WO2022036196A2 - Use of vista agonist for treatment/prevention of cytokine storm or crs or sepsis and/or acute or chronic respiratory distress syndrome (rds) - Google Patents

Abstract

The use of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment, for preventing, stabilizing or reducing cytokine storm and/or acute or chronic respiratory distress syndrome and symptoms associated therewith such as lung damage in patients comprising or suspected of comprising a coronavirus infection, e.g., COVID-19 or other bacterial or viral infection which is known to cause cytokine storm and/or acute or chronic respiratory distress syndrome by administering to said subject a prophylactically or therapeutically effective amount of a VISTA agonist are provided. In exemplary embodiments the methods are used in patients known or suspected to be infected with COVID-19 wherein optionally the patient may already exhibit signs of lung damage and/or or pneumoniae and/or may be on a respirator or ventilator. These treatments may be effected in combination with one or more other treatments for cytokine storm, or acute or chronic respiratory distress syndrome, pneumonia and/or viral or bacterial infection such as steroids, anti-virals and antibiotics or other checkpoint molecule agonists and antagonists.

Description

Use of VISTA Agonist for Treatment/Prevention of Cytokine Storm or CRS or Sepsis and/or

Acute or Chronic Respiratory Distress Syndrome (RDS)

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the priority benefit of U.S. Provisional Application Ser. Nos. 63/065,676 filed August 14, 2020, 63/079,255 filed September 16, 2020, and 63/080,063 filed September 18, 2020 each and all of which are incorporated by reference herein in their entirety.

FIELD

[002] This invention is directed to treating inflammatory diseases that are mediated by overexpression of innate derived cytokines and chemokines such as IL-la, IL-6, TNF-a, IFN-y, and granulocyte-monocyte colony stimulating factor (GM-CSF), IP-10 and others, many of which are driven by the IFNI response. In particular the invention relates to treatment and prevention of conditions associated with the overexpression of innate derived cytokines and chemokines including cytokine storm, septic shock, and sepsis acute respiratory distress syndrome which may result from different causes, such as infectious and non-infectious diseases including graft-versus-host disease (GVHD), coronavirus disease 2019 (COVID-19), sepsis, H1N1 flu, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS).

BACKGROUND

[003] A cytokine storm, also called hypercytokinemia, is a physiological reaction in humans and other animals in which the innate immune system causes an uncontrolled and excessive release of pro-inflammatory signaling molecules called cytokines. Normally, cytokines are part of the body's immune response to infection, but their sudden release in large quantities can cause multisystem organ failure and death. Cytokine storms can be caused by a number of infectious and non-infectious etiologies, especially viral respiratory infections such as H5N1 influenza, SARS-CoV-1, and SARS-CoV-2 (COVID-19 agent). Other causative agents include the Epstein-Barr virus, cytomegalovirus, and group A streptococcus, and non- infectious conditions such as graft-versus-host disease. [004] Cytokine storm syndrome is diverse set of conditions that can result in cytokine storm. Cytokine storm syndromes include familiar hemophagocytic lymphohistiocytosis, Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis, systemic or non- systemic juvenile idiopathic arthritis-associated macrophage activation syndrome, NLRC4 macrophage activation syndrome, cytokine release syndrome and sepsis.

[005] For example in its most severe form, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), leads to a lifethreatening pneumonia and acute respiratory distress syndrome (ARDS). The mortality rate from COVID-19 ARDS can approach 40% to 50%.

[006] Based on the foregoing new and improved treatments for preventing or treating cytokine storm or CRS and/or preventing or treating acute or chronic respiratory distress syndrome and side effects thereof such as lung and other organ and tissue damage caused thereby, specifically that caused by infectious and non-infectious diseases including graft- versus-host disease (GVHD), coronavirus disease 2019 (COVID-19), sepsis, H1N1 flu, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS) and other conditions associated with cytokine storm and/or acute or chronic respiratory distress syndrome are urgently needed.

BRIEF DESCRIPTION OF THE FIGURES

[007] Figures 1A to ID shows that an agonistic anti-VISTA antibody suppresses CD14 and CD16 (Fcgr3a) expression in human monocytes. (A) Boxplot depicting the CD14 gene expression difference between Anti-VISTA (803) and hlgG2 isotype control treated CD14+ human monocytes after 24 hours of treatment. (B) Flow cytometry plot showing the CD14 protein expression between Anti-VISTA (803) and hlgG2 isotype control treated CD14+ human monocytes. (C) Boxplot depicting the Fcgr3a gene expression difference between Anti-VISTA (803) and hlgG2 treated CD14+ human monocytes. (D) Flow cytometry plot showing the CD16 protein expression between Anti-VISTA (803) and hlgG2 treated CD14+ human monocytes.

[008] Figures 2A to 2B shows that anti-VISTA (803) treatment reduces antigen presentation pathway in human monocytes. (A) Dot plot showing the antigen presenting associated genes difference between anti-VISTA (803) and hlgG2 isotype control treated CD14+ human monocytes. (B) GSEA plot showing the enrichment of antigen presenting pathway between anti-VISTA (803) and h IgG treated CD14⁺ human monocytes. [009] Figures 3A to 3H shows that an agonist anti-VISTA antibody suppresses CXCL10 expression in human monocytes by suppression of IFN-I response pathway genes. (A) Dot plot depicting the CD14 gene expression difference between Anti-VISTA (803) and hlgG2 isotype control treated CD14+ human monocytes. (B) Human CD14+ monocytes were either treated with anti-VISTA agonist or hlgG2 isotype control for 24 hours and CXCL10 supernatant levels was determined via multiplex analysis (C) Dot plot showing the interferon response associated genes difference between Anti-VISTA (803) and hlgG2 isotype treated CD14+ human monocytes. (D) GSEA plot showing the enrichment of interferon response pathway between Anti-VISTA (803) and hlgG treated CD14+ human monocytes. (E-F) Supernatant levels of CXCL10 determined by multiplex analysis after anti-VISTA agonist or control hlgG2 treatment of monocytes in the presence of Flage I li n, B-glucan, LPS or Poly( I :C). (G-H) The Venn diagram showing the significant enrichment between Anti-VISTA (803) treated and COVID-19 CD14+ human monocytes.

[0010] Figures 4A to 4D shows that an anti-VISTA agonist strikingly changes the CD14⁺ monocyte state and induces novel archetypes associated with the anti-inflammatory phenotype. (A) Uniform manifold approximation and projection (UMAP) plot showing the cluster distribution of Anti-VISTA (803) and hlgG2 isotype control treated CD14⁺ human monocytes. The biological annotation of each cluster is presented in the table on the right. (B) Pie chart indicating the composition of cluster difference in anti-VISTA (803) and hlgG treated CD14⁺ human monocytes. (C-D) Feature plot showing the expression of CD14 and Fcgr3a across different clusters in anti-VISTA (803) and hlgG treated CD14⁺ human monocytes. [0011] Figures 5A to 5Z contains the CDR and variable heavy and light chain polypeptide sequences of exemplary agonistic anti-human VISTA antibodies.

SUMMARY

[0012] This invention is directed to treating inflammatory diseases that are mediated by overexpression of innate derived cytokines and chemokines such as IL-la, IL-6, TNF-a, IFN-y, and granulocyte-monocyte colony stimulating factor (GM-CSF), IP-10 and others, many of which are driven by the IFNI response. In particular the invention relates to treatment and prevention of conditions associated overexpression of innate derived cytokines and chemokines including cytokine storm, CRS, septic shock or sepsis and acute or chronic respiratory distress syndrome which may result from different causes, such as infectious and non-infectious diseases including graft-versus-host disease (GVHD), coronavirus disease 2019 (COVID-19), sepsis, H1N1 flu, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS) by the administration of a VISTA agonist, e.g., an agonistic anti-VISTA antibody or antibody fragment or a VISTA fusion protein.

[0013] This invention specifically relates to the use of VISTA agonists to prevent, stabilize or treat cytokine storm or CRS in a subject in need thereof, e.g., a subject with a condition such as chronic or acute viral or bacterial infectious condition corelated with cytokine storm or CRS and/or in a subject exhibiting signs of cytokine storm or CRS.

[0014] This invention also specifically pertains to the use of VISTA agonists, e.g., agonistic anti-VISTA antibodies or antibody fragments or VISTA fusion proteins, to prevent, stabilize or treat acute or chronic respiratory distress syndrome (ARDS or CRDS) and symptoms thereof such as lung damage in patients comprising or at risk of developing acute or chronic respiratory distress syndrome, e.g., as a result of infectious and non-infectious diseases including graft-versus-host disease (GVHD), coronavirus disease 2019 (COVID-19), sepsis, H1N1 flu, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS).

[0015] This invention more specifically relates to the use of VISTA agonists to prevent, stabilize or treat cytokine storm in a subject infected with coronavirus 2 (SARS-CoV-2) or COVID-19.

[0016] In exemplary embodiments the methods are used in patients who may already exhibit signs of lung or heart damage and/or or pneumoniae and/or may be on a respirator or ventilator. These treatments may be effected in combination with one or more other treatments for cytokine storm or CRS or sepsis and/or acute or chronic respiratory distress syndrome, pneumoniae and/or viral or bacterial infection such as steroids, anti-virals and antibiotics.

[0017] This invention further relates to the use of VISTA agonists, e.g., agonistic VISTA fusion proteins, agonistic anti-VISTA antibodies or agonistic anti-VISTA antibody fragments for the treatment or prevention of acute respiratory distress syndrome (ARDS) or chronic respiratory distress syndrome (CRDS) and the amelioration of side effects associated therewith such as lung damage in patients with a condition, e.g., an infectious or other condition associated with acute respiratory distress syndrome (ARDS) or chronic respiratory distress syndrome (CRDS) such as a virus or bacterial infection, e.g., influenza, Ebola, SARS, COVID-19, among others. [0018] It is another object of the invention to provide novel protocols for treating or preventing the treatment or prevention of respiratory distress syndrome and the amelioration of side effects associated therewith by the administration of VISTA agonists, e.g., agonistic VISTA fusion proteins, agonistic anti-VISTA antibodies and antibody fragments wherein aberrant (reduced or increased) levels of cytokines, immune signaling molecules and/or molecules associated with hyperinflammation are detected in the treated patient, e.g., elevated IL-6, gamma interferon, IL-2, and/or CRP levels are detected prior, during or after VISTA agonist administration.

[0019] It is another specific object of the invention object of the invention to provide therapeutic protocols for treating or preventing respiratory distress syndrome and for ameliorating pathogenic side effects associated therewith such as lung damage and cytokine storm comprising administering to said subject a prophy lactica I ly or therapeutically effective amount of an agonistic anti-VISTA antibody or antibody fragment, e.g., wherein the antibody or antibody fragment comprises a variable light chain polypeptide and a variable heavy chain polypeptide comprising the CDRs any one of the antibodies having the sequences contained in the table in Figure 5 and optionally comprises a human lgG2 Fc region.

[0020] It is another specific object of the invention object of the invention to provide therapeutic protocols for treating or preventing the treatment or prevention of acute or chronic respiratory distress syndrome and the amelioration of side effects associated therewith such as lung damage and cytokine storm in patients infected or suspected of being infected by the administration of an agonistic anti-VISTA antibody or antibody fragment, e.g., wherein the antibody or antibody fragment comprises a variable light chain polypeptide and a variable heavy chain polypeptide comprising the CDRs any one of the antibodies having the sequences contained in the table in Figure 5, wherein the patient is evaluated prior, during or after treatment to detect whether aberrant levels of cytokines, immune signaling molecules and/or molecules associated with hyperinflammation are detected, e.g., IL-6, gamma interferon, IL-2, and/or CRP levels are detected prior, during or after VISTA agonist administration.

[0021] This invention also provides methods of preventing, stabilizing or reducing cytokine storm or cytokine release syndrome (CRS) and/or sepsis or the symptoms thereof in a subject in need thereof comprising administering to said subject a prophylactically or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein.

[0022] This invention also provides methods of decreasing the levels of at least one of LPS- induced IL-12p40, IL-6, CXCL2 and TNF in a subject in need thereof, optionally a subject having or at risk of developing cytokine storm or septic shock in a subject in need thereof wherein this is effected by administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein.

[0023] A method of increasing the expression of mediators involved in macrophage tolerance induction, wherein said mediators optionally include at least one of IRG1, miR221, A20, and IL-10 and/or increasing the expression of anti-inflammatory transcription factors which drive an anti-inflammatory profile optionally including at least one of IRF5, IRF8, and NFKB1 thereby preventing, stabilizing or reducing the risk or severity of cytokine storm or septic shock, sepsis or CRS in a subject in need thereof, wherein this is effected by administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein.

[0024] This invention also provides methods of preventing, stabilizing or reducing the risk or severity of cytokine storm or septic shock, sepsis or CRS in a subject in need thereof by (i) reducing the level of CXCR2 and/or CXCL10; (ii) reducing neutrophil/lymphocyte ratios, (iii) reducing FcgRIII levels or a combination thereof, wherein this is effected by administering to said subject a prophylactical ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment. [0025] This invention also provides methods of preventing, stabilizing or reducing acute or chronic respiratory distress syndrome or the symptoms associated therewith in a subject, optionally a subject with an infection further optionally coronavirus infection, optionally COVID-19, or other viral or bacterial infection associated with acute or chronic respiratory distress syndrome comprising administering to said subject a prophy lactical ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein.

[0026] This invention also provides methods of decreasing the levels of at least one of LPS- induced IL-12p40, IL-6, CXCL2 and TNF in a subject in need thereof, thereby preventing, stabilizing or reducing acute or chronic respiratory distress syndrome or the symptoms associated therewith in a subject wherein this is effected by administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein.

[0027] This invention also provides methods of increasing the expression of mediators involved in macrophage tolerance induction, wherein said mediators optionally include at least one of IRG1, miR221, A20, and IL-10 and/or increasing the expression of antiinflammatory transcription factors which drive an anti-inflammatory profile optionally including at least one of IRF5, IRF8, and NFKB1 thereby preventing, stabilizing or reducing acute or chronic respiratory distress syndrome or the symptoms associated therewith in a subject in a subject in need thereof, wherein this is effected by administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein.

[0028] This invention also provides methods of preventing, stabilizing or reducing the risk or severity of cytokine storm or septic shock, sepsis or CRS or ARDS or other respiratory syndrome in a subject in need thereof by (i) reducing the level of CXCR2 and/or CXCL10; (ii) reducing neutrophil/lymphocyte ratios, (iii) reducing FcgRIII levels or a combination thereof, wherein this is effected by administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein.

[0029] Any of the above methods may optionally include the administration of another active, optionally selected from a PD-1 agonist, a CTLA-4 agonist, a TNF antagonist optionally an anti-TNF antibody or TNF-receptor fusion such as Embrel, an IL6 antagonist such as an anti-IL-6 or anti-l L-6R antibody, a corticosteroid or other anti-inflammatory agent.

[0030] In any of the above methods the patient may comprise or be suspected of comprising a coronavirus infection or another condition, e.g., a bacterial and/or viral infection correlated with an increased risk of cytokine storm, CRS, sepsis, septic shock or ARDS, optionally coronavirus disease 2019 (COVID-19), sepsis, H1N1 flu, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS).

[0031] In any of the above methods the patient comprises or is suspected of comprising COVID-19 infection.

[0032] In any of the above methods the agonistic anti-VISTA antibody or antibody fragment specifically binds to human VISTA, optionally wherein the agonistic anti-VISTA antibody or antibody fragment comprises a variable light chain and a variable heavy chain polypeptide comprising the same CDRs any one of the antibodies having the sequences contained in the table in Figure 5 and further optionally a human lgG2 Fc or constant region, optionally wherein FcR binding is maintained intact compared to an endogenous human lgG2 Fc or constant region.

[0033] In any of the above methods the agonistic anti-VISTA antibody or antibody fragment comprises a variable light chain and a variable heavy chain polypeptide comprising the same CDRs any one of the antibodies having the sequences contained in the table in Figure 5 and the variable light chain and the variable heavy chain polypeptide of said antibody or antibody fragment respectively each possess at least 90% sequence identity to the variable light chain and the variable heavy chain polypeptides of the same anti-human VISTA antibody having the sequences contained in the table in Figure 5 and further optionally a human lgG2 Fc or constant region, optionally wherein FcR binding is maintained intact compared to an endogenous human lgG2 Fc or constant region..

[0034] In any of the above methods the agonistic anti-VISTA antibody or antibody fragment optionally comprises a variable light chain and a variable heavy chain polypeptide comprising the same sequences as any one of the anti-human VISTA antibodies having the sequences contained in the table in Figure 5, further optionally wherein FcR binding is maintained intact compared to an endogenous human lgG2 Fc or constant region.

[0035] In any of the above methods the e VISTA agonist optionally comprises a human VISTA fusion polypeptide, e.g., a human VISTA-lg fusion protein and/or a human VSIG3 fusion polypeptide, e.g., a human VSIG3-lg fusion protein.

[0036] In any of the above methods the VISTA agonist optionally comprises a human Fc region, e.g., a human IgGl, lgG2, lgG3 or lgG4 Fc region.

[0037] In any of the above methods the VISTA agonist optionally, comprises a human lgG2 Fc region, further optionally wherein FcR binding is maintained intact compared to an endogenous human lgG2 Fc or constant region.

[0038] T In any of the above methods the VISTA agonist optionally may comprise a human Fc region, e.g., a human IgGl, lgG2, lgG3 or lgG4 Fc region, that has been mutated to alter (increase or decrease) at least one effector function, e.g., FcR binding, complement binding, glycosylation, or ADCC.

[0039] In any of the above methods the VISTA agonist optionally comprises a human lgG2 Fc region which binds to all or at least one Fc receptor bound by an endogenous human lgG2 Fc region.

[0040] In any of the above methods the VISTA agonist optionally (i) increases the ratio of lymphocytes to neutrophils and/or (ii) results in a more normal (anti-inflammatory) cytokine profile and/or (iii) decreases CXCL10 and/or CXCR2 levels, (iv) eliminates or reduces the CD14+ classical monocyte phenotype in favor of a more anti-inflammatory cell state characterized e.g., by downregulation of CD14, IFN receptors, Fcgr3a (CD16) and CSF1R, (v) upregulation of CDllb, M-CSF (Csfl), Cyclin-dependent kinase inhibitor (Cdknla) and the anti-inflammatory cytokines IL1RA and GDF15 or a combination of any of the foregoing.

[0041] In any of the above methods the treatment optionally prevents the patient from progressing to a clinical endpoint consistent with an ARDS diagnosis or which results in a much less severe form of ARDS, i.e., which does not progress to cytokine storm, sepsis and/or organ failure.

[0042] In any of the above methods the treatment optionally the patient has a coronavirus infection caused by COVID-19.

[0043] In any of the above methods the treatment optionally the patient is confirmed to be COVID-19 positive prior to treatment.

[0044] In any of the above methods the treatment optionally the patient is confirmed to be COVID-19 after starting treatment.

[0045] In any of the above methods the treatment optionally the patient shows at least one symptom of ARDS or pulmonary problems prior to or after treatment, optionally herein said symptoms or pulmonary problems include one or more of the following: barotrauma (volutrauma), pulmonary embolism (PE), pulmonary fibrosis, ventilator-associated pneumonia (VAP); gastrointestinal bleeding (ulcer), dysmotility, pneumoperitoneum, bacterial translocation; Hypoxic brain damage; abnormal heart rhythms, myocardial dysfunction; acute kidney failure, positive fluid balance; vascular injury, pneumothorax, tracheal injury/stenosis; malnutrition (catabolic state), electrolyte abnormalities;

Atelectasis, blood clots, weakness in muscles used for breathing, stress ulcers, depression or other mental illness; single or multiple organ failure; pulmonary hypertension or increase in blood pressure in the main artery from the heart to the lungs.

[0046] In any of the above methods the treatment optionally the levels of at least one cytokine or anti-inflammatory molecule or proinflammatory molecule, e.g., CXCL10, CXCR2, IL-6, CRP, gamma interferon, IL-1, TNF, IFN-y, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-lalpha, and CXCL11/I-TAC in the patient are detected prior to treatment, further optionally wherein the levels of said cytokine or proinflammatory molecule in the patient are detected and confirmed to be aberrant (consistent with a diagnosis of the onset of or increased risk of cytokine storm) prior to treatment.

[0047] In any of the above methods the treatment optionally the levels of VISTA in the patient are detected prior to and/or after treatment.

[0048] In any of the above methods the treatment optionally the levels of at least one of CXCL10, CXCR2, IL-6, CRP, IFN-y, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-lalpha, and CXCL11/I- TAC in the patient are detected prior to and/or after treatment. [0049] In any of the above methods the treatment optionally the levels of IL-6 and/or CRP and/or any of I FN-y, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-lalpha, and CXCL11/I-TAC in the patient are detected and confirmed to be elevated prior to treatment.

[0050] In any of the above methods the treatment optionally the VISTA agonist is administered at a dose ranging from .01-5000 mg, 1-1000 mg, 1-500 mg, 5mg - 50mg or about 1-25 mg.

[0051] In any of the above methods the treatment optionally the VISTA agonist is administered biweekly, weekly, every 2 or 3 weeks, or every 4 weeks intravenously or via subcutaneous injection.

[0052] In any of the above methods the treatment optionally the patient is administered another active or another therapeutic regimen used to treat coronavirus infection and/or treat or prevent ARDS or CRDS.

[0053] In any of the above methods the treatment optionally the patient receives another treatment for ARDS, optionally one or more of corticosteroids; inhaled nitric oxide (NO); extracorporeal membrane oxygenation (venovenous or venoarterial) or another immunosuppressive agent, optionally thymoglobulin, basiliximab, mycophenolate mofetil, tacrolimus, an anti-CD20 mAb such as rituximab, or a corticosteroid.

[0054] In any of the above methods the treatment optionally the patient is additionally treated with an antiviral or antibiotic or another anti-inflammatory agent, optionally another biologic, e.g. another antibody that targets a checkpoint protein such as PD-1, PD- Ll, PD-L2, CTLA-4 or an IL-6 antagonist.

[0055] In any of the above methods the treatment optionally the patient has pneumonia, optionally caused by COVID-19 or caused by another pathogen, e.g., another virus, a bacterium or a fungus.

[0056] In any of the above methods the treatment optionally the patient has one or more risk factors for developing cytokine storm, ARDS or a poor ARDS prognosis; e.g., the patient is over 60, 65, 70 years of age, the patient has type 1 or type 2 diabetes, the patient has high blood pressure, the patient has cancer, the patient has an inflammatory lung condition, e.g., asthma, COPD or cystic fibrosis, the patient has arteriosclerosis, the patient has another inflammatory or autoimmune condition or a combination of any of the foregoing.

[0057] The method of any of the previous claims, wherein the patient is on a ventilator or respirator prior to agonist administration. [0058] In any of the above methods the treatment optionally the patient has improved or normal lung function after agonist treatment.

[0059] In any of the above methods the treatment optionally the patient is further treated with any of the following:

(i) azathioprine,

(ii) calcineurin inhibitors (CNIs),

(iii) mycophenolate mofetil (MMF)/mycophenolic acid (MPA),

(iv) mTOR inhibitors (e.g., tacrolimus, everolimus, sirolimus),

(v) low dose corticosteroids (e.g., prednisone/prednisolone),

(vi) antihypertensive agents (e.g., angiotensin converting enzyme inhibitors (ACEIs),

(vii) angiotensin II receptor blockers (ARBs),

(viii) cyclosporine,

(ix) antidiabetogenic agents;

(x) a pulse steroid such as oral prednisone;

(xi) or a combination of any of the foregoing.

[0060] In any of the above methods optionally the anti-VISTA antibody or antibody fragment contains an Fc region that has been modified to alter effector function, half-life, proteolysis, and/or glycosylation.

[0061] In any of the above methods optionally the anti-VISTA antibody is selected from a humanized, single chain, or chimeric antibody and the antibody fragment is selected from a Fab, Fab', F(ab')2, Fv, or scFv.

[0062] In any of the above methods optionally the VISTA agonist dose is between about 0.001 and 100 mg/kg of body weight of recipient patient.

[0063] In any of the above methods the treatment optionally promotes survival and/or improves or restores normal lung function.

[0064] In any of the above methods the treatment optionally eliminates the need for the need for the patient to go on a ventilator or reduces the time the patient is on a ventilator.

[0065] In any of the above methods the treatment optionally administration of the VISTA agonist (i) increases the ratio of lymphocytes to neutrophils and/or (ii) results in a more normal (anti-inflammatory) cytokine profile and/or (iii) decreases CXCL10 and/or CXCR2 levels, (iv) eliminates or reduces the CD14+ classical monocyte phenotype in favor of a more anti-inflammatory cell state characterized e.g., by downregulation of CD14, IFN receptors, Fcgr3a (CD16) and CSF1R, (v) upregulates one or more of CDllb, M-CSF (Csfl), Cyclin- dependent kinase inhibitor (Cdknla) and the anti-inflammatory cytokines IL1RA and GDF15 or a combination of any of the foregoing.

DETAILED DESCRIPTION

[0067] Prior to disclosing the invention in more detail the following definitions are provided.

[0068] DEFINITIONS

[0069] As used herein "VISTA" or "V-domain Ig suppressor of T cell activation (VISTA)" refers to a type I transmembrane protein that functions as an immune checkpoint and is encoded by the C10orf54 gene. VISTA is produced at high levels in tumor-infiltrating lymphocytes, such as myeloid-derived suppressor cells and regulatory T cells, and its blockade with an antibody results in delayed tumor growth in mouse models of melanoma and squamous cell carcinoma. Monocytes from HIV-infected patients produce higher levels of VISTA compared to uninfected individuals. Increased VISTA levels correlated with an increase in immune activation and a decrease in CD4-positive T cells. VISTA further includes human, non-human primate, murine and other mammalian forms of VISTA.

[0070] As used herein, a "VISTA Agonist" refers to any molecule which specifically and directly agonizes (promotes) the expression of VISTA and/or which promotes or increases at least one functional activity of VISTA, e.g., its suppressive effects on T cell immunity (CD8+ T cell or CD4+ T cell immunity) and its suppressive effect on Foxp3 expression and/or its suppressive or promoting effect on the expression of cytokines, anti-inflammatory and proinflammatory molecules, particularly VISTA's modulatory (decrease or increase) effect on the expression of specific cytokines, activation markers and other immune molecules, e.g., those where expression is by or regulated by T cells. VISTA's effects on specific immune molecules including specific cytokines as shown in Figures 1-4. These molecules include by way of example IFN-y, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-lalpha, and CXCL11/I-TAC. VISTA agonists herein specifically include VISTA fusion proteins, agonist anti-VISTA antibodies and antibody fragments which directly promote VISTA's effects on one or more of these molecules. Exemplary agonist anti-VISTA antibodies will comprise the CDRs and/or variable regions of any of the anti-VISTA antibodies having the sequences shown in Figure 5 and will contain human lgG2 Fc regions, preferably a human lgG2 or Fc region wherein FcR binding is intact, i.e., the same as a native or endogenous human lgG2 Fc or constant region. Also, as VSIG3 reportedly is a ligand for VISTA (see Jinghua Wang, Guoping Wu, Brian Manick, Vida Hernandez, Mark Renelt, Ming Bi, Jun Li and Vassilios Kalabokis, J Immunol May 1, 2017, 198 (1 Supplement) 154.1); which when bound to VISTA promotes its activity, VISTA agonists herein further include compounds (VSIG3 fusion proteins, anti-VSIG3 antibodies and antibody fragments) which directly promote VISTA's effects on one or more of these molecules. Herein VSIG-3 also referred to as IGSF11 includes human, non-human primate, murine and other mammalian forms of VSIG-3. Also VISTA agonists include other moieties that provide for increased VISTA expression or amounts in a subject, e.g., cells engineered to express VISTA, e.g., under controllable conditions or compounds which promote the expression of VISTA. Also, VISTA agonists include anti-VISTA or anti-PSLGl antibodies and antibody fragments and PSGL1 fusion proteins and small molecules which agonize the VISTA/PSGL1 binding interaction. In this regard PSGL1 has been reported to be a binding partner of VISTA (see WO 2018/169993 filed by Bristol Myers and Robert J. Johnston et al., "VISTA is an acidic pH-selective ligand for PSGL-1", Nature (2019) 574: 565-570.

[0071] As used herein, "Cytokine Storm" or "Hypertyrosinemia" or Cytokine Release Syndrome" or "CRS" refers to a severe immune reaction in which the body releases too many cytokines into the blood too quickly. Cytokines play an important role in normal immune responses, but having a large amount of them released in the body all at once can be harmful. A cytokine storm can occur as a result of an infection, autoimmune condition, or other disease. It may also occur after treatment with some types of immunotherapy. Signs and symptoms include high fever, inflammation (redness and swelling), and severe fatigue and nausea. Sometimes, a cytokine storm may be severe or life threatening and lead to multiple organ failure. The pathogenesis is complex but includes loss of regulatory control of proinflammatory cytokine production, both at local and systemic levels. The disease progresses rapidly, and the mortality is high. For example COVID-19 infection has been closely associated with dysregulated and excessive cytokine release or "cytokine storm". [0072] As used herein, "Acute respiratory distress syndrome" or "ARDS" refers to a type of respiratory failure characterized by rapid onset of widespread inflammation in the lungs often the result of an infection. Symptoms include shortness of breath, rapid breathing, and bluish skin coloration. Among those who survive, a decreased quality of life is relatively common. Known causes may include sepsis, pancreatitis, trauma, pneumonia, and aspiration. The underlying mechanism involves diffuse injury to cells which form the barrier of the microscopic air sacs of the lungs, surfactant dysfunction, activation of the immune system, and dysfunction of the body's regulation of blood clotting. In effect, ARDS impairs the lungs' ability to exchange oxygen and carbon dioxide. Diagnosis is based on a PaO2/FiO2 ratio of less than 300 mmHg despite a PEEP of more than 5 cm H2O and heart related pulmonary edema, as the cause, must be excluded. The primary treatment involves mechanical ventilation together with treatments directed at the underlying cause.

Ventilation strategies include using low volumes and low pressures. If oxygenation remains insufficient, lung recruitment maneuvers and neuromuscular blockers may be used. If this is insufficient, ECMO may be an option. Current treatments for ARDS include treatment with corticosteroids; inhaled nitric oxide (NO); extracorporeal membrane oxygenation which comprises mechanically applied prolonged cardiopulmonary support.

[0073] As used herein, "chronic respiratory distress syndrome" or "Chronic respiratory disease" refers to long-term diseases of the airways and other structures of the lung. They are characterized by a high inflammatory cell recruitment (neutrophil) and/or destructive cycle of infection, (e.g. mediated by Pseudomonas aeruginosa). Some of the most common are asthma, chronic obstructive pulmonary disease, and acute respiratory distress syndrome. CRDS is not curable; however, various forms of treatment that help dilate major air passages and improve shortness of breath can help control symptoms and increase the quality of life.

[0074] As used herein "pneumonia" refers to an inflammatory condition of the lung affecting primarily the small air sacs known as alveoli. Typically, symptoms include some combination of productive or dry cough, chest pain, fever and difficulty breathing. The severity of the condition is variable. Pneumonia is usually caused by infection with viruses or bacteria and less commonly by other microorganisms, certain medications or conditions such as autoimmune diseases. Coronavirus and specifically COVID-19 may cause pneumonia. Risk factors for pneumonia include cystic fibrosis, chronic obstructive pulmonary disease (COPD), asthma, diabetes, heart failure, a history of smoking, a poor ability to cough such as following a stroke and a weak immune system.

[0075] As used herein, "improved," "improvement," and other grammatical variants, includes any beneficial change resulting from a treatment. A beneficial change is any way in which a patient's condition is better than it would have been in the absence of the treatment. "Improved" includes prevention of an undesired condition, slowing the rate at which a condition worsens, delaying the development of an undesired condition, and increasing the rate at which a desired condition is reached. For example, improvement in an ARDS patient encompasses any decrease in inflammatory cytokines as any increase in the amount or rate at which inflammatory cytokines are prevented, removed or reduced. For another example, improvement in a ARDS patient or patient at risk of ARDS encompasses any prevention, decrease, delay or slowing in the rate of the condition and cytokine mediated damage or loss of function, e.g., to lung function.

[0076] The term "antibody" or "Ab," or "immunoglobulin" is used herein in the broadest sense and encompasses various antibody structures which specifically binds with an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and/or antibody fragments (also referred to as "antigen-binding antibody fragments"). Typically, a full-size Ab (also referred to as an intact Ab) comprises two pairs of chains, each pair comprising a heavy chain (HC) and a light chain (LC). A HC typically comprises a variable region and a constant region. A LC also typically comprises a variable region and constant region. The variable region of a heavy chain (VH) typically comprises three complementarity-determining regions (CDRs), which are referred to herein as CDR 1, CDR 2, and CDR 3 (or referred to as CDR-H1, CDR-H2, CDR-H3, respectively). The constant region of a HC typically comprises a fragment crystallizable region (Fc region), which dictates the isotype of the Ab, the type of Fc receptor the Ab binds to, and therefore the effector function of the Ab. Any isotype, such as IgGl, lgG2a, lgG2b, lgG3, lgG4, IgM, IgD, IgE, IgGAl, or lgGA2, may be used. Fc receptor types include, but are not limited to, FcaR (such as FcaRI), Fca/mR, FceR (such as FceRI, FceRII), FcgR (such as FcgRI, FcgRIlA, FcgRIlBl, FcgRI I B2, FcgRIIIA, FcgRII IB), and FcRn and their associated downstream effects are well known in the art. The variable region of a light chain (VL) also typically comprises CDRs, which are CDR 1, CDR 2, and CDR 3 (or referred to as CDR-L1, CDR- L2, CDR-L3, respectively). In some embodiments, the antigen is ACVR1C (also referred to as ALK7). Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources. A portion of an antibody that comprises a structure that enables specific binding to an antigen is referred to "antigen-binding fragment," "AB domain," "antigen-binding region," or "AB region" of the Ab.

[0077] Certain amino acid modifications in the Fc region are known to modulate Ab effector functions and properties, such as, but not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), and half -life (Wang X. et al., Protein Cell. 2018 Jan; 9(1): 63- 73; Dall'Acqua W. F. et al., J Biol Chem. 2006 Aug 18;281(33):23514-24. Epub 2006 Jun 21; Monnet C. et al, Front Immunol. 2015 Feb 4;6:39. doi: 10.3389/fimmu.2015.00039. eCollection 2015). The mutation may be symmetrical or asymmetrical. In certain cases, antibodies with Fc regions that have asymmetrical mutation(s) (i.e., two Fc regions are not identical) may provide better functions such as ADCC (Liu Z. et al. J Biol Chem. 2014 Feb 7; 289(6): 3571-3590).

[0078] An IgGl-type Fc optionally may comprise one or more amino acid substitutions. Such substitutions may include, for example, N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E233P, L234V, G236-deleted, P238A, A327Q, A327G, P329A, K322A, L234F, L235E, P331S, T394D, A330L, P331S, F243L, R292P, Y300L, V305I, P396L, S239D, I332E, S298A, E333A, K334A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, G236A, K326W, S239D, E333S, S267E, H268F, S324T, E345R, E430G, S440Y, M428L, N434S, L328F, M252Y, S254T, T256E, and/or any combination thereof (the residue numbering is according to the EU index as in Kabat) (Dall’Acqua W. F. et al., J Biol Chem. 2006 Aug 18;281(33):23514-24. Epub 2006 Jun 21; Wang X. et al., Protein Cell. 2018 Jan; 9(1): 63-73), or for example, N434A, Q438R, S440E, L432D, N434L, and/or any combination thereof (the residue numbering according to EU numbering). The Fc region may further comprise one or more additional amino acid substitutions. Such substitutions may include but are not limited to A330L, L234F, L235E, P3318, and/or any combination thereof (the residue numbering is according to the EU index as in Kabat). Specific exemplary substitution combinations for an IgGl-type Fc include, but not limited to: M252Y, S254T, and T256E ("YTE" variant); M428L and N434A ("LA" variant), M428L and N434S ("LS" variant); M428L, N434A, Q438R, and S440E ("LA-RE" variant); L432D and N434L ("DEL" variant); and L234A, L235A, L432D, and N434L ("LALA-DEL" variant) (the residue numbering is according to the EU index as in Kabat). In particular embodiments, an IgGl-type Fc variant may comprise the amino acid sequence of SEQ ID NOS: 11, 12, 13, 14, 15, 16, or 17.

[0079] When the Ab is an lgG2, the Fc region optionally may comprise one or more amino acid substitutions. Such substitutions may include but are not limited to P238S, V234A, G237A, H268A, H268Q, H268E, V309L, N297A, N297Q, A330S, P331S, C232S, C233S, M252Y, S254T, T256E, and/or any combination thereof (the residue numbering is according to the EU index as in Kabat). The Fc region optionally may further comprise one or more additional amino acid substitutions. Such substitutions may include but are not limited to M252Y, S254T, T256E, and/or any combination thereof (the residue numbering is according to the EU index as in Kabat).

[0080] An lgG3-type Fc region optionally may comprise one or more amino acid substitutions. Such substitutions may include but are not limited to E235Y (the residue numbering is according to the EU index as in Kabat). As noted previously, in exemplary embodiments the human lgG2 Fc or constant region will comprise a human lgG2 or Fc region wherein FcR binding is intact, i.e., the same as a native or endogenous human lgG2 Fc or constant region

[0081] An lgG4-type Fc region optionally may comprise one or more amino acid substitutions. Such substitutions may include but are not limited to, E233P, F234V, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q, and/or any combination thereof (the residue numbering is according to the EU index as in Kabat). The substitution may be, for example, S228P (the residue numbering is according to the EU index as in Kabat).

[0082] In some cases, the glycan of the human-like Fc region may be engineered to modify the effector function (for example, see Li T. et al., Proc Natl Acad Sci USA. 2017 Mar 28;114(13):3485-3490. doi: 10.1073/pnas.1702173114. Epub 2017 Mar 13).

[0083] The term "antibody fragment" or "Ab fragment" as used herein refers to any portion or fragment of an Ab, including intact or full-length Abs that may be of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The term encompasses molecules constructed using one or more potions or fragments of one or more Abs. An Ab fragment can be immunoreactive portions of intact immunoglobulins. The term is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), diabodies, and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intra bodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di- scFv, tandem tri-scFv. In a specific embodiment, the antibody fragment is a scFv. Unless otherwise stated, the term "Ab fragment" should be understood to encompass functional antibody fragments thereof. A portion of an Ab fragment that comprises a structure that enables specific binding to an antigen is referred to as "antigen-binding Ab fragment," "AB domain," "antigen-binding region," or "antigen-binding region" of the Ab fragment.

[0084] The term "humanization" of an Ab refers to modification of an Ab of a nonhuman origin to increase the sequence similarity to an Ab naturally produced in humans. The term "humanized antibody" as used herein refers to Abs generated via humanization of an Ab. Generally, a humanized or engineered antibody has one or more amino acid residues from a source which is non-human, e.g., but not limited to mouse, rat, rabbit, non-human primate or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable, constant or other domain of a known human sequence. Known human Ig sequences are disclosed, e.g., www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.atcc.org/phage/hdb.html, each entirely incorporated herein by reference. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. Generally part or all of the non- human or human CDR sequences are maintained while part or all of the non-human sequences of the framework and/or constant regions are replaced with human or other amino acids. Antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties using three-dimensional immunoglobulin models that are known to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, framework (FR) residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in, for example, Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J.

Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824514, 5,817483, 5,814476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,187,287, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, each entirely incorporated herein by reference, included references cited therein.

[0085] An "isolated" biological component (such as an isolated protein, nucleic acid, vector, or cell) refers to a component that has been substantially separated or purified away from its environment or other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0086] The term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).

[0087] The term "nucleic acid" and "polynucleotide" refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism.

[0088] The term "gene" is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

[0089] The term "pharmaceutically acceptable excipient," "pharmaceutical excipient," "excipient," "pharmaceutically acceptable carrier," "pharmaceutical carrier," or "carrier" as used herein refers to compounds or materials conventionally used in pharmaceutical compositions during formulation and/or to permit storage. Excipients included in the formulations will have different purposes. Examples of generally used excipients include, without limitation: saline, buffered saline, dextrose, water-for- infection, glycerol, ethanol, and combinations thereof, stabilizing agents, solubilizing agents and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricating agents.

[0090] The term "recombinant" means a polynucleotide, a protein, a cell, and so forth with semi-synthetic or synthetic origin which either does not occur in nature or is linked to another polynucleotide, a protein, a cell, and so forth in an arrangement not found in nature.

[0091] The term "scFv," "single-chain Fv," or "single-chain variable fragment" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. The linker may comprise portions of the framework sequences. In scFvs, the heavy chain variable domain (HC V, HCV, or VH) may be placed upstream of the light chain variable domain (LC V, LCV, or VL), and the two domains may optionally be linked via a linker (for example, the G4S X3 linker). Alternatively, the heavy chain variable domain may be placed downstream of the light chain variable domain, and the two domains may optionally be linked via a linker (for example, the G4S X3 linker).

[0092] The term "subject" as used herein may be any living organisms, preferably a mammal. In some embodiments, the subject is a primate such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some examples, the patient or subject is a validated animal model for disease and/or for assessing toxic outcomes. The subject may also be referred to as "patient" in the art. The subject may have a disease or may be healthy.

[0093] As used herein, the term "treat," "treatment," or "treating" generally refers to the clinical procedure for reducing or ameliorating the progression, severity, and/or duration of a disease or of a condition, or for ameliorating one or more conditions or symptoms (preferably, one or more discernible ones) of a disease. In other embodiments "treat", "treatment," or "treating" may result in the inhibition of the progression of a disease, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. Additionally, the terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete cure or prevention. Rather, there are varying degrees of treatment effects or prevention effects of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention effects of a disease in a mammal. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

[0094] DETAILED DESCRIPTION [0095] This invention is based in part on the discovery that VISTA negatively regulates innate inflammation through the transcriptional and epigenetic re-programming of macrophages. Representative of VISTA re-programming is the ability of VISTA agonistic antibodies to augment LPS tolerance and reduce septic shock lethality in mice. This antiinflammatory effect of anti-VISTA was mimicked in vitro demonstrating that anti-VISTA treatment caused a significant reduction in LPS-induced IL-12p40, IL-6, CXCL2 and TNF; all hallmark proinflammatory mediators of endotoxin shock.

[0096] Even under conditions that typically "break" LPS tolerance, VISTA agonists sustained a macrophage anti-inflammatory profile. Analysis of the proteomic and transcriptional changes imposed by anti-VISTA show that macrophage re-programming was mediated by a composite profile of mediators involved in both macrophage tolerance induction (IRG1, miR221, A20, IL-10) as well as transcription factors central to driving an anti-inflammatory profile (e.g. IRF5, IRF8, NFKB1).

[0097] These findings underscore a novel and new activity of VISTA as a negative checkpoint regulator that induces both tolerance and anti-inflammatory programs in macrophages and controls the magnitude of innate inflammation in vivo.

[0098] Macrophage plasticity plays an important role in controlling both the amplitude and quality of the inflammatory response in a wide variety of physiological and pathological conditions, as well as the resolution of inflammation and tissue repair. To achieve this, macrophages undergo extensive transcriptional and epigenetic reprogramming in response to various environmental cues. These cues allow macrophages to rapidly respond to danger signals by inducing pro-inflammatory mediators on one extreme or to exist in a regulatory state for the purpose of tissue repair and/or maintenance. Two prominent re-programming mechanisms in macrophages that mitigate inflammation are those that mediate the development of tolerance to endotoxin [1] and the alternative differentiation of macrophages to a non-inflammatory phenotype [2],

[0099] Lipopolysaccharide (LPS) tolerance is an example of transcriptional and epigenetic reprogramming that prevents macrophage overactivation through development of refractoriness to repeated stimulation resulting in reduced capacity of macrophages to mediate septic shock. LPS tolerance has been extensively studied in vivo and in vitro with well-documented changes in transcriptional and epigenetic landscapes that abrogates release of the prototypic inflammatory cytokines secreted by activated macrophages, including TNFa, IL-6, IL-1 and IL-12p40. Several mediators including IRAK-M [3], NF-KBl(p50) [4, 5], mir221/222 [6], IRG1 and A20 [7] have been implicated in mediating or enhancing LPS tolerance.

[00100] A second example of macrophage plasticity is historically exemplified by the extremes of Ml (classical) and M2 (alternative) reprogramming of macrophages in response to environmental cues including TLR ligands, cytokines, and other soluble mediators such as corticosteroids and immune complexes (reviewed in [8]). Compared to the Ml state which is characterized by high production of I L12, TNFa, IL-6 and I LI; various M2 activation states are defined by attenuated production of I L12 and increased production of IL-10 and TGF|3. Key mediators of the Ml program include ST ATI , IRF5 [9, 10] and NFKB [8, 11] whereas the M2 programs variably depend on IRF4 [12], NFIL3 [13] and the inhibitory NF-KB homodimers of NF-KBl(p50) and NF-KB2 (p52) [4, 5],

[00101] It is clear that the development of the tolerance and anti-inflammatory transcriptional programs have overlapping functional consequences as macrophages polarized towards a regulatory state endow potent protection against LPS-induced lethality [14], In addition, regulatory polarization of macrophages can suppress subsequent proinflammatory polarization, and augment tolerance to inflammatory stimuli [2, 14-17], [00102] Despite extensive investigations of these two phenomena for many years, little is known about this overlap and how these processes are coordinately regulated in vivo to produce a unified macrophage response to a given stimulus. Porta et al. Proc. Natl. Acad. Sci., USA, "Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor KB", Proc. Natl. Acad. Sci., USA, , 2009, 106(35): 14978- 14983) validated that tolerance and alternative macrophage polarization are overlapping transcriptionally regulated processes and showed that N F-KB1 (p50) is central to establishing an "M2-like" state in LPS tolerized macrophages.

[00103] Amongst negative checkpoint regulators, VISTA (also known as PD-1H, DDla, Diesl) is unique in its high levels of constitutive expression on resting myeloid cells, including monocytes and macrophages [18],

[00104] VISTA is an immunoglobulin superfamily receptor broadly expressed by cells of the hematopoietic compartment (both T cells and myeloid cells) with well-defined roles as a negative immune checkpoint of T cell responses [19, 20], In this study, we show that VISTA agonists functionally and transcriptionally re-program macrophages by negatively regulating macrophage responses to proinflammatory stimuli.

[00105] Anti-VISTA alone induced mediators involved in both M2 polarization and LPS tolerance including IL-10, miR-221, IRG1, A20, and MerTK and suppressed mediators of Ml polarization (reduced IRF5 and IRF8 expression at both the transcriptional and protein levels). The VISTA-mediated reduction in these transcription factors (TFs) diminished the expression of inflammatory genes including IL-12 family members, IL-6 and TNFa.

Furthermore, anti-VISTA upregulated key mediators of LPS tolerance resulting in the enhanced survival of mice from endotoxin shock.

[00106] As discussed below we show herein that negative checkpoint regulation by VISTA agonists of innate immunity is mediated by the induction of transcriptional reprogramming of both tolerance and anti-inflammatory programs to mitigate innate inflammation in vivo. Based on these discoveries and observations promoting VISTA activity or expression, e.g., by administering a VISTA agonist, may provide novel means for treating and preventing cytokine storm, CRS and/or ARDS or CRDS in subjects in need thereof, e.g., those with an infectious or non-infectious diseases associated therewith such as

GVHD, coronavirus disease 2019 (COVID-19), sepsis, H1N1 flu, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS) among others wherein the disease pathology is characterized by the heightened production of cytokines including IL-la, IL-6, TNF-a, IFN-y, and granulocyte-monocyte colony stimulating factor (GM- CSF), IP-10 and others, many of which are driven by the IFNI response.

[00107] The following Materials and Methods were used in the experiments the results of which are shown in Figures 1-4 and further discussed below.

[00108] MATERIALS AND METHODS

[00109] Cell culture

[00110] Primary Bone marrow-derived macrophages (BMDMs) were generated by isolation and culture of mouse bone marrow in complete RPMI supplemented with 20 ng/ml recombinant murine M-CSF (Peprotech, 315-02) for up to 7 days. For cell stimulation, 10 ng/ml LPS (Sigma L8274) or 100 ng/ml recombinant mouse I FNy (Biolegend, 575306) were used. For tolerization experiments, BMDMs (1 x 10⁶ cells/ml per well in a 6 well plate) were stimulated with 10 ng/ml LPS for 15 hours, washed 5 times with lx PBS, then allowed to rest for 2 hours in LPS-free complete medium. BMDMs were then stimulated with 1 pg/ml LPS for 4 h (for total RNA-seq) or 12 hours (for Luminex) or as indicated.

[00111] For human monocyte and macrophage experiments, Ficoll-Paque (GE Healthcare) was used to isolate PBMCs from healthy volunteers by differential centrifugation. The RPMI 1640 medium (Sigma-Aldrich) was supplemented with 10 mM L- glutamine and 10 mM pyruvate (Life Technologies). Monocytes were obtained by depletion of CD3, CD19 and CD56 positive cells from PBMCs obtained upon Ficoll isolation of a buffy coat. CD3 MicroBeads (130-050- 101), CD19 MicroBeads (130-050-301) and CD56 (130-050- 401) were purchased from Miltenyi Biotec and used according to the manufacturer protocol. For RNA-seq analysis of the monocytes, additional CD14 positive cells selection was performed on the CD3-, CD19- and CD56- population using CD14 MicroBeads (130- 050- 201) from Miltenyi Biotec. For human monocyte-derived macrophage differentiation, isolated monocytes were cultured at 2 x 10⁶ cells/ml in 6-well plates (Corning, 3506) in RPMI supplemented with 10% human pooled serum and 20 ng/ml recombinant human M-CSF (Peprotech, 300-25) for 6 days prior to treatment with anti-VISTA for 24 hours followed by LPS (1 pg/ml) stimulation. For time-time course RNA-seq analysis, cells were isolated at each time-point, and RNA was extracted as described below.

[00112] Mice

For BMDM generation, hVISTA knock-in mice of 8-10 weeks of age were used [28], unless otherwise noted. Both male and female mice were used in experiments. For tolerance and septic shock experiments, C57BI/6 mice (Charles River) of 8-10 weeks of age were used. LPS (Escherichia coli O55:B5; Sigma L2880) and d-(+)-galactosamine hydrochloride (Sigma G0500) were re-suspended in sterile PBS and filter-sterilized before intraperitoneal injection. Mice were maintained under specific-pathogen-free conditions in the Dartmouth Center for Comparative Medicine and Research. The Animal Care and Use Committee of Dartmouth College approved all animal experiments [00113] Antibodies

Anti-VISTA agonist antibodies used in this study were anti-human VISTA clone 803 and antimouse VISTA clone 8G8 [28], [00114] Cytokine analysis

[00115] Simultaneous determination of multiple cytokine concentrations was carried out using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel— Premixed 32 Plex (EMD M i 11 i pore, Billerica, MA) on a Bio-Rad Bio-Plex Array Reader. Samples were diluted in cell culture medium to the dynamic range of each kit.

[00116] Proteomic Analysis

[00117] Control and anti-VISTA-treated BMDM protein lysate (10⁶ cells per replicate) were sent for global proteomic quantification (Thermo Fisher Scientific Center for Multiplexed Proteomics at Harvard). In brief, sample were reduced with TCEP, alkylated with iodoacetimide, then quenched with DTT. The proteins were precipitated using methanol/chloroform and sequentially digested with LysC (1:50) and trypsin (1:100) based on protease to protein ratio. 500 mg of peptides were labelled for enrichment. Peptides were separated using a gradient of 3 to 25% acetonitrile in 0.125% formic acid over 180 minutes prior to detection (MSI), sequencing (MS2) in the Ion trap, and quantification (MS3) in the Orbitrap. MS2 spectra were searched using the SEQUEST algorithm against a Uniprot composite database derived from the Mouse proteome containing its reversed complement and known contaminants. Peptide searches were performed using a 20 ppm precursor ion tolerance, 1 Da fragment ion tolerance, Max Internal Cleavage Site: 2, Max differential/Sites: 4, static modifications for TMT tags (+229.163 Da) on Lysine residues and N-terminus peptide, carbamidomethylation (+57.021 Da) on Cysteine residues and a variable modification for oxidation (+15.995 Da) on Methionine residues. For Phosphopeptide searches, another variable modification was considered for phosphorylation (+79.966 Da) on Serine (S), Threonine (T) and Tyrosine (Y) residues. Peptide spectral matches were filtered to a 1% false discovery rate (FDR) using the target-decoy strategy combined with linear discriminant analysis. The proteins were filtered to a <1% FDR. Proteins were quantified only from peptides with a summed SN threshold of >100 and MS2 isolation specificity of 0.5. Quantified proteins were hierarchically clustered using the Euclidean distance, average linkage. Multiple sample test with FDR <0.05 revealed about 1581 proteins that are significantly changing between two study groups.

[00118] RNA-seq

[00119] RNA was extracted using the Kapa Hyperprep with RiboErase kit, according to the manufacturer's instructions. Samples were sequences on the NextSeq500 machine in 75-bp paired-end runs. The quality of the runs was confirmed using the FastQC software [29], Sequencing output files were aligned to GRCh38 and GRCm38 for human and mouse data, respectively. Transcripts were counted by the Spliced Transcripts Alignment to a Reference (STAR) algorithm using the "--quantMode" option [30], The count data matrix was then processed in R and differentially expressed genes (DEGs) were identified using DESeq2 [31], In brief, the data were filtered by removing transcripts that were not detected in all replicates. Differential expression analysis was performed contrasting anti-VISTA- treated samples to the IgG-treated condition. Unless noted otherwise, DEGs were considered to be those with an FDR-adjusted P value of less than 0.05. The count data were transformed to Iog2-transformed transcripts per million (TPM) for downstream analyses and heatmap displays.

[00120] Genes differentially expressed throughout the BMDM and human monocyte time-course were selected by three complementary approaches: (1) DESeq2 [31] DEG identification at each time point comparing anti-VISTA to IgG-treatment, (2) EDGE [32, 33] DEG identification comparing the expression dynamics between anti-VISTA to IgG- treatment, (3) ANOVA DEG identification modeled by time and treatment. We selected all genes that were deemed significant by at least two of these methods as differentially expressed throughout the time course.

[00121] scATAC-seq

[00122] Nuclei from BMDMs were isolated following the 10X Genomics protocol for scATAC-seq. The CellRanger ATAC vl.1.0 pipeline [34] as used for initial processing. Raw base call (BCL) files were demultiplexed into FASTQ files using "mkfastq". Reads were aligned to the mouse mmlO reference genome using "count". Peak count matrices were aggregated into one file using the "aggr" function. Downstream analyses were conducted using the Signac R package (vO.2.4) [35], Only cells considered to be of sufficient quality were retained; cells with at least 3000 detected fragments, with less than 5% of fragments originating from blacklisted regions, with more than 20% of all fragments mapping to gene peaks, with nucleosome binding patterns present (nucleosome_signal < 10) and with a transcriptional start site (TSS) enrichment score of at least 2 were considered of high quality. The remaining cells were normalized for sequencing depth using frequency-inverse document frequency (TF-IDF) normalization. Singular value decomposition (SVD) was used to reduce the dimensionality of the data. Since the first reduced component was highly correlated with sequencing depth (Pearson correlation coefficient = -0.97), only the second to 30th components were retained for further analyses. Unsupervised clustering using Uniform Manifold Approximation and Projection (UMAP) [36] was used for all visual presentations of the data using the "RunllMAP" function on SVD-reduced data and the aforementioned components. Cell clusters were identified using the find "FindClusters" function using resolution 0.3. Cluster marker genes were obtained by the "FindAIIMarkers" function using a logistic regression framework to determine differentially expressed genes. Markers with a Bonferroni corrected p-value of < 0.001 were considered true marker genes. For global comparisons between treatment groups, the "FindAIIMarkers" function was similarly used after using "Setldent" to specify the treatment identify for each cell. A gene activity matrix was generated to evaluate gene-level differences between treatments. Gene coordinates for the mouse genome were obtained from EnsembleDB with the EnsDb.Mmusculus.v79 R package (v2.99.0) [37], Gene regions were extended to include the 2kb upstream promoter region. Gene activities were assigned based on the number of fragments that mapped to each of the gene regions using the "FeatureMatrix" function. Gene activity scores were log normalized using the "NormalizeData" function. The gene activity scores were utilized for all presented heatmaps.

[00123] GSEA, TF enrichment and network display

[00124] Gene Set Enrichment Analysis (GSEA) was performed using the GSEA software provided by the Broad [38, 39] (v4.3.0). Pathway gene sets were downloaded from the C2 and C7 category of the Molecular Signatures Database (MSigDB v7.0) database [38, 39], Only gene sets with at least 10 effective genes (i.e., the number of genes presented in a gene expression dataset) were retained. Transcription factor (TF) target genes were obtained from TRRUST (v.2), a manually curated database of human and mouse transcriptional regulatory networks [40], In addition, TF targets were added manually based on a literature investigation of TFs of interest. The TF network was displayed using Cytoscape [41],

RESULTS AND OBSERVATIONS koVISTA is a pleiotropic myeloid cell checkpoint

[00125] While the groundwork for a central role of VISTA in controlling T cell biology has been created, emerging data show an equally important and global role of VISTA in controlling innate inflammation. Studies discussed will show a role of VISTA in controlling myeloid chemotaxis, fate determination, and antigen presentation functions.

[00126] Unique to VISTA, as an NCR, is its role in the regulation of chemotaxis. It was shown that the genetic loss of VISTA reduced the expression of C5aRl on monocytes and macrophages and inhibited their migration to the cognate chemoattractant ligand C5a [2], A subsequent study revealed that the regulatory impact of VISTA was not limited to the C5a/C5arl axis, but exerted a broad impact on the expression of several chemokines and chemokine receptors [17], VISTA deficiency and targeting was shown to reduce CCR2 and CX3CR1 expression on monocytes; two hallmark receptors for Classical and Patrolling murine monocytes, respectively [17] (and unpublished observations). Of note, VISTA targeting also strikingly reduces CD14 and CD16 (Fcgllla) expression; two hallmark receptors for Classical and Patrolling human monocytes, respectively as noted from flow cytometry and RNA-seq analyses [Fig. 1], In addition, loss of VISTA enhanced the levels of the chemokine CCL2, CCL3, CCL4 and CCL5 by macrophages at steady-state [17], The authors attributed this enhancement to reduced consumption of these chemokines by VISTA- deficient macrophages owing to reduced steady-state CCR2 expression and enhanced CCR5 downregulation in response to their cognate chemokines. As a result, these cells had selective profound deficits in the migratory responses towards these chemokines. Very recent work also demonstrated an impact of VISTA targeting on reducing CXCR2 expression on neutrophils, with the virtual ablation of their migratory responses to the CXCR2 ligand in vitro and in vivo: CXCL2 (Li et al., unpublished observations). These result highlight VISTA as an important checkpoint regulating the response towards multiple chemokine/chemokine receptor networks. The migratory response of immune cells represents the earliest checkpoint towards inflammatory stimuli, and suggest that interfering with this pathway may eliminate or modulate immune responses prior to their exacerbation. These intriguing results also present the prospect of VISTA targeting being crucial for regulating myeloid cell responses in the context of inflammatory diseases where neutrophils and monocytes play dominant roles.

[00127] Accumulating evidence from multiple systems suggests that VISTA may play a role in the regulation of antigen presentation cell (APC) activity. At the level of expression, VISTA has been reported to colocalize with MHC-II, and VISTA overexpression in myeloid cells reduced MHC-II expression levels [18], In a melanoma tumor model, VISTA blockade enhanced the activation state of CDCs, upregulating the expression of MHC-II and CD80, as well as augmented the production of IL-12 and TNFa [19], In contrast, studies with VISTA agonists has revealed that agonist treatment of human monocytes induced a profound and broad time-dependent downregulation of MHC-II genes as well as CD80 [Fig. 2A], This is also supported by pathway analysis where the antigen presentation pathway was significantly downregulated in anti-VISTA agonist treated monocytes [Fig. 2B], Therefore, published studies and studies presented herein are providing documentation that VISTA plays an early and central role in the control myeloid migration and fate determination; in this context, a primary target for controlling innate inflammation.

[00128] VISTA, unlike other NCRs, is emerging as a major immunoregulatory factor in the regulation of myeloid fate determination. Loss of VISTA exacerbated psoriasis and the investigators attributed this effect [in part] to enhanced TLR7 signaling on DCs. More recent mechanistic insights into VISTA regulation of myeloid biology revealed a role for VISTA in modulating the ubiquitination and expression of the TLR-MyD88 effector TRAF6, and by consequence, the negative regulation of TLR signaling and the downstream MARK and NFkB axes [16], As a result, loss of VISTA on macrophages enhanced cytokine responses towards multiple TLR agonists including TLR2, TLR3, TLR4, TLR7 and TLR9. This agrees with our recent work showing that VISTA-/- macrophages showed enhanced cytokine responses to TLR4 stimulation [17], As one would anticipate, overexpression of VISTA in a monocytic-cell line (THP-1) dampened responses to TLR2 stimulation [16], To gain a global perspective of the impact of VISTA targeting on myeloid fate, a more comprehensive assessment of transcriptional reprogramming by VISTA on human monocyte transcriptome was performed and is presented.

VISTA induce myeloid reprogramming: Evidence for profound reprogramming and a target in COVID cytokine storm management

[00129] Single-cell RNA-seq of anti-VISTA agonist treated human monocytes revealed a profound shift and almost complete elimination of the CD14⁺ classical monocyte phenotype in favor of a more anti-inflammatory cell state characterized by a striking downregulation of CD14, IFN receptors, Fcgr3a (CD16) and CSF1R whereas the major VISTA- induced cluster 1 upregulated CDllb, M-CSF (Csfl), Cyclin-dependent kinase inhibitor (Cdknla) and the anti-inflammatory cytokines IL1RA and GDF15 [Figure 4A and B], There were 3 other cell states (clusters) specifically induced by anti-VISTA but these present a minority of the total cells. In agreement with the flow cytometry data on anti-VISTA agonist treated monocytes [Fig. 1], a hallmark of the VISTA agonist induced monocyte cell state was a near-complete downregulation of CD14 and CD16 [Fig. 4C and 4D], Additional independent analysis of the anti-VISTA agonist impact on CD14+ human monocytes at steady-state (unactivated) revealed a complete suppression of CXCL10 production, even in the presence of potent stimulatory pattern recognition ligands [Fig. 3], reproducible among heterogeneous donors. This suppression was manifested at the transcriptional [Fig. 3A] and proteomic levels [Fig. 3B] . We argue that the suppression of CXCL10 is a consequence of a penetrant downregulation of the IFN-I pathway genes, including its upstream effector ST ATI [Fig. 3C and 3D], Even after stimulation with multiple Pattern recognition receptor (PRR) ligands, the anti-VISTA suppression of CXCL10 was maintained [Fig. 3E and 3F] . These findings suggest that VISTA has an immunoregulatory role in COVID-induced inflammation and pathology.

[00130] These observations are clinically relevant e.g., in the context of COVID-19 or other conditions that may result in cytokine storm or CRS or sepsis or acute respiratory distress syndrome. In the case of COVID-19 in particular despite the recovery of most infected individuals, a significant number of COVID-19 patients presented with severe respiratory distress in addition to complications including a hyperinflammatory response [20, 21], Recent immunophenotyping analysis of the peripheral blood from a large heterogeneous pool of COVID-19 patients revealed a core consensus immune signature [22], Within this signature, sustained overexpression of the IFN-I/II inducible chemokine CXCL10 had a striking positive correlation with evolving disease severity, and was the most reliable prognostic biomarker. This immune signature highlighting CXCL10 chronic upregulation was further supported by two independent studies [23, 24], It is worth noting that CXCL10 was also highly upregulated with other coronaviruses SARS1 [25] and MERS [26-28], also positively correlating with disease severity.

[00131] Enrichment analysis of the COVID-19 immune profile revealed that anti-VISTA agonist downregulated over 40% of the hallmark genes defining the COVID-19 immune signature [Fig. 3G], This suggests that a VISTA agonist can suppress the COVID-19 inflammatory signature or inflammatory signatures seen in other conditions associated with cytokine storm or CRS and/or ARDS.

[00132] Therefore, VISTA intersects with the CXCL10 induction pathway which is of relevance to COVID-19 immunopathology. It is also critical to highlight that the reduction of FcgRIIIa by VISTA targeting is of significant interest as hyperinflammatory Fc receptor responses have been reported as a immunopathologic manifestation of COVID-19 infection [29],

[00133] Beyond the striking impact on transcriptional reprogramming exerted by VISTA, the impact of VISTA on myeloid chemotaxis may also play an important therapeutic role in controlling innate inflammation in COVID and in other infectious or non-infectious conditions associated with cytokine storm or CRS and/or ARDS.

[00134] With particular respect to COVID the high neutrophil to lymphocyte ratio in critically ill COVID-19 patients has been predictive in hospital mortality [30], Recent reports and commentaries implicate neutrophils as critical components of the hyperinflammatory responses to COVID-19, and suggest that impeding neutrophil recruitment via CXCR2 may be a promising treatment in this setting [31, 32], Our recent analysis of anti-VISTA agonist impact on neutrophil biology demonstrates a clear suppression of CXCR2 expression, and by consequence, their migratory responses, in both murine and human neutrophils. This would indicate a potential mechanism whereby VISTA targeting could suppress neutrophil chemotaxis and shut down the inflammatory circuit.

[00135] Therefore, we posit that VISTA agonists may be of valuable therapeutic relevance in a broad spectrum of inflammatory settings associated with cytokine storm or CRS or sepsis and/or acute or chronic respiratory associated syndrome and respiratory conditions. In particular, given the COVID-19 viral infection pandemic, and the observations reported herein we hypothesize that VISTA agonists may be utilized to normalize innate and adaptive immune responses in subjects in need thereof, particularly COVID-19 infected subjects, e.g., those showing signs of and/or who are at increased risk of cytokine storm and/or acute respiratory distress syndrome because of underlying risk factors or comorbidities which often result in poor outcomes and most seriously death in these subjects. [00136] Therefore, based on these discoveries this invention is directed to treating inflammatory diseases that are mediated by overexpression of innate derived cytokines and chemokines such as IL-la, IL-6, TNF-a, IFN-y, and granulocyte-monocyte colony stimulating factor (GM-CSF), IP-10 and others, many of which are driven by the IFNI response using VISTA agonists. In particular the invention relates to treatment and prevention of conditions associated overexpression of innate derived cytokines and chemokines including cytokine storm, CRS, septic shock, and sepsis acute respiratory distress syndrome which may result from different causes, such as infectious and non-infectious diseases including graft-versus- host disease (GVHD), coronavirus disease 2019 (COVID-19), sepsis, H1N1 flu, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS) by the administration of a VISTA agonist, e.g., an agonistic anti-VISTA antibody or antibody fragment or a VISTA fusion protein.

[00137] Particularly the present invention addresses these needs by the treatment of such subjects with an amount of a VISTA agonist effective to treat or prevent cytokine storm or CRS and the symptoms associated therewith , e.g., wherein the VISTA agonist comprises a VISTA fusion protein, e.g., a VISTA-lg fusion protein or an agonistic anti-VISTA antibody or antibody fragment. In some exemplary embodiments the agonistic anti-VISTA or antibody fragment will comprise variable light and heavy chain polypeptide comprising the CDRs of any one of the anti-human VISTA antibodies having the sequences contained in the table in Figure 5.

[00138] Also the present invention addresses these needs by treating, inhibiting or reversing ARDS or CRDS and symptoms associated therewith in a subject in need thereof, e.g., a patient with an infection that may result in ARDS or CRDS, by administering an effective amount of a VISTA agonist e.g., wherein the VISTA agonist comprises a VISTA fusion protein, e.g., a VISTA-lg fusion protein or an agonistic anti-VISTA antibody or antibody fragment, e.g., wherein the agonistic anti-VISTA or antibody fragment comprises a variable light and heavy chain polypeptide comprising the CDRs of any one of the anti-human VISTA antibodies having the sequences contained in the table in Figure 5, wherein such treatment effectively treats, inhibits or reverses ARDS or CRDS and symptoms associated therewith, e.g., cytokine storm and lung damage.

[00139] This invention specifically relates to the use of specific agonistic anti-VISTA antibodies or antibody fragments to treat or prevent acute or chronic respiratory distress syndrome and ameliorate side effects associated therewith such as lung damage and cytokine storm in patients infected or suspected of being infected by COVID-19 or another viral or bacterial or other pathogen. In particular the invention pertains to methods of improving lung function and/or reversing or preventing lung damage and/or cytokine storm and improving survival and quality of life in COVID-19 infected patients who have or exhibit signs of ARDS or CRDS or are at risk for developing COVID-19 associated ARDS or CRDS, e.g., because of other risk factors such as advanced age (over 60 or 70 years of age), other conditions such as other lung conditions such pneumonia, asthma, COPD, cystic fibrosis, cancer, diabetes, high blood pressure or other inflammatory or autoimmune conditions, especially those which adversely affect lung function.

[00140] Particularly the present invention addresses these needs by the use of specific anti-VISTA antibodies and antibody fragments wherein the antibody or antibody fragment comprises a variable light chain polypeptide and a variable heavy chain polypeptide comprising the same CDRs as one of the anti-human VISTA antibodies having the sequences shown in the table in Figure 5 that effectively treat, inhibit or reverse cytokine storm or ARDS or CRDS and symptoms associated therewith, e.g., lung or heart damage.

[00141] In some instances the treated patients who are treated with specific VISTA agonists according to the invention are individuals suspected of having COVID-19 infection, i.e., a definitive test result is not yet available, but they are suspected because of contact with other individuals and/or symptoms associated with COVID-19 infection such as fever, dry cough, breathing difficulties, et al.

[00142] In some instances the patients who are treated with specific VISTA agonists according to the invention are individuals who are already exhibiting signs of cytokine storm and/or lung damage associated with ARDS or CRDS.

[00143] In some instances the patients who are treated with specific VISTA agonists according to the invention are individuals who have or are individuals suspected of having an infection, e.g., COVID-19 infection, who have another respiratory or lung condition such as pneumonia caused by COVID-19 or another bacterial or viral pathogen which further disposes the patient to developing ARDS or CRDS, e.g., so severe it may require use of a ventilator or respirator.

[00144] In some instances the patients who are treated specific VISTA agonists according to the invention are individuals who have or are individuals suspected of having an infection, e.g., COVID-19 infection, who have another respiratory or lung condition, including those who have lung problems so severe that they are on a ventilator or respirator.

[00145] In some instances the patients who are treated with specific VISTA agonists according to the invention are individuals who have or are individuals suspected of having an infection, e.g., COVID-19 infection, or other condition associated with ARDS who are being treated with other therapeutics or regimens used to treat ARDS or CRDS or infection such as steroids, other immunosuppressives, e.g., thymoglobulin, basiliximab, mycophenolate mofetil, tacrolimus, an anti-CD20 mAb such as rituximab, corticosteroids, antivirals, antibiotics, et al.

[00146] In particular the invention provides novel therapeutic protocols for treating or preventing ARDS or CRDS and symptoms associated therewith in patients in need thereof by the use of agonistic anti-human VISTA antibodies comprising the same CDRs and/or variable heavy and light polypeptides as any one of the anti-human VISTA antibodies in Figure 5, e.g., wherein such agonists comprise human Fc regions, typically human lgG2 Fc regions.

[00147] Treatment with the subject VISTA agonists may prevent, inhibit or treat ARDS and CRDS and associated symptoms such as breathing difficulties, reduced lung function, increased IL-6 and/or CRP levels, cytokine storm, etc.

[00148] Pharmaceutical compositions for use in methods according to the invention can contain any pharmaceutically acceptable excipient. Examples of excipients include but are not limited to starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emulsifiers, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.

[00149] In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. This may include e.g., aerosol, nasal, oral, transmucosal, transdermal, parenteral or enteral.

[00150] "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Typically, the compositions are administered by injection. Methods for these administrations are known to one skilled in the art.

[00151] Pharmaceutical compositions according to the invention can contain any pharmaceutically acceptable carrier. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.

[00152] In order to further describe the invention the following examples are provided.

EXAMPLES

EXAMPLE 1: Use of Vista Agonist to Prevent Cytokine Storm and/or ARDS in COVID-19 Infected Patients

[00153] An agonistic anti-VISTA antibody according to the invention may be used as a primary therapeutic for preventing ARDS and symptoms thereof including cytokine storm in COVID-19 infected patients or patients suspected to comprise COVID-19 infection. As aforementioned coronaviruses, e.g., COVID-19, in severe cases, cause Acute Respiratory Distress Syndrome (ARDS) and is associated with cytokine storm, in many cases leading to death. [00154] An agonistic anti-VISTA antibody will be administered to COVID-19 infected patents at risk of developing ARDS or cytokine storm at a dose ranging from 1 to 100 mg administered by intravenous infusion or subcutaneous injection. Each dose is administered as a 1 mL injection of antibody (1 to 100 mg/mL).

[00155] Before and after treatment and blood is drawn from the treated patients and the levels of clinically relevant molecules including proinflammatory or anti-inflammatory cytokines and other important molecules, e.g., IFN-1, IFN-2, CXCL10 and CXCR2, CD14, IFN receptors, Fcgr3a (CD16), CSF1R, CDllb, M-CSF (Csfl), Cyclin-dependent kinase inhibitor (Cdknla) as well as anti-inflammatory cytokines IL1RA and GDF15 and other pro- inflammatory cytokines such as IL-6. In particular levels of CXCL10 and CXCR2 may be detected before and after treatment. Based on the detected levels in the patient, i.e., if they are aberrant and suggest that cytokine storm is developing the patient may immediately be treated with an additional dose of the VISTA agonist in order to promote a normal cytokine profile. EXAMPLE 2: Use of the VISTA agonist antibody in patients with life-threatening COVID-19 infection exhibiting pulmonary or respiratory difficulties and/or signs of cytokine storm

[00156] VISTA agonist antibody is administered to patients with life-threatening COVID-19 infection who exhibit pulmonary or respiratory difficulties and/or signs of cytokine storm. These patients may include those who do not exhibit pulmonary or respiratory difficulties so extreme that they require exogenous oxygen or high levels of oxygen as well as patients with more serious pulmonary or respiratory difficulties who already require exogenous oxygen and/or are on a ventilator. These patients will be monitored before and after VISTA agonist administration to assess the effects on proinflammatory cytokines and the levels of other markers correlated with a poor prognosis such as CXCL10, CXCR2 et al.

[00157] In particular before and after VISTA agonist antibody administration patients will be assessed to detect levels of one or more markers such as CXCL10, CXCR2 , 1 FN-y, IFN- 3, 1 L-2, 1 L-6, IL-17, CCL5/Rantes, CCL3/MIP-lalpha, and CXCL11/I-TAC. If following the first VISTA agonist dose the inflammatory markers do not decrease or normalize within 24-48 hours of VISTA agonist antibody administration then the patients will be administered a second VISTA agonist antibody dose immediately or within 24-48 hours again administered by intravenous infusion.

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Laing, A.L., Irene Del Molino Del Barrio, Abhishek Das, Matthew Fish, Leticia Monin, Miguel Munoz-Ruiz, Duncan Mckenzie, Thomas Hayday, Isaac Francos Quijorna, Shraddha Kamdar, Magdalene Joseph, Daniel Davies, Richard Davis, Aislinn Jennings, Iva Zlatareva, Pierre Vantourout, Yin Wu, Vasiliki Sofra, Florencia Cano, Maria Greco, Efstathios Theodoridis, Joshua Freedman, Sarah Gee, Julie Nuo En, Chan, Sarah Ryan, Eva Bugallo Blanco, Part Peterson, Kai Kisand, Liis Haljasmagi, Lauren Martinez, Blair Merrick, Karen Bisnauthsing, Kate Brooks, Mohammad Ibrahim, Jeremy Mason, Federico Lopez Gomez, Kola Babalola, Sultan Abdul- Jawad, John Cason, Christine Mant, Katie Doores, Jeffrey Seow, Carl Graham, Francesca di Rosa, Jonathan Edgeworth, Manu Shankar Hari, Adrian Hayday, A consensus Covid- 19 immune signature combines immuno-protection with discrete sepsis-like traits associated with poor prognosis. MedRxiv, 2020. Mathew, D., et a I., Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications. Science, 2020. Wilk, A.J., et al., A single-cell atlas of the peripheral immune response in patients with severe COVID-19. Nat Med, 2020. 26(7): p. 1070-1076. Tang, N.L., et al., Early enhanced expression of interferon-inducible protein-10 (CXCL- 10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome. Clin Chem, 2005. 51(12): p. 2333-40. Abadier, M. and K. Ley, P-selectin glycoprotein ligand-1 in T cells. Curr Opin Hematol, 2017. 24(3): p. 265-273. Faure, E., et al., Distinct immune response in two MERS-CoV-infected patients: can we go from bench to bedside? PLoS One, 2014. 9(2): p. e88716. Kim, E.S., et al., Clinical Progression and Cytokine Profiles of Middle East Respiratory Syndrome Coronavirus Infection. J Korean Med Sci, 2016. 31(11): p. 1717-1725. Willianne Hoepel, H.-J.C., Sona Allahverdiyeva, Xue Manz, Jurjan Aman, Amsterdam UMC COVID-19 Biobank, Peter Bonta, Philip Brouwer, Steven de Taeye, Tom Caniels, Karlijn van der Straten, Korneliusz Golebski, Guillermo Griffith, Rene Jonkers, Mads Larsen, Federica Linty, Annette Neele, Jan Nouta, Frank van Baarle, Cornells van Drunen, Alexander Vlaar, Godelieve de Bree, Rogier Sanders, Lisa Willemsen, Manfred Wuhrer, Harm Jan Bogaard, Marit van Gils, Gestur Vidarsson, Menno de Winther, Jeroen den Dunnen, Anti-SARS-CoV-2 IgGfrom severely ill COVID-19 patients promotes macrophage hyper-inflammatory responses. BioRxiv, 2020. Fu, J., et al., The clinical implication of dynamic neutrophil to lymphocyte ratio and D- dimer in COVID-19: A retrospective study in Suzhou China. Thromb Res, 2020. 192: p. 3-8. Koenig, L.M., et al., Blocking inflammation on the way: Rationale for CXCR2 antagonists for the treatment of COVID-19. J Exp Med, 2020. 217(9). Laforge, M., et al., Tissue damage from neutrophil-induced oxidative stress in COVID- 19. Nature Reviews Immunology, 2020.

Claims

Claims: A method of preventing, stabilizing or reducing cytokine storm or cytokine release syndrome (CRS) and/or sepsis or the symptoms thereof in a subject in need thereof comprising administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein, further optionally wherein any of the aforementioned agonists comprises a human lgG2 Fc or human lgG2 constant region, optionally one where FcR binding is intact compared to a endogenous human lgG2 Fc or human lgG2 constant region. A method of decreasing the levels of at least one of LPS-induced IL-12p40, IL-6, CXCL2 and TNF in a subject in need thereof, optionally a subject having or at risk of developing cytokine storm or septic shock in a subject in need thereof wherein this is effected by administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein, further optionally wherein any of the aforementioned agonists comprises a human lgG2 Fc or human lgG2 constant region, optionally one where FcR binding is intact compared to a endogenous human lgG2 Fc or human lgG2 constant region. A method of increasing the expression of mediators involved in macrophage tolerance induction, wherein said mediators optionally include at least one of IRG1, miR221, A20, and IL-10 and/or increasing the expression of anti-inflammatory transcription factors which drive an anti-inflammatory profile optionally including at least one of IRF5, IRF8, and NFKB1 thereby preventing, stabilizing or reducing the risk or severity of cytokine storm or septic shock, sepsis or CRS in a subject in need thereof, wherein this is effected by administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein

42 or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein, further optionally wherein any of the aforementioned agonists comprises a human lgG2 Fc or human lgG2 constant region, optionally one where FcR binding is intact compared to a endogenous human lgG2 Fc or human lgG2 constant region. A method of preventing, stabilizing or reducing the risk or severity of cytokine storm or septic shock, sepsis or CRS in a subject in need thereof by (i) reducing the level of CXCR2 and/or CXCL10; (ii) reducing neutrophil/lymphocyte ratios, (iii) reducing FcgRIII levels or a combination thereof, wherein this is effected by administering to said subject a prophylactical ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment, further optionally wherein any of the aforementioned agonists comprises a human lgG2 Fc or human lgG2 constant region, optionally one where FcR binding is intact compared to a endogenous human lgG2 Fc or human lgG2 constant region. A method of preventing, stabilizing or reducing acute or chronic respiratory distress syndrome or the symptoms associated therewith in a subject, optionally a subject with an infection further optionally coronavirus infection, optionally COVID-19, or other viral or bacterial infection associated with acute or chronic respiratory distress syndrome comprising administering to said subject a prophy lactica I ly or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein, further optionally wherein any of the aforementioned agonists comprises a human lgG2 Fc or human lgG2 constant region, optionally one where FcR binding is intact compared to a endogenous human lgG2 Fc or human lgG2 constant region. A method of decreasing the levels of at least one of LPS-induced IL-12p40, IL-6, CXCL2 and TNF in a subject in need thereof, thereby preventing, stabilizing or reducing acute or chronic respiratory distress syndrome or the symptoms associated therewith in a subject

43 wherein this is effected by administering to said subject a prophylactically or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti- VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein, further optionally wherein any of the aforementioned agonists comprises a human lgG2 Fc or human lgG2 constant region, optionally one where FcR binding is intact compared to a endogenous human lgG2 Fc or human lgG2 constant region. A method of increasing the expression of mediators involved in macrophage tolerance induction, wherein said mediators optionally include at least one of IRG1, miR221, A20, and IL-10 and/or increasing the expression of anti-inflammatory transcription factors which drive an anti-inflammatory profile optionally including at least one of IRF5, IRF8, and NFKB1 thereby preventing, stabilizing or reducing acute or chronic respiratory distress syndrome or the symptoms associated therewith in a subject in a subject in need thereof, wherein this is effected by administering to said subject a prophylactically or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein, further optionally wherein any of the aforementioned agonists comprises a human lgG2 Fc or human lgG2 constant region, optionally one where FcR binding is intact compared to a endogenous human lgG2 Fc or human lgG2 constant region. A method of preventing, stabilizing or reducing the risk or severity of cytokine storm or septic shock, sepsis or CRS or ARDS or other respiratory syndrome in a subject in need thereof by (i) reducing the level of CXCR2 and/or CXCL10; (ii) reducing neutrophil/lymphocyte ratios, (iii) reducing FcgRIII levels or a combination thereof, wherein this is effected by administering to said subject a prophylactically or therapeutically effective amount of a VISTA agonist, optionally an agonistic VISTA fusion protein or an agonistic anti-VISTA antibody or agonistic anti-VISTA antibody fragment or an agonistic VSIG3 fusion protein or an agonistic anti-VSIG3 antibody or agonistic anti-

44 VSIG3 antibody fragment or an agonistic PSGL1 antibody, antibody fragment or PSGL1 fusion protein, further optionally wherein any of the aforementioned agonists comprises a human lgG2 Fc or human lgG2 constant region, optionally one where FcR binding is intact compared to a endogenous human lgG2 Fc or human lgG2 constant region. The method of any of claims 1-8, which includes the administration of another active, optionally selected from a PD-1 agonist, a CTLA-4 agonist, a TNF antagonist optionally an anti-TNF antibody or TN F-receptor fusion such as Embrel, an IL6 antagonist such as an anti-IL-6 or anti-l L-6R antibody, a corticosteroid or other anti-inflammatory agent. The method of any of claims 1-9, wherein the patient comprises or is suspected of comprising a coronavirus infection or another condition, e.g., a bacterial and/or viral infection correlated with an increased risk of cytokine storm, CRS, sepsis, septic shock or ARDS, optionally coronavirus disease 2019 (COVID-19 or SARS-COV-2), SARS-COV-1, MERS, sepsis, H1N1 flu, Ebola, avian influenza, smallpox, and systemic inflammatory response syndrome (SIRS). The method of any of claims 1-9, wherein the patient comprises or is suspected of comprising COVID-19 infection. The method of any of the foregoing claims, wherein the subject prior to treatment shows signs of sepsis and/or ARDS. The method of any of the foregoing claims, wherein the subject prior to treatment comprises a viral or bacterial mediated respiratory infection. The method of any of the foregoing claims, wherein the agonistic anti-VISTA antibody or antibody fragment specifically binds to human VISTA. The method of claim 14, wherein the agonistic anti-VISTA antibody or antibody fragment comprises a variable light chain and a variable heavy chain polypeptide comprising the same CDRs any one of the antibodies having the sequences contained in the table in Figure 5 and optionally comprises a human lgG2 Fc or human lgG2 constant region. The method of any of the foregoing claims, wherein the agonistic anti-VISTA antibody or antibody fragment comprises a variable light chain and a variable heavy chain polypeptide comprising the same CDRs any one of the antibodies having the sequences contained in the table in Figure 5 and the variable light chain and the variable heavy chain polypeptide of said antibody or antibody fragment respectively each possess at least 90% sequence identity to the variable light chain and the variable heavy chain polypeptides of the same anti-human VISTA antibody having the sequences contained in the table in Figure 5 and optionally comprises a human lgG2 Fc or human lgG2constant region. The method of any of the foregoing claims, wherein the agonistic anti-VISTA antibody or antibody fragment comprises a variable light chain and a variable heavy chain polypeptide comprising the same sequences as any one of the anti-human VISTA antibodies having the sequences contained in the table in Figure 5 and optionally comprises a human lgG2 Fc or human lgG2 constant region. The method of any of the foregoing claims, wherein the VISTA agonist comprises a human VISTA fusion polypeptide, e.g., a human VISTA-lg fusion protein and/or a human VSIG3 fusion polypeptide, e.g., a human VSIG3-lg fusion protein. The method of any of the foregoing claims wherein the VISTA agonist, optionally an antihuman VISTA or anti-human VSIG3 antibody, comprises a human Fc region, e.g., a human IgGl, lgG2, lgG3 or lgG4 Fc region, and further optionally comprises a human lgG2 Fc or human lgG2 constant region. The method of any of the foregoing claims wherein the VISTA agonist comprises a human lgG2 Fc region optionally having intact or unmodified FcR binding. The method of any of the foregoing claims wherein the VISTA agonist comprises a human Fc region, e.g., a human IgGl, lgG2, lgG3 or lgG4 Fc region, that has been mutated to alter (increase or decrease) at least one effector function, e.g., FcR binding, complement binding, glycosylation, or ADCC. The method of any of the foregoing claims wherein the VISTA agonist, optionally an anti-human VISTA antibody, comprises a human lgG2 Fc region which binds to all or at least one Fc receptor bound by an endogenous human lgG2 Fc region. The method of any of the foregoing claims wherein the VISTA agonist (i) increases the ratio of lymphocytes to neutrophils and/or (ii) results in a more normal (antiinflammatory) cytokine profile and/or (iii) decreases CXCL10 and/or CXCR2 levels, (iv) eliminates or reduces the CD14⁺ classical monocyte phenotype in favor of a more antiinflammatory cell state characterized e.g., by downregulation of CD14, IFN receptors, Fcgr3a (CD16) and CSF1R, (v) upregulation of CDllb, M-CSF (Csfl), Cyclin-dependent kinase inhibitor (Cdknla) and the anti-inflammatory cytokines IL1RA and GDF15 or a combination of any of the foregoing. The method of any of the foregoing claims, which prevents the patient from progressing to a clinical endpoint consistent with an ARDS diagnosis or which results in a much less severe form of ARDS, i.e., which does not progress to cytokine storm, sepsis and/or organ failure. The method of any of the foregoing claims, wherein the patient has a coronavirus infection caused by COVID-19. The method of any of the foregoing claims, wherein the patient is confirmed to be COVID-19 positive prior to treatment. The method of any of the foregoing claims, wherein the patient is confirmed to be COVID-19 after starting treatment. The method of any of the foregoing claims, wherein the patient shows at least one symptom of ARDS or pulmonary problems prior to or after treatment. The method of claim 28, wherein said symptoms or pulmonary problems include one or more of the following: barotrauma (volutrauma), pulmonary embolism (PE), pulmonary fibrosis, ventilator-associated pneumonia (VAP); gastrointestinal bleeding (ulcer), dysmotility, pneumoperitoneum, bacterial translocation; Hypoxic brain damage; abnormal heart rhythms, myocardial dysfunction; acute kidney failure, positive fluid

47 balance; vascular injury, pneumothorax, tracheal injury/stenosis; malnutrition (catabolic state), electrolyte abnormalities; Atelectasis, blood clots, weakness in muscles used for breathing, stress ulcers, depression or other mental illness; single or multiple organ failure; pulmonary hypertension or increase in blood pressure in the main artery from the heart to the lungs. The method of any of the previous claims, wherein the levels of at least one cytokine or anti-inflammatory molecule or proinflammatory molecule, e.g., CXCL10, CXCR2, IL-6, CRP, gamma interferon, IL-1, TNF, IFN-y, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-lalpha, and CXCL11/I-TAC in the patient are detected prior to treatment. The method of claim 30, wherein the levels of said cytokine or proinflammatory molecule in the patient are detected and confirmed to be aberrant (consistent with a diagnosis of the onset of or increased risk of cytokine storm) prior to treatment. The method of any of the previous claims, wherein the levels of VISTA in the patient are detected prior to and/or after treatment. The method of any of the previous claims, wherein the levels of at least one of CXCL10, CXCR2, IL-6, CRP, IFN-y, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-lalpha, and CXCL11/I-TAC in the patient are detected prior to and/or after treatment. The method of claim 32, wherein the levels of IL-6 and/or CRP and/or any of IFN-y, IL-2, IL-17, CCL5/Rantes, CCL3/MIP-lalpha, and CXCL11/I-TAC in the patient are detected and confirmed to be elevated prior to treatment. The method of any of the previous claims, wherein the VISTA agonist is administered at a dose ranging from .01-5000 mg, 1-1000 mg, 1-500 mg, 5mg - 50mg or about 1-25 mg. The method of any of the previous claims, wherein the VISTA agonist is administered biweekly, weekly, every 2 or 3 weeks, or every 4 weeks intravenously or via subcutaneous injection.

48 The method of any of the previous claims, wherein the patient is administered another active or another therapeutic regimen used to treat coronavirus infection and/or treat or prevent ARDS or CRDS. The method of any of the previous claims, wherein the patient receives another treatment for ARDS, optionally one or more of corticosteroids; inhaled nitric oxide (NO); extracorporeal membrane oxygenation (venovenous or venoarterial) or another immunosuppressive agent, optionally thymoglobulin, basiliximab, mycophenolate mofetil, tacrolimus, an anti-CD20 mAb such as rituximab, or a corticosteroid. The method of any of the previous claims, wherein the patient is additionally treated with an antiviral or antibiotic or another anti-inflammatory agent, optionally another biologic, e.g. another antibody that targets a checkpoint protein such as PD-1, PD-L1, PD-L2, CTLA-4 or an IL-6 antagonist. The method of any of the previous claims, wherein the patient has pneumonia, optionally caused by COVID-19 or caused by another pathogen, e.g., another virus, a bacterium or a fungus. The method of any of the previous claims, wherein the patient has one or more risk factors for developing cytokine storm, ARDS or a poor ARDS prognosis; e.g., the patient has a respiratory infection, the patient is over 60, 65, 70 years of age, the patient has type 1 or type 2 diabetes, the patient has high blood pressure, the patient has cancer, the patient has an inflammatory lung condition, e.g., asthma, COPD or cystic fibrosis, the patient has arteriosclerosis, the patient has another inflammatory or autoimmune condition or a combination of any of the foregoing. The method of any of the previous claims, wherein the patient is on a ventilator or respirator prior to agonist administration. The method of any of the previous claims, wherein the patient has improved or normal lung function after agonist treatment. The method of any of the previous claims wherein the patient optionally is further treated with any of the following:

49 (i) azathioprine,

(ii) calcineurin inhibitors (CNIs),

(iii) mycophenolate mofetil (MMF)/mycophenolic acid (MPA),

(iv) mTOR inhibitors (e.g., tacrolimus, everolimus, sirolimus),

(v) low dose corticosteroids (e.g., prednisone/prednisolone),

(vi) antihypertensive agents (e.g., angiotensin converting enzyme inhibitors (ACEIs),

(vii) angiotensin II receptor blockers (ARBs),

(viii) cyclosporine,

(ix) antidiabetogenic agents;

(x) a pulse steroid such as oral prednisone;

(xi) or a combination of any of the foregoing. The method of any of the previous claims wherein the anti-VISTA antibody or antibody fragment contains an Fc region that has been modified to alter effector function, halflife, proteolysis, and/or glycosylation. The method of any of the previous claims wherein the anti-VISTA antibody is selected from a humanized, single chain, or chimeric antibody and the antibody fragment is selected from a Fab, Fab', F(ab')2, Fv, or scFv. The method of any of the previous claims wherein the VISTA agonist dose is between about 0.001 and 100 mg/kg of body weight of recipient patient. The method of any of the previous claims which promotes survival and/or improves or restores normal lung function. The method of any of the previous claims which eliminates the need for the need for the patient to go on a ventilator or reduces the time the patient is on a ventilator. The method of any of the foregoing claims wherein administration of the VISTA agonist

(i) increases the ratio of lymphocytes to neutrophils and/or (ii) results in a more normal (anti-inflammatory) cytokine profile and/or (iii) decreases CXCL10 and/or CXCR2 levels, (iv) eliminates or reduces the CD14⁺ classical monocyte phenotype in favor of a more anti-inflammatory cell state characterized e.g., by downregulation of CD14, IFN receptors, Fcgr3a (CD16) and CSF1R, (v) upregulates one or more of CDllb, M-CSF (Csfl), Cyclin-dependent kinase inhibitor (Cdknla) and the anti-inflammatory cytokines IL1RA and GDF15 or a combination of any of the foregoing. The method of any of the previous claims, wherein a VISTA agonist is administered concurrent, prior or shortly after the subject receives an anti-viral vaccine, optionally a coronavirus vaccine, further optionally a SARS-COV-2, SARS-CoV-1 or MERS vaccine. The method of claim 51, wherein the vaccine comprises the Moderna, Pfizer or Janssen COVID-19 vaccine or comprises another coronavirus vaccine that comprises a coronavirus spike protein or immunogenic fragment thereof, nucleic acid encoding said spike protein or immunogenic fragment thereof, or antibodies specific to the coronavirus spike protein, optionally a SARS-COV-2, SARS-COV or MERS spike protein.

51