (登録商標) ), brolucizumab (Boevu (登録商標) ), pegaptanib (Macugen (登録商標) ), abicipar pegol (e.g., (See Moisseiev, E., Loewenstein, A. Abicipar pegol-a novel anti-VEGF therapy with a long duration of action. Eye (2019). JPEG2024501582000002.jpg659, ranibizumab biosimilars FYB201 (Bioeq GMBH, Munich, GE), PF582 (Pfenex Inc.), SB11 (Samsung Bioepis Co. Ltd), and Xlucane (Xbrane Biopharma), aflibercept biosimilar MYL-1701P / M-710 (Momenta Pharmaceuticals, Inc.), or Conbercept (Liu K et al.; PHOENIX study group. Conbercept for treatment of neovascular age-related macular degeneration: Results of the Randomized phase 3 PHOENIX study. Am J Ophthalmol.2019;197:156-167); Faricimab / RG7716 (bispecific antibody VEGF-A + Ang-2) (Mojunder, MI et al., The Mechanism of the Bispecific Antibody Faricimab Retinal Physician, Volume: 16, March 2019, 32- 35), OPT-302 (VEGF-C / 'trap') (see Dugel above), KS-301 (anti-VEGF polymer conjugate biologic) (Kodiak Sciences; Wykoff CC. Extended durability in exudative retinal diseases using the novel intravitreal anti-VEGF antibody biopolymer conjugate KSI-301: Results from the Phase 1b study in patients with AMD, DME and RVO. Presented at American Academy of Ophthalmology Subspecialty Day Retina 2019;October 11, 2019; San Francisco, CA) KS-501 (anti-VEGF trap + anti-IL6 antibody fusion) (Kodiak Sciences; see https: / / www.prnewswire.com / news-releases / kodiak-sciences-announces-emerging-durability-data-from-ongoing-phase-1b-study-of-ksi-301-in-wet-amd-patients-at- the-retina-society-annual-meeting-300918262).

[0059] In some embodiments, the VEGF antagonist useful in the combination is selected from those described in U.S. Patent Application Publication No. 20190381087, including: ranibizumab (registered trademark of Genentech, San Francisco, Calif.; 登録商標 For heavy and light chain variable region sequences, see Figure 1 in U.S. Pat. No. 7,060,269), bevacizumab (Avastin (登録商標) (Genentech, San Francisco, Calif.; for heavy and light chain variable region sequences, see FIG. 1 of U.S. Pat. No. 6,054,297); aflibercept [Eylea (登録商標) (Regeneron, Tarrytown, NY)], KH902 VEGF receptor-Fc fusion protein (see Zhang et al. (2008) Mol Vis. 14:37-49), 2C3 antibody (U.S. Pat. No. 6,342,221, Column 8, lines 48-67, Column 9, lines 1-21), ORA102 (available from Ora Bio, Ltd.), pegaptanib (e.g., pegaptanib sodium; trade name Macugen 登録商標:Valeant Pharmaceuticals, Bridgewater, NJ; see Figure 1 in U.S. Pat. No. 6,051,698), bevasiranib (see Dejneka et al. (2008) Mol Vis. 14:997-1005), SIRNA-027 (Shen et al. (2006) Gene Ther. 13:225-34), decursin (see U.S. Pat. No. 6,525,089 (Column 3, lines 5-16)), decursinol (see Ahn et al. (1997) Planta Med. 63:360-1), picropodophyllin (see Economou (2008) Investigative Ophthalmology & Visual Science. 49:2620-6), guggulsterone (see Kim et al. (2008) Oncol. Rep.20:1321-7), PLG101 (see Ahmadi and Lim (2008) Expert Opin Pharmacother.9:3045-52), PLG201 (see Ahmadi and Lim (2008)), the eicosanoid LXA4 (see Baker et al. (2009) J Immun.182:3819-26), PTK787 (trade name Vitalanib 登録商標; see Barakat and Kaiser (2009) Expert Opin Investig Drugs 18:637-46), pazopanib (see Takahashi et al. (2009) Arch Ophthalmol. 127:494-9), axitinib (see Hu-Lowe et al. (2008) Clin Cancer Res. 14:7272-83), CDDO-Me (see Sogno et al. (2009) Recent Results Cancer Res. 181:209-12), CDDO-Imm (see Sogno et al. (2009)), shikonin (see Hisa et al. (1998) Anticancer Res. 18:783-90), beta-hydroxyisovaleryl shikonin (see Hisa et al. (1998)), Ganglioside GM3 (Chung et al. (2009) Glycobio.19:229-39), DC101 antibody (see U.S. Patent No. 6,448,077, Column 2, lines 61-65), Mab25 antibody (see U.S. Patent No. 6,448,077, Column 2, lines 61-65), Mab73 antibody (see U.S. Patent No. 6,448,077, Column 2, lines 61-65), 4A5 antibody (see U.S. Patent No. 6,383,484, Column 12, lines 50-54), 4E10 antibody (see U.S. Patent No. 6,383,484, Column 10, lines 66-67, Column 11, lines 1-2), 5F12 antibody (see U.S. Patent No. 6,383,484, Column 10, lines 62-65), VA01 antibody (see U.S. Pat. No. 5,730,977, Column 6, lines 26-30), BL2 antibody (see U.S. Pat. No. 5,730,977, Column 6, lines 30-32), VEGF-related protein (see U.S. Pat. No. 6,451,764, FIG. 1), sFLT01 (see Pechan et al. (2009) Gene Ther. 16:10-6), sFLT02 (see Pechan et al. (2009)), Peptide B3 (see Lacal et al. (2008) Eur J Cancer 44:1914-21), TG100801 (see Palanki et al. (2008) J Med Chem.51:1546-59), sorafenib (brand name Nexavar. 登録商標; see Kernt et al. (2008) Acta Ophthalmol. 86:456-8), G6-31 antibody (see Crawford et al. (2009) Cancer Cell 15:21-34), ESBA1008 (see U.S. Pat. No. 8,349,322), tivozanib (see U.S. Pat. No. 6,821,987), all of which are incorporated herein by reference; Campas et al. (2009) Drugs Fut 2009, 34(10): 793), or pharma- ceutically acceptable salts or prodrugs thereof.

[0060] In another embodiment, the VEGF antagonist is an antibody or antibody fragment that binds to epitope VEGF-A or VEGF-B, or any portion of the epitopes cited above, all of which are incorporated by reference. In one embodiment, the VEGF antagonist is an antibody or antibody fragment that binds to one or more epitopes of VEGF. In another embodiment, the VEGF antagonist is an antibody or antibody fragment that binds to an epitope of VEGF, such as an epitope of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E. In some embodiments, the VEGF antagonist binds to an epitope of VEGF such that the binding of VEGF and VEGFR is inhibited. In one embodiment, the epitope includes a component of the three-dimensional structure of VEGF that is displayed such that the epitope is exposed on the surface of the folded VEGF molecule. In one embodiment, the epitope is a linear amino acid sequence derived from VEGF.

[0061] In some embodiments, the inhibitory antibodies against VEGF are known in the art, e.g., those described in U.S. Pat. U.S. Patent Nos. 6,524,583 and 6,451,764 (VRP antibodies); U.S. Patent Nos. 6,448,077, 6,416,758, 6,403,088 (to VEGF-C); U.S. Patent No. 6,383,484 (to VEGF-D); U.S. Patent No. 6,342,221 (anti-VEGF antibody); U.S. Patent Nos. 6,342,219, 6,331,301 (VEGF-B antibodies) and U.S. Patent No. 5,730,977, and WO 96 / 30046, WO 97 / 44453, and WO 98 / 45331. (the contents of which are incorporated herein by reference in their entireties).

[0062] Other non-antibody VEGF antagonists include antibody mimetics (e.g., Affibody (登録商標) molecules, affilins, affitins, anticalins, avimers, Kunitz domain peptides, and monobodies). This includes a recombinant binding protein containing an ankyrin repeat domain, which binds to VEGF-A and prevents it from binding to VEGFR-2. One example is AGN 150998 (DARPin (登録商標) Also known as MP0112. In yet other embodiments, recombinant binding proteins containing ankyrin repeat domains that bind VEGF-A and prevent binding to VEGFR-2, as described in more detail in WO2010 / 060748 and WO2011 / 135067, are useful. In yet another embodiment, specific antibody mimetics with VEGF antagonist activity are the 40 kD pegylated anticalin PRS-050 and the monobody angiocept (CT-322).

[0063] The aforementioned non-antibody VEGF antagonists can be modified to further improve their pharmacokinetic properties or bioavailability. For example, a non-antibody VEGF antagonist can be chemically modified (eg, pegylated) to extend its in vivo half-life. Alternatively, or in addition, it can be modified by glycosylation or the addition of additional glycosylation sites that are not naturally occurring in the protein sequence of the native protein from which the VEGF antagonist is derived. Another non-antibody VEGF antagonist immunoadhesin currently in preclinical development is a recombinant human soluble VEGF receptor fusion protein, similar to VEGF Trap, that contains extracellular ligand-binding domains 3 and 4 from VEGFR2 / KDR and domain 2 from VEGFR1 / Flt-1; these domains are fused to a human IgG Fc protein fragment (Li et al., 2011 Molecular Vision 17:797-803). This antagonist binds to the isoforms VEGF-A, VEGF-B and VEGF-C. The molecule is prepared using two different production processes, resulting in different glycosylation patterns on the final protein. The two glycoforms are designated KH902 (conbercept) and KH906. The fusion protein can exist as a dimer. This fusion protein and related molecules are further characterized in US Patent Application Publication No. 2010 / 0215655. All of the above disclosures of suitable VEGF or VEGFr inhibitors or antagonists are incorporated herein by reference.

[0064] xi. IL-6 specific inhibitors Interleukin 6 (IL-6), also known as B cell stimulating factor 2 (BSF2) or interferon β2, is a cytokine. IL-6 is a differentiation factor involved in the activation of cells of the B lymphocyte lineage and is a multifunctional cytokine that affects the functions of various cells. Various IL-6 inhibitors are known in the art, including, but not limited to, MH166, SK2, MR16-1, PM-1 antibody, AUK12-20 antibody, AUK64-7 antibody, AUK146-15 antibody, and IL6 receptor antibodies including tocilizumab. Methods for treating choroidal neovascularization by administration of IL-6 inhibitors, including anti-IL-6 antibodies, anti-IL-6 receptor antibodies, anti-gp130 antibodies, IL-6 mutants, soluble IL-6 receptor mutants, and partial peptides of IL-6 or IL-6 receptor and low molecular weight compounds exhibiting similar activity, are described in U.S. Pat. No. 8,771,686 B2, which is incorporated herein by reference.

[0065] H. Effective Dose, Route of Administration and Related Definitions A "therapeutically effective amount" of a compound or pharmaceutical composition refers to an amount effective to prevent, inhibit, treat, or reduce the symptoms of a particular disorder or disease. For example, a "therapeutically effective amount" can refer to an amount sufficient to reduce abnormal angiogenesis or edema in the eye of a treated subject having an ocular disorder.

[0066] "Pharmaceutically acceptable excipient or carrier" refers to, but is not limited to, a diluent, adjuvant, excipient, adjuvant, or vehicle with which an active agent of the invention is administered. Pharmaceutically acceptable carriers are those approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more specifically, humans, and can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences by E. W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0067] As used herein, the term "treatment" refers to any method used that can provide a benefit to a subject, i.e., result in the alleviation of, delaying the onset of, reducing the severity or incidence of, or preventing one or more symptoms or aspects of an ocular disease, disorder, or condition. For purposes of the present invention, treatment can be administered before, during, and / or after the onset of symptoms. In certain embodiments, the treatment is administered after the subject has undergone a conventional treatment. In some embodiments, the term "treating" includes arresting, substantially inhibiting, slowing, or reversing the progression of a condition, substantially ameliorating a clinical or cosmetic symptom of a condition, or substantially preventing the appearance of a clinical or cosmetic symptom of a condition, or reducing the severity and / or frequency of one or more symptoms attributable to a disease.

[0068] As used herein, the term "preventing" refers to a prophylactic treatment of a subject at risk of developing a condition, resulting in a reduced likelihood that the subject will develop a condition.

[0069] The term "treatment regimen" (also referred to as therapeutic regimen) as used herein refers to the particular sequence, timing, duration, route, and interval of administration of one or more therapeutic agents. In one embodiment, the treatment regimen is subject specific. In another embodiment, the treatment regimen is disease specific. In another embodiment, the treatment regimen varies as the subject responds to the treatment. In another embodiment, the treatment regimen is fixed until certain treatment milestones are met.

[0070] In one embodiment, the methods described herein comprise inhibiting pathological neovascularization in a subject. The method, in one embodiment, comprises removing or inhibiting IDVC in the retina of the subject. In another embodiment, the method comprises administration of a composition that blocks or inhibits expression, induction, activity, and / or signaling of an ISR node. In one embodiment, the ISR node is selected from GCN2, CHOP, and ATF4.

[0071] In yet other embodiments, the methods described herein include administering a combination of a composition that blocks or inhibits expression, induction, activity, and / or signaling of an ISR node and a composition that blocks or inhibits expression, induction, activity, and / or signaling of one or more of a VEGF subtype or isoform, or a VEGFR subtype or isoform, and the treatment regimen includes one or more doses of the administered composition.

[0072] As used herein, "treatment effect" or "treatment benefit" refers to an improvement or reduction in the severity of a disease system. By "effective amount" is meant an amount of a composition sufficient to provide a therapeutic benefit or effect after a suitable course of administration.

[0073] It should be understood that an "effective amount" of a composition comprising at least one IDO inhibitor, VEGF or VEGFR inhibitor, or ISR node inhibitor (e.g., GCN2, CHOP, or ATF4) will vary depending on the inhibitor / antagonist selected for use in the method. With respect to dosage, it should be understood that "small molecule" drugs are typically dosed in flat doses rather than on a mg / kg basis. For injections, a doctor or nurse can inject the calculated amount by loading this amount into a syringe from a vial. In contrast, tablets and some biologics are provided in fixed dosage forms. Some dose range studies with small molecules use mg / kg, but other doses can be used by those skilled in the art based on the teachings herein. In one embodiment, an effective amount of the inhibitor of the composition includes, but is not limited to, about 0.001 to about 25 mg / kg of subject body weight. In one embodiment, the effective amount ranges from In another embodiment, the effective dose ranges from 0.001 to 0.01 mg / kg body weight. In another embodiment, the effective dose ranges from 0.001 to 0.1 mg / kg body weight. In another embodiment, the effective dose ranges from 0.001 to 1 mg / kg body weight. In another embodiment, the effective dose ranges from 0.001 to 10 mg / kg body weight. In another embodiment, the effective dose ranges from 0.001 to 20 mg / kg body weight. In another embodiment, the effective amount ranges from 0.01 to 25 mg / kg body weight. In another embodiment, the effective dose ranges from 0.01 to 0.1 mg / kg body weight. In another embodiment, the effective dose ranges from 0.01 to 1 mg / kg body weight. In another embodiment, the effective dose ranges from 0.01 to 10 mg / kg body weight. In another embodiment, the effective dose ranges from 0.01 to 20 mg / kg body weight. In another embodiment, the effective dose ranges from 0.1 to 25 mg / kg body weight. In another embodiment, the effective dose ranges from 0.1 to 1 mg / kg body weight. In another embodiment, the effective dose ranges from 0.1 to 10 mg / kg body weight. In another embodiment, the effective dose ranges from 0.1 to 20 mg / kg body weight. In another embodiment, the effective dose ranges from 1 to 25 mg / kg body weight. In another embodiment, the effective dose ranges from 1 to 5 mg / kg body weight. In another embodiment, the effective dose ranges from 1 to 10 mg / kg body weight. In another embodiment, the effective dose ranges from 10 to 20 mg / kg body weight. In another embodiment, the effective dose ranges from 20 to 30 mg / kg body weight. In another embodiment, the effective dose range is: In another embodiment, the effective dose range is: 1 to 50 mg / kg body weight. Other doses within these ranges are expected to be useful.

[0074] An "effective amount" of a composition that blocks or inhibits expression, induction, activity, and / or signaling of one or more VEGF subtypes or isoforms, or VEGFR subtypes or isoforms, is achieved when the composition comprises at least one VEGF or VEGFR inhibitor and is administered to a subject in an amount appropriate to achieve a therapeutic effect. These amounts may vary based on the specific inhibitor selected and the route of administration. An "effective amount" for a composition containing at least one VEGF or VEGFR antagonist or blocker can vary depending on the inhibitor / antagonist selected for use in the method. When the VEGF inhibitor is a small molecule, the VEGF inhibitor may be delivered in the same dosages as described above for the compositions comprising at least one ISR node inhibitor (eg, GCN2, CHOP, or ATF4) inhibitor described above. When the VEGF antagonist is a protein, such as an antibody, an antibody fragment, or a recombinant protein or peptide, the effective amount may be similar to that approved for VEGF monotherapy, i.e., 0.01-25 mg antibody / injection (also called infusion). In one embodiment, the effective amount is: In another embodiment, the effective amount is: In another embodiment, the effective amount is: In another embodiment, the effective amount is: 0.2, 0.5, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0 up to more than mg antibody / injection.

[0075] Still other doses within these ranges are expected to be useful. In one embodiment, the dosage and method of administration of the composition suitable for administration to a particular patient can be determined by a physician, taking into account the patient's age, sex, weight, general medical condition, and the particular condition and severity with which the composition is co-administered. The physician may also consider the route of administration of the agent, the pharmaceutical carrier with which the agent(s) may be combined, and the biological activity of the agent.

[0076] "Administration" or "route of administration" includes any known route of administration that is appropriate for the selected inhibitor or composition and capable of delivering an effective amount to a subject. In one embodiment of the methods described herein, the routes of administration of the compositions are the same. In another embodiment, the compositions are administered by different routes. Routes of administration useful in the methods of the present invention include one or more of oral, parenteral, intravenous, intranasal, sublingual, intraocular injection, intravitreal injection, via a depot formulation or device, via eye drops, and by inhalation. In one embodiment, the inhibitor may be administered orally, or potentially intravitreally, simultaneously with another inhibitor, or potentially co-administered in the same device (syringe or depot device) as another inhibitor treatment, or administered via eye drops, intranasally, or sublingually.

[0077] Throughout the specification, the words "comprise," "comprises," and "comprising" are to be interpreted inclusively rather than exclusively. "Consist," "consisting," and variations thereof are to be interpreted exclusively rather than inclusively. Although various embodiments herein are presented using the term "comprising," it will be understood that under various circumstances the relevant embodiments may also be described using "consisting" or "consisting essentially of" language.

[0078] The terms "a" or "an" refer to one or more biomarkers, e.g., "biomarker" is understood to represent one or more biomarkers. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. As used herein, the term "about" means a 10% variation from the given standard, unless otherwise specified.

[0079] II. Pharmaceutical Preparations The compositions described herein may be contained in a single composition that includes at least one carrier (eg, a pharma- ceutically acceptable carrier). Alternatively, the agents may be administered separately (eg, in separate compositions) comprising at least one carrier. Pharmaceutical preparations containing the compositions may be conveniently formulated for administration with an acceptable vehicle, such as water, buffered saline, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), dimethylsulfoxide (DMSO), oils, detergents, suspending agents, or suitable mixtures thereof. The concentration of the drug in the selected vehicle can vary, and the vehicle can be selected based on the desired route of administration of the pharmaceutical formulation. Except insofar as any conventional media or agent is incompatible with the inhibitor or composition being administered, its use in the pharmaceutical preparation is contemplated.

[0080] In certain embodiments, when two or more compositions are administered, the compositions can be administered sequentially and / or simultaneously. For example, a composition containing an ISR node inhibitor can be administered before, after, and / or simultaneously with a composition containing an anti-VEGF inhibitor or antagonist. If the compositions are not administered at the same time, they should be administered sufficiently close in time so that two or more compositions can act synergistically, additively, or in a manner to achieve a therapeutic benefit in the subject.

[0081] Selection of the appropriate pharmaceutical formulation will depend on the method of administration chosen. For example, the composition can be administered by direct injection into the eye or into certain tissues of the eye, in which case the pharmaceutical formulation will contain the agent dispersed in a vehicle compatible with ocular delivery. Agents may also be administered parenterally, by intravenous injection into the bloodstream, or by subcutaneous, intramuscular, or intraperitoneal injection. Pharmaceutical formulations for parenteral injection are known in the art. If parenteral injection is chosen as the method for administering antibodies, steps must be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect. The lipophilicity of the drugs, or of the pharmaceutical formulation in which they are delivered, can be increased to enable the molecules to better reach their target location.

[0082] Pharmaceutical compositions containing the inhibitors / antagonists described herein as the active ingredient in intimate admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. Carriers can take a wide variety of forms depending on the form of preparation desired for administration. In preparing drugs in oral dosage form, any of the usual pharmaceutical media can be used, such as water, polyethylene glycol, oil, alcohol, flavoring agent, preservative, coloring agent, etc. for oral liquid preparations (e.g., suspensions, elixirs, solutions, etc.); or starch, sugar, diluent, granulating agent, lubricant, binder, disintegrating agent, etc. for oral solid preparations (e.g., powders, capsules, tablets, etc.). Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques. For parenteral compositions, the carrier will usually comprise sterile water, though other ingredients may be included, for example, to aid solubility or for preservation purposes. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.

[0083] The pharmaceutical formulations of the invention can be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit of pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining appropriate dosage units are well known to those skilled in the art. Dosage units may be proportionally increased or decreased based on the patient's body weight. Appropriate concentrations for the alleviation of a particular pathological condition may be determined by dose-concentration curve calculations, as known in the art.

[0084] According to the present invention, the appropriate dosage unit for administration of the composition of the present invention can be determined by assessing the toxicity of the active therapeutic inhibitor in an animal model. Various concentrations of the above inhibitors, including in combination, can be administered to mouse models of ocular disease (e.g., oxygen-induced retinopathy (OIR)), and minimum and maximum doses can be determined based on the results of significant reduction in angiogenesis or edema and side effects as a result of treatment. An appropriate dosage unit may also be determined by assessing the effectiveness of the inhibitor composition in combination with other standard drugs for the treatment of ocular disorders. Dosage units of the inhibitors can be determined individually or in combination for treatment of each eye having a selected condition.

[0085] The compositions containing the combined inhibitors of the present invention may be administered at appropriate intervals, for example at least twice a day or more, until the pathological symptoms are alleviated or ameliorated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case will usually depend on the condition of the patient.

[0086] III. Specific Methods and Compositions The methods provided herein relate to the inventors' identification of IDVCs and their role in pathological neovascularization associated with ocular diseases. In one embodiment, provided herein is a method of treating retinopathy or inhibiting pathological neovascularization in a subject, the method comprising ablating or inhibiting the activity of IDO (indoleamine 2,3-dioxygenase)-dependent angiogenic cells (IDVCs) in the eye of the subject. IDVCs have been functionally characterized as having a role in neovascularization, the establishment and maintenance of which requires the induction of indoleamine 2,3-dioxygenase 1 (IDVC1) in IDVCs.

[0087] In one embodiment, IDVCs are inhibited by inhibiting the activity of IDO1. IDO1 inhibitors are described herein. In another embodiment, IDVCs are inhibited via antibodies against cell surface markers of IDVCs. In one embodiment, the antibody is an ADC or an antibody drug conjugate. The cell surface markers can be selected from any that distinguish the IDVCs from other non-targeted cell populations, for example, in one embodiment, the cell surface markers are selected from CD33, CD11b, CD15, and CD66.

[0088] In another embodiment, the method involves blocking or inhibiting the integrated stress response (ISR) in IDVC cells in an ocular tissue (eg, the retina or choroid). The present inventors have shown that inhibition of key ISR nodes such as GCN2, CHOP, and ATF4 reduces neovascularization in an OIR (oxygen-induced retinopathy) mouse model. Thus, in one embodiment, the method comprises blocking or inhibiting expression, induction, activity, and / or signaling of an ISR pathway node. Inhibitors of an ISR pathway node include small molecules, biological molecules, and nucleic acid molecules that inhibit the translation or transcription of said ISR pathway node. These molecules may be known in the art or may yet to be discovered. Non-limiting examples are provided herein.

[0089] In one embodiment, the method comprises blocking or inhibiting GCN2 expression, induction, activity, or signaling. In one embodiment, the method comprises blocking or inhibiting ATF4 expression, induction, activity, or signaling. In one embodiment, the method involves blocking or inhibiting CHOP expression, induction, activity, or signaling. In one embodiment, the method further comprises blocking or inhibiting IL-6.

[0090] In one embodiment, the method comprises administering an effective amount of GCN2-IN-1 (A-92). In another embodiment, the method comprises administering an effective amount of GCN2iA. In another embodiment, the method comprises administering an effective amount of GZD824. In another embodiment, the method includes the disclosure of administering an effective amount of an inhibitor based on a triazolo[4,5-d]pyrimidine scaffold as described by Lough et al. (Triazolo[4,5-d]pyrimidines as validated General Control Nonderepressible 2 (GCN2) protein kinase inhibitors reduce proliferation of leukemia cells, Vol. 16, September 2018, pp. 350-360).

[0091] In another embodiment, the method is as described by Klar et al. (Abstract 3275:LNAplus TM Inhibition of the ER stress factor C / EBP homologous protein (Chop) with antisense oligonucleotides, DOI: 10.1158 / 1538-7445.AM2019-3275, published July 2019) to improve immunotherapy of cancer. In another embodiment, the method comprises administering an effective amount of ISRIB.

[0092] In another embodiment, the method comprises administering an effective amount of ursolic acid. In another embodiment, the method comprises administering an effective amount of tomatidine. In another embodiment, the method comprises administering an effective amount of GSK2606414. In another embodiment, the method comprises administering an effective amount of TRIB3.

[0093] In another aspect, a method for treating retinopathy or inhibiting pathological neovascularization in a subject is provided, comprising blocking or inhibiting a signaling molecule downstream of the integrated stress response. Downstream of the ISR, certain signaling molecules are known, including the cytokine IL-6.

[0094] Another embodiment of the method further comprises blocking or inhibiting any form of vascular endothelial growth factor (VEGF) expression, induction, activity, or signaling. In one embodiment, the VEGF inhibitor is ranibizumab (Lucentis (登録商標) ), bevacizumab (Avastin (登録商標) ), aflibercept (Eylea (登録商標) ), brolucizumab (Boevu (登録商標) ), pegaptanib (Macugen (登録商標) ), abicipar pegol, ranibizumab biosimilars FYB201, PF582, SB11, and Xlucane, aflibercept biosimilar MYL-1701P / M-710, or conbercept, faricimab / RG7716 (bispecific antibody VEGF-A + Ang-2), OPT-302 (VEGF-C / D 'trap': (VEGF-C / 'trap')), KS301 (anti-VEGF polymer conjugated biologic) (Kodiak Sciences), KS501 (anti-VEGF trap plus anti-IL6 Antibody Fusion) (Kodiak Sciences - anti-VEGF trap plus anti-IL6 Antibody Fusion).

[0095] Another embodiment of the method further comprises blocking or inhibiting the expression, induction, activity, or signaling of any form of indoleamine 2,3 dioxygenase-1 (IDO1). In one embodiment, the blocker or inhibitor of IDO-1 comprises at least one of the following: 1-methyl-D-tryptophan (indoximod), 1-methyl-L-tryptophan, a racemic mixture of 1-methyl-D-tryptophan and 1-methyl-L-tryptophan, epacadostat, navoximod (GDC-919), and NLG802, or their salts, enantiomers, or prodrugs; 1-RD-tryptophan or 1-RL-tryptophan, where R is C1-C12 alkyl; Methylthiohydantoin-DL-tryptophan (MTH-Trp), β-(3β)-DL-alanine, β-(3-benzo(b)thienyl)-DL-alanine, 6-nitro-L-tryptophan, indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-DL-tryptophan, 5-bromoindoxyl diacetate, naphthoquinones, S-allylbrassinin, S-benzylbrassinin, 5-bromobrassinin, phenylimidazoles, 4-phenylimidazole, exiguamine A, and NSC 401366; or BMS-986205 / ONO-7701, PF-06840003 / EOS200271, MK-7162 / IOM2983, LY3381916, KHK2455, HTI-1090 / SHR9146, DN1406131, RG70099, Roxyl-WL, TPST-8844, ethyl pyruvate, Amg-1 or DX-03-12, or a salt, enantiomer or prodrug thereof, or any therapeutically active formulation thereof.

[0096] In another embodiment, the ISR pathway blocker is selected from GSK-2606414, RPT-GCN2i, AMG-PERK44, and trans-ISRIB.

[0097] Certain combination therapies are provided herein. For example, in one embodiment, an ISR node inhibitor is administered together with an IL-6 inhibitor. In another embodiment, the ISR node inhibitor is administered with a VEGF inhibitor. According to this combination therapy treatment method, the subject receives one or more advantageous treatments or treatment benefits. Among these are treatment effects or treatment benefits that are enhanced compared to administration of a single agent. Another advantage is that it produces synergistic treatment effects or treatment benefits. Yet another advantage of combination therapy is the improved tolerability of one or more of the administered compositions.

[0098] In certain embodiments, the compositions described herein are administered in an integrated treatment regimen. Based on the selection of compounds included in the compositions (i.e., inhibitors of ISR nodes, IL-6, IDO, VEGF / VEGFR, etc. identified herein), the route of administration may be the same and / or the two compositions may be administered in a single formulation. In certain embodiments, the compositions described herein are administered continuously. In certain embodiments, the compositions described herein are administered simultaneously. In certain embodiments, the route of administration of the compositions is the same. In certain embodiments, the route of administration of the composition varies, including at least one of oral administration, intravenous injection, intranasal administration, sublingual administration, intravitreal injection, intraocular injection, administration via a depot formulation or device, or administration via eye drops.

[0099] Another advantage of combination therapy is that the coadministration of inhibitors allows for the use of doses of any one administered inhibitor that are lower than the dose approved for monotherapy. Alternatively, coadministration of inhibitors utilizes dosages of two or more administered inhibitors that are lower than the doses approved for monotherapy. Yet another advantage is that co-administration of the compounds described herein enhances the duration of the treatment effect or treatment benefit achieved with any one composition administered alone. For example, in one embodiment, the combination therapy allows for a VEGF inhibitor or antagonist to be administered by intraocular injection in a treatment regimen that includes at least 5% to at least 20% less frequently than the VEGF inhibitor or antagonist is administered as the sole treatment agent. In another embodiment, the combination therapy allows the VEGF inhibitor or antagonist to be administered by intraocular injection in a treatment regimen that is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, up to 20% or less frequent than when the VEGF inhibitor or antagonist is administered as the sole treatment agent.

[0100] In one embodiment, the IDVCs are localized to the retina of the eye, hi another embodiment, the IDVCs are localized to the choroid of the eye. In yet another embodiment, the methods of the present invention provide for the use of compositions in which an active therapeutic inhibitor or antagonist penetrates the retina, distributes to ocular tissues, and achieves an ocular concentration that is greater than the concentration in the serum of the subject.

[0101] In any of the above methods, one or more of the compositions further comprises a pharma- ceutically acceptable excipient or carrier. Formulations for any of the compositions can be designed according to the choice of active inhibitor or agent, as well as the route and dosage of administration.

[0102] IV. Kits The present invention also includes pharmaceutical kits useful for treating or preventing the ocular diseases or disorders referred to herein, or for administration according to a desired treatment regimen, for example comprising one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of an ISR node inhibitor. Such kits can, if desired, further include one or more of a variety of conventional pharmaceutical kit components, as would be readily apparent to one of skill in the art, e.g., a container with one or more pharma- ceutically acceptable carriers, additional containers, etc. Instructions, either as a package insert or label, can also be included in the kit indicating the amounts of the components to be administered, guidelines for administration, and / or guidelines for mixing the components.

[0103] Measures of treatment improvement using these methods can be determined by assessment of visual acuity, measurement of retinal fluid and / or vascular leakage, and reduction in side effects from administration of the treatment. Components that allow for these efficacy tests can also be included in the kits.

[0104] The following examples are provided to illustrate various embodiments of the invention and are not intended to limit the invention in any way. EXAMPLES

[0105] Example 1 - Materials and Methods Transgenic mouse strains: Congenic Ido1 null (Ido1 - / - ) Mice were provided by A. Mellor. WT BALB / c mice and congenic Ifng and Il6 null mice on a BALB / c strain background were obtained from Jackson Laboratory. Gcn2 null mice established by D. Ron (15) on the 129svev (Taconic) strain background were obtained from A. Mellor and backcrossed in house for 10 generations to obtain congenic BALB / c strains. Genotyping was performed using specific primer sets (Tables 1-3) designed to distinguish between transgenic and wild-type alleles (Fig. 8A). Table 1: List of siRNAs * WT BALB / c mice [Table 1] *SEQ ID NOs: 1-8 are listed in descending order. Table 2: List of DNA primers * [Table 2] *SEQ ID NOs: 9-20 are listed in descending order. Table 3: In vivo siRNAs [Table 3]

[0106] Ido1 - / - The lack of IDO1 protein expression in the Il6 mouse line has been previously verified (9, 16). - / - Lack of Il6 induction and Ifng in the strain - / - IFN-γ induction in strains was confirmed by assaying lung biopsies 24 h after intranasal instillation of 25 μg LPS (Figure 8B). All studies involving mice were approved by the Lankenau Institute for Medical Research IACUC and conformed to AAALAC guidelines.

[0107] 4T1 tumor cell metastasis: 1 × 10 cells of primary orthotopic (also called orthotopic) 4T1 mouse carcinoma in 50 μl of serum-free medium 4 Cells were established by injection into the mammary fat pad of 8-week-old female BALB / c mice and then metastasized to the lungs. Lung metastatic burden was visualized by inflating the lungs with 15% India Ink dye (reconstituted with 1x PBS and 1 drop of ammonium hydroxide), lavaging, and bleaching in Fekete's solution (40 ml glacial acetic acid, 32 ml (37%) formalin, 700 ml (100%) ethanol stock reconstituted with ddH2O to 1 liter). For immunohistochemistry analysis, 4T1 metastasis-bearing lungs were inflated with 50% OCT, frozen in OCT blocks, and subsequently sectioned at 4 μm using the CryoJane tape transfer system. Antibodies and staining reagents are listed in Table 4. Table 4: List of antibodies [Table 4]

[0108] Analysis of the vasculature in lung metastatic lesions was performed by immunofluorescence staining with rabbit anti-Caveolin1 polyclonal antibody. Vascular density within metastatic nodes was quantified by acquiring multiple fields per mouse lung on a Zeiss inverted microscope with a 40X objective and 1.0 ovoid. The pixel density corresponding to positive CAV1 signal per field was determined in Adobe Photoshop, and these values ​​were then averaged to determine an overall mean value per mouse. To examine protein expression in 4T1 lung metastatic nodes, slides were blocked with 40 μg / mL goat anti-mouse IgG-Fab (H+L) (Jackson ImmunoResearch) followed by 10% normal goat serum (Jackson ImmunoResearch) and immunostained with α-IDO1 (clone 4B7), α-CAV1, α-CD45 (FITC conjugated), α-Gr-1, or α-CD11b (biotinylated) antibodies. Slides were washed and incubated with goat anti-mouse secondary antibody (Cy3 conjugated) to detect IDO1, goat anti-rabbit secondary antibody (FITC or Cy3 conjugated) to detect CAV1, goat anti-rat (FITC conjugated) to detect Gr-1, and streptavidin (Alexa 488 conjugated) to detect CD11b. Slides were then mounted with Prolong Gold with DAPI (Life Technologies; Cat. No. P36935) and imaged under a confocal microscope (Nikon Eclipse Ti) using a 60X objective with NIS-Elements AR software.

[0109] Oxygen-induced retinopathy: The OIR model of neovascularization was performed as described (17). Neonatal mice were housed in a chamber containing 75% oxygen (OxyCycler) from postnatal day 7 to 12 (P7-P12). Neonatal mice were removed from the chamber at P12 and then maintained in normoxia for 5 days (P17), at which time neovascularization in neonates reaches its peak level. Quantification of retinal neovascularization was performed by the same confocal microscopy analysis method as previously reported (7) using the antibodies and reagents listed in Table 4. Eyes were fixed (4% paraformaldehyde and methanol) and isolated retinas were stained with isolectin B4-Alexa488 and flat mounted. Where indicated, additional IDO1 staining in retinas was performed after isolectin B4-Alexa488 staining. Retinas were blocked in 40 μg / mL goat anti-mouse IgG-Fab(H+L) (Jackson ImmunoResearch) followed by 10% normal goat serum (Jackson ImmunoResearch). Anti-mouse IDO1, α-CD45α (FITC conjugated) and α-Gr-1 (FITC conjugated) were incubated overnight at 4°C. Retinas were washed and incubated with goat anti-mouse secondary antibody (Cy3 conjugated) to detect IDO1 and then flat mounted. Confocal microscope (Nikon Eclipse Ti) imaging was performed with a 20x objective using NIS-elements AR software.

[0110] Intravitreal injection: Localized delivery of siRNA and antibody treatments in OIR studies was performed by intravitreal injection. Prior to injection, the local anesthetic alcaine (0.5% proparacaine hydrochloride eye drops) was applied to the eyelids. Eyes were injected with 1 μl of siRNA or antibody in PBS through the cornea into the vitreous humor under a dissecting microscope using a 33-gauge needle attached to a 2.5 μl glass syringe. For siRNA (siGcn2, siAtf4, siChop and siAhr; Tables 1-3), a single injection was administered at P14, with targeted siRNA injected into one eye and non-targeted control siRNA injected into the contralateral eye. For anti-Gr-1 antibody (BioXCell), a single injection was administered at P14, with the experimental antibody injected into one eye and the isotype control antibody injected into the contralateral eye. All studies were terminated at P17 for evaluation of effects on retinal neovascularization, quantified by confocal microscopy analysis as described above.

[0111] Cell culture: 4T1 (mouse mammary carcinoma; ATCC catalog number CRL-2539) and U937 (human histiocytic lymphoma; ATCC catalog number CRL-1593.2) cell lines were cultured in DMEM supplemented with penicillin, streptomycin, 10% FBS and 55 μM β-mercaptaethanol. The HL60 (human promyelocytic leukemia; ATCC Cat# CCL-240) cell line was maintained in IMDM medium supplemented with 10% FCS + 55 μM β-mercaptaethanol, 2 mM Glutamax (Life Technologies), and 50 μg / ml gentamicin. A negative IMPACT I pathogen test result (RADIL) was obtained for the 4T1 cell line prior to its use in mice. No mycoplasma testing was performed on U937 or HL60 cell lines. For 4T1 tumor engraftment studies, cells from the original stock vials were expanded and immediately frozen, and each experiment was performed with cells taken directly from the freeze. In vitro experiments were performed using U937 and HL60 cells maintained at passage 6 or less. U937 and HL60 cells (2x10 6) were stimulated with IFNγ (100ng / mL; ThermoFisher Scientific, Cat. No. PHC4031) and LPS (100ng / mL; E. coli K-235; Cat. No. L2018) with either the IDO1 inhibitor Epacadostat (1μM +0.01% DMSO; Chemietek Cat. No. CT-EPAC), the integrated stress response inhibitor ISRIB (1μM +0.01% DMSO; Sigma-Aldrich Cat. No. SML843), the AHR inhibitor BAY-218 (1μM +0.01% DMSO; Selleckchem Cat. No. 2162982) or 0.01% DMSO vehicle alone. At 24 h, mRNA was prepared from treated cells for qPCR-based analysis of gene expression, and cell culture medium was analyzed for levels of kynurenine to assess IDO1 enzymatic activity.

[0112] Viability assay: Cell viability in U937 and HL60 cells was measured using the LDI Cell CountEZ™ Toxicity, Proliferation and Survival (TPS) Assay Kit (LDI1201) according to the manufacturer's protocol. (商標) ) were measured. Both cell lines were seeded at a density of 10,000 cells / well in 96-well plates in 100 μl of DMEM medium containing 10% FBS. Assay groups included untreated cells and IFNγ+LPS stimulation with or without Epacadostat, ISRIB, and BAY-218 according to gene expression analysis. Bortezomib (20 nM; LC Laboratories, catalog number B-1408) was used as a positive control for inducing cell apoptosis. The treated cells were incubated for 24 hours at 37° C. in a humidified CO2 incubator. The TPS assay was then performed with cell viability assessed by reading the absorbance at 412 nm in a microtiter plate reader.

[0113] Kynurenine Assay: Kynurenine levels in U937 and HL60 cell supernatants were measured using Ehrlich reagent (p-dimethylaminobenzaldehyde). After treatment, the supernatant was collected, transferred to a V-bottom 96-well plate, and mixed with 40 μL of 50% (w / V) trichloroacetic acid (TCA; LabChem) to precipitate any proteins. The plate was then incubated at 65° C. for 30 min, followed by centrifugation at 1250×g for 10 min. After centrifugation, 100 μL of the clarified supernatant was transferred to a new flat-bottom 96-well plate and mixed with an equal volume of 2% (w / v) Ehrlich's reagent in acetic acid. The resulting reaction was measured at 490 nm using a Synergy HT microtiter plate reader (Bio-Tek). Vitreous fluid was collected from the eyes of mice that underwent the OIR process. The levels of kynurenine were measured using the IDK (登録商標) Kynurenine was measured using an ELISA kit (Immundiagnostik AG, Bensheim, Germany) according to the manufacturer's protocol. Furthermore, kynurenine levels were confirmed by high-performance liquid chromatography (HPLC) coupled to electrospray ionization liquid chromatography / tandem mass spectrometry (LC / MS-MS) as described ( 18 ).

[0114] IL6 measurement: To measure levels of IL6 from the OIR model, vitreous fluid from neonatal retinas was collected immediately after eye harvest and analyzed for cytokine levels using the BD Biosciences Mouse Cytokine Bead Array IL6 Kit at a 1:4 dilution ratio of vitreous fluid to assay diluent samples. Samples were prepared according to the manufacturer's guidelines. Samples were read using a FACS Canto II flow cytometer (BD Biosciences) and FACSDIVA software (BD Biosciences), and concentrations were obtained using FCAP array software. Cytokine concentrations were calculated by comparison to standard curves provided by the manufacturer (BD Biosciences) in the cytokine bead array kit.

[0115] Quantitative real-time PCR analysis: Total cellular RNA was prepared from tissue culture cells using Trizol (Life Technologies) reagent according to the manufacturer's protocol. cDNA was prepared using the Transcription First Strand cDNA Synthesis Kit (Applied biosystems; catalog no. 4368814). Quantitative PCR (QPCR) reactions were performed using Fast Start Universal SYBR Green Mix (Roche Applied Science) with an Eppendorf Realplex 4 Mastercycler QPCR instrument. mRNA expression levels detected with primers specific for Atf4, Chop Il6, and Cyp1a1 (Tables 1–3) were normalized to 18S mRNA levels, and differences between treatment groups were analyzed using the delta-delta cycle threshold method.

[0116] Cell sorting: Gr-1+CD11b lo and Gr-1+CD11b hi Cells were analyzed and sorted using a flow cytometry cell sorter (BD FACSAria III) with the antibodies and staining reagents listed in Table 4. After 4.5 weeks of 4T1 injection (WT, Ifng - / - ) ~ 5.5 weeks (Ido1 - / - , Il6 - / - , Gcn2 - / -), tumor-bearing lungs were harvested and subsequently dissociated using GentreMACS tissue dissociator (Miltenyi Biotec) according to the manufacturer's guidelines, followed by RBC lysis and blocking with Trustay FcxTM (anti-mouse CD16 / 32) (Biolegend; Cat#101320). From the dissociated lungs, cells were stained for anti-CD45, anti-CD11b and anti-Gr-1 antibodies and then Gr-1+ CD11b+ CD45+ CD11b ... lo and Gr-1+CD11b hi The population was further selected. Autofluorescence (AF) from the Gr-1+ CD11blo gated population hi ) and Low (AF lo ) Populations of cells were sorted and the level of autofluorescence was confirmed by confocal microscopy using 495 / 519 nm excitation and emission channels. High or low combined CD11c and asialo-GM1 expression (CD11c hi Asialo-GM1 hi and CD11c lo Asialo-GM1 lo ) with Gr-1+ CD11b lo AF hi Further sorting was also performed to analyze cells from the gated population. To assess IDO1 expression levels in the different sorted cell populations, 50,000 cells were centrifuged onto slides at 800 rpm using a Shandon Cytospin 3. Slides were dried overnight, fixed with acetone at -20°C, immunostained for IDO1 (clone 4B7), and subsequently imaged by confocal microscopy (Nikon Eclipse Ti). hi Asialo-GM1 hi and CD11c lo Asialo-GM1 loThe cellular morphology of the cells was determined by staining with a Differential Quick Staining kit (Modified Giemsa, Electron Microscopy Sciences Cat# 26096-25) according to the manufacturer's protocol.

[0117] Matrigel Plug Inspection: To assess the in vivo neovascularization capacity of selected immune cell populations isolated from 4T1 lung metastases, 5x10 4 ~2x10 6 Sorted cells were isolated by fluorescence-activated cell sorting (BD FACSAria III) in RPMI + 10% FCS (as indicated in the corresponding figure legends) and kept on ice. Immediately after sorting, isolated immune cell populations were suspended in 250 μl of PBS and plated with 250 μl of Matrigel (Corning) per plug on ice for in vivo injection. (登録商標) Catalog number 356237). Plugs were established by subcutaneous injection into the dorsal region of recipient mice using an ice-cold syringe with a 21 gauge needle. To study IDO1 inhibition association, mice were administered 50 mg / kg epacadostat in 100 μl vehicle (3% N,N-dimethylacetamide, 10% 1-hydroxypropyl-β-cyclodextrin) by oral gavage bid for 72 hours starting on day 6 after injection of the Matrigel plug. Nine days after injection, the Matrigel plugs were harvested and imaged using a 2x lens with an additional 10x macro lens attachment (Moment) on a Samsung Galaxy S6 or iPhone. 登録商標 The image was recorded using either X. For quantification of blood vessels, Matrigel plugs were blocked in OCT and cryosectioned using tape transfer (>4 sections per plug). Following sectioning, neovascularization was visualized by fluorescent staining with rabbit anti-mouse for caveolin-1 and quantitatively analyzed using Adobe Photoshop as described above. Statistical analysis: Statistical analysis and graphing were performed using Prism 7 (GraphPad Software Inc.). Scatter plots are presented as means ± SEM, and statistical significance was determined by unpaired two-tailed Student's t test or by one-way ANOVA with either Dunnett's or Sidak's multiple comparison test (as indicated in the respective figure legends). The significance of Kaplan-Meier survival curves was determined by the two-group log-rank test. P-value ranges are indicated as follows: ****; P < 0.0001; ***; P < 0.01; **; P < 0.01;*; P < 0.05: ns; not significant.

[0118] Example 2: Results Genetic loss of GCN2 phenocopies the anti-angiogenic effects of both IDO1 and IL6 loss:

[0119] GCN2, a serine kinase activated in response to amino acid deficiency including tryptophan catabolism by IDO1, is one of four mammalian kinases that feed into the ISR, which has been implicated in the regulation of IL6 expression, although there are conflicting reports as to whether the regulatory effect is positive or negative (11,12,19). To examine the biological relevance of this signaling pathway as a possible mechanistic link bridging the IFNγ-dependent effects on neovascularization shared by both IDO1 and IL6 (7), we analyzed Gcn2 - / - Studies were carried out in mice. Phenotype Gcn2 challenged in metastatic 4T1 tumors - / - Mice, Ido1 - / - mice and showed a similar primary tumor growth rate as WT mice ( Fig. 9 ), but had a significantly reduced metastatic burden in the lungs ( Fig. 1 A ). This difference in transfer is due to Ido1 - / - Similar to mouse, Gcn2 - / - This was associated with a significant survival advantage that Gcn2 mice showed compared to WT mice (Figure 1B). - / - Immunofluorescence staining of 4T1 lung metastases from mice shows Ido1 - / - We found evidence of a significant reduction in vascular density comparable to that observed in mice (FIGS. 1C and 1D). These data are consistent with the hypothesis that GCN2 is required for IDO1 to promote tumor growth, neovascularization, and IL6 induction in this metastatic setting.

[0120] IDO1 signals through the integrated stress response pathway to induce IL6: IDO1-mediated tryptophan catabolism can lead to activation of AHR signaling through ISR signaling and production of the endogenous ligand kynurenine in response to tryptophan depletion. To directly address whether the observed link between IDO1 activity and IL6 induction was via either of these two downstream signaling pathways, we examined the effects of inhibiting both pathways in isolated cell cultures. We have previously shown that IFNγ+LPS-mediated induction of IDO1 activity in both U937 and HL60 cells was accompanied by a corresponding increase in IL6, and both could be blocked with the IDO1 enzyme inhibitor MTH-Trp (9). In this study, we used a different IDO1 inhibitor, Epacadostat, which blocked 80-90% of the kynurenine produced by the cells (Figure 2A) and led to a ≥ 99% reduction in IL6 gene expression (Figures 2B-2C), further supporting the link between IDO1 activity and IL6 induction. To assess the activation status of signaling pathways downstream of IDO1, we similarly assessed genes induced upon ISR and AHR activation. CHOP (C / EBP homologous protein / Ddit3 DNA damage-inducible transcript 3) and ATF4 (activator of transcription 4) are two downstream signaling elements induced upon ISR activation ( 20 ), whereas CYP1A1 (cytochrome P450, family 1, subfamily a, polypeptide 1) is a downstream target gene of AHR ( 21 ). qPCR analysis revealed elevated levels of CHOP, ATF4, and CYP1A1 message following IDO1 induction, and all three were effectively blocked by Epacadostat treatment (Figures 2B-2C). To investigate which IDO1 signaling pathway is involved in the regulation of IL6 expression, we used the inhibitors ISRIB, which blocks ISR signaling (22), and BAY-218, which blocks AHR signaling (23). As expected, ISRIB blocked the induction of both CHOP and ATF4, but not CYP1A1, and BAY-218 blocked the induction of CYP1A1, but not CHOP or ATF4 (Figures 2B-2C), and neither had a significant effect on the levels of kynurenine produced (Figure 2A). IL6 gene expression was effectively blocked by ISRIB, whereas BAY-218 had no significant effect (Figure 2B-2C), indicating that IL6 induction is controlled by signaling through the ISR and not the AHR. Neither ISRIB, Epacadostat, nor BAY-218 had a significant effect on cell viability at the concentrations used to explain the observed effects (Figure 10). Taken together, these data argue that ISR signaling is a downstream regulatory link connecting IDO1 activity to the induction of IL6.

[0121] ISR signaling acts downstream of IDO1 to promote neovascularization in a mouse model of oxygen-induced retinopathy:

[0122] To examine the effect of IDO1 on neovascularization separately from its other effects on tumor development and immune evasion, we previously used a mouse OIR (oxygen-induced retinopathy) model (7) that reproducibly and quantitatively produces abnormal blood vessels similar to those seen in tumors (17) (Figure 3A and Figure S11). In the OIR model, IDO1 is important for promoting neovascularization in a manner that opposes IFNγ and phenocopies IL6 (7). To assess the relevance of ISR signaling in this biological context, we analyzed OIR-associated neovascularization by Gcn2 - / - was evaluated in mice. Ido1 - / - Similar to our observations in mice, neovascularization was found to be significantly reduced in mice lacking GCN2 (FIG. 3B). This effect is due to Gcn2 - / - and Ido1 - / - This coincided with the decrease in IL6 levels in the vitreous humor of mice (Fig. 3C). - / - Unlike mice, Gcn2 - / - Mice did not show a significant reduction in kynurenine levels in the vitreous humor, indicating that IDO1 activity was unaffected in these animals ( Fig. 3D ).

[0123] Further evidence for a role for ISR signaling in promoting OIR-associated neovascularization came from studies using siRNA specifically directed to the eye to target key signaling components of this pathway, an approach previously used to successfully target Ido1 and Vegfa (7). Consistent with the germline knockout data, siRNA-mediated targeting of Gcn2 significantly reduced OIR-associated neovascularization (Figure 3E). Similarly, targeting the ISR signaling components Atf4 or Chop in this manner resulted in a similar reduction in OIR-associated neovascularization compared to non-targeting control siRNA injected into the contralateral eye. In contrast, using siRNA against Ahr to disrupt this alternative IDO1 signaling pathway, no reduction in OIR-associated neovascularization was observed. Instead, Ahr targeting was associated with a smaller but significant increase in neovascularization (Figure 3E), suggesting that AHR signaling may exert a subtle counterregulatory effect in this context, consistent with the trend for increased Il6 expression with AHR inhibition in U937 and HL60 cell-based assays.

[0124] In conjunction with immunofluorescent staining of blood vessels, retinal whole mounts (slides) were also stained for IDO1, and IDO1 expression was detected only in cells closely associated with neovascular tufts (Figure 3F). No IDO1 staining was observed outside the neovascular areas in OIR mouse retinas or throughout the retinas of mice maintained under constant normoxia (data not shown). - / - No positive staining for IDO1 was detected in neovascular tufts from OIR mouse retinas, confirming the specificity of anti-IDO1 antibody staining ( Fig. 3F ). In contrast, IDO1 positive staining cells were - / - Despite showing reduced levels of angiogenesis comparable to those observed in mice, Gcn2 - / - was detectable in OIR retinas from mice ( Fig. 3F ). This observation is consistent with GCN2 acting as a key signaling component of the ISR signaling pathway downstream of IDO1. - / -We argue against the concern that the lack of IDO1 positive staining observed in mouse retinas could be a spurious result of reduced neovascularization. Similarly, IDO1 expression was detected in OIR retinas of mice treated with siRNA against Gcn2, Chop and Atf4, as well as Ahr (FIG. 3G). Collectively, these data are consistent with the ISR being a biologically relevant signaling pathway through which IDO1 acts to promote pathological neovascularization in OIR.

[0125] Gr-1+ CD11b lo IDO1 Expression in Immune Cell Populations: Staining of 4T1 lung metastases for IDO1 revealed that similar to OIR retinas, IDO1+ cells associated with blood vessels in established 4T1 metastases in WT hosts ( Fig. 4A ). Importantly, evidence of IDO1 staining indicates that Ido1 - / - This was not observed in 4T1 metastases established in the host ( Fig. 4A ), confirming both the specificity of the antibody against IDO1 as well as the lack of IDO1 expression by transplanted 4T1 tumor cells in this setting, despite the retention of the WT Ido1 allele. On the other hand, IDO1+ cells express Gcn2 - / - This was observed in established 4T1 metastases in the host, despite the significantly reduced neovascularization observed in these tumors (Figure 4A), again consistent with GCN2 acting downstream of IDO1.

[0126] IDO1-positive cells were closely associated with areas of neovascularization in both the OIR and 4T1 lung metastasis models, although staining for IDO1 did not appear to directly overlap with staining for endothelial cell markers used to identify blood vessels. Instead, the Ido1 gene was derived from the WT. - / -IDO1 staining detected in 4T1 metastatic lesions, but not in the host, overlapped with a subset of cells expressing the common leukocyte marker CD45 ( Fig. 4B ). In contrast, IDO1 expression was not detected in CD45+ cells present in primary 4T1 tumors or in corresponding spleens from WT mice (Fig. 4B). 4T1 tumors induce a massive proliferation of MDSCs (24), which are primarily composed of a granulocytic polymorphonuclear subset termed G- or PMN-MDSCs (25). Further analysis of IDO1-expressing immune cell populations in 4T1 metastases revealed that IDO1 positivity colocalized with a subset of cells expressing Gr-1, a differentiation marker commonly used to identify MDSCs, but not with CD11b, another commonly used MDSC marker (Figure 4C). Similar to 4T1 metastases, IDO1-expressing cells in the retinas of OIR mice were identified as both CD45+ and Gr-1+ (Fig. 4D). Consistent with these staining data, antibody-mediated depletion of Gr-1+ cells resulted in a significant decrease in OIR-associated neovascularization compared to the isotype antibody-treated control group (Figure 4E), implying that IDO1 is a biologically relevant cell promoter of neovascularization.

[0127] The IDO1-expressing subpopulation of Gr-1+ CD11blo cells is functionally distinct from conventional MDSCs in its ability to promote neovascularization:

[0128] When analyzed by flow cytometry, the majority of CD45+ cells isolated from lungs bearing 4T1 metastases were positive for both Gr-1 and CD11b. Because the anti-IDO1 antibody used in these studies could not adequately discriminate between IDO1 expressing and non-expressing cells directly by flow cytometry, Gr-1+ cells from dissociated 4T1 lung metastases were separated based on their CD11b expression levels for microscopic analysis (Figure 5A and Figure 12). Immunofluorescence staining revealed CD11b IgG1 expression, which comprises < 5% of the total CD45+ Gr-1+ population. lo Within the cell fraction, IDO1+ cells were highly enriched, but the majority were CD45+ Gr-1+ CD11b hi No evidence of IDO1 expression was detected in the population. To assess whether the IDO1-expressing subpopulation could functionally promote neovascularization, we used sorted Gr-1+ CD11b IDO1-expressing subpopulations isolated from established 4T1 lung metastases in WT hosts. lo The cells were mixed with Matrigel and implanted subcutaneously into naive WT mice. For comparison, Gr-1+ CD11b, which represents conventional MDSCs hi Cells were similarly isolated for matrigel implantation. When the Matrigel plugs were excised after 9 days, IDO1-expressing CD11b lo It was visually evident that the plugs incorporating the cells were highly vascularized internally, but did not express IDO1, CD11b hi This was not the case for plugs that contained cells (Fig. 5C).

[0129] Immunofluorescence analysis of stained sections revealed that CD11b lo Plug and CD11b hi We observed a >10-fold difference in mean vascular density between the CD11b and CD11b plugs (Figure 5C,D). lo The CD11b population can independently promote neovascularization, and these cells are hiWe show that they can be functionally distinguished from MDSCs. In contrast, Ido1 - / - CD11b lo Matrigel plugs loaded with cells (Ido1 - / - The tumor cells (isolated from established 4T1 metastases in mice) showed minimal evidence of vascular invasion (Fig. 5C), which was consistent with the corresponding CD11b hi It was quantitatively indistinguishable from plugs that incorporated cells and was only slightly elevated above Matrigel plugs that did not incorporate any cells (FIG. 5C, D). These results suggest that IDO1 expression promotes neovascularization in Gr-1+ CD11b lo We show that it is not only relevant but important for cellular performance.

[0130] Inhibition of IDO1 inhibits isolated Gr-1+ CD11b lo Reduces cell-induced neovascularization:

[0131] CD45+ Gr-1+ CD11b lo To directly test the role of IDO1 enzyme activity in enabling cells to promote neovascularization, WT Gr-1+ CD11b lo or Gr-1+ CD11b hi Matrigel plugs incorporating the cells were allowed to establish for 6 days, after which mice were treated with the IDO1 inhibitor epacadostat for 3 days. Evaluation of excised Matrigel plugs by immunofluorescence staining demonstrated that epacadostat treatment reduced the expression of CD11b lo The extent of neovascularization promoted by CD11b hi We found that the expression of β-lactam seroconverted β-lactams was reduced to a level that was quantitatively indistinguishable from that of normal cells (Fig. 5E, F). Consistent with our previous findings that epacadostat treatment reverses angiogenesis within established 4T1 metastases (7), these results suggest that ongoing IDO1 activity is essential for the regulation of Gr-1+ CD11 lo We demonstrate that it is required to maintain, not just initiate, cell-driven neovascularization.

[0132] Neovascularization by isolated Gr-1+ CD11blo cells requires IL6 and GCN2:

[0133] Results from both the 4T1 lung metastasis and OIR models indicated that the ability of IDO1 to promote neovascularization is dependent on downstream induction of IL6, which is enhanced via activation of the ISR. To assess whether this association also holds true in isolated Gr-1+ CD11blo cells, loss of IL6 and GCN2 was assessed genetically. Il6 - / - or Gcn2 - / - Gr-1+ CD11b isolated from either mouse lo Matrigel plugs incorporating cells showed minimal evidence of vascular invasion (Figure 6A, B). These results suggest that both IL6 and GCN2 are involved in the expression of IDO1-expressing Gr-1+ CD11b lo These results confirm that IDO1 is required for promoting neovascularization, consistent with the unique in vivo relevance of this IDO1-driven signaling pathway at the cellular level.

[0134] Withdrawal of host IFNγ restores the ability of IDO1-deficient Gr-1+ CD11blo cells to promote neovascularization:

[0135] IFNγ is a major inducer of IDO1 expression but also exerts antiangiogenic activity. Our previous finding that ablation of IFNγ expression in mice rendered IDO1 insignificant as a determinant of neovascularization (7) led us to hypothesize that induction of IDO1 may act as a counterregulatory feedback mechanism controlling the antiangiogenic effects of IFNγ. To explicitly test this hypothesis, the present inventors carried out studies using mice genetically deficient in IFNγ. Evaluation of neovascular areas in OIR retina and 4T1 metastasis was performed using Ifng - / - We revealed a lack of IDO1-positive staining in specimens from mice ( Figures 6C,D ), indicating that IFNγ is required for induction of IDO1 expression in these settings, consistent with the hypothesis that IDO1 functions as a downstream counterregulatory element.

[0136] To directly assess whether the role of Ido1 in neovascularization depends on the presence of exogenous IFNγ, we tested Ido1, which failed to effectively induce neovascularization when introduced into WT mice. - / - Gr-1+ CD11b isolated from mice lo Ifng cells - / - was introduced into mice. In this context, Ido1 - / - The cells were transduced with CD11b lo This resulted in highly vascularized Matrigel plugs with vessel density comparable to plugs incorporating cells (Figure 6E,F). On the other hand, Gr-1+ CD11b hi Cells were cultured in WT or Ido1 - / - Ifng, whether derived from mouse or not - / - However, it remained unable to effectively promote neovascularization in mice (FIGS. 6E, F). These data indicate that Gr-1+ CD11b loThis supports the conclusion that IDO1 expression in cells does not determine their functional ability to promote neovascularization per se, but rather is important for protecting induced neovasculature from the anti-angiogenic effects of local IFNγ.

[0137] The IDO1+ angiogenic subpopulation is characterized by high levels of autofluorescence and surface expression of CD11c and asialo-GM1:

[0138] Gr-1+ CD11b lo Within conditions, further flow cytometric and fluorescence microscopy analyses revealed that IDO1 expression was associated with a subset of cells identifiable by high levels of autofluorescence (FIGS. 7A and 13A). Gr-1+ CD11b lo The populations were divided into high and low autofluorescence (AF hi and A.F. lo ), the ability to promote neovascularization within the Matrigel plugs was separated by a high autofluorescent signal (Figure 7B). The autofluorescence signal was particularly strong in the three excitation / emission channels 488 / 530, 450 / 407 and 488 / 585, but was weak in the 633 / 660 and 633 / 780 channels (Figure 13B).

[0139] Using the weaker autofluorescence channel, we performed AF with a series of antibodies to detect different cell surface markers. hi was evaluated in cell populations. Two antibodies from this panel, directed against CD11c and asialo-GM1, showed clearly marked distinct subpopulations with strong fluorescence intensity shifts above background (FIG. 14A). Examining these two markers, AF hi The population is CD11c hi Asialo-GM1 hi and CD11c lo Asialo-GM1 lo The cells branched into two predominant groups (FIG. 7C and FIG. 14B). IDO1 expression was morphologically more heterogeneous than CD11c lo Asialo-GM1 lo In contrast to the CD11c population, which appeared as a morphologically homogenous population hi Asialo-GM1 hi population almost exclusively (Fig. 7D). Consistent with our previous tissue staining data, IDO1 expression was significantly increased in Ido1 - / - Or Ifng - / - CD11c isolated from mice hi Asialo-GM1 hi Although it was not observed in cells, Il6 - / - and Gcn2 - / - was retained in cells from the mouse (FIG. 14C). CD11c hi Asialo-GM1 hi It was confirmed that the positivity for CD11c and asialo-GM1 as well as CD45 in the cell population was not due to the high background autofluorescence inherent to these cells (Figure 15), demonstrating the association of these markers with the IDO1-expressing cell population.

[0140] Unexpectedly, transfer of WT cells into WT recipients resulted in CD11c hi Asialo-GM1 hi (IDO1+) and CD11c lo Asialo-GM1 lo Both (IDO1-) populations were able to promote neovascularization in a Matrigel assay (FIG. 7E and FIG. 16A). This activity was shown to reduce the number of cells incorporated into the plug by CD11c hi Asialo-GM1 hi and CD11c lo Asialo-GM1 lo The corresponding reduction in vascular density between the CD11c and CD11c populations was too similar to support this explanation. lo Asialo-GM1 lo This was argued to be due to a small residual population of IDO1+ cells present within the population (Figure 16B). Instead, Ido1 - / - By performing a Matrigel assay with cells from donors to WT recipients, we were able to confirm that CD11c lo Asialo-GM1 lo The CD11c population no longer requires IDO1 to promote neovascularization. hi Asialo-GM hi The population appeared to remain IDO1 dependent (FIG. 7E and FIG. 16A). Similarly, CD11c lo Asialo-GM1 lo The population lost the requirement for both GCN2 and IL6, but not CD11c hi Asialo-GM hi population was not (Fig. 16C, D). These data suggest that AF promotes neovascularization. hi Consistent with IDO1+ cells being part of a larger population of cells, distinguishable by their ability to trigger IFNγ-dependent elimination of these newly formed blood vessels, which in turn is suppressed by their concomitant ability to induce IDO1. Ido1 - / - , Il6 - / - and CD11c from Gcn2 / mice hi Asialo-GM hi The requirement that donor cells have the presence of host IFNγ and exhibit an impaired ability to promote neovascularization is - / - This was confirmed by performing a Matrigel assay using the recipient (FIG. 7E and FIG. 17). Utilizing CD11c and asialo-GM1 markers for the initial identification of IDVC (IDO1-dependent angiogenic cell) populations allowed for further characterization of their CD11b and Gr-1 status. CD11b was determined to be low but absent (Figure 18), whereas levels of the composite marker Gr1, along with its component molecules, Ly6C and Ly6G, were all found to be present at intermediate levels (Figure 19), further distinguishing the IDVC surface marker profile from conventional MDSC (Table 5). Table 5: Surface marker comparison between IDVC populations and the majority PMN-MDSC population [Table 5]

[0141] Example 3: Observations In contrast to the detailed insights gained from studies of the ability of IDO1 to suppress T cell responses (26), the case for a role for IDO1 as an essential mediator of inflammatory neovascularization has thus far been largely conceptual (7). In the present invention, we have elucidated the molecular and cellular basis of this previously unrecognized aspect of IDO1 biology. Detection of IDO1-expressing cells associated with areas of neovascularization by immunofluorescence microscopy led to the identification of a unique subset of immune cells present, but distinct from conventional MDSCs, that are able to counter the antiangiogenic activity of IFNγ and promote the in vivo formation of new blood vessels via induction of IDO1. Examination of how IDO1 induction in these cells counteracts IFNγ delineates downstream involvement of signaling initiated by GCN2 and propagated through the ISR pathway leading to the induction of IL6, which is required to promote neovascularization. Overall, these findings reveal fundamental new insights into this novel aspect of IDO1 biology. By initiating the breakdown of tryptophan, IDO1 could potentially signal through both tryptophan depletion and the production of catabolites, and debate is ongoing as to which of these two possible signaling mechanisms is most relevant (27, 28). Biological evidence has been reported for AHR-mediated signaling in response to the catabolite kynurenine and GCN2-mediated signaling in response to tryptophan depletion (15, 29). With regard to the effect of IDO1 on neovascularization, our data clearly favor tryptophan depletion. Targeting key components of the ISR in vivo, including GCN2, CHOP, and ATF4, all phenotype-dependent effects of loss of either IDO1 or IL6 on neovascularization. Importantly, disrupting key components of the ISR was phenotypically comparable to ablating IDO1, although IDO1 expression and activity remained elevated. In the debate over mechanisms of immune regulation, one principled objection to the tryptophan depletion hypothesis has been that systemic levels of tryptophan are too high for IDO1-expressing DCs to exogenously affect T cell function via a depletion-based mechanism of action ( 30 ). However, in the case of IDO1's effects on neovascularization, this concern is made more important by evidence, both in vitro and in vivo, that IFNγ-induced induction of IDO1-mediated tryptophan catabolism acts constitutively within cells to initiate ISR pathway activation and downstream IL6 induction. Given the massive expansion of MDSCs occurring in 4T1 tumor-bearing mice, our determination that Gr-1, a lineage marker for MDSCs and neutrophils, overlaps with IDO1 expression in 4T1 metastases led us to initially hypothesize that these IDO1-expressing cells might be conventional MDSCs. However, the lack of overlap between IDO1 expression and CD11b, another characteristic lineage marker of both M-MDSCs and M-MDSCs, differentiated IDO1 + cells (called IDO1-dependent angiogenic cells or IDVCs in this study) from the majority of currently defined MDSCs ( 31 ). High degrees of intrinsic autofluorescence were subsequently identified as a defining physical feature associated with the IDO1+ cell population, which enabled further refinement of IDO1+ cells but also presented additional complications for flow cytometry-based characterization. Utilizing the channel with the lowest autofluorescence signal, IDO1-expressing cells were further delineated as nearly homogeneous with CD11c and asialo-GM1, two cell surface markers typically used to identify dendritic cells and natural killer cells, respectively. Both AF hi The cell population was able to induce neovascularization in a Matrigel assay, but CD11c hi Asiaro GM1 hi Only a subset required IDO1 or the downstream components GCN2 and IL6. Isolated CD11c lo Asialo-GM1 lo Cells no longer require IDO1 to effectively promote Matrigel neovascularization, but rather CD11c lo Asialo-GM1 lo and CD11c hi Asialo-GM1 hi In this context, a mixed population of cells expressing IDO1, CD11c hi Asialo-GM1 hi These results suggest that this population is involved not only in counteracting but also in inducing IFNγ-mediated antiangiogenic responses.

[0142] Conventional Gr-1+ CD11b hi The lack of detectable IDO1 expression in the broader population of MDSCs is consistent with our previous findings ( 9 ), although published evidence of IDO1 expression in MDSCs has also been reported. In the case of murine MDSCs, evidence generally consists of expression analysis within cell populations by PCR or Western blot analysis ( 32 – 34 ). By not examining IDO1 expression at the individual cell level, such studies run the risk of being misconstrued by contamination of traditional MDSC populations with the IDVCs identified here. There is also concern that many of the commercially available antibodies for detecting IDO1 expression lack specificity, which could result in false-positive results (35), an issue that was addressed in this study by developing an antibody that specifically recognizes IDO1 in mouse tissues using germline knockout lines for validation (36). Therefore, the broad notion that elevated expression of IDO1 occurs in conventionally defined murine MDSCs should be viewed with skepticism until more conclusive evidence can be provided.

[0143] Functionally, the ability to promote angiogenesis is currently one of the defining characteristics of conventional MDSCs (4), however, our data suggest that a reevaluation of this attribution may also be necessary. AF identified in the present invention hi Although a small subpopulation of cells was highly effective in promoting neovascularization within engrafted Matrigel plugs, conventional MDSCs were, in comparison, quite ineffective. This is because the proangiogenic activity that is generally attributed to conventional MDSCs is not related to AF. hi This raises the possibility that it may be restricted to this minor subset of cells. For example, studies performed using the Matrigel plug assay have clearly associated isolated MDSCs with angiogenic activity. However, when MDSCs were isolated solely on the basis of their Gr-1+ (or Ly6C / Ly6G) status (37), Gr-1+ CD11b lo AF hi The potential for inadvertent contamination with cells clearly exists. Even when MDSCs were isolated based on their combined Gr-1+ CD11b+ status (38), CD11blo AF hiCellular cross-contamination can still be a problem due to insufficient stringency of the CD11b cutoff value or inadvertent gating on non-specific autofluorescence. Based on these findings, a careful reevaluation of the angiogenic activity of conventional MDSCs is clearly necessary. These studies demonstrate that IDO1 is not simply a marker that distinguishes IDVCs from MDSCs, but rather is functionally required by these cells to promote neovascularization. However, IDO1 is not an angiogenic factor that directly promotes the formation of new blood vessels, like VEGF. Rather, our results demonstrated that the primary role of IDO1 in IDVC is to counteract the antiangiogenic effects of IFNγ. Mechanistically, how IFNγ exerts its antiangiogenic effects remains poorly understood. Recent studies have demonstrated that targeted deletion of IFNGR in endothelial cells is sufficient to protect the vasculature ( 39 ), whereas downregulation of D114 (Delta-like ligand 4) / Notch signaling in endothelial cells contributes to this process ( 40 ). These data suggest that a potential key to elucidating the mechanism may lie in the observed ability of IDVC-produced IL6 to counter IFNγ. Although it has been demonstrated that IL6 stimulates new blood vessel formation through a VEGF-independent mechanism ( 41 ), our data suggest that IL6 production by IDVC is more relevant as a check on the anti-neovascular activity of IFNγ than as a direct promoter of angiogenesis. Furthermore, although the present invention focuses on neovascularization, IL6 is a pleiotropic cytokine that has also been implicated in inducing functionally suppressive MDSCs (42). Our previous study showed that Ido1 - / -showed that MDSCs obtained from mice exhibited reduced suppressive activity that could be restored by transplantation of modified tumor cells ectopically expressing IL6 ( 9 ). Future studies are required to address whether IDO1-dependent production of IL6 by IDVCs may also contribute to the development of a full suppressive capacity of MDSCs in conjunction with promoting neovascularization.

[0144] While genetic studies have demonstrated an essential role for IDO1 in inflammatory neovascularization, further studies with epacadostat extend this concept further by showing that an IDO1 inhibitor is able to reverse the neovascularization process once initiated, consistent with a continuing role for IDO1 in maintaining vascular integrity. One of the challenges of antiangiogenic therapy with VEGF-targeting agents is the development of acquired resistance. In this regard, both MDSC recruitment and IL6 signaling have been implicated in the failure of VEGF-directed antibody therapy in patients (43,44). These observations are consistent with the existence of a human equivalent to the murine IDVC identified in this study that contributes to acquired resistance to VEGF antibody therapy.

[0145] Each and every patent, patent application, and publication, including websites and other publications, cited throughout this specification is hereby incorporated by reference. Although the invention has been described with reference to specific embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

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Claims

1. A medical treatment agent for a disease caused by IDO-dependent neovascularization cells (IDVCs) in the eye of a subject, comprising administering an inhibitor or remover of pathological neovascularization, wherein the establishment and maintenance of neovascularization is due to the induction of IDO1 (indoleamine 2,3-dioxygenase-1) in IDVCs.

2. The medical treatment agent according to claim 1, wherein the inhibitor achieves inhibition of IDVC activity by inhibiting IDO1.

3. 3. The medical treatment agent of claim 1 or 2, wherein the IDVCs are inhibited or removed using an antibody against a cell surface marker of the IDVCs.

4. The cell surface marker is CD33, CD11b, CD15, and CD66 The medical treatment agent according to claim 3, which is selected from the group consisting of

5. The cell surface marker is CD274(PDL1), MHC class II, CD4, CD31 (PECAM-1), CD202B(TIE2), CD205 (DEC-205), Siglec 8, and EMR1 The medical treatment agent according to claim 3, which is selected from the following:

6. The medical treatment agent according to any one of claims 1 to 5, comprising administering an agent that blocks or inhibits the Integrated Stress Response (ISR) in IDVCs.

7. 7. The medical treatment of claim 6, comprising blocking or inhibiting the integrated stress response (ISR) in ocular tissue, or in the retina or choroid.

8. The medical treatment agent of claim 6 or 7, wherein the blocking or inhibiting agent is an agent that blocks or inhibits expression, induction, activity, and / or signal transduction of an ISR pathway node.

9. The medical treatment agent according to claim 8, wherein the blocking or inhibiting agent has the following function: (a) blocking or inhibiting the expression, induction, activity or signal transduction of GCN2 (general control nonderepressible 2); and / or (b) blocking or inhibiting expression, induction, activity or signal transduction of activating transcription factor 4 (ATF4); and and / or (c) Blocking or inhibiting the expression, induction, activity or signal transduction of CHOP (C / EBP homologous protein / Ddit3 DNA-damage inducible transcript 3).

10. The medical treatment method according to any one of claims 1 to 9, further comprising administering an agent that blocks or inhibits IL-6.

11. 7. The medical treatment agent of claim 6, which blocks or inhibits downstream signaling molecules of the integrated stress response.

12. 12. The medical treatment of claim 11, wherein the signaling molecule is a cytokine.

13. The medical treatment agent of claim 12, wherein the cytokine is IL-6.

14. The medical treatment of any one of claims 1 to 13, further comprising administering an agent that blocks or inhibits any form of vascular endothelial growth factor (VEGF) expression, induction, activity, or signaling.

15. 15. The medical treatment of claim 14, wherein the VEGF inhibitor is administered less frequently than it would be administered as the sole treatment.

16. The medical treatment agent according to any one of claims 1 to 15, further comprising administering in combination an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) expression, induction, activity, or signal transduction.

17. 17. The medical treatment agent according to any one of claims 1 to 16, wherein the IDVCs are localized in the retina or choroid of the eye.

18. 18. The medical treatment of any one of claims 1 to 17, wherein the route of administration of the treatment comprises oral administration, intravenous injection, intranasal administration, sublingual administration, intravitreal injection, intraocular injection, administration via a depot preparation (also called a delayed action preparation) or device, or administration via eye drops.

19. The medical treatment agent according to any one of claims 1 to 18, wherein the blocker or inhibitor of IDO-1 comprises at least one of the following: i. 1-methyl-D-tryptophan (indoximod), 1-methyl-L-tryptophan, A racemic mixture of 1-methyl-D-tryptophan and 1-methyl-L-tryptophan, Epacadostat, Naboximo (GDC-0919), and NLG802, or a salt, enantiomer or prodrug thereof; ii. 1-RD-tryptophan or 1-RL-tryptophan, where R is C1-C12 alkyl; iii. Methylthiohydantoin-DL-tryptophan (MTH-Trp), β-(3β)-DL-alanine, β-(3-benzo(b)thienyl)-DL-alanine, 6-nitro-L-tryptophan, Indole 3-carbinol, 3,3'-diindolylmethane, Epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, Acemetacin, 5-bromo-DL-tryptophan, 5-bromoindoxyl diacetate, Naphthoquinones, S-allyl-brassinin, S-benzyl-brassinin, 5-bromo-brassinine, Phenylimidazoles, 4-phenylimidazole, Exiguamine A, and NSC401366; or iv. BMS-986205 / ONO-7701, PF-06840003 / EOS200271, MK-7162 / IOM2983, LY3381916, KHK2455, HTI-1090 / SHR9146, DN1406131, RG70099, Roxyl-WL, TPST-8844, Ethyl pyruvate, Amg-1 or DX-03-12, or A salt, enantiomer or prodrug thereof or any therapeutically active formulation thereof.

20. 16. The medical treatment of claim 14 or 15, wherein the VEGF inhibitor comprises one or more of the following: Ranibizumab (Lucentis (登録商標) ), Bevacizumab (Avastin (登録商標) ), Aflibercept (Eylea (登録商標) ), Brolucizumab (Boev (登録商標) ), Pegaptanib (Macugen (登録商標) ), Abicipar Pegol, ranibizumab biosimilars FYB201, PF582, SB11, and Xlucane; Aflibercept biosimilar MYL-1701P / M-710, or Conbercept, Faricimab / RG7716 (bispecific antibody VEGF-A + Ang-2), OPT-302 (VEGF-C / 'trap'), KS301 (Kodiak Sciences anti-VEGF polymer-linked biologic); KS501 (Kodiak Sciences anti-VEGF trap + anti-IL6 antibody fusion).

21. The medical treatment agent of claim 8 or 9, wherein the ISR pathway node is inhibited by a small molecule that inhibits translation or transcription of the ISR pathway node.

22. 10. The medical treatment agent of claim 8 or 9, wherein the ISR pathway node is inhibited by a biological molecule that inhibits translation or transcription of the ISR pathway node.

23. 23. The medical treatment of claim 22, wherein the biological molecule is an antibody or a fragment thereof.

24. 10. The medical treatment agent of claim 8 or 9, wherein the ISR pathway node is inhibited by a nucleic acid molecule that inhibits translation or transcription of the ISR pathway node.

25. 25. The medical treatment of claim 24, wherein the nucleic acid molecule is an siRNA or an shRNA.

26. The medical treatment method according to any one of claims 1 to 25, comprising administering a GCN2 inhibitor.

27. The GCN2 inhibitor, GCN2-IN-1(A-92), GCN2iA, GZD824, Triazolo[4,5-d]pyrimidine scaffold-based inhibitors 27. The medical treatment agent of claim 26, selected from the group consisting of

28. The medical treatment method according to any one of claims 1 to 27, comprising administering a CHOP inhibitor.

29. The medical treatment method according to any one of claims 1 to 28, comprising administering an ATF4 inhibitor.

30. The ATF4 inhibitor, Ursolic acid, Tomatidine, GSK2606414, and TRIB3 30. The medical treatment of claim 29, wherein the medical treatment is selected from the group consisting of

31. The ISR pathway blocker is GSK-2606414, RPT-GCN2i, AMG-PERK44 and trans-ISRIB 22. The medical treatment of claim 21, wherein the medical treatment is selected from the group consisting of