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TNX-1800 is a preclinical stage synthetic-derived live attenuated chimeric horsepox virus vaccine engineered to express the SARS-CoV-2 spike (S) gene. The objectives of this study were to assess the safety, tolerability, and immunogenicity of TNX-1800 administration in Syrian golden hamsters and New Zealand white rabbits. Animals were vaccinated at three doses via percutaneous inoculation. The data showed that the single percutaneous administration of three TNX-1800 vaccine dose levels was well tolerated in both hamsters and rabbits. At all dose levels, rabbits were more decerning regarding vaccine site reaction than hamsters. Lastly, no TNX-1800 genomes could be detected at the site of vaccination. Post-vaccination, all animals had anti-SARS-CoV-2 spike protein IgG specific antibody responses. These data demonstrate that TNX-1800 infection was limited, asymptomatic, and cleared by the end of this study, and a single dose was able to generate immune responses.

The ongoing global Monkeypox outbreak that started in the spring of 2022 has reinforced the importance of protecting the population using live virus vaccines based on the vaccinia virus (VACV). Smallpox also remains a biothreat and although some U.S. military personnel are immunized with VACV, safety concerns limit its use in other vulnerable groups. Consequently, there is a need for an effective and safer, single dose, live replicating vaccine against both viruses. One potential approach is to use the horsepox virus (HPXV) as a vaccine. Contemporary VACV shares a common ancestor with HPXV, which from the time of Edward Jenner and through the 19th century, was extensively used to vaccinate against smallpox. However, it is unknown if early HPXV-based vaccines exhibited different safety and efficacy profiles compared to modern VACV. A deeper understanding of HPXV as a vaccine platform may allow the construction of safer and more effective vaccines against the poxvirus family. In a proof-of-concept study, we vaccinated cynomolgus macaques with TNX-801, a recombinant chimeric horsepox virus (rcHPXV), and showed that the vaccine elicited protective immune responses against a lethal challenge with monkeypox virus (MPXV), strain Zaire. The vaccine was well tolerated and protected animals from the development of lesions and severe disease. These encouraging data support the further development of TNX-801.

Convalescent sera, rich in pathogen-specific antibodies, offers passive immunity to patients with infectious diseases. Screening assays using convalescent sera are crucial for evaluating therapeutic efficacy, selecting suitable serum donors, and standardizing assays. They measure antibody levels, neutralizing potential, and specificity against viruses like SARS-CoV-2, ensuring therapeutic serum contains potent antibodies. Standardized procedures enable reliable results and wider adoption of serum therapy for COVID-19. We have developed a high-content image-based assay for screening convalescent sera against SARS-CoV-2 variants. Using various cell lines, we identified optimal candidates, employed immunofluorescence to visualize infected cells, and assessed neutralizing antibody efficacy. Screening convalescent sera for therapeutic potential identified neutralizing activity against SARS-CoV-2 variants. Dose–response analysis showed variable neutralizing activity, with some sera exhibiting broad neutralization. Additionally, we explored the synergy between neutralizing sera and β-d-N4-hydroxycytidine (NHC), an initial metabolite of molnupiravir. These assays enhance serum therapy’s benefits for COVID-19 treatment and aid in understanding neutralizing activity against SARS-CoV-2 variants, addressing viral challenges.

TNX-1800 is a synthetically derived live recombinant chimeric horsepox virus (rcHPXV) vaccine candidate expressing Wuhan SARS-CoV-2 spike (S) protein. The primary objective of this study was to evaluate the immunogenicity and efficacy of TNX-1800 in two nonhuman primate species challenged with USA-WA1/2020 SARS-CoV-2. TNX-1800 vaccination was well tolerated with no serious adverse events or significant changes in clinical parameters. A single dose of TNX-1800 generated humoral responses in African Green Monkeys and Cynomolgus Macaques, as measured by the total binding of anti-SARS-CoV-2 S IgG and neutralizing antibody titers against the USA-WA1/2020 strain. In addition, a single dose of TNX-1800 induced an interferon-gamma (IFN-γ)-mediated T-cell response in Cynomolgus Macaques. Following challenge with SARS-CoV-2, African Green and Cynomolgus Macaques exhibited rapid clearance of virus in the upper and lower respiratory tract. Future studies will assess the efficacy of TNX-1800 against newly emerging variants and demonstrate its safety in humans.

Background Isometheptene is a sympathomimetic drug effective in acute migraine treatment. It is composed of two enantiomers with diverse pharmacological properties. This study investigated in pithed rats the cardiovascular effects of ( S )- isometheptene and ( R )-isometheptene, and the pharmacological profile of the more potent enantiomer. Methods The effects of i.v. bolus injections (0.03, 0.1, 0.3, 1 and 3 mg/kg) of isometheptene racemate, ( S )-isometheptene or ( R )-isometheptene on heart rate and blood pressure were analyzed in control experiments. The enantiomer producing more pronounced tachycardic and/or vasopressor responses was further analyzed in rats receiving i.v. injections of prazosin (0.1 mg/kg), rauwolscine (0.3 mg/kg), propranolol (1 mg/kg) or intraperitoneal reserpine (5 mg/kg, -24 h). Results Compared to ( R )-isometheptene, ( S )-isometheptene produced greater vasopressor responses, whilst both compounds equipotently increased heart rate. The tachycardic responses to ( S )-isometheptene were abolished after propranolol, but remained unaffected by the other antagonists. In contrast, the vasopressor responses to ( S )-isometheptene were practically abolished after prazosin. Interestingly, after reserpine, the tachycardic responses to ( S )-isometheptene were abolished, whereas its vasopressor responses were attenuated and subsequently abolished by prazosin. Conclusions The different cardiovascular effects of the isometheptene enantiomers are probably due to differences in their mechanism of action, namely: (i) a mixed sympathomimetic action for ( S )-isometheptene (a tyramine-like action and a direct stimulation of α 1 -adrenoceptors); and (ii) exclusively a tyramine like action for ( R )-isometheptene. Thus, ( R )-isometheptene may represent a superior therapeutic benefit as an antimigraine agent.

BackgroundPolymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) is pathologically activated immature neutrophil that exerts immunosuppressive functions. PMN-MDSC is short-lived and constantly replenished through bone marrow (BM) myelopoiesis. Within the BM, histidine decarboxylase (HDC) expressing immature neutrophil constitute a histaminergic niche that enforces hematopoietic stem cell quiescence and inhibits pathological myelopoiesis.1 Trefoil factor family 2 (TFF2), a partial agonist for CXCR4, inhibits CXCL12-dependent chemotaxis.2 Here we engineered a stabilized TFF2 peptide linked to mouse serum albumin (TFF2-MSA), and investigated its effect on PMN-MDSC and combination with PD1 inhibitor in advanced gastric cancer mouse models.MethodsA syngeneic gastric cancer cell line ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) was grafted subcutaneously into HDC-GFP transgenic mice to trace HDC-GFP+MDSCs. Once tumors reach 200 mm3, mice received TFF2-MSA or/and anti-PD-1 antibody. The CXCR4 antagonist AMD3100 served as comparison to TFF2-MSA. We further established a spontaneous lung metastasis model in which mice bearing ACKP tumors received surgical tumor resection and subsequent TFF2-MSA/anti-PD-1 treatments. In another orthotopic model, ACKP-luciferase cells were implanted to stomach submucosa, and the treatments started after 1 week. At 1 month, flow cytometry and single cell RNA sequencing (scRNA-seq) were performed to study treatment induced immune profile changes.ResultsEither TFF2-MSA or anti-PD-1 monotherapy showed little inhibition of subcutaneous ACKP tumor (15% and 25%, N=10, p>0.05), but their combination dramatically suppressed ACKP growth by 78% in a synergistic manner (p<0.0001). In the lung metastasis model, the combination completely abrogated metastasis in 80% mice (N=10, p<0.0001) and prolonged mice survival (p<0.0001), in contrast to minimal effect of either monotherapy (p>0.05). In the orthotopic model, although either monotherapy only slightly delayed tumor growth, their combination successfully eradicated tumor in 80% mice (N=5, p<0.001). Mechanistically, TFF2-MSA systematically reduced HDC+ PMN-MDSCs in the TME, spleen and blood, and its combination with anti-PD-1 profoundly increased polyfunctional Tim-3-Lag-3-CD8+ T cells in the TME and TCF1+ T cells in the TDLN. Inside BM, TFF2-MSA decreased PMN-MDSC generation from its precursors (MPP3/CMP/GMP/GP) to a level similar to tumor-free mice, and restored hematopoietic stem cell quiescence via histamine feedback loop. Importantly, flow cytometry, scRNA-seq and functional analysis suggested TFF2-MSA treated PMN-MDSCs display a more mature profile with less immunosuppressive property. In contrast, AMD3100 plus anti-PD-1 failed to decrease tumor growth and TME MDSCs possibly due to destruction of BM hematopoietic homeostasis.ConclusionsTFF2-MSA systematically reduced PMN-MDSC, and its immunosuppression and generation in BM, thereby sensitizing advanced gastric cancer to PD-1 inhibitors.ReferencesChen X, Deng H, Churchill MJ, Luchsinger LL, Du X, Chu TH, Friedman RA, Middelhoff M, Ding H, Tailor YH, Wang ALE, Liu H, Niu Z, Wang H, Jiang Z, Renders S, Ho SH, Shah SV, Tishchenko P, Chang W, Swayne TC, Munteanu L, Califano A, Takahashi R, Nagar KK, Renz BW, Worthley DL, Westphalen CB, Hayakawa Y, Asfaha S, Borot F, Lin CS, Snoeck HW, Mukherjee S, Wang TC. Bone Marrow Myeloid Cells Regulate Myeloid-Biased Hematopoietic Stem Cells via a Histamine-Dependent Feedback Loop. Cell Stem Cell. 2017 Dec 7;21(6):747–760.e7.Dubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, Wang TC. Secreted trefoil factor 2 activates the CXCR4 receptor in epithelial and lymphocytic cancer cell lines. J Biol Chem. 2009 Feb 6;284(6):3650–62.Ethics ApprovalFor experiments in mouse models, all procedures for mice were approved by the Institutional Animal Care and Use Committee of Columbia University in accordance with institutional and NIH guidelines (protocol number AC-AABV0664).

BackgroundMyeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are potential therapeutic targets in immune checkpoint cancer therapy, particularly for cancers that are unresponsive to anti-PD-1 therapy. It has previously been demonstrated that trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can partially suppress MDSC expansion and partially activate tumor immunity through agonism of the CXCR4 receptor.1–3 We investigated whether a novel fusion protein, murine TFF2-murine serum albumin (mTFF2-MSA), designated mTNX-1700, has single agent activity and can improve on the therapeutic effects of anti-PD-1 in the Pan02 syngeneic mouse model of advanced pancreatic cancer.MethodsA syngeneic pancreatic mouse model was developed using the Pan02 pancreatic cell line grafted subcutaneously into C57BL/6 mice. We generated a recombinant fusion protein, designated mTFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing the frequency of dosing. Mice subsequently received mTFF2-MSA, anti-PD-1 antibody (clone RMP1–14) or combination of mTFF2-MSA and anti-PD-1, and tumor volume was measured. At the endpoint, flow cytometry was performed on the tumor, axillary lymph node, blood, and bone marrow, to examine treatment-induced effects on cellular immune profiles.ResultsIn the Pan02 model, on Day 19 of treatment, tumor growth was suppressed (TGI) by mTFF2-MSA alone, anti-PD-1 alone, and by the combination mTFF2-MSA and anti-PD-1 by 23%, 28% and 36% TGI, respectively. In the blood, as measured by flow cytometry, there was a significant increase in total macrophages and M2 macrophages between the control and all treatment groups, and a decrease in monocytes and neutrophils in the groups dosed with anti-PD-1. In the axillary lymph node, there was a significant decrease in VISTA+ cells in both the CD4+ and CD8+ T-cells, and an increase in T regulatory cells in all treatment groups as compared to the control. In addition, an increase in PD-1+ and Lag3+ in both the CD4+ and CD8+ T-cells in the anti-PD-1 and combination treated groups was observed.ConclusionsmTFF2-MSA exhibits single agent activity and is additive to anti-PD-1 antibody checkpoint inhibition in treating the Pan02 syngeneic mouse model of advanced pancreatic cancer.ReferencesDubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, Wang TC. Secreted trefoil factor 2 activates the CXCR4 receptor in epithelial and lymphocytic cancer cell lines. J Biol Chem. 2009;284:3650–3662.Dubeykovskaya Z, Si Y, Chen X, Worthley DL, Renz BW, Urbanska AM, Hayakawa Y, Xu T, Westphalen CB, Dubeykovskiy A, Chen D, Friedman RA, Asfaha S, Nagar K, Tailor Y, Muthupalani S, Fox JG, Kitajewski J, Wang TC. Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer. Nat Commun. 2016;7:1–11.Dubeykovskaya Z, Duddempudi PK, Deng H, Valenti G, Cuti, KL, Nagar K, Tailor Y, Guha C, Kitajewski J, Wang TC. Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer. Cancer Gene Ther. 2019;26:48–57.

BackgroundRacemic isometheptene [(RS)-isometheptene] is an antimigraine drug that due to its cardiovascular side-effects was separated into its enantiomers, (R)- and (S)-isometheptene. This study set out to characterize the contribution of each enantiomer to its vasoactive profile. Moreover, rat neurogenic dural vasodilatation was used to explore their antimigraine mechanism of action.MethodsHuman blood vessel segments (middle meningeal artery, proximal and distal coronary arteries, and saphenous vein) were mounted in organ baths and concentration response curves to isometheptene were constructed. Calcitonin gene-related peptide (CGRP)-induced neurogenic dural vasodilation was elicited in the presence of the enantiomers using a rat closed cranial window model.ResultsThe isometheptene enantiomers did not induce any significant contraction in human blood vessels, except in the middle meningeal artery, when they were administered at the highest concentration (100 μM). Interestingly in rats, (S)-isometheptene induced more pronounced vasopressor responses than (R)-isometheptene. However, none of these compounds affected the CGRP-induced vasodilator responses.ConclusionThe isometheptene enantiomers displayed a relatively safe peripheral vascular profile, as they failed to constrict the human coronary artery. These compounds do not appear to modulate neurogenic dural CGRP release, therefore, their antimigraine site of action remains to be determined.

BackgroundMyeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are potential therapeutic targets in immune checkpoint cancer therapy, particularly for cancers that are unresponsive to anti-PD-1 therapy. It has previously been demonstrated that trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can partially suppress MDSC expansion and activate tumor immunity through agonism of the CXCR4 receptor.1–3 We investigated whether a novel fusion protein, murine TFF2-murine serum albumin (mTFF2-MSA), has single agent activity and can improve on the therapeutic effects of anti-PD-1 in CT26.wt subcutaneous and CT26-Luciferase (CT26-Luc) orthotopic syngeneic mouse models of advanced colorectal cancer (CRC).MethodsTwo syngeneic colon carcinoma mouse models were developed using the CT26.wt and CT26-Luc CRC cell lines grafted subcutaneously and orthotopically, respectively, into BALB/C mice. We generated a recombinant fusion protein, designated mTFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing the frequency of dosing. Mice subsequently received mTFF2-MSA, anti-PD-1 antibody (clone 29F.1A12 for subcutaneous study; clone RMP-1–14 for orthotopic study) or combination of mTFF2-MSA and anti-PD-1. Tumor volume, and survival were measured. At the endpoint, flow cytometry was performed on the blood, bone marrow, tumor, and lymph nodes, to examine treatment-induced effects on cellular immune profiles.ResultsIn the CT26.wt model, tumor growth was suppressed by mTFF2-MSA, anti-PD-1 and by the combination of mTFF2-MSA/anti-PD-1 by 16%, 40% and 60%, respectively. Survival in the CT26.wt model on Day 30 treated with vehicle, mTFF2-MSA, anti-PD1 and the combination of mTFF2-MSA and anti-PD-1 was 0%, 40%, 60% and 60%, respectively. In the CT26-Luc model, mTFF2-MSA, anti-PD-1, and the combination of mTFF2-MSA and anti-PD-1 suppressed tumor growth by 42%, 94%, and 94%, respectively. In the CT26-Luc model, neutrophils were significantly reduced in the blood in all treatment groups by flow cytometry. In the bone marrow, a significant reduction in total macrophages, M2 macrophages, and neutrophils was also observed but only in the group treated with anti-PD-1/mTFF2-MSA. In the axillary lymph node, there was a significant reduction in TOX+ cells in both CD4+ and CD8+ T-cells in all treatment groups. In the tumor, there was a significant reduction in total macrophages and M2 macrophages in all treatment groups, while NK cells were also increased, but only in the combination anti-PD-1/mTFF2-MSA treated group.ConclusionsmTFF2-MSA has single agent activity and is additive to anti-PD-1 antibody checkpoint inhibition in treating two syngeneic (subcutaneous and orthotopic) mouse models of advanced colorectal cancer.ReferencesDubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, Wang TC. Secreted trefoil factor 2 activates the CXCR4 receptor in epithelial and lymphocytic cancer cell lines. J Biol Chem. 2009;284:3650–3662.Dubeykovskaya Z, Si Y, Chen X, Worthley DL, Renz BW, Urbanska AM, Hayakawa Y, Xu T, Westphalen CB, Dubeykovskiy A, Chen D, Friedman RA, Asfaha S, Nagar K, Tailor Y, Muthupalani S, Fox JG, Kitajewski J, Wang TC. Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer. Nat Commun. 2016;7:1–11.Dubeykovskaya Z, Duddempudi PK, Deng H, Valenti G, Cuti, KL, Nagar K, Tailor Y, Guha C, Kitajewski J, Wang TC. Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer. Cancer Gene Ther. 2019;26:48–57.