Galectin-1 for treatment of bacterial pneumonia

Abstract

Stabilized forms of Gal-1, and a method of treating infection by S. pneumoniae in a subject, comprising administering a therapeutically effective amount of a Gal-1 polypeptide or modified Gal-1 polypeptide to the subject, are described.

Description

Attorney Docket No. PHC-034110 WO ORDGALECTIN-1 FOR TREATMENT OF BACTERIAL PNEUMONIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 718,845, filed on November 11, 2024, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Bacteria are a common cause of pneumonia in adults. Many types of bacteria cause pneumonia, but Streptococcus pneumoniae is the most common cause. It poses a significant healthcare challenge, contributing to substantial morbidity and mortality worldwide. The condition is marked by inflammation of the lung parenchyma and alveolar spaces and clinically manifests with a constellation of symptoms including fever, cough, dyspnea, and chest pain. Complications can be severe, leading to necrotizing pneumonia, empyema, extending to systemic effects such as sepsis, meningitis, and multiple organ failure.

[0004] When the bacteria invade and proliferate in the lung parenchyma and alveolar spaces, the subsequent inflammatory response leads to clinical manifestations. Upon bacterial invasion, alveolar macrophages initiate an inflammatory response to contain and limit bacterial spread. Cytokines released during this process initiate a cascade of events. Interleukin- 1 (IL-1) and tumor necrosis factor (TNF) are released. The chemokines interleukin-8 (IL-8) and colony-stimulating factors like granulocyte colony-stimulating factor (G-CSF) enhance chemotaxis and neutrophil maturation and lead to leukocytosis. Interleukin- 9 (IL-9) and interleukin- 13 (IL- 13) increase mucin production, resulting in purulent secretions. The pro-inflammatory cytokines contribute to leakage of the alveolar-capillary membrane at the site of inflammation. The leakage impairs gas exchange and leads to fibrosis, resulting in decreased lung compliance and severe dyspnea. Inflammation in the pleura activates somatic receptors of the phrenic nerve within the parietal pleura, causingpleuritic chest pain. Additionally, localized necrosis and damage to the lung parenchyma, along with tissue extravasation leads to hemoptysis.

[0005] Streptococcus pneumoniae is the most prevalent cause of community acquired pneumonia (CAP) globally, in both adults and children accounting for 60 to 70 percent of all bacterial CAP cases. The rate of resistance to many commonly used antibiotics in treating the condition is of great concern. Penicillin-resistant .S', pneumoniae was uncommon in the early 1990s - since that time it has become prevalent. Worldwide, the 5. pneumoniae serotypes most commonly associated with antimicrobial resistance are serotypes 6B, 9V, 19F, 14, 23 F, 6A and 19A.

[0006] Galectins are a family of carbohydrate-binding proteins having an affinity for [3- galactosides. Galectin-1 (Gal-1) is differentially expressed by various normal and pathological tissues. It is functionally polyvalent and has a wide range of biological activity. Gal-1 and its ligands appear to be one of the master regulators of immune responses such as T-cell homeostasis and survival, T-cell immune disorders, inflammation and allergies as well as host-pathogen interactions.

[0007] Galectin-1 is a 14-kDa protein that contains 135 amino acids. It is encoded by the LGALS1 gene. See Kadoya, T.; Horie, H. Cun. Drug Targets 2005, 6, 375-383. Human galectin-1 is soluble and exists in a dimeric form maintained by non-covalent binding. See Harrison, F.L., J. Cell Sci. 1991, 100, 9-14. It is composed of a 22-strand anti-parallel [3- sandwich, and each monomer contains a CRD. See Liao, D.I.; Kapadia, G.; Ahmed, H.; Vasta, G.R.; Herzberg, O„ Proc. Natl. Acad. Sci. USA 1994, 91, 1428-1432.

[0008] Galectin-1 is found in the cytoplasm and is also present on the cell membrane and can be secreted into the extracellular matrix. Each monomer of galectin-1 contains six cysteine residues (Cys2, Cysl6, Cys42, Cys60, Cys88, and Cysl30), and the reduced or oxidized states of them play a significant impact on the function of this protein. See Guardia et al., Glycobiology 2014, 24, 428-441.

[0009] Galectin-1 is broadly expressed in a wide range of tissues as well as cell types and exert sits effects intra- and extracellularly. Since its discovery, galectin-1 has been demonstrated to mediate diverse physiological and pathological processes. It is involved in cell growth and migration, inflammation, angiogenesis and promoting nervous systemdevelopment, muscle differentiation, and tumor progression, mediating evasion of cancer immune surveillance, immune tolerance in early pregnancy and cell adhesion. See for example, Yang et al., Expert Rev. Mol. Med. 2008, 10, el7; Ramirez Hernandez et al., Expert Rev. Neurother. 2020, 20, 439-448; Tirado-Gonzalez, I.; Freitag et al., Mol. Hum. Reprod. 2013, 19, 43-53; and Barrientos et al., Hum. Reprod. Update 2014, 20, 175-193.SUMMARY OF THE INVENTION

[0010] The inventors show that galectin-1 (Gal-1) possess the ability to specifically kill a distinct strain of Streptococcus pneumoniae, Serotype 14. Their data demonstrate that Gal-1 and variants thereof can eliminate this specific strain of microbe in vitro and in vivo, providing a unique strategy to specifically eliminate a pathogen as opposed to current antimicrobial approaches that kill a broad spectrum of microbes.

[0011] Accordingly, in one aspect, the invention includes a method of treating a Streptococcus pneumoniae infection in a patient, comprising administering an effective amount of a Gal-1 polypeptide to the patient. Alternatively, or additionally, a modified (i.e., variant) Gal-1 polypeptide may be administered. Such Gal-1 variants include any modification designed to enhance the stability, solubility, or bactericidal potency of the molecule. These modifications may be achieved through amino-acid substitutions (e.g., replacement of cysteine residues), chemical modifications such as nitrosylation, alkylation (e.g., with iodoacetamide), or other covalent attachments to stabilize free sulfhydryl groups. Additional stabilizing modifications may include conjugation with small molecules, peptides, polymers, or other agents known to reduce protein aggregation or enhance structural resilience under physiological conditions. In certain embodiments, the Gal-1 variants may also be generated using computational or artificial-intelligence-assisted design, machinelearning algorithms, or directed-evolution methods that identify amino-acid substitutions predicted to improve protein stability, folding, or bacterial-binding selectivity while maintaining [3-galactoside recognition. These approaches allow the development of nextgeneration Gal-1 analogues with optimized physicochemical or antimicrobial properties independent of specific modification routes. In further embodiments, the Gal-1 polypeptide or variant may be administered in combination with one or more antibacterial agents, including but not limited to antibiotics, antimicrobial peptides, or bacteriophage-derived lysins, to achieve additive or synergistic therapeutic effects. Such combination therapy may enhance bacterial clearance, reduce the required antibiotic dose, and mitigate thedevelopment of antibiotic resistance. The invention finds particular efficacy in treating S. pneumoniae serotype 14 infection, including antibiotic-resistant strains, in human adults and children. The invention further encompasses pharmaceutical compositions comprising Gal-1 variants or analogues formulated with pharmaceutically acceptable carriers for use in preventing or treating bacterial infections.BRIEF DESCRIPTION OF THE FIGURES

[0012] FIGS. 1A-1E provide graphs and images showing Galectin-1 and Galectin-3 are the Predominant Lectins Expressed in the Lung and Exhibit Specific Binding to Streptococcus pneumoniae on a Whole-Bacteria Microarray (a) Schematic workflow of proteomic analysis of lung tissue. Lung tissue samples were harvested from the lungs of mice or human slides sections. Tissues were homogenized and subjected to sonication. Proteins were extracted, separated by SDS-PAGE, and processed via in-gel digestion. The resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). (b-c) Mouse and human lung lectin expression profile. (Left) Bar graph showing normalized intensity (%) of detected lectins in mouse lung tissue, with individual data points overlaid. (Right) Bar graph showing normalized intensity (%) of detected lectins in human lung tissue, (d-e) (Left) Heatmaps representing relative fluorescence unit (RFU) values for Gal-1 (d) and Gal-3 (e) binding to 24 distinct Streptococcus pneumoniae serotypes displayed on the wholebacteria microarray platform. Galectins were applied in a concentration series from 0.16 pM to 10 pM. Darker blue indicates higher RFU values, while white indicates no detectable binding. (Right) Binding isotherms of Gal-1 and Gal-3 to serotype 14 (Spl4; red) and serotype 2 (Sp2; blue) are shown as representative examples. RFU signals were plotted against galectin concentrations to demonstrate specificity and binding strength. Error bars represent mean ± SD.

[0013] FIGS. 2A-2G provide graphs and images showing Galectin-1 is essential for in vivo clearance of .S'. pneumoniae serotype 14, whereas Galectin-3 has a limited role, (a-c) In vitro quantification of viable 5. pneumoniae following galectin treatment, (a, b) Percentage of CFU remaining for S. pneumoniae serotype 14 (Spl4, left) and serotype 2 (Sp2, right) after 2-hour incubation with increasing concentrations of Gal-1 (a) or Gal-3 (b). (c) Quantification of viable bacteria after incubation with PBS (control), 5 pM GaLl or Gal-3, with or without 25 mM TDG, as indicated. Data shown correspond to the mean of triplicate experiments and error bars represent means ± SD. (d-h) In vivo infection model using wild-type (WT), Gal-lknockout (Gal-1 KO), and Gal-3 knockout (Gal-3 KO) mice, (d) Schematic of the infection protocol (top left). Mice were intranasally infected with Spl4. After 12 hours, lungs were harvested, homogenized, and bacterial burden was quantified by CFU plating (bottom). Data represent mean ± standard deviation, n > 7 mice per group. Statistical significance determined by one-way ANOVA with Kruskal -Wallis test; ns, not significant; *P < 0.05. (e- g) Left panels: Excised lungs or lung sections from uninfected WT, Gal-1 KO, and Gal-3 KO mice. Right panels: Lungs or lung sections from the same genotypes following Spl4 infection, (e) Gross morphological examination, (f) Immunofluorescence staining of Gal-1 (green), S. pneumoniae Spl4 (red), and nuclei (DAPI, blue). Scale bars, 60 pm. (g) Hematoxylin and eosin (H&E) staining. Scale bars, 60 pm. (h) Relative protein abundance of Gal-1 and Gal-3 in lung tissue as determined by LC-MS / MS proteomics, as described in Fig. la. Data represent mean ± standard deviation, n = 4-6 mice per group. Statistical significance determined by one-way ANOVA with Kruskal-Wallis test; ns, not significant; *P < 0.05.

[0014] FIGS. 3A-3G provide graphs and images showing Galectin-3 exhibits minimal synergistic antibacterial activity with galectin-1 in vitro and is dispensable for host defense against .S', pneumoniae serotype 14 infection in vivo, (a-b) In vitro bacterial killing assays against .S'. pneumoniae serotype 14. (a) Dose-response analysis of combined galectins treatment. CFU remaining (%) after a 2-h incubation with varying Gal-1 (0-10 pM) at fixed Gal-3 levels: 0.3 pM (brown), 0.6 pM (blue), 1.2 pM (purple), 2.5 pM (cyan), 5.0 pM (orange), and 10 pM (green). In all panels, the gray curve denotes Gal-3 = 0 pM (GaLl tested at 0-10 pM). (b) Quantitative assessment of bacterial viability at fixed GaLl concentration (1.2 pM) combined with increasing Gal-3 concentrations (0.3-10 pM). Data shown correspond to the mean of triplicate experiments and error bars represent means ± SD. (c-d) In vivo bacterial burden quantification in lung homogenates 12 hours post-intranasal infection, (c) CFU enumeration in wild-type (WT), GaLl knockout (GaLl KO), GaL3 knockout (Gal-3 KO), and Gal- 1 / 3 double knockout (DKO) mice infected with S. pneumoniae serotype 14 (Spl4, left) or serotype 2 (Sp2, right). Data represent mean ± standard deviation, n > 7 mice per group. Statistical significance determined by one-way ANOVA with Kruskal-Wallis test; ns, not significant; *P < 0.05. (d) Representative gross lung morphology from DKO mice at 12 hours post-infection with Spl4 (bottom) compared to uninfected controls (top), (e-f) Histopathological analysis of lung tissue from DKO mice, (e) Immunofluorescence microscopy showing nuclei (DAPI, blue) and S. pneumoniae distribution in uninfected (top) and Spl4-infected (bottom) lung sections. Scale bars, 60 pm.(f) Hematoxylin and eosin staining demonstrating inflammatory infiltration and tissue architecture in uninfected (top) and infected (bottom) DKO lung tissue. Scale bars, 60 pm. (g) Comparative proteomic analysis of galectin family members in lung tissue. Relative protein abundance of Gal-1, Gal-3, galectin-9, galectin-7, and galectin-8 determined by LC-MS / MS across genotypes (WT, Gal-1 KO, Gal-3 KO, DKO) under uninfected and Spl4- infected conditions. Data expressed as normalized intensity relative to internal standards. Data represent mean ± standard deviation, n = 4-6 mice per group. Statistical significance determined by one-way ANOVA with Kruskal-Wallis test; ns, not significant.

[0015] FIGS. 4A-4N provide graphs and images showing chemically stabilized galectin- 1 enhances its bactericidal activity against Spl4 without altering binding specificity (a) Lactose affinity chromatography profiles of wild-type Gal-1 , carbamidomethylated galectin-1 (Gal-1 (IAM)), and nitrosylated galectin-1 (Gal-1 (Nitro)), (b) Differential scanning fluorimetry (DSF) analysis of Gal-1, Gal-1 (IAM), and Gal-1 (Nitro), monitoring light scattering at increasing temperatures to determine the onset temperature of protein aggregation (T_agg, onset) for each variant, (c) Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) analysis of Gal-1, Gal-1 (IAM), and Gal-1 (Nitro), showing refractive index (RI) and molar mass profiles; "D" indicates dimeric forms, (d) Heatmaps of relative fluorescence unit (RFU) values for Gal-1 (IAM) and Gal-1 (Nitro) binding to a panel of Streptococcus pneumoniae serotypes on whole-bacteria microarrays across a concentration range (0.16-10 pM). (e) Flow cytometric analysis of Gal-1 (IAM) and Gal-1 (Nitro) binding to .S'. pneumoniae serotype 14 (Sp 14), with or without 25 mM thiodigalactoside (TDG) competition, (f) Quantification of viable Sp 14 bacteria after incubation with Gal-1, Gal-1 (IAM), or Gal-1 (Nitro) at various concentrations, (g) Colony -forming unit (CFU) quantification of Sp 14 after treatment with PBS control, 5 pM Gal-1 (IAM), 5 pM Gal-1 (Nitro), or the respective protein plus 25 mM TDG. (h) Lactose affinity chromatography profiles of galectin- 1 V5D mutant (Gal-1 V5D) and its carbamidomethylated form (Gal-1 V5D (IAM)). (i) DSF analysis of Gal-1 V5D and Gal-1 V5D (IAM), monitoring light scattering to determine T_agg, onset for each mutant, (j) SEC-MALS analysis of GaLl V5D and GaLl V5D (IAM), showing RI and molar mass profiles; "D" and "M" indicate dimeric and monomeric forms, respectively, (k) Heatmaps of RFU values for Gal-l V5D and Gal-l V5D (IAM) binding to .S'. pneumoniae serotypes on whole-bacteria microarrays across a concentration range (0.16-10 pM). (1) Flow cytometric analysis of Gal-l V5D and Gal-l V5D (IAM) binding to Sp 14, with or without 25 mM TDG. (m) Quantification of viable Sp14 bacteria after incubation with Gal-1 V5D or Gal-1 V5D (IAM) at various concentrations, (n) CFU quantification of Sp 14 after treatment with PBS control, 5 pM Gal-1 V5D, 5 pM Gal-1 V5D (IAM), or the respective protein plus 25 mM TDG. Error bars represent standard deviation from three experimental replicates. Results are representative of three independent experiments. CFU, colony-forming unit.

[0016] FIGS. 5A-5L provide graphs and images showing genetically stabilized galectin-1 promotes its bactericidal activity against Spl4 without altering binding specificity (a) Lactose affinity chromatography profiles of Gal-1 with all cysteine replaced by serine (Gal 1 (All C to S)). (b) Differential scanning Huorimetry (DSF) analysis of (Gal 1 (All C to S)) monitoring light scattering at increasing temperatures to determine the onset temperature of protein aggregation (T_agg, onset) for each variant, (c) Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) analysis of (Gal 1 (All C to S)), showing refractive index (RI) and molar mass profiles; "D" indicates dimeric forms, (d) Heatmaps of relative fluorescence unit (RFU) values for (Gal 1 (All C to S)) binding to a panel of Streptococcus pneumoniae serotypes on whole-bacteria microarrays across a concentration range (0.16-10 pM). (e) Flow cytometric analysis of (Gal 1 (All C to S)) binding to .S', pneumoniae serotype 14 (Sp 14), with or without 25 mM thiodigalactoside (TDG) competition, (f) Quantification of viable Sp 14 bacteria after incubation with (Gal 1 (All C to S)) at various concentrations, (g) Colony-forming unit (CFU) quantification of Sp 14 after treatment with PBS control, 5 pM (Gal 1 (All C to S)), or (Gal 1 (All C to S)) plus 25 mM TDG. (h-1) In vivo bacterial burden quantification in lung homogenates 12 hours post-intranasal infection, (h) Schematic of the infection protocol (top left). Mice were intranasally infected with Spl4. After 12 hours, lungs were harvested, homogenized, and bacterial burden was quantified by CFU plating (bottom). CFU enumeration in wild-type (WT), Gal-1 knockout (Gal-1 KO) and Gal- 1 all C to S knock-in mice (Gal-1 KI) infected with S. pneumoniae serotype 14 (Spl4, left) or serotype 2 (Sp2, right). Data represent mean ± standard deviation, n > 7 mice per group. Statistical significance determined by one-way ANOVA with Kruskal-Wallis test; ns, not significant; *P < 0.05. (i) Representative gross lung morphology from Gal-1 KI mice at 12 hours post-infection with Spl4 (bottom) compared to uninfected controls (top), (j and k) Histopathological analysis of lung tissue from Gal-1 KI mice, (j) Immunofluorescence microscopy showing nuclei (DAPI, blue) and S. pneumoniae distribution in uninfected (top) and Spl4-infected (bottom) lung sections on Gal-1 KI mice. Scale bars, 60 pm. (k) Hematoxylin and eosin staining demonstrating inflammatory infiltration and tissuearchitecture in uninfected (top) and infected (bottom) Gal- 1 KI lung tissue. Scale bars, 60 pm. (g) Comparative proteomic analysis of galectin family members in lung tissue in Gal-1 KI mie. Relative protein abundance of Gal-1, Gal-3, galectin-9, galectin-7, and galectin-8 determined by LC-MS / MS across genotypes (WT, WT with spl4, Gal-1 KI, and Gal-1 KI) under uninfected and Spl4-infected conditions. Data expressed as normalized intensity relative to internal standards. Data represent mean ± standard deviation, n = 4—6 mice per group. Statistical significance determined by one-way ANOVA with Kruskal-Wallis test; ns, not significant.

[0017] FIG. 6 provides an image showing in vivo therapeutic efficacy of galectin-1 variants against S. pneumoniae serotype 14 pulmonary infection (a) Experimental timeline. Mice were infected intranasally with Spl 4 and held for 12 h, then received three intraperitoneal injections of PBS, Gal-1, or stabilized Gal-1 (Gal-1 all C— >S) at 4-h intervals over 12 h. Lungs were collected 12 h after the final dose for CFU quantification, immunofluorescence, and H&E histology, (b) Gal-1 knockout (Gal-1 KO) mice treated as in (a). Left: gross lung morphology. Middle: immunofluorescence showing bacteria (red), Gal-1 (green), and nuclei (DAPI, blue). Right: H&E sections and CFU quantification, (c) Gal-l / Gal-3 double knockout (DKO) mice treated as in (a). Layout as in (b). (d) Wild-type (WT) mice treated as in (a). Layout as in (b); both Gal-1 and stabilized Gal-1 reduce CFU relative to PBS, with stabilized Gal-1 showing the largest effect. Data are mean ± s.d.; each point represents one mouse. Statistical comparisons in CFU panels by one-way ANOVA with Kruskal-Wallis test; P < 0.05, P < 0.01, **P < 0.0001. Scale bars, 60 pm (immunofluorescence and H&E).DETAILED DESCRIPTION OF THE INVENTION

[0018] The inventors have provided stabilized forms of Gal-1, and a method of treating infection by S. pneumoniae in a subject, comprising administering a therapeutically effective amount of a Gal-1 polypeptide or modified GaLl polypeptide to the subject.Definitions

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. Allpublications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0020] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0021] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. Unless otherwise specified, "a," "an," "the," and "at least one" arc used interchangeably. Furthermore, as used in the description of the invention and the appended claims, the singular forms “a", “an", and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

[0022] A “subject,” as used herein, can be any animal, and may also be referred to as the patient. Preferably the subject is a vertebrate animal, and more preferably the subject is a mammal, such as a domesticated farm animal (e.g„ cow, horse, pig) or pet (e.g., dog, cat). In some embodiments, the subject is a human.

[0023] As used herein, "a subject in need" refers to a subject who has, or has an increased risk for developing an infection by an intracellular pathogen, an increased susceptibility to infection by an intracellular pathogen, or an increased susceptibility to developing bacterial inflammation. A subject may have an increased risk due to being immunosuppressed or having been exposed to a bacterial pathogen, for example.

[0024] The terms “therapeutically effective” and “pharmacologically effective” are intended to qualify the amount of each agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies. The therapeutically effective amount may be administered in one or more doses. An effective amount, on the other hand, is an amount sufficient to provide a significant chemical effect.

[0025] “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0026] “Treat”, “treating”, and “treatment”, etc., as used herein, refer to any action providing a benefit to a patient at risk for or afflicted with a disease, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, etc.

[0027] The terms "peptide(s)", "protein(s)" and "polypeptide(s)" are used interchangeably herein. As used herein, “polypeptide” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds (i.e., peptide isomers). “Polypeptide(s)” refers to both short chains, commonly referred as peptides, oligopeptides or oligomers, and to longer chains generally referred to as proteins.

[0028] The term "fragment" refers to any subject peptide having an amino acid residue sequence shorter than that of a polypeptide whose amino acid residue sequence is shown herein. Fragments lack one or more amino acids from one or both ends of the peptide. For example, a fragment can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 fewer amino acids than the non-fragmented peptide.

[0029] The following abbreviations for amino acids are used throughout the application: A = Ala = Alanine, T = Thr = Threonine, V = Vai = Valine, C = Cys = Cysteine, L = Leu = Leucine, Y = Tyr = Tyrosine, I = He = Isoleucine, N = Asn = Asparagine, P = Pro = Proline, Q = Gin = Glutamine, F = Phe = Phenylalanine, D = Asp = Aspartic Acid, W = Trp = Tryptophan, E = Glu = Glutamic Acid, M = Met = Methionine, K = Lys = Lysine, G = Gly = Glycine, R = Arg = Arginine, S = Ser = Serine, H = His = Histidine.Treatment of S. pneumoniae Infection

[0030] In one aspect, the invention provides a method of treating infection by S. pneumoniae in a subject. The method includes administering a therapeutically effective amount of a Gal- 1 polypeptide to the subject. In some embodiments, the subject is human.

[0031] As shown in the examples provided herein, Galectin-1 (Gal-1), and in particular stabilized Galectin-1 (sGal-1), a Gal-1 variant having all six of the cysteine residues changed to serine, possesses the ability to kill multidrug resistant Streptococcus pneumoniae, thereby providing a useful strategy not only to specifically target this microbe, but also having an advantage over currently employed antimicrobial approaches. Native human Galectin-1 has the amino acid sequenceMACGLVASNLNLKPGECLRVRGEVAPDAKSFVLNLGKDSNNLCLHFNPRFNAHGD ANTIVCNSKDGGAWGTEQREAVFPFQPGSVAEVCITFDQANETVKEPDGYEFKFPNR LNLEAINYMAADGDFKIKCVAFD (SEQ ID NO: 1).

[0032] Galectin-1 has been tested in vitro and in a model of pneumonia in vivo. sGal-1 was able to successfully treat Streptococcus pneumoniae infection. Additionally tested were various other stabilized forms of Gal-1. In some embodiments, a Gal-1 polypeptide that has been modified to have improved stability is provided. These included sGal-1, in which all cysteines have been replaced with serines to prevent disulfide-mediated inactivation, Gal-1 IAM, a variant having iodoacetamide alkylation of free sulfhydryls, Gal-1 Nitro, a variant having free sulfhydryls modified by nitrosylation using S -nitrosocysteine, and Gal-1 V5D, a variant wherein valine at position 5 was substituted with aspartic acid. Further stabilization methods applied to Gal-1 variants may include additional amino acid substitutions, chemical conjugations, or covalent attachments aimed at enhancing molecular stability. These methods collectively highlight potential modifications applicable to Gal-1 for therapeutic use in treating S. pneumoniae infections. Accordingly, in some embodiments, the Gal-1 polypeptide is selected from the group consisting of sGal-1, Gal-1 IAM, Gal-1 Nitro, Gal-1 V5D, and Gal-1 V5D IAM.

[0033] sGal-1 has the amino acid sequenceMASGLVASNLNLKPGESLRVRGEVAPDAKSFVLNLGKDSNNLSLHFNPRFNAHGDA NTIVSNSKDGGAWGTEQREAVFPFQPGSVAEVSITFDQANLTVKLPDGYEFKFPNRL NLEAINYMAADGDFKIKSVAFD (SEQ ID NO: 2). In some embodiments, the sGal-1 polypeptide comprises an amino acid sequence having 95% homology with SEQ ID NO: 2. In further embodiments, the sGal-1 polypeptide consists of SEQ ID NO: 2.

[0034] In some embodiments, the Gal-1 polypeptides are unmodified peptides. In other embodiments, the Gal-1 polypeptides can be subject to various changes, substitutions,insertions, and deletions to provide modified peptides, where such changes provide for certain advantages in its use. In this regard, targeting peptides that bind to and / or complex with glycans can be substantially homologous with, rather than be identical to, the sequence of a recited peptide where one or more changes are made and it retains the ability to function as specifically binding to glycans.

[0035] In some embodiments, analogs of the Galectin-1 polypeptide can be used. The term "analog" includes any peptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and that specifically binds to glycans. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue, such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

[0036] The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite binding activity.

[0037] In some modified Gal-1 polypeptides, chemical derivatives of the Gal-1 polypeptides are used. "Chemical derivative" refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-benzylhistidine. Also included as chemical derivatives arc those polypeptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.

[0038] In some embodiments, the Gal-1 analogue or variant engineered by computational design, directed evolution, or artificial-intelligence-assisted methods to enhance stability,solubility, oxidation resistance, or bactericidal potency relative to wild- type Gal-1, while retaining [3-galactoside-binding activity.

[0039] In some embodiments, the Gal-1 polypeptide is selected from the group consisting of wild type Gal-1, sGal-1, Gal-1 IAM, Gal-1 Nitro, Gal-1 V5D, and Gal-1 V5D IAM, while in further embodiments the Gal-1 polypeptide is sGal-1.

[0040] Gal-1 polypeptides can be prepared using recombinant technology and is commercially available from, for example, Beta Lifescience™. In addition, Gal-1 polypeptides can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a solid-phase Merrifield-type synthesis, can be used for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production and the like. A summary of the many techniques available can be found in Steward et al., "Solid Phase Peptide Synthesis", W. H. Freeman Co., San Francisco, 1969; Bodanszky et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976; J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969; Fields et al., int. J. Peptide Protein Res., 35:161-214, 1990; and U.S. Pat. No. 4,244,946 for solid phase peptide synthesis, and Schroder et al., "The Peptides", Vol. 1, Academic Press (New York), 1965 for classical solution synthesis, each of which is incorporated herein by reference. Appropriate protective groups usable in such synthesis are described in the above texts and in J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973, which is incorporated herein by reference.

[0041] In some embodiments, the subject has been diagnosed as having pneumonia, or pneumococcal pneumonia. Pneumonia is an inflammatory condition of the lung primarily affecting the small air sacs known as alveoli. Symptoms typically include some combination of productive or dry cough, chest pain, fever, and difficulty breathing Diagnosis of pneumonia is typically by obtained using a chest x-ray and blood tests, sputum culture and sputum Gram staining. In further embodiments, the subject is diagnosed as having pneumonia caused by S. pneumoniae infection.

[0042] Streptococcus pneumoniae is a Gram-positive, spherical bacteria, alpha-hemolytic member of the genus Streptococcus. Streptococcus pneumoniae is part of the normal upper respiratory tract flora. As with many natural flora, it can become pathogenic under the rightconditions, typically when the immune system of the host is suppressed. Invasins, such as pneumolysin, an antiphagocytic capsule, various adhesins, and immunogenic cell wall components are all major virulence factors.

[0043] In some embodiments, the 5. pneumoniae is serotype 14. Spl4 is among the most clinically significant and historically prevalent S. pneumoniae serotypes, accounting for roughly 10-30% of invasive pneumococcal disease cases, especially in children. See Garcia- Vidal, J Infect 65:521-527 (2012), the disclosure of which is incorporated herein. It is characterized by high invasiveness and strong antibiotic resistance, making it a major global pathogen of concern.

[0044] In some embodiments, the 5. pneunoniae is antibiotic resistant. Antibiotics, as defined herein, are bactericidal or bacteriostatic compounds already known in the art. Examples of known antibiotics include agents that target the bacterial cell wall, such as penicillins, cephalosporins, agents that target the cell membrane such as polymixins, agents that interfere with essential bacterial enzymes, such as quinolones and sulfonamides, and agents that that target protein synthesis such as the aminoglycosides, macrolides and tetracyclines. Additional known antibiotics include cyclic lipopeptides, glycylcyclines, and oxazolidinones. Antibiotic resistance represents the ability of intracellular pathogens to decrease (i.e., resist) the cytotoxic and cytostatic effects of antibiotics. In some embodiments, the 5. pneumoniae has developed resistance to multiple different antibiotics.

[0045] In some embodiments, the Gal-1 polypeptide is used together with another antibacterial agent to provide combinational therapy of the intracellular pathogen. The combination of agents can provide additive or synergistic effects. The Gal-1 polypeptide may be administered before, simultaneously, or after administration of an additional agent useful for treating infection with an intracellular pathogen. In some embodiments, the effects of the drugs overlap one another in time.

[0046] A variety of different antibacterial agents can be used in combination with the Gal- 1 polypeptide. Examples include quinolones (e.g., fluoroquinolones) such as ciprofloxacin, ansamycins, macrolides, and tetracyclines such as tigecyclin, and more exotic therapies such as the use of antisense oligonucleotides.Administration and Formulation

[0047] The invention also provides pharmaceutical compositions that can be used for the administration of active agent (i.e., the Gal-1 polypeptide or modified Gal-1 polypeptide) used in the method of the invention to a subject in need thereof. In some embodiments, the Gal-1 polypeptide or modified Gal-1 polypeptide is administered with a pharmaceutically acceptable carrier. The pharmaceutical acceptable carrier can have many forms, including tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels. An oral dosage form may be formulated such that the polypeptide is released into the intestine after passing through the stomach. Such formulations are described in U.S. Patent No. 6,306,434 and in the references contained therein.

[0048] Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.

[0049] The active agents can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical compositions suitable for rectal administration can be prepared as unit dose suppositories. Suitable carriers include saline solution and other materials commonly used in the art.

[0050] In some embodiments, the Gal-1 polypeptide or modified Gal-1 polypeptide is administered by pulmonary administration (e.g., intranasal). For administration by inhalation, active agents can be conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromcthanc, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

[0051] Alternatively, for administration by inhalation or insufflation, the active agents may take the form of a dry powder composition, for example, a powder mix of a modulator and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator. For intranasal administration, the active agents may be administered via a liquid spray, such as via a plastic bottle atomizer.

[0052] Active agents can be formulated for transdermal administration. The active agents can also be formulated as an aqueous solution, suspension or dispersion, an aqueous gel, a water-in-oil emulsion, or an oil-in-water emulsion. A transdermal formulation may also be prepared by encapsulation of an active agent within a polymer, such as those described in U.S. Pat. No. 6,365,146. The dosage form may be applied directly to the skin as a lotion, cream, salve, or through use of a patch. Examples of patches that may be used for transdermal administration are described in U.S. Pat. Nos. 5,560,922 and 5,788,983.

[0053] It will be appreciated that the amount of active agent required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately the attendant health care provider may determine proper dosage. In addition, a pharmaceutical composition may be formulated as a single unit dosage form, or it may be administered in multiple doses.

[0054] The amount of active agent that is delivered to the subject will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the subject has undergone. Ultimately, the attending physician will decide the amount of active agent with which to treat each individual patient. For example, the attending physician can administer low doses of the active agent of the present invention and observe the patient's response. Larger doses of the active agent may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ng to about 100 mg (preferably about 0.1 [tg to about 10 mg, more preferably about 0.1 pg to about 1 mg) of active agent per kg body weight.

[0055] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. The examples demonstrate the efficacy and utility in treating an S. pneumoniae infection in vitro and in vivo.EXAMPLESExample 1 : Galectin-1 Selectively Targets Drug-Resistant Streptococcus pneumoniae Serotype 14

[0056] Antibiotic-resistant Streptococcus pneumoniae, particularly serotype 14 (Spl4), represents a serious global health threat, causing severe pneumonia and meningitis that are difficult to treat in vulnerable populations due to rising resistance to multiple antibiotics. This urgent clinical challenge underscores the need for alternative therapeutic strategies that can effectively target antibiotic-resistant pathogens. One such strategy involves leveraging host glycan-binding proteins (lectins), which can recognize and bind specific glycans on microbial surfaces, potentially triggering downstream signahng that leads to microbial killing. However, many pathogenic bacteria - including Spl4 - use molecular mimicry to evade immune detection by decorating themselves with host- like antigens, making them particularly difficult to eliminate through standard adaptive immune responses.

[0057] In this context, galectins are especially interesting because they are innate immune lectins specialized in recognizing microbial glycans that resemble host antigens. Galectins can effectively target microbes that exploit host mimicry for immune evasion. To explore this potential defense mechanism, we first focused on the primary infection site for 5. pneumoniae - the lung - by performing a comprehensive proteomics analysis to identify lectins abundantly expressed in human and mice lung tissues. Interestingly, Galectin-1 (Gal- 1) and Galectin-3 (Gal-3) emerged as the most abundant lectins in the both human and mice lung tissues (figure la, lb and 1c). We then used a whole-bacteria microarray assay to examine the ability of these lectins to recognize S. pneumoniae strains. This binding analysis revealed that Gal-1 exhibited strong and selective recognition of Spl4, whereas Gal-3 also bound Spl4 along with other serotypes such as 33F and 16B, but with much weaker overall affinity compared to Gal-1 (figure Id and le). These findings suggest that Gal-I is not only abundantly present in the lung but also exhibits a unique ability to distinguish Spl4 fromother serotypes, hinting at a previously underappreciated role for Gal-1 in host-pathogen interactions at the respiratory mucosa.

[0058] To investigate Gal-l’s glycan-binding preferences at high resolution, we employed the Consortium for Functional Glycomics (CFG) mammalian glycan microarray (-600 glycans), testing multiple concentrations to derive dose-response curves. Consistent with its classification as a [3-galactoside-binding lectin, Gal-1 displayed strong affinity for N- acetyllactosamine (LacNAc)-containing glycans, with robust binding to repeating poly- LacNAc structures on multi-antennary N-glycan scaffolds. In addition, Gal-1 displayed detectable binding to ABO(H) blood group antigens only when these motifs were embedded within particular N-glycan scaffolds, indicating that its recognition of these antigens is conditional and scaffold-dependent. Furthermore, sialylation modulated Gal-1 binding in a linkage-specific manner, with a strong preference for a2-3-linked sialic acids, whereas a2-6- linked sialic acids exhibited minimal to no detectable binding. Quantitative nested-pie analysis confirmed that Gal-1 exhibited saturated binding only to specific LacNAc-containing glycans (IDs 376, 472, and 546), while other glycans - including blood group antigens - elicited only partial binding. It also preferentially recognized a2-3-linked Sialyl-LacNAc, selected galactose-3 -sulfated structures, and GM3, with minimal to no binding to a2-6-linked sialic acids or other gangliosides. These data establish Gal-1 as a lectin with a narrow but potent preference for human-like host glycans, raising the possibility that it might also engage molecular mimics displayed by pathogens.

[0059] To directly assess Gal-l’s ability to recognize bacterial surface glycans relevant to infection, we next screened it on a microbial glycan microarray (MGM) containing 313 distinct bacterial carbohydrate antigens. Strikingly, Gal-1 showed highly selective, high- affinity binding to the capsular polysaccharide of Streptococcus pneumoniae serotype 14 (Spl4), with binding saturation observed at sub-micromolar concentrations (-0.12 pM). Apart from Spl4, Gal-1 exhibited only minor binding to Providencia alcalifaciens 05 (PAO5), requiring much higher concentrations (-30 pM) to observe saturation, and showed minimal binding across most other bacterial glycans. The exquisite selectivity of Gal-1 for Spl4 therefore mirrors its preference for LacNAc-terminated host glycans, suggesting that Spl4 may exploit molecular mimicry to evade other arms of innate immunity.Example 2: Galectin-1 exerts carbohydrate-dependent bactericidal activity and is required for effective pulmonary clearance of Streptococcus pneumoniae serotype 14

[0060] To assess whether Galectin-1 (Gal-l)’s selective binding to S. pneumoniae serotype 14 (Spl4) leads to functional antimicrobial activity, we first performed a two-hour killing assay. Gal-1 significantly reduced Spl4 colony-forming units (CFUs) with an ECso of ~1.5 pM, whereas Galectin-3 (Gal-3) required -8 pM to achieve similar effects. Addition of thiodigalactoside (TDG), a galectin carbohydrate -binding inhibitor, reversed Gal-l’s effect, confirming that the killing is carbohydrate binding-dependent (Figure 2a-c). To determine whether Gal-l ’s antimicrobial activity is relevant in vivo, we performed infection experiments using wild-type (WT), Gal-1 knockout (Gal-1 KO), and Gal-3 knockout (Gal-3 KO) mice, each mock-treated or intranasally infected with .S', pneumoniae serotype 14 (Spl4). Upon gross inspection, Gal-1 KO mice infected with Spl4 displayed markedly more severe lung pathology - including swollen, hemorrhagic, and mottled tissue - compared to WT and Gal-3 KO animals (Figure 2e). Immunofluorescence analysis further revealed that red fluorescence, representing Spl4 bacterial burden, was substantially increased in Gal-1 KO lungs relative to control groups, consistent with more severe and poorly controlled infection (Figure 2f). In contrast, in infected WT and Gal-3 KO lungs, Gal-1 (green fluorescence) was found to co-localize with Spl4 (red), producing distinct yellow signals, indicating direct physical engagement between Gal-1 and the pathogen (Figure 2f). Hematoxylin and eosin (H&E) staining corroborated these findings: Gal-1 KO lungs exhibited dense inflammatory infiltrates and alveolar consolidation, further supporting the presence of uncontrolled pneumonia (Figure 2g). Finally, to rule out compensatory expression as a confounding factor, we analyzed galectin expression profiles in all groups using EC-MS / MS. Proteomic analysis confirmed that knockout of one galectin did not induce overexpression of the other, suggesting that the observed phenotypes were not due to altered galectin stoichiometry (Figure 2h). Together, these results demonstrate that Galectin-1 plays a non-redundant role in controlling S. pneumoniae serotype 14 infection in vivo, and its absence leads to impaired bacterial clearance and exacerbated lung pathology.Example 3: Galectin-3 exhibits minimal synergistic antibacterial activity with galectin-1 in vitro and is dispensable for host defense against 5. pneumoniae serotype 14 infection in vivo.

[0061] To determine whether Galectin-3 can reinforce the Galectin-l-mediated control of Streptococcus pneumoniae serotype 14 (Spl4), we first carried out two-hour combination killing assays in vitro. To evaluate potential synergistic effects between Galectin-1 and Galectin-3, we performed dose-response killing assays using a fixed concentration of Gal-3 and a titration series of Gal-1 (0-10 pM). At lower concentrations of Gal-3 (0.3-5 pM), the killing curves closely overlapped with the Gal-l-alone condition, with EC50 values remaining in the 1.2-1.4 pM range, indicating minimal to no synergistic effect. However, at 10 pM Gal- 3, we observed a synergistic enhancement, with the EC50 for Gal-1 dropping to approximately 0.95 pM. This result suggests that Gal-3 can enhance Gal-l-mediated bacterial killing, but only at relatively high concentrations, nearly an order of magnitude above Gal-1 ’s effective range (Figure 3a). In Figure 3b, Gal-1 concentration was fixed at 1.2 pM, while Gal-3 was tested at concentrations ranging from 0.3 to 10 pM. Again, we did not observe a synergistic antibacterial effect until Gal-3 reached 10 pM. Together, these results indicate that Gal-3 can provide a modest synergistic enhancement, but only at concentrations significantly higher than those required for Gal-1 ’s maximal activity.

[0062] We next tested whether Gal-3 can compensate for Gal-1 deficiency in vivo. Using the same intranasal infection protocol described for Figure 2d, we compared wild-type mice with animals lacking Gal-1, Gal-3, or both Galectins (Gal-1 and Gal-3 double knock out). Twelve hours after inoculation, lung homogenates from Gal-1 knockout animals contained approximately ten-fold more bacteria than those from wild-type controls (Figure 3c). Strikingly, double-knockout mice lacking both Gal-1 and Gal-3 displayed bacterial burdens indistinguishable from the Gal-1 knockout group, whereas Gal-3 knockout mice resembled wild-type (Figure 3c). Macroscopic inspection of the lungs mirrored these quantitative data: the organs of Gal-1 knockout and double-knockout mice were swollen and mottled with hemorrhage, while those from wild-type and Gal-3 knockout cohorts retained grossly normal architecture (Figure 3d). Immunofluorescence staining provided a direct visual correlate: in wild-type and Gal-3 knockout lungs, abundant yellow overlay signified co-localization of host lectin (green) with bacteria (red); in contrast, the absence of Gal-1 in the knockout and double-knockout groups eliminated this overlap, leaving a profusion of unopposed red bacterial signal (Figure 3e). Histopathological analysis reinforced this pattern. Hematoxylin- and-eosin sections from Gal- 1 -deficient and double-knockout lungs showed dense neutrophilic infiltrates and widespread alveolar consolidation, whereas Gal-3 knockout tissue was largely clear (Figure 3f). Because gene deletions can sometimes trigger compensatoryexpression of related proteins, we profiled the full galectin repertoire in lung homogenates by LC-MS / MS. No significant up-regulation of Gal-7, Gal-8, Gal-9 or any other family member was detected in the single- or double-knockout mice, whether infected or not (Figure 3g). These proteomic data rule out compensatory reshaping of the pulmonary lectome as an explanation for the heightened susceptibility of Gal- 1 -deficient animals. Taken together, the experiments in Figure 3 show that Gal-1 is the principal, non-redundant lectin effector against antibiotic-resistant Spl4. Gal-3 provides only a limited, high-dose enhancement in vitro and, when removed in vivo, adds no further detriment beyond that caused by the loss of Gal-1 alone.Example 4: Chemically Stabilized galectin-1 enhances its bactericidal activity against Spl4 without altering binding specificity

[0063] Having established Galectin-1 (Gal-1) as the non-redundant lectin effector against Streptococcus pneumoniae serotype 14 (Spl4), we next asked whether the molecule itself could be engineered into a practical antimicrobial. A major obstacle is its intrinsic lability: the protein contains six surface-exposed cysteines, and even modest oxidative stress rapidly converts the canonical non-covalent homodimer into higher-order disulfide-linked aggregates that precipitate and lose activity. To counter this, we alkylated the cysteine thiols with iodoacetamide (Gal-1 (IAM)) or subjected them to selective nitrosylation (Gal-1 (Nitro)), reasoning that either modification would block unwanted disulfide formation without perturbing the carbohydrate recognition domain. To assess whether the protein could withstand oxidative stress, we subjected all samples - either with or without cysteine modification - to hydrogen peroxide (H2O2) treatment for two hours. Following treatment, the proteins were passed through a lactosyl affinity column to evaluate their functional binding activity. The unmodified Galectin-1 showed only -33% retention on the column, indicating a loss of nearly 67% of its binding function. In contrast, the cysteine-modified versions (IAM- Gal-land Nitro-Gai- 1) retained over 92% binding, suggesting that these modifications confer substantial protection against oxidative damage and enhance protein stability (figure 4a). Differential scanning fluorimetry revealed that cysteine capping increased the aggregation onset temperature (Tagg, onset) of Galectin-1 from 59.93 ± 0.1 °C for the wild-type protein to 66.50 + 0.15 °C for IAM-Gal-1 and 63.95 + 0.20 °C for Nitro-Gal-1, indicating enhanced thermal stability (figure 4b). Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) demonstrated that Gal-1, IAM-Gal-1, and Nitro-Gai- 1 allpredominantly exist as dimers, as indicated by the 'D' peak. The consistent refractive index and molar mass profiles across all variants suggest that chemical modifications preserve Gal- 1 dimerization (figure 4c). Critically, these biochemical gains did not come at the cost of specificity. On the whole-bacterium microarray, both modified proteins reproduced the wildtype (WT) binding profile: strong signal for Spl4 and weak or absent binding to the other 23 pneumococcal serotypes, suggesting that these chemical modifications do not alter binding specificity (Figure 4d). Flow cytometry showed that both IAM- and Nitro-Gai- 1 bound to live Spl4, and this binding was abolished by TDG treatment, indicating glycan-dependent recognition (Figure 4e). The functional consequence was clear in the 2-h killing assay: the FCso dropped from ~1.5 pM for WT Gal-1 to -0.87 pM with IAM-Gal-1 and -0.93 pM with Nitro-Gai- 1, suggesting that cysteine modification may stabilize the protein and thereby enhance killing efficiency (figure 4f). This activity was abolished by TDG, indicating glycan- dependent recognition (Fig. 4g).

[0064] To probe the importance of dimerization itself, we prepared monomeric V5D Gal-1 mutant (Gal-1 V5D) that lacks the dimerization interface. Also, we also apply the same stabilization strategy on a monomeric V5D Gal-1 mutant to make a stabilized but monomeric Gal-1 (Gal-1 V5D (IAM)). To assess oxidative tolerance, we subjected Gal-1 V5D and Gal-1 V5D (IAM) to hydrogen peroxide (H2O2) for 2 h. After treatment, proteins were applied to a lactosyl-affinity column to evaluate carbohydrate-binding activity. Unmodified Gal-1 V5D showed -53% retention, indicating a -47% loss of binding. By contrast, the cysteine- modified Gal-1 V5D (IAM) retained >93% binding, suggesting that this modification confers substantial protection against oxidative damage and enhances protein stability (Fig. 4h). Differential scanning fluorimetry revealed that cysteine capping increased the aggregation onset temperature (Tagg, onset) of Gal-1 V5D from 52.05 ± 0.28 °C to 57.98 ± 0.04 °C for Gal-1 V5D (IAM), indicating enhanced thermal stability (figure 4i). SEC-MALS showed that Gal-1 V5D elutes predominantly as a monomer (M) with only a small dimer shoulder (D). In contrast, Gal-1 V5D (IAM) displays a clear monomer-dimer equilibrium, evidenced by a pronounced D-M region alongside the monomer peak (figure 4j). On the wholebacterium microarray, both Gal-1 V5D and Gal-1 V5D (IAM) still show strong signal for Spl4 and weak or absent binding to the other 23 pneumococcal serotypes, suggesting these modifications won’t change their binding specificity (Figure 4k). Flow cytometry showed that both Gal-1 V5D and Gal-1 V5D (IAM) bound to live Spl4, but the binding is weaker than Gal-1 (IAM), suggesting monomeric form could potentially decrease Gal-l’s bindingaffinity. And this binding was abolished by TDG treatment, indicating glycan-dependent recognition (Figure 4e). The functional consequence was clear in the 2-h killing assay: the EC.50 increased to ~3.8 pM for Gal-1 V5D, whereas it decreased to ~1.8 pM for Gal- 1 V5D (IAM), indicating that the predominantly monomeric form is less potent in the bacterial killing assay and that cysteine modification may stabilize the protein and thereby enhance killing efficiency (Fig. 4m). This activity was abolished by TDG, indicating glycan- dependent recognition (Fig. 4n). Conversely, enforcing a monomeric state undermines antimicrobial function even when the protein is thermally safeguarded, highlighting dimerization as an essential structural prerequisite. These findings nominate chemically stabilized, dimeric Gal-1 - particularly the Nitro-Gai- 1 and IAM-Gal-1 variants - as realistic therapeutic candidates for combating drug-resistant pneumococcal infections and provide a clear blueprint for their further development in vivo.Example 5: Genetically stabilized galectin-1 promotes its bactericidal activity against Spl4 without altering binding specificity10065 J Guided by the success of chemically hardened Gal-1, we next asked whether a fully oxidation-proof lectin could be created by substituting every surface-exposed cysteine with serine. The resulting Gal-1 (all C— >S) withstood oxidative challenge: after a 2-h H2O2 exposure it still displayed robust lactose-elutable binding on a lactosyl-affinity column (Fig. 5a). Differential scanning fluorimetry showed a defined aggregation onset (Tagg, onset = 55.38 ± 0.06 °C), indicating a stable protein fold (Fig. 5b). SEC-MALS revealed that the variant elutes predominantly as a dimer, preserving the native oligomeric architecture (Fig. 5c). On the whole-bacterium microarray, all C— >S reproduced the WT specificity — strong recognition of Spl4 with weak / absent binding to the other 23 pneumococcal serotypes (Fig. 5d). Flow cytometry likewise showed clear binding to live Spl4 that was abolished by TDG, confirming glycan-dependent recognition (Fig. 5e). Functionally, the genetically stabilized lectin showed enhanced killing, with an EC50 -0.47 pM versus -1.5 pM for WT Gal-1 (Fig. 5f). As with WT, this bactericidal activity was blocked by TDG, demonstrating carbohydrate dependence (Fig. 5g). To determine whether these improvements matter in vivo, we generated knock-in (KI) mice in which the endogenous Lgalsl allele was replaced with the all-C— >S variant. Using the same intranasal challenge protocol, KI lungs harvested 12 h after Spl4 infection contained ~10-100x fewer CFU than wild-type (WT) littermates and were dramatically lower than Gal-1 KO cohorts (Fig. 5h, left). By contrast, the Sp2 challengeshowed no significant differences among groups (Fig. 5h, right), indicating a serotypespecific protective effect of Gal-1. Gross inspection mirrored the CFU data: KI lungs retained normal color and turgor after Spl4 exposure (Fig. 5i), in contrast to the hemorrhagic, swollen appearance typical of high bacterial burden in Gal-1 KO mice (Fig. 2e) and Gal-1 / 3 double KO mice (Fig. 3d). Immunofluorescence confirmed a lower red bacterial signal in KI lungs. In the Gal-1 (all C— >S) KI + Spl4 group, we observed clear co-localization of Gal-1 (green) with bacteria (red), producing a yellow overlap, consistent with direct bacterial targeting by Gal-1. The red signal in KI lungs was weak overall (Fig. 5j), indicating a reduced bacterial burden, especially when compared with the strong red signal seen in Gal-1 KO lungs in Fig. 2f and in Gal-1 / 3 double-knockout lungs in Fig. 3e. Histology likewise revealed reduced inflammatory infiltrates and alveolar consolidation in KI tissue (Fig. 5k). Finally, LC-MS / MS profiling showed no compensatory changes in other galectins (Gal-3, -7, -8, -9) across groups, indicating that the protection arises from stabilizing Gal-1 rather than remodeling the pulmonary lectomc (Fig. 51). Together, these data demonstrate that genetic stabilization of Gal-1 enhances bactericidal activity against Spl4 in vivo without altering binding specificity or the overall lung lectin landscape.Example 6: In vivo therapeutic efficacy of galectin-1 variants against S. pneumoniae serotype 14 pulmonary infection

[0066] Treated therapeutically rather than prophylactically, Gal-1 retains - and even amplifies - its power against drug-resistant S. pneumoniae serotype 14. We first tested a stringent setting in which endogenous lectins are absent: Gal- 1 -knockout (Gal-1 KO) and Gal- 1 / Gal-3 double-knockout (DKO) mice were intranasally infected with Spl4 and left for 12 h to allow robust pulmonary colonization. At that point, animals received three intratracheal doses (q8h over the next 12 h) of either phosphate-buffered saline (PBS), wildtype Gal-1, or the oxidation-proof all-C^S Gal-1 (Fig. 6a). Twelve hours after treatment, PBS cohorts in both KO backgrounds still harbored heavy bacterial burdens, with dark, hemorrhagic lungs and dense inflammatory infiltrates (Fig. 6b-c). Therapeutic WT Gal-1 significantly reduced CFU and partially normalized lung appearance and histology, but the stabilized Gal-1 produced the most pronounced benefit - driving CFU down by roughly an order of magnitude relative to PBS, with near-normal gross morphology, minimal red bacterial signal by immunofluorescence, and markedly improved alveolar architecture (Fig. 6b-c). Statistical comparisons confirmed that stabilized Gal-1 outperformed WT Gal-1 inboth KO and DKO mice (**•** in CPU panels). We then asked whether added benefit could be detected on top of the intact endogenous lectin system. In wild-type mice, PBS-treated animals maintained substantial bacterial loads and lung injury, WT Gal-1 yielded a modest, non-significant CFU reduction, whereas stabilized Gal-1 achieved a significant drop in CFU together with visibly healthier lungs and cleaner histology (Fig. 6d). Together, these experiments show that therapeutic delivery of galectin-1 is effective after infection is established, and that genetic stabilization (all-C^S) consistently enhances in vivo bactericidal efficacy across lectin-deficient and wild-type hosts.

[0067] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating infection by S. pneumoniae in a subject, comprising administering a therapeutically effective amount of a Gal-1 polypeptide or modified Gal-1 polypeptide to the subject.

2. The method of claim 1, wherein the Gal-1 polypeptide or modified Gal-1 polypeptide is selected from the group consisting of wild type Gal-1, sGal-1, Gal-1 IAM, Gal-1 Nitro, Gal-1 V5D, and Gal-1 V5D IAM.

3. The method of claim 1, wherein the modified Gal-1 polypeptide is sGal-1.

4. The method of any one of claims 1-3, wherein the subject is human.

5. The method of any one of the preceding claims, wherein the subject has been diagnosed as having pneumonia.

6. The method of any one of the preceding claims, wherein the 5. pneumoniae is serotype 14.

7. The method of any one of the preceding claims, wherein the 5. pneumoniae is antibiotic resistant.

8. The method of any one of the preceding claims, wherein the Gal-1 polypeptide or modified Gal- 1 is administered with a pharmaceutically acceptable carrier.

9. A Gal-1 polypeptide that has been modified to have improved stability.

10. The Gal-1 polypeptide of claim 9, wherein the Gal-1 polypeptide is selected from the group consisting of sGal-1, Gal-1 IAM, Gal-1 Nitro, Gal-1 V5D, and Gal-1 V5D IAM.

11. The Gal-1 polypeptide of claim 9, wherein the polypeptide comprises an amino acid sequence having 95% homology with SEQ ID NO: 2.

12. The Gal-1 polypeptide of claim 9, wherein the polypeptide consists of SEQ ID NO: 2.

13. A Gal-1 polypeptide comprising one or more modifications that increase its stability, solubility, or resistance to oxidation, aggregation, or degradation relative to wild-type Gal-1, regardless of the specific modification method employed.

14. The Gal-1 polypeptide of claim 13, wherein the modification is achieved by a method selected from the group consisting of chemical modification, amino-acid substitution, fusion protein formation, cross-linking, polymer conjugation, or domain engineering.

15. The Gal-1 polypeptide of any one of claims 9-14, wherein the polypeptide retains |3- galactoside-binding activity and exhibits enhanced thermal or chemical stability as measured by a differential scanning fluorimetry (DSF) assay or an oxidation-resistance assay relative to wild-type Gal- 1.

16. A pharmaceutical composition comprising the Gal-1 polypeptide of any one of claims 9- 15 and a pharmaceutically acceptable carrier for use in treating infection by . pneumoniae.

17. A method of treating a bacterial infection, comprising administering to a subject in need thereof a therapeutically effective amount of a Gal- 1 variant according to any one of claims 9-16 in combination with at least one additional antibacterial agent selected from antibiotics, antimicrobial peptides, or bacteriophage-derived lysins, wherein the combined administration results in additive or synergistic reduction of bacterial load.

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