Siglec-9 inhibitors and methods of using them to enhance the efficacy of immunotherapy

Abstract

Disclosed herein are compositions comprising Siglec-9 inhibitors, fragments thereof, variants thereof, or combinations thereof, and methods of using them to enhance immune responses. Also disclosed herein are therapeutic compositions comprising a combination of a Siglec-9 inhibitor and an anti-SARS-CoV-2 antibody, or a bispecific anti-Siglec-9 / anti-SARS-CoV-2 antibody, and methods of using them to treat or prevent COVID-19.

Description

[Technical Field]

[0001] Federal Research and Development Statement This invention was made with government support under Grant DK123733 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 477,088, filed December 23, 2022, the entire text of which is incorporated herein by reference. [Background technology]

[0003] Background of the Invention COVID-19, following Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), is a rapidly evolving global public health crisis due to the rise of coronavirus-related diseases that have jumped from animals to humans. There are at least seven coronaviruses that infect humans. Symptoms range from mild flu-like illness to severe, life-threatening pneumonia (Huang et al., 2020, Lancet, 395:497-506). While most people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) experience mild illness, many require hospitalization (Guan WJ, et al., 2020, N Engl J Med 382:1708-1720; Gandhi RT, et al., 2020, N Engl J Med).

[0004] The mechanisms underlying the progression of coronavirus disease 2019 (COVID-19) are likely multifactorial, and immune dysfunction may be one of the underlying mechanisms. Severe COVID-19 has been associated with altered profiles of several immune cells (Schulien I, et al., 2021, Nat Med 27:78-85; Stephenson E, et al., 2021, Nat Med 27:904-916), including natural killer (NK) cells (Leem G, et al., 2021, J Allergy Clin Immunol 148:996-1006 e18; Maucourant C, et al., 2020, Sci Immunol 5; Osman M, et al., 2020, Blood Adv 4:5035-5039). NK cells are innate effector immune cells that play a central role in antiviral immunity through direct cytotoxicity and / or antibody-dependent cellular cytotoxicity (ADCC) (Hammer Q, et al., 2018, Nat Immunol 19:800-808). However, it remains unclear whether severe COVID-19 impairs the anti-SARS-CoV-2 functions of NK cells and what host factors regulate these functions.

[0005] The cytotoxic potential of NK cells is determined by the balance of opposing signals generated by multiple activating receptors (e.g., NKG2C) and inhibitory receptors (e.g., NKG2A) expressed on their surface (Lanier LL. 2005, Annu Rev Immunol 23:225-74; Cerwenka A, et al., 2001, Nat Rev Immunol 1:41-9; Wu J, et al., 2003, Adv Cancer Res 90:127-56). Among the inhibitory receptors, NK cells express Siglec-7 and Siglec-9, two receptors that belong to a new immune checkpoint family called Siglecs (Nicoll G, et al., 1999, J Biol Chem 274:34089-95; Adeniji OS, et al., 2021, PLoS Pathog 17:e1010034). Siglecs are immunoglobulin-like lectins that bind to sialic acid and inhibit immune function by interacting with sialoglycans (sialic acid-containing sugar chain structures) on target cells and signaling through intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (Duan S, et al., 2020, Annu Rev Immunol 38:365-395). In cancer, Siglec-sialoxylcan interactions help tumor cells evade NK immune surveillance (Hudak JE, et al., 2014, Nat Chem Biol 10:69-75; Jandus C, et al., 2014, J Clin Invest 124:1810-20; Laubli H, et al., 2014, Proc Natl Acad Sci USA 111:14211-6). Recently, it has also been suggested that these interactions help HBV- and SARS-CoV-2-infected cells evade NK immune surveillance (Adeniji OS, et al., 2021, PLoS Pathog 17:e1010034; Zhao D, et al., 2018, Front Immunol 9:1124).Despite the growing appreciation of Siglecs as glycoimmune negative checkpoints in cancer, the role of Siglecs in helping SARS-CoV-2 evade immune surveillance has not previously been investigated.

[0006] Thus, there remains a pressing need for new immunotherapeutic tools to treat or prevent serious disease associated with viral infections such as COVID-19. The present invention addresses this unmet need. Summary of the Invention

[0007] In one embodiment, the present invention relates to a therapeutic composition comprising a Siglec-9 inhibitor as an adjuvant for enhancing an immune response to a target antigen. In one embodiment, the Siglec-9 inhibitor comprises an anti-Siglec-9 antibody. In one embodiment, the anti-Siglec-9 antibody comprises a variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. In one embodiment, the anti-Siglec-9 antibody comprises a sequence having at least 95% identity to the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a sequence having at least 95% identity to the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. In one embodiment, the anti-Siglec-9 antibody comprises a fragment comprising at least 80% of the full-length sequence of the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a fragment comprising at least 80% of the full-length sequence of the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40.

[0008] In one embodiment, the therapeutic composition further comprises one or more additional antibodies targeting the antigen. In one embodiment, the antigen is a viral antigen. In one embodiment, the antigen is a SARS-CoV-2 antigen.

[0009] In one embodiment, the therapeutic composition comprises a bispecific antibody comprising an inhibitory Siglec-9 antibody domain and a SARS-CoV-2 antigen binding domain.

[0010] In one embodiment, a therapeutic composition comprises one or more inhibitory nucleic acid molecules specific for binding to Siglec-9 or a fragment thereof. In one embodiment, a therapeutic composition comprises one or more mRNA molecules encoding a Siglec-9 inhibitor. In one embodiment, a therapeutic composition comprises one or more DNA molecules encoding a Siglec-9 inhibitor. In one embodiment, a nucleic acid molecule comprises a nucleotide sequence encoding the variable heavy chain sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26. In one embodiment, a nucleic acid molecule comprises a nucleotide sequence encoding the variable light chain sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28. In one embodiment, a nucleic acid molecule comprises a nucleotide sequence having at least 95% identity to the variable heavy chain sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26. In one embodiment, a nucleic acid molecule comprises a nucleotide sequence having at least 95% identity to the variable light chain sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28. In one embodiment, the nucleic acid molecule comprises a fragment comprising at least 80% of the full-length sequence of the variable heavy chain sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26. In one embodiment, the nucleic acid molecule comprises a fragment comprising at least 80% of the full-length sequence of the variable light chain sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28.

[0011] In one embodiment, the therapeutic composition further comprises one or more additional nucleic acid molecules comprising a nucleotide sequence encoding an antibody or fragment thereof that targets the antigen. In one embodiment, the antigen is a viral antigen. In one embodiment, the antigen is a SARS-CoV-2 antigen.

[0012] In one embodiment, the therapeutic composition comprises a nucleotide sequence encoding a bispecific antibody comprising an inhibitory Siglec-9 antibody domain and a SARS-CoV-2 antigen binding domain.

[0013] In one embodiment, the present invention relates to a method for enhancing the effectiveness of immunotherapy in a subject, comprising administering to the subject a therapeutic composition comprising a Siglec-9 inhibitor as an adjuvant to enhance the immune response to a target antigen in the subject. In one embodiment, the Siglec-9 inhibitor comprises an anti-Siglec-9 antibody. In one embodiment, the anti-Siglec-9 antibody comprises the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. In one embodiment, the anti-Siglec-9 antibody comprises a sequence having at least 95% identity to the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a sequence having at least 95% identity to the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. In one embodiment, the anti-Siglec-9 antibody comprises a fragment comprising at least 80% of the full-length sequence of the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a fragment comprising at least 80% of the full-length sequence of the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40.

[0014] In one embodiment, the method further comprises administering one or more additional antibodies targeting the antigen. In one embodiment, the antigen is a viral antigen. In one embodiment, the antigen is a SARS-CoV-2 antigen.

[0015] In one embodiment, the present invention relates to a method for increasing the level of antibody-dependent cellular cytotoxicity (ADCC) activity against a target antigen, the method comprising administering to a subject a therapeutic composition comprising a Siglec-9 inhibitor as an adjuvant to enhance the immune response to the target antigen. In one embodiment, the Siglec-9 inhibitor comprises an anti-Siglec-9 antibody. In one embodiment, the anti-Siglec-9 antibody comprises the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. In one embodiment, the anti-Siglec-9 antibody comprises a sequence having at least 95% identity to the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a sequence having at least 95% identity to the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. In one embodiment, the anti-Siglec-9 antibody comprises a fragment comprising at least 80% of the full-length sequence of the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a fragment comprising at least 80% of the full-length sequence of the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40.

[0016] In one embodiment, the method further comprises administering one or more additional antibodies targeting the antigen. In one embodiment, the antigen is a viral antigen. In one embodiment, the antigen is a SARS-CoV-2 antigen.

[0017] In one embodiment, the present invention relates to a method for preventing or treating a disease or disorder associated with a viral infection in a subject, the method comprising administering to the subject a therapeutic composition comprising a Siglec-9 inhibitor as an adjuvant to enhance the immune response to a target antigen. In one embodiment, the Siglec-9 inhibitor comprises an anti-Siglec-9 antibody. In one embodiment, the anti-Siglec-9 antibody comprises the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. In one embodiment, the anti-Siglec-9 antibody comprises a sequence having at least 95% identity to the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a sequence having at least 95% identity to the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. In one embodiment, the anti-Siglec-9 antibody comprises a fragment comprising at least 80% of the full-length sequence of the variable heavy chain sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39. In one embodiment, the anti-Siglec-9 antibody comprises a fragment comprising at least 80% of the full-length sequence of the variable light chain sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40.

[0018] In one embodiment, the method further comprises administering one or more additional antibodies targeting the antigen. In one embodiment, the antigen is a viral antigen. In one embodiment, the antigen is a SARS-CoV-2 antigen. In one embodiment, the viral infection is a SARS-CoV-2 infection. [Brief explanation of the drawings]

[0019] The following detailed description of the embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings, It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0020] [Figure 1A] Figures 1A–1M present data demonstrating that hospitalized COVID-19 is associated with decreased degranulation of CD56dimNK cells against SARS-CoV-2 spike-expressing target cells. Figure 1A shows a schematic diagram of the experiments used to assess direct cytotoxicity and ADCC-mediated degranulation of CD56dimNK cells at different severities of COVID-19 infection. To examine direct cytotoxicity-mediated degranulation, PBMCs from each donor in three COVID-19 status groups (n = 8, SARS-CoV-2 negative; n = 12, mild COVID-19; and n = 21, hospitalized COVID-19) were cocultured with SARS-CoV-2 spike-expressing 293T target cells (2) or not (1) (E:T ratio 10:1). To examine ADCC-mediated degranulation, the same cocultures were performed in the presence of negative (3) or positive (4) antibody pools. Direct cytotoxicity was assessed by subtracting (1) from (2) (E:T ratio 10:1). ADCC was assessed by subtracting (1) from (4) from the result obtained by subtracting (1) from (3). [Figures 1B-1I]Figure 1B shows graphs of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dim NK cell population, assessed as the percentage of CD107a+IFN-γ+ cells. Median and interquartile range (IQR) are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 1C shows graphs of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dim NK cell population, assessed as the percentage of CD107a+TNF-α+ cells. Median and interquartile range (IQR) are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 1D shows graphs of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dim NK cell population, assessed as the percentage of IFN-γ+ cells. Median and interquartile range (IQR) are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 1E shows graphs of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dim NK cell population, assessed as the percentage of IFN-γ+TNF-α+ cells. Median and interquartile range (IQR) are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 1F shows graphs of ADCC-mediated degranulation and cytokine production in the CD56dim NK cell population, assessed as the percentage of CD107a+IFN-γ+ cells. Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 1G shows graphs of ADCC-mediated degranulation and cytokine production in the CD56dim NK cell population, assessed as the percentage of CD107a+TNF-α+ cells. Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's correction for multiple comparisons. Figure 1H shows a graph of ADCC-mediated degranulation and cytokine production in the CD56dim NK cell population, assessed as the percentage of IFN-γ+ cells. Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's correction for multiple comparisons.Figure 1I shows a graph of ADCC-mediated degranulation and cytokine production in the CD56dim NK cell population, assessed as the percentage of IFN-γ+TNF-α+ cells. Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. [Figure 1J-1M] Figure 1J shows a regression graph plotting Spearman's rank correlation between plasma N antigen levels and the percentage of CD56dim NK cells expressing CD107a+ in a direct cytotoxicity assay. Only samples from COVID-19-positive donors were used (n=33). Figure 1K shows a regression graph plotting Spearman's rank correlation between plasma N antigen levels and the percentage of CD56dim NK cells expressing IFN-γ+ in a direct cytotoxicity assay. Only samples from COVID-19-positive donors were used (n=33). Figure 1L shows a regression graph plotting Spearman's rank correlation between plasma N antigen levels and the percentage of CD56dim NK cells expressing CD107a+IFN-γ+ in a direct cytotoxicity assay. Only samples from COVID-19-positive donors were used (n=33). Figure 1M shows a regression graph plotting Spearman's rank correlation between plasma N antigen levels and the percentage of CD56dim NK cells expressing IFN-γ+TNF-α+ in a direct cytotoxicity assay. Only samples from COVID-19-positive donors were used (n=33). [Figure 2A] Figure 2 shows data measuring the mean fluorescence intensity of CD107a, IFN-γ, and TNF-α in CD56dimNK cells after coculture with SARS-CoV-2 spike-expressing 293T cells. Figure 2A shows the gating strategy for the experiments shown in Figures 1, 2, 3, 5F-5H, and 6A-6E. Direct cytotoxicity and ADCC were calculated within each COVID-19 status group (n = 8, SARS-CoV-2 negative; n = 12, mild COVID-19; and n = 21, hospitalized COVID-19) as described in Figure 1. [Figures 2B-2G]Figure 2B shows a graph plotting the mean fluorescence intensity (MFI) of CD107a to assess direct cytotoxicity. The median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 2C shows a graph plotting the mean fluorescence intensity (MFI) of IFN-γ to assess direct cytotoxicity. The median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 2D shows a graph plotting the mean fluorescence intensity (MFI) of TNF-α to assess direct cytotoxicity. The median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 2E shows a graph plotting the mean fluorescence intensity (MFI) of CD107a to assess ADCC. The median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 2F shows a graph plotting the mean fluorescence intensity (MFI) of IFN-γ to assess ADCC. The median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 2G shows a graph plotting the mean fluorescence intensity (MFI) of TNF-α to assess ADCC. The median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. [Figure 3A]Figure 3 shows representative data on gender-specific differences in NK cell cytotoxicity against SARS-CoV-2 spike-expressing 293 T cells. The direct cytolytic and ADCC activities of CD56dim NK cells from SARS-CoV-2 negative controls (n = 8; gray dots), mild COVID-19 donors (n = 12; orange dots), and hospitalized COVID-19 donors (n = 21; maroon dots) were compared based on gender (female; n = 18, and male; n = 23). Figure 3A shows a graph assessing direct cytotoxicity, calculated using the method described in Figure 1. Direct cytotoxicity was measured as the percentage of cells expressing CD107a+, IFN-γ+, TNF-α+, CD107a+IFN-γ+, CD107a+TNF-α+, and IFN-γ+TNF-α+, as well as the mean fluorescence intensity (MFI) of CD107a, IFN-γ, and TNF-α. Median and IQR are shown. Statistical analysis was performed using the Mann-Whitney U test. [Figure 3B] Figure 3B shows a graph assessing ADCC, calculated as described in Figure 1. ADCC was measured as the percentage of cells expressing CD107a+, IFN-γ+, TNF-α+, CD107a+IFN-γ+, CD107a+TNF-α+, and IFN-γ+TNF-α+, as well as the mean fluorescence intensity (MFI) of CD107a, IFN-γ, and TNF-α. Median and IQR are shown. Statistical analysis was performed using the Mann-Whitney U test. [Figures 4A-4F]Figures 4A–4F show representative data demonstrating that hospitalized COVID-19 is associated with reduced NK cell dysfunction. Figure 4A shows representative data from an experiment examining ADCC-mediated lysis of SARS-CoV-2 spike-expressing CHO target cells by PBMCs from mild COVID-19 donors (n=8) and hospitalized COVID-19 donors (n=9). CHO-K1 target cells were incubated with a positive or negative SARS-CoV-2 antibody pool for 15 minutes. After 15 minutes, cells were co-cultured at an E:T ratio of 10:1 for 5 hours. SARS-CoV-2 S CHO-K1 cells stably express the SARS-CoV-2 spike (S) protein and the HaloTag-HiBit protein. Upon target cell lysis by ADCC, the intracellular HaloTag-HiBit protein interacts with an extracellular detection reagent, generating a luminescent signal that quantitatively measures the extent of target cell lysis. Target cell lysis values ​​were obtained by subtracting the luminescence values ​​obtained from each negative pool from those obtained from each donor in the positive pool. Median and IQR are shown. Statistical analysis was performed using the Mann-Whitney U test. Figures 4B–4F show representative data from an experiment demonstrating that hospitalized COVID-19 is associated with decreased degranulation of CD56dim NK cells against K562 target cells. PBMCs from three COVID-19 status groups (n = 11, SARS-CoV-2 negative; n = 9, mild COVID-19; and n = 16, hospitalized COVID-19) were cocultured with K562 at an E:T ratio of 5:1. Direct cytotoxicity was assessed by subtracting the degranulation / cytokine production of PBMCs cultured alone from that of PBMCs cocultured with target cells. Figure 4B shows a graph plotting the percentage of CD107a+ cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dimNK cell population against K562 target cells. Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 4C shows a graph plotting the percentage of IFN-γ+ cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dimNK cell population against K562 target cells. Median and IQR are shown.Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 4D shows a graph plotting the percentage of TNF-α+ cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dim NK cell population against K562 target cells. Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 4E shows a graph plotting the percentage of CD107a+IFN-γ+ cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dim NK cell population against K562 target cells. Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 4F shows a graph plotting the percentage of CD107a+TNF-α+ cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production in the CD56dim NK cell population against K562 target cells. Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's correction for multiple comparisons. [Figures 5A-5F]Figures 5A-5I show representative data from experiments demonstrating that the Siglec-9+CD56dim NK cell subpopulation exhibits higher SARS-CoV-2-specific ADCC than the Siglec-9-CD56dim NK cell subpopulation. Figure 5A shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing CD107a. Wilcoxon signed-rank tests were used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5B shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing IFN-γ. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5C shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing TNF-α. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5D shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing CD107a and IFN-γ. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 status group. Figure 5E shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 status group, assessed by the percentage of cells expressing CD107a and TNF-α.The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5F shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, as assessed by the percentage of cells expressing IFN-γ and TNF-α. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5J shows a Spearman correlation heatmap showing the association between direct cytotoxicity of Siglec-9+ and Siglec-9-CD56dim NK cells, ADCC-mediated NK degranulation, and plasma N antigen levels. The color of the squares indicates the strength of correlation, with blue representing a negative correlation and red representing a positive correlation. Only samples from COVID-19 positive donors were used (n=33). [Figure 5G-5J]Figures 5A-5I show representative data from experiments demonstrating that the Siglec-9+CD56dim NK cell subpopulation exhibits higher SARS-CoV-2-specific ADCC than the Siglec-9-CD56dim NK cell subpopulation. Figure 5A shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing CD107a. Wilcoxon signed-rank tests were used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5B shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing IFN-γ. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5C shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing TNF-α. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5D shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing CD107a and IFN-γ. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 status group. Figure 5E shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 status group, assessed by the percentage of cells expressing CD107a and TNF-α.The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5F shows a graph plotting ADCC-mediated degranulation and / or cytokine production of Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group, as assessed by the percentage of cells expressing IFN-γ and TNF-α. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. Figure 5J shows a Spearman correlation heatmap showing the association between direct cytotoxicity of Siglec-9+ and Siglec-9-CD56dim NK cells, ADCC-mediated NK degranulation, and plasma N antigen levels. The color of the squares indicates the strength of correlation, with blue representing a negative correlation and red representing a positive correlation. Only samples from COVID-19 positive donors were used (n=33). [Figures 6A-6F]Figures 6A-6J show representative data from experiments demonstrating that the Siglec-7+CD56dim NK cell subpopulation exhibits higher SARS-CoV-2-specific direct cytolytic and ADCC activity than the Siglec-7-CD56dim NK cell subpopulation. Figures 6A-6C show graphs plotting direct cytolytic degranulation and / or cytokine production of Siglec-7+ and Siglec-7-CD56dim NK cells within each COVID-19 condition group, as assessed by the percentage of cells expressing (A) CD107a, (B) TNF-α, and (C) CD107a and TNF-α. Wilcoxon signed-rank tests were used to compare Siglec-7+CD56dim NK cells with Siglec-7-CD56dim NK cells within each disease group. Figure 6A shows a graph plotting the percentage of cells expressing CD107a. Figure 6B shows a graph plotting the percentage of cells expressing TNF-α. Figure 6C shows a graph plotting the percentage of cells expressing CD107a and TNF-α. Figures 6D-6I show graphs plotting ADCC-mediated degranulation and / or cytokine production of Siglec-7+ and Siglec-7-CD56dim NK cells within each COVID-19 condition group, assessed by the percentage of cells expressing CD107a, IFN-γ, TNF-α, CD107a and IFN-γ, CD107a and TNF-α, or IFN-γ and TNF-α. Wilcoxon signed-rank tests were used to compare Siglec-7+ and Siglec-7-CD56dim NK cells within each disease group. Figure 6D shows the percentage of cells expressing CD107a. Figure 6E shows the percentage of cells expressing IFN-γ. Figure 6F shows the percentage of cells expressing TNF-α. [Figure 6G-6J]Figure 6G shows the percentage of cells expressing CD107a and IFN-γ. Figure 6H shows the percentage of cells expressing CD107a and TNF-α. Figure 6I shows the percentage of cells expressing IFN-γ and TNF-α. Figure 6J shows a Spearman correlation heatmap showing the association between direct cytotoxicity and ADCC-mediated NK degranulation of Siglec-7+ and Siglec-7-CD56dim NK cells and plasma N antigen levels. The color of the squares indicates the strength of correlation, with blue indicating a negative correlation and red indicating a positive correlation. Only samples from COVID-19-positive donors were used (n=33). [Figure 7A-7C] Figures 7A-7C show representative data from experiments demonstrating that the Siglec-7+CD56dim NK cell subpopulation exhibits higher SARS-CoV-2-specific ADCC activity than the Siglec-7-CD56dim NK cell subpopulation. ADCC-mediated degranulation of Siglec-7+ and Siglec-7-CD56dim NK cells within each COVID-19 status group (n = 8, SARS-CoV-2 negative; n = 12, mild COVID-19; and n = 21, hospitalized COVID-19) was also assessed by mean fluorescence intensity (MFI). Figure 7A shows a graph plotting the MFI of CD107a. Siglec-9+ and Siglec-9-CD56dim NK cells within each disease group were compared using the Wilcoxon signed-rank test. Figure 7B shows a graph plotting the MFI of IFN-γ. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each disease group. Figure 7C shows a graph plotting the MFI of TNF-α. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each disease group. [Figure 8] FIG. 8 shows the gating strategy for the experiments in FIG. 12 and FIGS. 13A-13E. [Figure 8-1] FIG. 8 shows the gating strategy for the experiments in FIG. 12 and FIGS. 13A-13E. [Figure 9A-9B]Figures 9A-9B show representative data from an experiment demonstrating that a Siglec-9 antibody marks the Siglec-9+ population with high expression of Siglec-9 mRNA transcripts. To verify the specificity of the Siglec-9 antibody (K8 clone) in identifying Siglec-9+ cells, primary NK cells from three healthy individuals were sorted based on Siglec-9 expression into cells with no, low, or high Siglec-9 expression. qPCR was then used to measure the relative copy number of Siglec-9 transcripts in the sorted populations. Figure 9A shows the gating strategy for the sorting experiment. Figure 9B shows a graph plotting the relative copy number of Siglec-9 transcripts measured by qPCR and normalized using a eukaryotic 18S rRNA endogenous control. Relative copy numbers were determined using the comparative Ct method. Mean values ​​and standard error of the mean (SEM) are shown. Paired t-tests were used for statistical analysis. [Figures 10A-10F]Figures 10A–10F show representative data from an experiment evaluating the gender-dependent expression of Siglec-7 and Siglec-9 on CD56dim NK cells. To examine the potential influence of gender on Siglec-9 and Siglec-7 expression on CD56dim NK cells, 79 individuals with three COVID-19 pathologies (negative, n=12 (gray dots); mild, n=26 (orange dots); hospitalized, n=41 (maroon dots)) were divided by gender (female, n=32; male, n=47). Figure 10A shows a graph plotting the percentage of Siglec-9+CD56dim NK cells. Median and IQR are displayed. Group comparisons were performed using the Mann-Whitney U test. Figure 10B shows a graph plotting the percentage of Siglec-7+CD56dim NK cells. Median and IQR are displayed. Group comparisons were performed using the Mann-Whitney U test. Figure 10C shows a graph plotting the percentage of Siglec-9-Siglec-7-CD56dim NK cells. Median and IQR are shown. Groups were compared using the Mann-Whitney U test. Figure 10D shows a graph plotting the percentage of Siglec-9+Siglec-7-CD56dim NK cells. Median and IQR are shown. Groups were compared using the Mann-Whitney U test. Figure 10E shows a graph plotting the percentage of Siglec-9-Siglec-7+CD56dim NK cells. Median and IQR are shown. Groups were compared using the Mann-Whitney U test. Figure 10F shows a graph plotting the percentage of Siglec-9+Siglec-7+CD56dim NK cells. Median and IQR are shown. Groups were compared using the Mann-Whitney U test. [Figures 11A-11C]Figures 11A-11C show representative data from experiments demonstrating that hospitalized COVID-19 is associated with decreased CD16 and Siglec-7 expression and increased CD57 expression on CD56dimNK cells. Figure 11A shows a graph plotting CD16 expression on CD56dimNK cells from n = 79 patients (n = 12, SARS-CoV-2 negative; n = 26, mild COVID-19; and n = 41, hospitalized COVID-19). Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 11B shows a graph plotting CD57 expression on CD56dimNK cells from n = 79 patients (n = 12, SARS-CoV-2 negative; n = 26, mild COVID-19; and n = 41, hospitalized COVID-19). Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. Figure 11C shows a graph plotting Siglec-7 expression on CD56dim NK cells from n = 79 patients (n = 12, SARS-CoV-2 negative; n = 26, mild COVID-19; and n = 41, hospitalized COVID-19). Median and IQR are shown. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparison correction. [Figures 12A-12B]Figures 12A-12B show representative data from experiments demonstrating that Siglec-9 marks activated and mature CD56dim NK cell populations in vivo. Figure 12A shows a graph plotting the comparative expression of CD16 (FcγRIII; a mediator of ADCC), CD57 (a maturation marker), NKG2C (an activating receptor), and NKG2A (an inhibitory receptor) on Siglec-9+ and Siglec-9-CD56dim NK cells obtained from 79 individuals with three COVID-19 pathologies (negative, n=12; mild, n=26; and hospitalized, n=41). Siglec-9+ cells exhibit higher levels of CD16, CD57, and NKG2C, and lower levels of NKG2A, compared with Siglec-9- cells. The Wilcoxon signed-rank test was used to compare Siglec-9+ and Siglec-9-CD56dim NK cells within each COVID-19 condition group. The Mann-Whitney U test was used to compare condition groups. Figure 12B shows a graph plotting the expression of CD16, CD57, NKG2C, and NKG2A on Siglec-7+ and Siglec-7-CD56dim NK cells. Siglec-7+ cells exhibit higher levels of CD16, NKG2C, and NKG2A than Siglec-7- cells. No difference in CD57 expression was observed between Siglec-7+ and Siglec-7- cells. As in panel A, the Wilcoxon signed-rank test was used to compare Siglec-7+ and Siglec-7-CD56dim NK cells within each COVID-19 condition group, and the Mann-Whitney U test was used to compare condition groups. [Figures 13A-13D]Figures 13A-H show representative data from experiments demonstrating that Siglec-9, but not Siglec-7, marks CD56dimNK cells with enhanced ADCC activity against SARS-CoV-2. Figure 13A shows a graph plotting the percentage of each Siglec-expressing cell subpopulation (Siglec-7-Siglec-9-, Siglec-7+Siglec-9-, Siglec-7-Siglec-9+, or Siglec-7+Siglec-9+) within CD56dimNK cells across all donors (n=79). Figure 13B shows a graph plotting the in vivo expression of CD16 in the indicated cell subpopulations (n=79). Medians and IQRs are displayed. Statistical analysis was performed using the Friedman test with Dunn's multiple comparisons correction. Figure 13C shows a graph plotting the in vivo expression of CD57 in the indicated cell subpopulations (n=79). Medians and IQRs are displayed. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison correction. Figure 13D shows a graph plotting in vivo expression of NKG2C in the indicated cell subpopulations (n=79). Median and IQR are displayed. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison correction. [Figures 13E-13H]Figure 13E shows a graph plotting in vivo expression of NKG2A in the indicated cell subpopulations (n ​​= 79). Medians and IQRs are displayed. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison correction. Figure 13F shows a graph plotting ADCC-mediated degranulation in the indicated NK cell subpopulations (n ​​= 41), assessed by the percentage of cells expressing CD107a. Medians and IQRs are displayed. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison correction. Figure 13G shows a graph plotting ADCC-mediated degranulation in the indicated NK cell subpopulations (n ​​= 41), assessed by MFI of CD107a. Medians and IQRs are displayed. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison correction. Figure 13H shows a graph plotting ADCC-mediated degranulation of the indicated NK cell subpopulations (n=41), assessed by the percentage of cells expressing CD107a and IFN-γ. Median and IQR are shown. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison correction. [Figures 14A-14F]Figures 14A-14F show representative data from an experiment demonstrating that blocking Siglec-9 interactions using a Siglec-9 blocking antibody enhances anti-SARS-CoV-2 ADCC of CD56dim NK cells. Figures 14A-14E show representative data from an experiment evaluating the effect of a Siglec-9 blocking antibody on ADCC-mediated degranulation and / or cytokine production against SARS-CoV-2 compared to an isotype control. PBMCs from six healthy controls were used as effector cells, and SARS-CoV-2 spike-expressing 293T cells were used as target cells. Cells were cocultured at an E:T ratio of 10:1 for 12 hours. Figure 14A shows a graph plotting NK degranulation and / or cytokine production, assessed by the percentage of cells expressing CD107a. Statistical analysis was performed using a paired t-test. Figure 14B shows a graph plotting NK degranulation and / or cytokine production as assessed by the percentage of cells expressing CD107a and IFN-γ. Paired t-tests were used for statistical analysis. Figure 14C shows a graph plotting NK degranulation and / or cytokine production as assessed by the percentage of cells expressing CD107a and TNF-α. Paired t-tests were used for statistical analysis. Figure 14D shows a graph plotting NK degranulation and / or cytokine production as assessed by the percentage of cells expressing TNF-α. Paired t-tests were used for statistical analysis. Figure 14E shows a graph plotting NK degranulation and / or cytokine production as assessed by the percentage of cells expressing IFN-γ and TNF-α. Paired t-tests were used for statistical analysis. Figure 14F shows a graph plotting representative data from an experiment evaluating the effect of Siglec-9 blocking antibodies compared to isotype controls on ADCC-mediated lysis of SARS-CoV-2 target cells. Purified NK cells isolated from PBMCs from five healthy donors were used as effector cells, and SARS-CoV-2 S CHO-K1 cells were used as target cells. The cells were co-cultured at an E:T ratio of 5:1 for 5 hours. SARS-CoV-2 S CHO-K1 cells stably express the SARS-CoV-2 spike (S) protein and HaloTag-HiBit protein.Upon target cell lysis by ADCC, the intracellular HaloTag-HiBit protein interacts with the extracellular detection reagent to generate a luminescent signal that can quantitatively measure target cell lysis. Statistical analysis was performed using a paired t-test. [Figure 15] Figure 15 shows a model demonstrating how Siglec-9 blocking antibodies enhance the cytotoxicity of Siglec-9+ NK cells. Left panel: Siglec-9- cells have low cytotoxicity. Middle panel: The Siglec-9+CD56dim NK cell subset has high ADCC activity, likely due to its higher expression of CD16 (FcγRIII; a mediator of ADCC activity), CD57 (a maturation marker), and NKG2C (an activating receptor), but lower expression of the inhibitory receptor NKG2A, compared with Siglec-9-CD56dim NK cells. However, the Siglec-9 molecule itself is an inhibitory receptor, which binds to sialic acid on the surface of target cells and suppresses the cytolytic activity of these highly cytotoxic Siglec-9+CD56dim NK cells. Right panel: Blocking the inhibitory receptor Siglec-9 using a blocking antibody can further enhance the ADCC activity of the Siglec-9+CD56dim subpopulation. [Figure 16] Figure 16 shows representative data from an experiment characterizing Siglec-9 blocking antibodies. Binding of recombinantly expressed anti-Siglec-9 antibodies at different dilutions to 1) recombinant Siglec-9 protein, 2) recombinant Siglec-7 protein (as a negative control), and 3) HIV-1-gp120 protein (as a negative control) was measured by ELISA. Each point represents the OD value (mean ± SEM).

[0021] Detailed Description In some embodiments, the present invention comprises a Siglec-9 inhibitor for use in enhancing the cytotoxic and antibody-dependent cellular cytotoxicity (ADCC) activity of natural killer (NK) cells in response to the presence of a target antigen. In some embodiments, the present invention relates to a composition comprising at least one anti-Siglec-9 antibody comprising a domain specific for binding to Siglec-9.

[0022] In some embodiments, the Siglec-9 inhibitors of the present invention are administered in combination with one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is a therapeutic antibody. In some embodiments, the Siglec-9 inhibitors of the present invention function as adjuvants that enhance NK cell cytotoxicity against the antigen targeted by the therapeutic antibody.

[0023] In one embodiment, the therapeutic antibody targets a viral antigen. In one embodiment, the viral antigen is a SARS-CoV-2 antigen. The antigen may be a SARS-CoV-2 viral antigen, or a fragment thereof, or a variant thereof. The SARS-CoV-2 antigen may be derived from factors that enable viral replication, infection, or survival. In some embodiments, the SARS-CoV-2 antigen may be a spike antigen or a fragment thereof.

[0024] In one embodiment, the present invention provides a composition comprising one or more bispecific antibodies comprising an anti-Siglec-9 domain for inhibiting Siglec-9 activity in NK cells and a target antigen-binding domain specific for binding to the target antigen. In one embodiment, the target antigen is a viral antigen. In one embodiment, the viral antigen is a SARS-CoV-2 antigen. The antigen can be a SARS-CoV-2 viral antigen, or a fragment thereof, or a variant thereof. The SARS-CoV-2 antigen can be derived from factors that enable viral replication, infection, or survival. In some embodiments, the SARS-CoV-2 antigen can be a spike antigen or a fragment thereof.

[0025] The present invention also relates, in part, to methods of enhancing natural killer (NK) cell cytolytic and antibody-dependent cellular cytotoxicity (ADCC) activity in response to the presence of a target antigen in a subject by administering a Siglec-9 inhibitor of the present invention. In some embodiments, the present invention relates to methods of treating or preventing a disease or disorder in a subject by administering a Siglec-9 inhibitor of the present invention.

[0026] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control. Preferred methods and materials are described below; however, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0027] As used herein, the terms "comprise," "include," "having," "has," "can," and "contain," and variations thereof, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0028] "Antibody" may refer to antibodies of the IgG, IgM, IgA, IgD, or IgE class, or fragments, or fragments or derivatives thereof (including Fab, F(ab')2, Fd, and single chain antibodies, and derivatives thereof). The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof that exhibits sufficient binding specificity for a desired epitope or sequences derived therefrom.

[0029] As used interchangeably herein, the terms "antibody fragment" and "antibody fragment" refer to a portion of an intact antibody that contains the antigen-binding site or variable region. This portion does not include the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides comprising only a light chain variable domain, a single-chain polypeptide comprising three CDRs of a light chain variable domain, a single-chain polypeptide comprising only a heavy chain variable region, and a single-chain polypeptide comprising three CDRs of a heavy chain variable region.

[0030] "Antigen" refers to a protein that has the ability to produce an immune response in a host. An antigen is capable of being recognized by and bound to an antibody. Antigens can originate from within the body or from the external environment.

[0031] As used herein, a "coding sequence" or "encoding nucleic acid" may refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding an antibody described herein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further comprise a sequence encoding a signal peptide.

[0032] As used herein, "complement" or "complementary" can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0033] As used herein, "endogenous antibodies" may refer to antibodies produced in a subject administered an effective amount of an antigen to induce a humoral immune response.

[0034] "Fragment" may refer to a polypeptide fragment of an antibody that is functional, i.e., capable of binding to a desired target and having the same intended effect as the full-length antibody. An antibody fragment is 100% identical to the full-length antibody except for the deletion of at least one amino acid from the N-terminus and / or C-terminus, in either case with or without a signal peptide and / or a methionine at position 1. A fragment may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of a particular full-length antibody, excluding any added heterologous signal peptide. Fragments include fragments of polypeptides having 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to an antibody and may further include an N-terminal methionine or a heterologous signal peptide, which are not included in the percent identity calculation. Fragments may further include an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide (e.g., an IgE or IgG signal peptide). The N-terminal methionine and / or signal peptide may be linked to the antibody fragment.

[0035] A fragment of a nucleic acid sequence encoding an antibody is 100% identical to the full length, except for the deletion of at least one nucleotide from the 5' and / or 3' end, in each case with or without the sequence encoding the signal peptide and / or methionine at position 1. A fragment can comprise 20% or more, 25% or more, 30% or more, 35%, or more, 40% or more, 45%, or more, 50% or more, 55%, or more, 60% or more, 65%, or more, 70%, or more, 75%, or more, 80%, or more, 85%, or more, 90%, or more, 91%, or more, 92%, or more, 93%, or more, 94%, or more, 95%, or more, 96%, or more, 97%, or more, 98%, or more, 99% or more of the length of the specified full-length coding sequence, excluding any added heterologous signal peptide. Fragments can include fragments that encode polypeptides having 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the antibody, and can optionally include sequences encoding an N-terminal methionine or a heterologous signal peptide that are not included in the percent identity calculation. Fragments can further include coding sequences for an N-terminal methionine and / or signal peptide (e.g., an immunoglobulin signal peptide, such as an IgE or IgG signal peptide). The coding sequence encoding the N-terminal methionine and / or signal peptide can be linked to a fragment of the coding sequence.

[0036] As used herein, "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein, such as an antibody. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, that are capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that contains the necessary regulatory elements operably linked to a coding sequence that encodes a protein, such that the coding sequence is expressed when present in the cells of an individual.

[0037] As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences can mean that the sequences have a specified percentage of the same residues over a specified region. This percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to determine the percentage of sequence identity. If the two sequences are of different lengths or if the alignment produces one or more kinked ends and only a single sequence is included in the designated comparison region, the residues of the single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity can be determined manually or using computer sequence algorithms such as BLAST and BLAST 2.0.

[0038] As used herein, "impedance" is used when discussing feedback mechanisms and can be converted to a current value according to Ohm's law, allowing comparison to a set current.

[0039] As used herein, "immune response" refers to activation of a host's immune system (e.g., a mammalian immune system) in response to the introduction of one or more nucleic acids and / or peptides. The immune response can take the form of a cellular response, a humoral response, or both.

[0040] As used herein, "nucleic acid," "oligonucleotide," or "polynucleotide" can refer to at least two nucleotides covalently linked together. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also includes the complementary strand of the depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a specific nucleic acid. Thus, a nucleic acid also includes a substantially identical nucleic acid and its complementary strand. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

[0041] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA (both genomic and cDNA), RNA, or hybrids, where the nucleic acid may contain a combination of deoxyribonucleotides and ribonucleotides, and may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis or recombinant methods.

[0042] As used herein, "operably linked" may mean that the expression of a gene is under the control of a promoter to which the gene is spatially linked. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without impairing the function of the promoter.

[0043] As used herein, "peptide," "protein," or "polypeptide" refers to a sequence of linked amino acids, which may be natural, synthetic, or a modified or combination of natural and synthetic.

[0044] As used herein, a "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter the spatial and / or temporal expression of a nucleic acid. A promoter can also contain distal enhancer or repressor elements located as far away as thousands of base pairs from the transcription start site. Promoters can be derived from sources such as viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of genetic components constitutively or differentially depending on the cell, tissue, or organ in which expression occurs, the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMVIE promoter, an SV40 early promoter or an SV40 late promoter, and a CMVIE promoter.

[0045] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that may be linked at the amino terminus of a protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. As used herein, a signal peptide / leader sequence preferably facilitates secretion of a protein from the cell in which it is produced. Upon secretion from the cell, the signal peptide / leader sequence is cleaved from the remainder of the protein, often referred to as the mature protein. The signal peptide / leader sequence is attached at the N-terminus of the protein.

[0046] As used herein, "stringent hybridization conditions" can refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will vary under different circumstances. Stringent conditions can be selected to be approximately 5-10°C lower than the melting temperature (Tm) of a particular sequence at a defined ionic strength and pH. The Tm can be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, 50% of the probes are occupied at Tm). Stringent conditions are those with a pH of 7.0 to 8.3, a salt concentration of less than about 1.0 M sodium ion, e.g., about 0.01 to 1.0 M sodium ion (or other salt), and a temperature of at least about 30°C for short probes (e.g., about 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times higher than background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5x SSC, and 1% SDS incubation at 42°C; or 5x SSC, 1% SDS incubation at 65°C followed by a wash in 0.2x SSC and 0.1% SDS at 65°C.

[0047] As used herein, "subject" and "patient" refer interchangeably to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, non-human primates (e.g., monkeys such as cynomolgus monkeys, rhesus monkeys, chimpanzees, and humans)). In some embodiments, the subject can be human or non-human. The subject or patient may also be undergoing other forms of therapy.

[0048] As used herein, "substantially complementary" means that a first sequence is substantially complementary to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides or amino acids. It can mean that a sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of the sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0049] As used herein, "substantially identical" means that the first and second sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, For nucleic acids, "at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of 10 or more nucleotides or amino acids, or where a first sequence is substantially complementary to the complement of a second sequence.

[0050] As used herein, "synthetic antibody" refers to an antibody encoded by a recombinant nucleic acid sequence described herein and produced in a subject.

[0051] As used herein, "treatment" or "treating" means protecting a subject from disease by means of preventing, suppressing, quelling, or completely eliminating the disease. Preventing disease involves administering an antibody of the invention to a subject before the onset of disease. Suppressing disease involves administering an antibody of the invention to a subject after the induction of disease but before clinical onset. Suppressing disease involves administering an antibody of the invention to a subject after clinical onset of disease.

[0052] As used herein, "variant" with respect to a nucleic acid means (i) a portion or fragment of a reference nucleotide sequence, (ii) the complement of a reference nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0053] With respect to a peptide or polypeptide, a "variant" refers to a protein that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. A variant can also refer to a protein having substantially the same amino acid sequence as a reference protein that retains at least one biological activity. Conservative amino acid substitutions, i.e., replacing one amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes. These minor changes can be identified, in part, by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is determined by considering its hydrophobicity and charge. It is known in the art that substitutions of amino acids with similar hydropathic indexes can maintain protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that will maintain the biological function of a protein. Considering the hydrophilicity of amino acids in a peptide, the maximum local average hydrophilicity of the peptide can be calculated, a useful index that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 (incorporated herein by reference in its entirety) Substitution of amino acids with similar hydrophilicity values ​​may result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitutions can be made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0054] A variant may be a nucleic acid sequence that is substantially identical over the entire length of the complete gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the gene sequence or fragment thereof. A variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or fragment thereof. The amino acid sequences can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence or a fragment thereof.

[0055] As used herein, the term "vector" may refer to a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA vector or an RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector that integrates into a host genome.

[0056] In the description of numerical ranges herein, each intervening value is expressly contemplated with the same degree of precision, e.g., for the range of 6 to 9, 7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0057] composition In one embodiment, the present invention relates to a composition comprising an inhibitor of the Siglec-9 signaling pathway for use in enhancing immune responses and improving the efficacy of immunotherapy. In some embodiments, the immunotherapy is SARS-CoV-2 immunotherapy.

[0058] In various embodiments, the present invention provides compositions and methods for inhibiting Siglec-9 to enhance the cytolytic and antibody-dependent cellular cytotoxicity (ADCC) activity of natural killer cells in response to the presence of a target antigen (e.g., a SARS-CoV-2 antigen).

[0059] In various embodiments, the present invention includes compositions and methods for treating a viral infection in a subject. In various embodiments, a composition for treating a viral infection includes an inhibitor of Siglec-9. In one embodiment, the inhibitor of the present invention reduces the amount of Siglec-9 polypeptide, the amount of Siglec-9 mRNA, the amount of Siglec-9 activity, or a combination thereof.

[0060] Based on the disclosure herein, those skilled in the art will understand that a reduction in Siglec-9 levels includes a reduction in expression, including transcription, translation, or both. After comprehending the teachings of the present invention, those skilled in the art will understand that a reduction in Siglec-9 levels also includes a reduction in Siglec-9 activity. Thus, a reduction in Siglec-9 levels or activity includes, but is not limited to, a reduction in the amount of Siglec-9 polypeptide, and a reduction in transcription, translation, or both, of a nucleic acid encoding Siglec-9, and also includes a reduction in Siglec-9 activity.

[0061] In one embodiment, the present invention provides a comprehensive concept for inhibiting Siglec-9 as an antitumor therapy. In one embodiment, the composition of the present invention comprises an inhibitor of Siglec-9. In one embodiment, the inhibitor is selected from the group consisting of small interfering RNA (siRNA), microRNA, antisense nucleic acid, ribozyme, expression vector encoding a transdominant negative mutant, intracellular antibody, peptide, and small molecule.

[0062] Based on the disclosure herein, those skilled in the art will understand that one way to reduce Siglec-9 mRNA and / or protein levels in a cell is to reduce or inhibit expression of a nucleic acid encoding Siglec-9. Thus, Siglec-9 protein levels in a cell can also be reduced using molecules or compounds that inhibit or reduce gene expression, such as, for example, siRNAs, antisense molecules, or ribozymes. However, the present invention is not limited to these examples.

[0063] In one embodiment, siRNA is used to reduce the level of Siglec-9. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a variety of organisms and cell types results in the degradation of complementary mRNA. Within the cell, long dsRNA is cleaved by a ribonuclease known as Dicer into short, 21-25 nucleotide small interfering RNAs (i.e., siRNAs). The siRNAs then assemble with protein components to form the RNA-induced silencing complex (RISC), unwinding in the process. The activated RISC then binds to complementary transcripts through base-pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, resulting in gene silencing through sequence-specific degradation of the mRNA. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); Gregory J. Hannon, ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe chemical modifications of siRNA to facilitate intravenous systemic delivery. Optimization of siRNAs takes into account the overall G / C content, terminal C / T content, Tm value, and nucleotide content of the 3' overhangs (see, e.g., Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216).Thus, the present invention also includes methods for reducing the level of Siglec-9 at the protein level using RNAi technology.

[0064] In other related aspects, the present invention includes isolated nucleic acids encoding inhibitors, such as siRNAs or antisense molecules, that inhibit Siglec-9, its derivatives, its regulators, or downstream effectors, operably linked to a nucleic acid comprising a promoter / regulatory sequence, preferably capable of directing expression of the protein encoded by the nucleic acid. Accordingly, the present invention encompasses expression vectors and methods for introducing foreign DNA into cells and simultaneously expressing the foreign DNA within the cells, such as those described in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and elsewhere herein. In another aspect of the present invention, Siglec-9 or its regulators can be inhibited by inactivating and / or sequestering one or more of Siglec-9 or its regulators. Accordingly, inhibition of Siglec-9 action can be achieved using transdominant-negative mutants.

[0065] In another aspect, the present invention includes a vector comprising an siRNA or antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of Siglec-9. Incorporation of a desired polynucleotide into a vector and selection of the vector are known in the art, for example, as described in Sambrook et al. (supra).

[0066] The siRNA or antisense polynucleotide can be cloned into various types of vectors, as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis.

[0067] To evaluate the expression of siRNA or antisense polynucleotides, the expression vector introduced into cells can also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a cell population targeted for transfection or infection with a viral vector. In other embodiments, the selection marker can be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selection marker gene and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selection markers are known in the art, and include, for example, antibiotic resistance genes such as neomycin resistance.

[0068] In one embodiment of the invention, Siglec-9 is inhibited using an antisense nucleic acid sequence expressed by a plasmid vector. The antisense expression vector is used to transfect mammalian cells or the mammal itself, thereby reducing the endogenous expression of Siglec-9.

[0069] Antisense molecules and their use in inhibiting gene expression are known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids, as defined elsewhere herein, are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize with the corresponding mRNA to form a double-stranded molecule, thereby inhibiting gene translation.

[0070] The use of antisense methods to inhibit gene translation is known in the art and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules can be provided to cells through gene expression using DNA encoding the antisense molecule, as taught in Inoue, 1993, U.S. Patent No. 5,190,931.

[0071] Alternatively, antisense molecules of the present invention can be synthetically produced and then delivered to cells. Antisense oligomers of about 10 to about 30 nucleotides, and more preferably about 15 nucleotides, are preferred because they can be easily synthesized and introduced into target cells. Synthetic antisense molecules contemplated by the present invention include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (see U.S. Pat. No. 5,023,243).

[0072] Compositions and methods for synthesizing and expressing antisense nucleic acids are described elsewhere herein.

[0073] Ribozymes and their use for inhibiting gene expression are also known in the art (see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933; Eckstein et al., WO 92 / 07065; Altman et al., U.S. Pat. No. 5,168,053). Ribozymes are RNA molecules that have the ability to specifically cleave other single-stranded RNA in a manner similar to DNA restriction enzymes. By modifying the nucleotide sequences encoding these RNAs, molecules can be engineered to recognize and cleave specific nucleotide sequences within RNA molecules (Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of this approach is that ribozymes are sequence-specific.

[0074] There are two basic types of ribozymes: Tetrahymena-type (Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-type ribozymes recognize sequences four bases long, while hammerhead-type ribozymes recognize sequences 11–18 bases long. The longer the sequence, the greater the likelihood that the sequence will be present exclusively in the target mRNA species. Therefore, for inactivating a specific mRNA species, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes, and 18-base recognition sequences are preferable to shorter recognition sequences that may occur randomly within various unrelated mRNA molecules.

[0075] In one embodiment of the present invention, ribozymes are used to inhibit Siglec-9. Ribozymes useful for inhibiting expression of target molecules can be designed by incorporating a target sequence complementary to the mRNA sequence of, for example, Siglec-9 of the present invention into the basic ribozyme structure. Ribozymes targeting Siglec-9 can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or can be genetically expressed from DNA encoding them.

[0076] When the inhibitors of the present invention are small molecules, small molecule antagonists can be obtained using standard methods known to those skilled in the art, including chemical and organic synthesis or biological means, including purification from biological sources, recombinant synthesis, and in vitro translation systems, using methods known in the art.

[0077] Combinatorial libraries of molecularly diverse compounds potentially useful in the treatment of various diseases and conditions are known in the art, as are methods for generating the libraries, which can use a variety of techniques known to those skilled in the art, including solid-phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear arrays, deconvolution strategies, tagging techniques, and the generation of unbiased molecular landscapes for lead discovery and biased structures for lead development.

[0078] A common approach to small library synthesis involves condensing an activated core molecule with a large number of building blocks, resulting in a combinatorial library of covalently linked core-building block assemblies. The shape and rigidity of the core determine the orientation of the building blocks in shape space. Libraries can be biased toward characterized biological structures by varying the core, linkages, or building blocks ("focused libraries"), or can be synthesized with reduced structural bias using flexible cores.

[0079] In another embodiment of the present invention, Siglec-9 can be inhibited by inactivating and / or sequestering Siglec-9. Thus, inhibition of the action of Siglec-9 can be achieved by using a transdominant-negative mutant. Alternatively, an antibody specific for Siglec-9 (e.g., a Siglec-9 antagonist) may be used. In one embodiment, the antagonist is a protein and / or compound with the desirable property of interacting with a binding partner of Siglec-9, thereby competing with the corresponding protein. In another embodiment, the antagonist is a protein and / or compound with the desirable property of interacting with Siglec-9, thereby sequestering Siglec-9.

[0080] As will be appreciated by those skilled in the art, any antibody capable of recognizing and binding to an antigen of interest is useful in the present invention. Methods for producing and using antibodies are known in the art. For example, polyclonal antibodies useful in the present invention can be generated by immunizing rabbits according to standard immunological techniques known in the art (see, e.g., Harlow et al., 1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY). Such techniques include immunizing animals with chimeric proteins containing a portion of another protein, such as a maltose-binding protein or glutathione (GSH) tag polypeptide portion, and / or a portion that renders the antigen protein of interest immunogenic (e.g., the antigen of interest conjugated to keyhole limpet hemocyanin (KLH)), and a portion containing amino acid residues from the respective antigen protein. Chimeric proteins can be generated by cloning an appropriate nucleic acid encoding a marker protein into a plasmid vector suitable for this purpose (such as, but not limited to, pMAL-2 or pCMX).

[0081] However, the present invention should not be construed as being limited solely to methods and compositions comprising these antibodies or to these portions of antigens. Rather, the present invention should be construed as including other antibodies directed against antigens or portions thereof, as that term is defined elsewhere herein. Furthermore, the present invention should be construed as encompassing, inter alia, antibodies that bind to a particular antigen of interest, and that are capable of binding to the antigen present in, for example, Western blots, in solution in enzyme-linked immunosorbent assays, fluorescence-activated cell sorting (FACS), magnetic affinity cell sorting (MACS), and immunofluorescence microscopy of cells transiently transfected with nucleic acid encoding at least a portion of the antigen protein.

[0082] Those skilled in the art will understand, based on the disclosure provided herein, that an antibody can specifically bind to any portion of an antigen and that the full-length protein can be used to generate antibodies specific to that portion. However, the present invention is not limited to using the full-length protein as an immunogen. Rather, the present invention includes using an immunogenic portion of a protein to generate antibodies that specifically bind to a particular antigen. That is, the present invention includes using an immunogenic portion or antigenic determinant of an antigen to immunize an animal.

[0083] Siglec-9 antibody In various embodiments, the inhibitor of Siglec-9 comprises an antibody, fragment thereof, or variant thereof specific for binding to Siglec-9. As used herein, the term "antibody" or "immunoglobulin" refers to a protein (including glycoproteins) belonging to the immunoglobulin (Ig) superfamily of proteins. An antibody or immunoglobulin (Ig) molecule may be a tetramer comprising two identical light chain polypeptides and two identical heavy chain polypeptides. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Each full-length Ig molecule contains at least two binding sites for a specific target or antigen.

[0084] Anti-Siglec-9 antibodies, or antigen-binding fragments thereof, include, but are not limited to, polyclonal antibodies, monoclonal fusion proteins, antibodies or fragments thereof, chimerized or chimeric fusion proteins, antibodies or fragments thereof, humanized fusion proteins, antibodies or fragments thereof, deimmunized human fusion proteins, antibodies or fragments thereof, fully human fusion proteins, antibodies or fragments thereof, single-chain antibodies, single-chain Fv fragments (scFv), Fv, Fd fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, diabodies or antigen-binding fragments thereof, minibodies or antigen-binding fragments thereof, triabodies or antigen-binding fragments thereof, domain fusion proteins, antibodies or fragments thereof, camelid fusion proteins, antibodies or fragments thereof, dromedary fusion proteins, antibodies or fragments thereof, phage-displayed fusion proteins, antibodies or fragments thereof, or antibodies or antigen-binding fragments thereof identified in a repetitive backbone array (e.g., repetitive antigen presentation).

[0085] The immune system produces several different classes of Ig molecules (isotypes), including IgA, IgD, IgE, IgG, and IgM, each distinguished by the class of heavy chain polypeptide present: alpha (α) in IgA, delta (δ) in IgD, epsilon (ε) in IgE, gamma (γ) in IgG, and μ (μ) in IgM. There are at least five different gamma heavy chain polypeptide isotypes for IgG. In contrast, there are only two light chain polypeptide isotypes, called kappa (κ) and lambda (λ). The characteristics of an antibody isotype are defined by the sequence of the constant domain of the heavy chain.

[0086] IgG molecules consist of two light chains (κ or λ) and two heavy chains (γ) linked by disulfide bonds. The κ and λ IgG light chains each have a variable region (V L- Area”, “V κ- Area”, “V λ- The amino acid sequence domains with relatively high variability are called "constant regions" (C L-Similarly, each IgG heavy chain contains a variable region (V H- A complete IgG heavy chain contains three constant domains (CH 1- Area”, “CH 2- Area”, “CH 3- Each V L- Area or V H- Within a region, hypervariable regions, also known as complementarity-determining regions ("CDRs"), are interspersed between relatively conserved framework regions ("FRs"). Typically, a light or heavy chain polypeptide variable region contains four FRs and three CDRs arranged in the following order along the polypeptide: NH2-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-COOH. Together, the CDRs and FRs determine the three-dimensional structure of the IgG binding site, and thus the specific target protein or antigen to which the IgG molecule binds. Each IgG molecule is a dimer and can bind two antigen molecules. Cleavage of the dimeric IgG with the protease papain produces two identical antigen-binding fragments ("Fab") and an "Fc" fragment or domain, so named because they are easily crystallized.

[0087] The term "antibody," as used throughout this disclosure, further refers to whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecules produced by any of a variety of methods known in the art and described herein. The term "antibody" includes polyclonal antibodies, monoclonal antibodies, chimerized or chimeric antibodies, humanized antibodies, deimmunized human antibodies, and fully human antibodies. Antibodies may alternatively be derived from various species, e.g., mammals such as humans, non-human primates (e.g., monkeys, baboons, chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Antibodies may be purified or recombinant.

[0088] As used herein, the term "epitope" refers to a site on a protein to which an antibody binds. "Overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residues.

[0089] In one embodiment, the antibody of the present invention specifically binds to a SARS-CoV-2 antigen. As used herein, the term "specific binding" or "specifically binds" refers to two molecules forming a complex that is relatively stable under physiological conditions. Typically, binding occurs when the binding constant (Ka) is greater than or equal to 10. 6 Antibodies are considered specific if they are greater than M-1. 6 (or more) (e.g., at least 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 , or more)M -1 The target can specifically bind to a target having a Ka of

[0090] Methods for determining whether an antibody binds to an antigen and / or the affinity between an antibody and an antigen are known in the art. For example, the binding of an antibody to a protein antigen can be detected and / or quantified using various techniques, including, but not limited to, Western blotting, dot blotting, surface plasmon resonance (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or enzyme-linked immunosorbent assay (ELISA). For example, Harlow and Lane (1988) “Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Benny KC Lo (2004) “Antibody Engineering: Methods and Protocols,” Humana Press (ISBN: 1588290921); Borrebaek (1992) “Antibody Engineering, A Practical Guide,” WH Freeman and Co., NY; Borrebaek (1995) “Antibody Engineering,” 2nd Edition, Oxford University Press, NY, Oxford; Johne et al. (1993) J. Immunol. Meth. 160: 191-198; Jonsson et al. (1993) Ann. Biol. Clin. 51: 19- 26; and Jonsson et al. (1991) Biotechniques 11 :620-627. See also U.S. Patent No. 6,355,245.

[0091] Immunoassays that can be used to analyze the immunospecific binding and cross-reactivity of antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIAs, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. These assays are routinely performed and known in the art.

[0092] Antibodies can also be assayed using any surface plasmon resonance (SPR)-based assay known in the art to characterize the kinetic parameters of the interaction of an antibody with its target or epitope. Any commercially available SPR instrument can be used in the methods described herein, including, but not limited to, BIAcore Instruments (Biacore AB; Uppsala, Sweden); IAsys Instruments (Affinity Sensors; Franklin, Massachusetts); IBIS system (Windsor Scientific Limited; Berks, UK), SPR-CELLIA systems (Nippon Laser and Electronics Lab; Hokkaido, Japan), and SPR Detector Spreeta (Texas Instruments; Dallas, Texas). See, for example, Mullett et al. (2000) Methods 22: 77-91; Dong et al. (2002) Reviews in Mol Biotech 82: 303-323; Fivash et al. (1998) Curr Opin Biotechnol 9: 97-101; Rich et al. (2000) Curr Opin Biotechnol 11:54-61.

[0093] In some embodiments, antibodies and fragments thereof can be "chimeric." Chimeric antibodies and antigen-binding fragments thereof contain portions derived from two or more different species (e.g., mouse and human). Chimeric antibodies can be generated by combining mouse variable regions with desired specificity with human constant domain gene segments (see, e.g., U.S. Pat. No. 4,816,567). In this manner, non-human antibodies can be modified to make them more suitable for human clinical applications (e.g., methods for treating or preventing complement-related diseases in human subjects).

[0094] Monoclonal antibodies of the present disclosure include "humanized" forms of non-human (e.g., murine) antibodies. Humanized or CDR-grafted monoclonal antibodies are particularly useful as human therapeutics because they do not clear from the circulation as rapidly as murine antibodies and typically do not provoke adverse immune responses. Methods for preparing humanized antibodies are generally known in the art. For example, humanization can be performed essentially according to the method of Winter and coworkers (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. See also, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In some embodiments, humanized forms of non-human (e.g., murine) antibodies are constructed by substituting hypervariable region (CDR) residues of a human antibody (recipient antibody) with hypervariable region residues from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and binding capacity. In some cases, framework region residues of the human immunoglobulin are also substituted with corresponding non-human residues (so-called "backmutations"). Furthermore, phage display libraries can be used to alter amino acids at selected positions within the antibody sequence. The properties of the humanized antibody are influenced by the choice of human framework. Furthermore, humanized and chimeric antibodies can be modified to include residues that are not found in the recipient or donor antibody to further improve antibody properties, such as affinity or effector function.

[0095] The present disclosure also provides fully human antibodies. The term "human antibody" includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies). Fully human antibodies or human antibodies can be obtained from transgenic mice containing human antibody genes (variable exon (V), diversity exon (D), joining exon (J), and constant exon (C)) or from human cells. For example, it is now possible to produce transgenic animals (e.g., mice) that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; and Duchosal et al. (1992) Nature 355:258). Transgenic mouse strains can be engineered to contain unrearranged human immunoglobulin gene sequences. The human sequences encode both human antibody heavy and light chains, which function correctly in the mouse and can be rearranged to provide a broad antibody repertoire similar to that in humans. Transgenic mice can be immunized with a target protein to produce a variety of specific antibodies and the RNA that encodes them. Nucleic acids encoding the antibody chain components of such antibodies can then be cloned from the animals into display vectors.Typically, separate populations of nucleic acids encoding heavy and light chain sequences are cloned, and these separate populations are combined upon insertion into a vector so that any copy of the vector receives a random combination of heavy and light chains. The vector is designed to express antibody chains, which are assembled and displayed on the outer surface of a display package containing the vector. For example, antibody chains can be expressed as fusion proteins with phage coat proteins from the outer surface of a phage. The display package can then be screened to display antibodies that bind to the target.

[0096] Thus, in some embodiments, the disclosure provides humanized, deimmunized, or primatized antibodies comprising one or more complementarity determining regions (CDRs) of, for example, a murine monoclonal antibody described herein, which retain the ability of the murine monoclonal antibody portion to bind to an antigen (e.g., at least 50, 60, 70, 80, 90, or 100%, or greater than 100%).

[0097] Additionally, human antibodies can be obtained from phage display libraries (Hoogenboom et al. (1991) J. Mol. Biol. 227:381; Marks et al. (1991) J. Mol. Biol, 222:581-597; and Vaughan et al. (1996) Nature Biotech 14:309 (1996)). Synthetic phage libraries can be generated using random combinations of synthetic human antibody V regions. Through antigen selection, fully human antibodies can be generated in which the V regions are essentially very similar to human V regions. See, e.g., U.S. Patent Nos. 6,794,132, 6,680,209, 4,634,666, and Ostberg et al. (1983) Hybridoma 2:361-367. The contents of each of these references are incorporated herein by reference in their entirety.

[0098] For the generation of human antibodies, see also Mendez et al. (1998) Nature Genetics 15:146-156 and Green and Jakobovits (1998) J. Exp. Med. 188:483-495, the disclosures of which are incorporated herein by reference in their entireties. For human antibodies, see U.S. Patent Nos. 5,939,598, 6,673,986, 6,114,598, 6,075,181, 6,162,963, 6,150,584, 6,713,610, and 6,657,103, and U.S. Patent Application Publication Nos. 2003-0229905A1, 2004-0010810A1, US2004-0093622A1, 2006-0040363A1, 2005-0054055A1, 2005-0076395A1, and 2005-0287630A1. See also International Publication Nos. WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, and European Patent No. EP 0 463 151 B1, the disclosures of each of the above patents, applications, and references being incorporated herein by reference in their entirety.

[0099] As an alternative approach, other companies, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by the integration of fragments (individual genes) from the Ig locus: one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) into a construct for insertion into an animal. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, and 5,814,318, 5,591,669, 5,612,226, and ... 05, 5,721,367, 5,789,215, 5,643,763, 5,569,825, 5,877,397, 6,300,129, 5,874,299, 6,255,458, and 7,041,871, the disclosures of which are incorporated herein by reference. See also European Patent 0546073B1, International Patent Publications WO92 / 03918, WO92 / 22645, WO92 / 22647, WO92 / 22670, WO93 / 12227, WO94 / 00569, WO94 / 25585, WO96 / 14436, WO97 / 13852, and WO98 / 24884, the disclosures of each of which are incorporated herein by reference in their entirety.Furthermore, Taylor et al. (1992) Nucleic Acids Res. 20: 6287; Chen et al. (1993) Int. Immunol. 5: 647; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-4; Choi et al. (1993) Nature Genetics 4: 117; Lonberg et al. (1994) Nature 368: 856-859; Taylor et al. (1994) International Immunology 6: 579-591; Tuaillon et al. (1995) J. Immunol. 154: 6453-65; Fishwild et al. (1996) Nature Biotechnology 14: 845; and Tuaillon et al. (2000) Eur. J. Immunol. 10: 2998-3005, the disclosures of each of which are incorporated herein by reference in their entireties.

[0100] Methods for producing antibodies are within the knowledge of those skilled in the art. Traditionally, recombinant production of bispecific antibodies has been based on the coexpression of two immunoglobulin heavy / light chain pairs, where the two heavy / light chain pairs have different specificities (Milstein and Cuello (1983) Nature 305:537-539). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Fusions of heavy chain variable regions are preferably with immunoglobulin heavy chain constant domains, comprising at least part of the hinge, CH2, and CH3 regions. DNAs encoding the immunoglobulin heavy chain fusions, and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details of exemplary currently known methods for generating bispecific antibodies, see, e.g., Suresh et al. (1986) Methods in Enzymology 121:210; PCT Publication No. WO96 / 27011; Brennan et al. (1985) Science 229:81; Shalaby et al, J Exp Med (1992) 175:217-225; Kostelny et al. (1992) J Immunol 148(5): 1547-1553; Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448; Gruber et al. (1994) J Immunol 152:5368; and Tutt et al. (1991) J Immunol 147:60. Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0101] Various techniques for producing and isolating antibody fragments directly from recombinant cell culture have also been reported. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol 148(5):1547-1553. The leucine zipper peptides from Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers can be reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 provides an alternative mechanism for making bispecific antibody fragments. These fragments comprise a heavy-chain variable domain (VH) and a light-chain variable domain (VL) connected by a linker that is too short to permit pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of the other fragment, forming two antigen-binding sites. Another strategy for generating bispecific antibody fragments using single-chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibody may be a "linear antibody," as described, for example, in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0102] Antibodies with more than two valencies (eg, trispecific antibodies) are also contemplated and are described, for example, in Tutt et al. (1991) J Immunol 147:60.

[0103] The present disclosure also encompasses variant forms of multispecific antibodies, such as the dual variable domain immunoglobulin (DVD-lg) molecules described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297. DVD-lg molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem by recombinant DNA techniques, either directly or via a short linker, followed by a light chain constant domain. Similarly, the heavy chain contains two different heavy chain variable domains (VH) linked in tandem, followed by a constant domain CH1 and an Fc region. Methods for generating DVD-Ig molecules from two parent antibodies are further described, for example, in PCT Publications WO08 / 024188 and WO07 / 024715.

[0104] The present disclosure also provides camel or dromedary antibodies (e.g., antibodies derived from Bactrian camels (Camelus bactrianus), dromedaries (Calelus dromaderius), or alpacas (lama paccos). Such antibodies generally lack light chains, unlike the typical two-chain (fragment) or four-chain (whole antibody) antibodies derived from most mammals. See U.S. Pat. No. 5,759,808; Stijlemans et al. (2004) J Biol Chem 279: 1256-1261; Dumoulin et al. (2003) Nature 424:783-788; and Pleschberger et al. (2003) Bioconjugate Chem 14:440-448.

[0105] Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example from Ablynx (Ghent, Belgium). As with other antibodies of non-human origin, the amino acid sequences of camelid antibodies can be modified using recombinant techniques to make them more similar to human sequences; i.e., nanobodies can be "humanized" to further reduce the potential immunogenicity of antibodies.

[0106] In one embodiment, the anti-Siglec-9 antibody, or fragment thereof, comprises a heavy chain variable region having a sequence selected from one or more of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39, or a fragment or variant thereof. In one embodiment, the anti-Siglec-9 antibody, or fragment thereof, comprises a light chain variable region having a sequence selected from one or more of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40, or a fragment or variant thereof.

[0107] In some embodiments, variants of the amino acid sequences described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared to the defined amino acid sequence. In some embodiments, variants of the amino acid sequences described herein share at least about 60% identity over the entire length of a variable heavy chain having the amino acid sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39, or a variable light chain having the amino acid sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40. , 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity.

[0108] In some embodiments, the present disclosure also provides antibodies or antigen-binding fragments thereof, which are variants of the peptides, proteins, or antibodies described herein. In some embodiments, such variant peptides, proteins, or antibodies retain the binding or inhibitory ability of the parent peptide, protein, or antibody. Methods for preparing variants of known proteins, peptides, or antibodies are known in the art. In some embodiments, such variants contain at least a single amino acid substitution, deletion, insertion, or other modification. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein contain two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein do not contain amino acid modifications in the CDRs. In some embodiments, the fusion proteins, antibodies, or fragments thereof described herein comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid modifications in a CDR.

[0109] As used herein, the terms "antibody fragment," "antigen-binding fragment," "antigen binding fragment," or similar terms refer to a fragment of an antibody that retains the ability to bind to an antigen, where the antigen-binding fragment can optionally include additional components not part of the original antibody (e.g., different framework regions or mutations) and fragment(s) from the original antibody. Examples include, but are not limited to, single-chain antibodies, single-chain Fv fragments (scFv), Fd fragments, Fab fragments, Fab' fragments, or F(ab')2 fragments. An scFv fragment is a single polypeptide chain that contains both the heavy and light chain variable regions of the antibody from which the scFv is derived. Additionally, diabodies (Poljak (1994) Structure 2(12): 1121-1123; Hudson et al. (1999) J. Immunol. Methods 23(1-2): 177-189, the disclosures of which are incorporated herein by reference in their entireties), minibodies, triabodies (Schoonooghe et al. (2009) BMC Biotechnol 9:70), and domain antibodies (also called "heavy chain immunoglobulins" or camelids; Holt et al. (2003) Trends Biotechnol 21(11):484-490), the disclosures of which are incorporated herein by reference in their entireties, which bind to complement component proteins, can be incorporated into compositions and used in the methods described herein. In some embodiments, any of the antigen-binding fragments described herein can be included under the term "antigen-binding fragment thereof" or equivalent terms when referring to a fragment related to an antibody, whether such a fragment is actually derived from the antibody or is an antigen-binding fragment that binds to the same epitope, an overhanging epitope, or an epitope encompassed by the epitope of the antibody. The antigen-binding fragment can include an antigen-binding fragment that binds to the same or an overhanging antigen as the original antibody, in which case the antigen-binding fragment includes a portion that is a fragment of the original antibody (e.g., one or more CDRs, one or more variable regions, etc.).

[0110] In some embodiments, the antibodies described herein comprise altered or mutated sequences that result in altered stability or half-life compared to the parent antibody. This includes, for example, increased stability or half-life due to improved affinity or increased clearance time in vitro or in vivo, or decreased stability or half-life due to decreased affinity or more rapid clearance. Additionally, the antibodies described herein contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid substitutions, deletions, or insertions that result in altered post-translational modifications, including, for example, altered glycosylation patterns (e.g., addition of one or more sugar moieties, loss of one or more sugar moieties, or a change in the composition of one or more sugar moieties).

[0111] In some embodiments, the antibodies described herein have reduced effector function (e.g., no effector function at all). Altered effector function includes modulation of one or more of the following activities: for example, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc receptors, and proinflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody comprising an altered constant region compared to the activity of the unaltered form of the constant region. In certain embodiments, modulation also includes situations in which an activity is terminated or completely lost.

[0112] Antibodies with altered or no effector function can be generated by engineering or generating antibodies with variant constant regions, Fc regions, or heavy chain regions. Recombinant DNA technology and / or cell culture and expression conditions can be used to generate antibodies with altered function and / or activity. For example, recombinant DNA technology can be used to engineer the substitution, deletion, or insertion of one or more amino acids in a region (e.g., Fc region or constant region) that affects antibody function, including effector function. Alternatively, changes in post-translational modifications, such as glycosylation patterns, can be achieved by manipulating the cell culture and expression conditions under which the antibody is produced. Suitable methods for introducing one or more substitutions, additions, or deletions into the Fc region of an antibody are known in the art and include, for example, standard DNA mutagenesis techniques, which are described, for example, in Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; Borrebaek (1992), supra; Johne et al. (1993), supra; PCT Publication No. WO 06 / 53301; and U.S. Patent No. 7,704,497.

[0113] In some embodiments, an antibody can comprise a set of heavy and light chain complementarity determining regions ("CDRs") and a set of light chain complementarity determining regions ("CDRs"), which are interposed between a set of heavy and light chain frameworks ("FRs"), respectively, to support the CDRs and define their relative spatial relationships to one another. A set of CDRs can include three hypervariable regions of a heavy chain V region or a light chain V region. From the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. Thus, an antigen-binding site can comprise six CDRs, comprising the sets of CDRs from each of the heavy and light chain V regions.

[0114] The proteolytic enzyme papain preferentially cleaves IgG molecules to generate multiple fragments, two of which (F(ab) fragments) constitute covalently linked heterodimers each containing an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to provide multiple fragments, including the F(ab')2 fragment, which contains both antigen-binding sites. Thus, an antibody can be Fab or F(ab')2. Fab can comprise heavy and light chain polypeptides. The heavy chain polypeptide of a Fab can comprise a VH region and a CH1 region. The light chain of a Fab can comprise a VL region and a CL region.

[0115] The antibody may be an immunoglobulin (Ig). The Ig may be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin may comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the immunoglobulin may comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin may comprise a VL region and a CL region.

[0116] The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single-chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody may be an antibody from a non-human species that binds to a desired antigen and has one or more complementarity-determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0117] The antibody may be a bispecific antibody, as described in more detail below. The antibody may be a bifunctional antibody, as described in more detail below.

[0118] As described above, administering the composition to a subject can generate antibodies in the subject. The antibody can have a half-life in the subject. In some embodiments, the antibody can be modified to extend or shorten its half-life in the subject. Such modifications are described in more detail below. The antibody may be defucosylated, as described in more detail below.

[0119] As described above, the inhibitors of the present invention can comprise anti-Siglec-9 antibodies, fragments thereof, variants thereof, or combinations thereof. In some embodiments, the anti-Siglec-9 antibodies inhibit Siglec-9 signaling. Thus, in some embodiments, the antibodies of the present invention comprise an inhibitory Siglec-9 binding domain.

[0120] An inhibitory Siglec-9 binding domain can comprise a set of heavy and light chain complementarity determining regions ("CDRs") and a set of light chain complementarity determining regions ("CDRs"), which are interposed between a set of heavy chain frameworks ("FRs") and a set of light chain frameworks ("FRs"), respectively, which support the CDRs and define the relative spatial relationship between the CDRs. The set of CDRs can comprise three hypervariable regions of the heavy chain V region or the light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. Thus, an antigen-binding domain can comprise six CDRs, comprising the sets of CDRs from each of the heavy and light chain V regions.

[0121] The proteolytic enzyme papain preferentially cleaves IgG molecules to generate multiple fragments, two of which (F(ab) fragments) constitute covalently linked heterodimers each containing an intact antigen-binding site. The enzyme pepsin cleaves IgG molecules to generate multiple fragments, including F(ab')2 fragments, which contain F(ab')2 fragments. Thus, the target antigen-binding domain of a BiTE can be Fab or F(ab')2. Fab can comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of a Fab can comprise a VH region and a CH1 region. The light chain of a Fab can comprise a VL region and a CL region.

[0122] The anti-Siglec-9 antibody may be an immunoglobulin (Ig). The Ig may be, for example, IgA, IgM, IgD, IgE, or IgG. The immunoglobulin may comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the immunoglobulin may comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin may comprise a VL region and a CL region.

[0123] The anti-Siglec-9 antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single-chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody may be an antibody derived from a non-human species that binds to a desired antigen and has one or more complementarity-determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0124] ScFv antibody In one embodiment, the inhibitor of the present invention is an ScFv antibody fragment. In one embodiment, the ScFv relates to a Fab fragment excluding the CH1 and CL regions. Accordingly, in one embodiment, the ScFv relates to a Fab fragment comprising a VH and a VL. In one embodiment, the ScFv comprises a linker between the VH and VL. In one embodiment, the inhibitor of the present invention is an ScFv-Fc. In one embodiment, the ScFv-Fc comprises a VH, a VL, and a CH2 and CH3 region. In one embodiment, the ScFv-Fc comprises a linker between the VH and VL. In one embodiment, the ScFv of the present invention has modified expression, stability, half-life, antigen binding, heavy-light chain pairing, tissue penetration, or a combination thereof, compared to the parent antibody.

[0125] In one embodiment, the ScFv of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold higher expression than the parent antibody.

[0126] In one embodiment, the ScFv of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold higher antigen binding than the parent antibody.

[0127] In one embodiment, the ScFv of the invention has a half-life that is at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold longer than the parent antibody.

[0128] In one embodiment, the ScFv of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold greater stability than the parent antibody.

[0129] In one embodiment, the ScFv of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold greater tissue penetration than the parent antibody.

[0130] In one embodiment, the ScFv of the invention has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, or more than 50 fold greater heavy chain-light chain pairing than the parent antibody.

[0131] bispecific antibody In some embodiments, the inhibitors of the present invention comprise a bispecific anti-Siglec-9 antibody, a fragment thereof, a variant thereof, or a combination thereof. In some embodiments, the bispecific anti-Siglec-9 antibody functions as a natural killer cell engager (NKE), bringing natural killer cells into close proximity with target cells expressing a target antigen (e.g., a viral antigen). Thus, in some embodiments, the NKE comprises an inhibitory Siglec-9 binding domain and a target antigen binding domain. In some embodiments, the target antigen binding domain is specific for binding to a viral antigen. In some embodiments, the target antigen binding domain is specific for binding to a SARS-CoV-2 viral antigen.

[0132] The target antigen-binding domain of an NKE may comprise an antibody, a fragment thereof, a variant thereof, or a combination thereof. The target antigen-binding domain of an NKE may comprise a set of heavy and light chain complementarity-determining regions ("CDRs") and a set of light chain complementarity-determining regions ("CDRs"), each interposed between a set of heavy chain frameworks ("FRs") and a set of light chain frameworks ("FRs") that support the CDRs and define the relative spatial relationship between the CDRs. The set of CDRs may comprise three hypervariable regions of the heavy chain V region or the light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. Thus, the antigen-binding domain may comprise six CDRs, including the sets of CDRs from each of the heavy and light chain V regions.

[0133] The proteolytic enzyme papain preferentially cleaves IgG molecules to generate multiple fragments, two of which (F(ab) fragments) constitute covalently linked heterodimers each containing an intact antigen-binding site. The enzyme pepsin cleaves IgG molecules to generate multiple fragments, including the F(ab')2 fragment, which contains both antigen-binding sites. Thus, the target antigen-binding domain of a BiTE can be Fab or F(ab')2. Fab can comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of a Fab can comprise a VH domain and a CH1 domain. The light chain of a Fab can comprise a VL domain and a CL domain.

[0134] The target antigen-binding domain of the NKE can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, or IgG. The immunoglobulin can comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can comprise a VL region and a CL region.

[0135] The target antigen-binding domain of NKE can be a polyclonal antibody or a monoclonal antibody. The antibody can be a chimeric antibody, a single-chain antibody, an affinity-matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody is an antibody derived from a non-human species that binds to a desired antigen and has one or more complementarity-determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule.

[0136] In one embodiment, at least one of the target antigen binding domain and the inhibitory Siglec-9 binding domain of the NKE is an scFv monoclonal antibody.

[0137] In one embodiment, the NKE is a bispecific antibody. In some embodiments, a bispecific antibody is a bivalent antibody comprising a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen.

[0138] A bispecific antibody molecule according to the invention may have two binding sites with any desired specificity.

[0139] In one embodiment, the synthetic antibody (e.g., NKE) is directed against Siglec-9 and one or more additional antigens, or fragments or variants thereof. The antigens can be nucleic acid sequences, amino acid sequences, polysaccharides, or combinations thereof. The nucleic acid sequences can be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The amino acid sequences can be proteins, peptides, variants thereof, fragments thereof, or combinations thereof. The polysaccharides can be polysaccharides encoded by nucleic acids.

[0140] In one embodiment, the synthetic bispecific NKE of the present invention targets two or more antigens. In one embodiment, at least one antigen targeted by the bispecific antibody is a viral antigen. The antigen can be a viral antigen. In one embodiment, the antigen can be a SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigen is a spike antigen.

[0141] Bifunctional antibody In one embodiment, the inhibitor of the present invention is a bifunctional antibody, a fragment thereof, a variant thereof, or a combination thereof. The bifunctional antibody is capable of inhibiting Siglec-9 as described elsewhere herein. The bifunctional antibody can also be modified to confer additional functions to the antibody beyond antigen recognition and binding. Such modifications include, but are not limited to, binding to factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and, therefore, can contribute to the immune response via complement-mediated lysis (CML).

[0142] Extending antibody half-life The antibodies of the invention may be modified to increase or decrease the half-life of the antibody in a subject, which may increase or decrease the half-life of the antibody in the serum of the subject.

[0143] The modification may be present in the constant region of the antibody. The modification may be one or more amino acid substitutions in the constant region of the antibody that extend the half-life of the antibody compared to the half-life of an antibody that does not contain one or more amino acid substitutions. The modification may be one or more amino acid substitutions in the CH2 domain of the antibody that extend the half-life of the antibody compared to the half-life of an antibody that does not contain one or more amino acid substitutions.

[0144] In some embodiments, the one or more amino acid substitutions in the constant region comprise substituting a methionine residue in the constant region with a tyrosine residue, a serine residue in the constant region with a threonine residue, a threonine residue in the constant region with a glutamic acid residue, or any combination thereof, thereby increasing the half-life of the antibody.

[0145] In some embodiments, the one or more amino acid substitutions in the constant region comprise a substitution of a methionine residue in the CH2 domain with a tyrosine residue, a substitution of a serine residue in the CH2 domain with a threonine residue, a substitution of a threonine residue in the CH2 domain with a glutamic acid residue, or any combination thereof, thereby increasing the half-life of the antibody.

[0146] Defucosylation The antibodies of the present invention may be defucosylated or nonfucosylated antibodies, fragments thereof, variants thereof, or combinations thereof. Fucosylation includes the addition of the sugar fucose to a molecule, for example, the addition of fucose to N-glycans, O-glycans, and glycolipids. Thus, in defucosylated antibodies, fucose is not added to the carbohydrate chains of the constant region. As a result, this lack of fucosylation may improve FcγRIIIa binding and antibody-induced cell-mediated cytotoxicity (ADCC) activity of the antibody compared to fucosylated antibodies. Thus, in some embodiments, nonfucosylated antibodies may exhibit increased ADCC activity compared to fucosylated antibodies.

[0147] Antibodies can be modified to prevent or inhibit antibody fucosylation. In some embodiments, such modified antibodies may exhibit increased ADCC activity compared to unmodified antibodies. The modifications can occur in the heavy chain, the light chain, or a combination thereof. The modifications can be one or more amino acid substitutions in the heavy chain, one or more amino acid substitutions in the light chain, or a combination thereof.

[0148] nucleic acid molecule The present invention provides polynucleotides encoding the Siglec-9 inhibitors, anti-Siglec-9 antibodies, or NKE antibodies of the present invention, or fragments thereof. In some embodiments, the polynucleotide also comprises a sequence encoding a signal peptide operably linked to the 5' end of the coding sequence. In some embodiments, the polynucleotide also comprises a sequence encoding a linker sequence.

[0149] In one embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26, or a fragment or variant thereof, encoding the heavy chain variable region. In one embodiment, the nucleic acid molecule comprises the nucleotide sequence having the sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28, or a fragment or variant thereof, encoding the light chain variable region.

[0150] In some embodiments, variants of the nucleotide sequences described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a designated region when compared to the defined nucleotide sequence. In some embodiments, variants of the nucleotide sequences described herein comprise at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity over the entire length of the nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26 encoding the variable heavy chain, or the nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28 encoding the variable light chain.

[0151] In some embodiments, fragments of the nucleotide sequences described herein comprise at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full-length sequence of the defined nucleotide sequence. In some embodiments, fragments of the nucleotide sequences described herein comprise at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of the nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26 encoding the variable heavy chain, or the full length of the nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28 encoding the variable light chain.

[0152] In one embodiment, the nucleic acid molecule comprises an RNA molecule corresponding to the nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26 encoding the variable heavy chain, or to the nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28 encoding the variable light chain, or to a fragment or variant thereof.

[0153] In one embodiment, the nucleic acid molecule comprises a DNA molecule corresponding to the nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26 encoding the variable heavy chain, or to the nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28 encoding the variable light chain, or to a fragment or variant thereof.

[0154] In some embodiments, variants of the nucleotide sequences described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a designated region when compared to the defined nucleotide sequence. In some embodiments, variants of the nucleotide sequences described herein comprise at least about 60% identity, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over the entire length of the nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26 encoding the variable heavy chain, or the nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28 encoding the variable light chain.

[0155] In some embodiments, fragments of the nucleotide sequences described herein comprise at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full-length sequence of the defined nucleotide sequence. In some embodiments, fragments of the nucleotide sequences described herein comprise at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the full length sequence of the nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26 encoding the variable heavy chain, or SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28 encoding the variable light chain.

[0156] The isolated nucleic acid can include any type of nucleic acid, including, but not limited to, DNA, cDNA, and RNA. For example, in one embodiment, the composition includes an isolated DNA molecule (e.g., including an isolated cDNA molecule) encoding a protein inhibitor or a functional fragment thereof. In one embodiment, the composition includes an isolated RNA molecule encoding a Siglec-9 inhibitor, an anti-Siglec-9 antibody, an NKE, or a functional fragment thereof.

[0157] The nucleic acid molecules of the present invention can be modified to improve stability. Modifications can be added to enhance the stability, functionality, and / or specificity of the nucleic acid molecules of the present invention and minimize their immunostimulatory properties. For example, to enhance stability, 3' residues can be stabilized against degradation, e.g., selected to consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, e.g., substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the function of the molecule.

[0158] In one embodiment of the invention, the nucleic acid molecule may contain at least one modified nucleotide analogue, for example, the termini may be stabilized by incorporating modified nucleotide analogues.

[0159] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., containing modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate ester group attached to an adjacent ribonucleotide is replaced with a modified group, such as a phosphothioate group.

[0160] Another example of modification is nucleobase-modified ribonucleotide, i.e., ribonucleotide containing at least one unnatural nucleobase instead of a natural nucleobase. The base can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deazaadenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine. The above modifications can also be combined.

[0161] In some cases, the nucleic acid molecule contains at least one of the following chemical modifications: 2'-H, 2'-O-methyl, or 2'-OH modifications of one or more nucleotides. In some embodiments, the nucleic acid molecules of the present invention have enhanced resistance to nucleases. To enhance nuclease resistance, the nucleic acid molecule can contain, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with many different "oxy" or "deoxy" substituents. To enhance nuclease resistance, the nucleic acid molecule of the present invention can contain 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Locked nucleic acids (LNA), ethylene nucleic acids (ENA), such as 2'-4'-ethylene bridged nucleic acids, and certain nucleobase modifications, such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, etc., can also enhance binding affinity to targets.

[0162] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and optionally, all nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0163] The nucleic acid materials discussed herein include naturally unmodified RNA and DNA, as well as RNA and DNA modified, for example, to improve efficacy, and polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the nucleic acid building blocks, i.e., sugar, base, and phosphate moieties, are identical or essentially identical to those found in nature (e.g., naturally occurring in the human body). Rare or unusual but naturally occurring RNAs are referred to in the art as modified RNAs. See, for example, Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs are often referred to as modified RNAs, typically the result of post-transcriptional modifications, and are included in the term "unmodified RNA" as used herein. As used herein, "modified RNA" refers to molecules in which one or more of the nucleic acid building blocks (i.e., sugar, base, and phosphate moieties) differ from those found in nature (e.g., found in the human body). While these are referred to as "modified RNAs," the term also includes molecules that are not strictly RNA due to their modification. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct, which allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with a ribophosphate backbone (e.g., an uncharged mimic of a ribophosphate backbone).

[0164] Modifications of the nucleic acids of the invention can be at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0165] The invention also includes vectors into which the isolated nucleic acids of the invention have been inserted. Suitable vectors useful in the present invention are abundant in the art.

[0166] Thus, in another aspect, the present invention relates to a vector comprising the nucleotide sequence of the present invention or the construct of the present invention. The choice of vector depends on the host cell into which it will subsequently be introduced. In some embodiments, the vector of the present invention is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In particular embodiments, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. The present invention uses systems based on prokaryotic and / or eukaryotic vectors to produce polynucleotides or their cognate polypeptides. Many such systems are commercially available and widely available.

[0167] In some embodiments, expression of a synthetic nucleic acid encoding a protein is typically achieved by operably linking the nucleic acid encoding the protein or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication in eukaryotic cells, and optionally for integration. Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0168] The recombinant nucleic acid sequence construct may contain one or more transcription termination regions. The transcription termination region may be located downstream of the coding sequence to provide efficient termination. The transcription termination region may be obtained from the same gene as the promoter or from one or more different genes.

[0169] A recombinant nucleic acid sequence construct can include one or more start codons. The start codon can be located upstream of the coding sequence. The start codon can be in-frame with the coding sequence. The start codon can be associated with one or more signals required for efficient translation initiation (such as, but not limited to, a ribosome binding site).

[0170] A recombinant nucleic acid sequence construct can contain one or more stop or termination codons. The stop codon can be downstream of the coding sequence. The stop codon can be in-frame with the coding sequence. The stop codon can be associated with one or more signals required for efficient translation termination.

[0171] The recombinant nucleic acid sequence construct can contain one or more polyadenylation signals. The polyadenylation signal can include one or more signals necessary for efficient polyadenylation of the transcript. The polyadenylation signal can be located downstream of the coding sequence. The polyadenylation signal can be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal can be a polyadenylation signal derived from the pCEP4 plasmid (Invitrogen, San Diego, CA).

[0172] The recombinant nucleic acid construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, such as, but not limited to, an IgG signal peptide and an IgE signal peptide.

[0173] The vectors of the present invention can also be used for nucleic acid immunization using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466 (which are incorporated herein by reference in their entirety).

[0174] The isolated nucleic acids of the present invention can be cloned into many types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0175] Furthermore, vectors can be provided to cells in the form of viral vectors.Viral vector technology is known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.In general, suitable vectors contain a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0176] Furthermore, the expression vector can be provided to cells in the form of a viral vector.Viral vector technology is known in the art and is described, for example, in Sambrook et al. (2012), and in Ausubel et al. (1997), and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.In general, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0177] By way of example, the vector into which the nucleic acid sequence is introduced may be a plasmid, which may or may not be integrated into the genome of the host cell when introduced into the cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequences of the invention or the genetic constructs of the invention can be inserted include tet-on inducible vectors for expression in eukaryotic cells.

[0178] The vector can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In a specific embodiment, the vector is a vector useful for transforming animal cells.

[0179] In one embodiment, the recombinant expression vector may also include a nucleic acid molecule encoding a peptide or protein of the invention as described elsewhere herein.

[0180] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene transfer systems. A selected gene is inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus is then isolated and delivered to the cells of a subject in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0181] For example, vectors derived from retroviruses, such as lentiviruses, are suitable tools for long-term gene transfer because they allow long-term and stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have the advantage over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, of being able to transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity. In one embodiment, the composition comprises a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become powerful gene delivery tools in the treatment of various disorders. AAV vectors possess many ideal characteristics for gene therapy, including lack of pathogenicity, minimal immunogenicity, and the ability to stably and efficiently transduce post-mitotic cells. Expression of a specific gene within an AAV vector can be specifically targeted to one or more cell types by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0182] In some embodiments, the vector also contains conventional control elements operably linked to the transgene in a manner that allows transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences contiguous with the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if necessary, sequences that promote secretion of the encoded product. Numerous expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known and available in the art.

[0183] A promoter is one that is naturally associated with a gene or polynucleotide sequence and can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exons. Such promoters are referred to as "endogenous." Similarly, an enhancer is naturally associated with a polynucleotide sequence located downstream or upstream of that sequence. Alternatively, certain advantages can be gained by placing a coding polynucleotide segment under the control of a recombinant or heterologous promoter (which generally refers to a promoter not associated with a polynucleotide sequence in its natural environment). A recombinant or heterologous enhancer also refers to an enhancer that is not associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers include promoters or enhancers of other genes, promoters or enhancers isolated from other prokaryotic, viral, or eukaryotic cells, and non-"naturally occurring" enhancers, i.e., promoters or enhancers containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically producing promoter and enhancer nucleic acid sequences, in the context of the compositions disclosed herein, sequences can also be produced using nucleic acid amplification techniques, including recombinant cloning and / or PCR (U.S. Patent Nos. 4,683,202 and 5,928,906). Furthermore, regulatory sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria and chloroplasts, may also be used.

[0184] Naturally, it is important to use a promoter and / or enhancer that effectively directs expression of the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the field of molecular biology generally know how to use combinations of promoters, enhancers, and cell types for protein expression. See, for example, Sambrook et al. (2012). The promoter used should be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to induce high-level expression of the introduced DNA segment, which is advantageous, for example, for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0185] The recombinant expression vector can also contain a selectable marker gene that facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof, e.g., the Fc region of immunoglobulins such as IgG. The selectable marker can be introduced into a vector separate from the nucleic acid of interest.

[0186] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30–110 bp upstream from the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. Spacing between promoter elements is often flexible, allowing elements to be inverted or moved relative to one another while still retaining promoter function. In the thymidine kinase (tk) promoter, activity begins to decline when spacing between promoter elements is increased to 50 bp. Depending on the promoter, individual elements appear to be able to activate transcription either cooperatively or independently.

[0187] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of conferring high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α) promoter. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (SARS-CoV-2) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters (such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter). Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0188] Enhancer sequences present on vectors also regulate the expression of genes contained therein. Typically, enhancers bind to protein factors to enhance gene transcription. Enhancers can be located upstream or downstream of the gene they regulate. Enhancers can also be tissue-specific to enhance transcription in specific cell or tissue types. In one embodiment, the vector of the present invention contains one or more enhancers to enhance the transcription of genes present in the vector.

[0189] To assess the expression of a protein inhibitor, the expression vector introduced into cells can also contain a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells targeted for transfection or infection with a viral vector. In other embodiments, the selectable marker can be carried on a separate DNA fragment and used in a cotransfection method. Both the selectable marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0190] Reporter genes are used to identify potentially transfected cells and evaluate the function of regulatory sequences. Generally, a reporter gene is a gene encoding a polypeptide that is not present in or expressed by the recipient organism or tissue, and whose expression is manifested by an easily detectable characteristic, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimal 5' flanking region that produces the highest reporter gene expression level is identified as the promoter. Such a promoter region can be linked to a reporter gene and used to evaluate drugs capable of modulating promoter-driven transcription.

[0191] Methods for introducing and expressing genes in cells are known in the art. In the case of expression vectors, the vectors can be easily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method known in the art. For example, the expression vector can be introduced into the host cells by physical, chemical, or biological means.

[0192] Physical methods for introducing peptides or proteins into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or foreign nucleic acids are known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0193] Biological methods for introducing a peptide or protein of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0194] Chemical means for introducing peptides or proteins into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An example of a colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0195] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed to a micelle or lipid nanoparticle, or associated with a lipid. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to a particular structure in solution. For example, they may exist in a bilayer structure, a micelle, or a "collapsed" structure. They may also simply be interspersed in a solution, forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a group of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0196] Suitable lipids for use are commercially available. For example, dimyristyl phosphatidylcholine ("DMPC") is obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") is obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids are obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles created by the formation of encapsulated lipid bilayers or aggregates. Liposomes are characterized by vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes contain multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess amount of aqueous solution. The lipid components reorganize and then form closed structures, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also contemplated. For example, lipids may form micelles or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0197] host cell Host cells (e.g., isolated cells, transient cell lines, and stable cell lines) for expressing the molecules described herein are also provided. Host cells can be prokaryotic or eukaryotic. Exemplary prokaryotic host cells include E. coli K12 strain 294 (ATCC No. 31446), E. coli B, E. coli X1776 (ATCC No. 31537), E. coli W3110 (F-, gamma-, prototrophic / ATCC No. 27325), Bacillus, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas bacteria. One suitable prokaryotic host cell is E. coli BL21 (Stratagene), which is deficient in OmpT and Lon proteases, potentially preventing isolation of intact recombinant proteins, and is useful for T7 promoter-driven vectors such as pET vectors. Another suitable prokaryotic cell is E. coli W3110 (ATCC No. 27325). Peptides expressed by prokaryotes typically contain only an N-terminal methionine or formyl methionine and are not glycosylated. In the case of fusion proteins, the N-terminal methionine or formyl methionine may be present at the amino terminus of the fusion protein or in the signal sequence of the fusion protein. Of course, these examples are illustrative and not limiting.

[0198] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi and yeast, are suitable cloning or expression hosts for fusion protein-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other microorganisms include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); European Patent No. 139,383, published May 2, 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio / Technology, 9:968-975 (1991)), such as Kluyveromyces lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), Kluyveromyces fragilis, and the like. (ATCC No. 12,424), Kluyveromyces bulgaricus (K. bulgaricus) (ATCC No. 16,045), Kluyveromyces wickeramii (K. wickeramii) (ATCC No. 24,178), Kluyveromyces waltii (ATCC No. 56,500), Kluyveromyces drosophilarum (ATCC No. 36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), Kluyveromyces thermotolerans, and Kluyveromyces marxianus (K. marxianus); Yarrowia (European Patent No. 402,226); Pichia pastoris (European Patent No. 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma reesia (European Patent No. 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces, e.g., Schwanniomyces occidentalis occidentalis (European Patent No. 394,538, published October 31, 1990); and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium (WO 91 / 00357, published January 10, 1991), and Aspergillus hosts, e.g., Aspergillus nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 (1984)), and Aspergillus niger (A. niger) (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Methylotrophic yeasts are suitable for the present invention and include, but are not limited to, yeasts capable of growing on methanol selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of exemplary species of this class of yeast is C.Anthony, The Biochemistry of Methylotrophs, 269 (1982). Host cells also include insect cells such as Drosophila S2 cells and Spodoptera Sf9 cells, as well as plant cells.

[0199] Examples of useful mammalian host cell lines include, but are not limited to, HeLa cells, Chinese hamster ovary (CHO cells), COS-7 cells, L cells, C127 cells, 3T3 cells, BHK cells, CHL-1 cells, NSO cells, HEK293 cells, WI38 cells, BHK cells, C127 cells, or MDCK cell lines. Another exemplary mammalian cell line is CHL-1. When CHL-1 is used, hygromycin is included as a eukaryotic selection marker. CHL-1 cells are derived from RPMI7032 melanoma cells, a readily available human cell line. Cells suitable for the present invention are commercially available from ATCC.

[0200] substrate In one embodiment, the present invention provides a scaffold, substrate, or device comprising a bispecific immune cell engager, a fragment thereof, or the nucleic acid molecules encoding them. For example, in some embodiments, the present invention provides tissue-engineered scaffolds, including but not limited to hydrogels, electrospun scaffolds, polymer matrices, etc., comprising a modulator. In certain embodiments, the bispecific immune cell engager, a fragment thereof, or the nucleic acid molecules encoding it can be coated along the surface of the scaffold, substrate, or device. In certain embodiments, the bispecific immune cell engager, a fragment thereof, or the nucleic acid molecules encoding it are encapsulated within the scaffold, substrate, or device.

[0201] Delivery Vehicle In one embodiment, the invention provides a composition comprising a delivery vehicle comprising a bispecific anti-SARS-CoV-2 immune cell-inducing antibody, fragment thereof, or nucleic acid molecule encoding it, as described herein. In one embodiment, the nucleic acid molecule encoding the bispecific anti-SARS-CoV-2 immune cell-inducing antibody comprises an mRNA molecule.

[0202] Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymerosomes, liposomes, and micelles. For example, in some embodiments, the delivery vehicle is a lipid nanoparticle loaded with a nucleic acid molecule encoding the bispecific anti-SARS-CoV-2 immune cell-inducing antibody of the present invention or a fragment thereof. In one embodiment, the nucleic acid molecule encoding the bispecific anti-SARS-CoV-2 immune cell-inducing antibody comprises an mRNA molecule. In one embodiment, the mRNA encoding the bispecific anti-SARS-CoV-2 immune cell-inducing antibody corresponds to or is transcribed from the DNA sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7. In one embodiment, the mRNA encoding the bispecific anti-SARS-CoV-2 immune cell-inducing antibody encodes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0203] In some embodiments, the delivery vehicle provides controlled, delayed, or continuous release of its payload. In some embodiments, the delivery vehicle includes a targeting moiety that targets the delivery vehicle to a treatment site.

[0204] In some cases, expressing proteins by delivering mRNA encoding the protein offers many advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence for the protein of interest is the only substance delivered to the cell, avoiding all side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, mRNA does not risk integration into the genome, and protein production begins immediately after mRNA delivery. For example, high levels of circulating protein have been measured within 15 to 30 minutes of injecting the encoding mRNA into the body. In certain embodiments, using mRNA rather than protein also offers many advantages. Proteins often have short half-lives in the circulation, necessitating frequent administration of protein therapeutics, while mRNA serves as a template for continuous protein production over several days. Protein purification can be problematic, as they can contain aggregates and other impurities that can cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0205] A variety of assays can be performed to confirm the presence of an mRNA sequence in a host cell, including, for example, "molecular biological" assays known to those skilled in the art, such as Northern blotting and RT-PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides by immunogenic means (ELISA and Western blot), or by the assays described herein to identify agents within the scope of the invention.

[0206] Combination with additional drugs In some embodiments, the Siglec-9 inhibitor of the present invention is administered in combination with one or more additional agents. Examples of additional agents include, but are not limited to, antibodies, siRNAs, miRNAs, shRNAs, small molecules, and chemical compounds. In some embodiments, the one or more additional agents are additional therapeutic agents. For example, in some embodiments, the present invention relates to a composition comprising a combination of an anti-Siglec-9 antibody and one or more additional antibodies. In some embodiments, the one or more additional antibodies are specific for binding to a viral antigen. In some embodiments, the viral antigen is a SARS-CoV-2 antigen.

[0207] In some embodiments, the present invention relates to a composition comprising one or more nucleic acid molecules (e.g., mRNA or DNA molecules) encoding an anti-Siglec-9 antibody in combination with one or more nucleic acid molecules (e.g., mRNA or DNA molecules) encoding one or more additional antibodies. In some embodiments, the one or more additional antibodies are specific for binding to a viral antigen. In some embodiments, the viral antigen is a SARS-CoV-2 antigen.

[0208] Treatment method The present invention provides methods for inhibiting Siglec-9 activity in natural killer cells of a target subject. In some embodiments, the present invention provides methods for diagnosing, treating, or preventing a disease or disorder, comprising administering an effective amount of a composition comprising one or more Siglec-9 inhibitors of the present invention. In some embodiments, the composition comprising one or more Siglec-9 inhibitors further comprises one or more adjuvants, one or more additional therapeutic agents, or a combination thereof. In some embodiments, the composition comprising one or more Siglec-9 inhibitors functions as an adjuvant to enhance the effectiveness of the immune response to a target antigen. In some embodiments, the composition comprising one or more Siglec-9 inhibitors comprises immunotherapy for SARS-CoV-2 infection or COVID-19.

[0209] In some embodiments, the method confers or enhances immunity in a target subject against an infection or a disease or disorder associated with an infectious agent. Thus, the present invention provides methods for treating or preventing an infectious disease or a disease or disorder associated with an infectious agent. For example, the method can be used to treat or prevent a viral infection, a bacterial infection, a fungal infection, or a parasitic infection, depending on the type of antigen in the administered composition. Exemplary antigens and associated infectious diseases, diseases, and tumors are described elsewhere herein.

[0210] In one embodiment, the composition is administered to a subject with a SARS-CoV-2 infection or COVID-19. In one embodiment, the composition is administered to a subject at risk of developing a SARS-CoV-2 infection or COVID-19.

[0211] In one embodiment, the method comprises administering an anti-Siglec-9 antibody for the treatment or prevention of a disease or disorder. In one embodiment, the antibody is administered to a subject with SARS-CoV-2 infection or COVID-19. In one embodiment, the antibody is administered to a subject at risk of developing SARS-CoV-2 infection or COVID-19.

[0212] In one embodiment, the method comprises administering a bispecific anti-Siglec-9 antibody for the treatment or prevention of a disease or disorder. In one embodiment, the bispecific anti-Siglec-9 antibody is administered to a subject with SARS-CoV-2 infection or COVID-19. In one embodiment, the antibody is administered to a subject at risk of developing SARS-CoV-2 infection or COVID-19.

[0213] In one embodiment, the compositions of the present invention can be administered in combination with an additional therapeutic agent, an adjuvant, or a combination thereof. For example, in one embodiment, the method comprises administering an LNP composition comprising a nucleic acid molecule encoding one or more anti-Siglec-9 antibodies. In one embodiment, the method comprises administering an LNP composition comprising a nucleic acid molecule encoding one or more bispecific anti-Siglec-9 antibodies.

[0214] In certain embodiments, the methods comprise administering to a subject a combination of a Siglec-9 inhibitor of the present invention and one or more additional therapeutic agents, hi some embodiments, the additional therapeutic agents are additional agents for the treatment of a target pathogen, adjuvants, or combinations thereof.

[0215] The administration of the compositions of the present invention in the therapeutic method can be achieved in various ways using methods known in the art. In one embodiment, the method of the present invention includes systemic administration to a subject, including, for example, enteral or parenteral administration. In certain embodiments, the method includes intradermal delivery of the composition. In another embodiment, the method includes intravenous delivery of the composition. In some embodiments, the method includes intramuscular delivery of the composition. In one embodiment, the method includes subcutaneous delivery of the composition. In one embodiment, the method includes inhalation of the composition. In one embodiment, the method includes intranasal delivery of the composition.

[0216] It will be understood that the compositions of the present invention can be administered to a subject alone or in combination with other agents.

[0217] Accordingly, the therapeutic and prophylactic methods of the present invention encompass the use of pharmaceutical compositions comprising the Siglec-9 inhibitors of the present invention, adjuvants, or combinations thereof described herein to practice the methods of the present invention. Pharmaceutical compositions useful for practicing the present invention can be administered to deliver a dose of 0.001 ng / kg / day to 100 mg / kg / day. In one embodiment, the present invention contemplates the administration of a dose that results in a concentration of the compound of the present invention in a mammal of 10 nM to 10 μM.

[0218] Typically, dosages that can be administered to a mammal, preferably a human, in the methods of the present invention range from 0.01 μg to about 50 mg per kg of mammalian body weight, although the exact dose administered will vary depending on a variety of factors, including, but not limited to, the type of mammal, the type of disease being treated, the mammal's age, and the route of administration. Preferably, the dosage of the compound ranges from about 0.1 μg to about 10 mg per kg of mammalian body weight. More preferably, the dosage ranges from about 1 μg to about 1 mg per kg of mammalian body weight.

[0219] The compositions can be administered to a mammal as frequently as several times daily, or less frequently, such as once daily, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even once a year or less. The frequency of administration will be readily apparent to one of skill in the art and will depend on a variety of factors, including, but not limited to, the type and severity of the disease being treated, and the species and age of the mammal.

[0220] In certain embodiments, administration of the Siglec-9 inhibitors or antibodies of the invention, or the nucleic acid molecules encoding them, can be by a single dose or multiple booster doses. [Example]

[0221] The present invention is further described in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are for illustrative purposes only. From the above discussion and these examples, those skilled in the art will be able to grasp the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Thus, various modifications of the present invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims.

[0222] Example 1: Blocking Siglec-9 interactions enhances NK cell cytotoxicity against SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters the immunological profile of natural killer (NK) cells, effector cells that play a key role in controlling viral infection. However, it remains unclear whether anti-SARS-CoV-2 NK functions are impaired in severe COVID-19 and what host factors regulate these functions. The results presented herein demonstrate that NK cells from hospitalized COVID-19 patients exhibit significantly lower direct cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) activity against SARS-CoV-2 antigen-expressing cells than NK cells from mild COVID-19 patients or negative controls. Lower NK cell cytotoxicity was associated with elevated plasma concentrations of SARS-CoV-2 nucleocapsid antigen. Detailed phenotypic and functional analyses revealed that NK cells expressing the glycoprotein-immune checkpoint Siglec-9 inhibit Siglec-9. - Consistently, Siglec-9 elicited higher SARS-CoV-2-specific ADCC than NK cells. + NK cells express Siglec-9 - CD56 dimCompared with NK cells, Siglec-9-expressing NK cells exhibited an activated and mature phenotype, with higher expression of CD16 (FcγRIII; a mediator of ADCC), CD57 (a maturation marker), and NKG2C (an activating receptor), and lower expression of the inhibitory receptor NKG2A. These data are consistent with the notion that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic to SARS-CoV-2. However, the Siglec-9 molecule itself is a well-established inhibitory receptor for suppressing NK cytotoxicity during cancer and other viral infections. Indeed, blocking Siglec-9 with an anti-Siglec-9 antibody significantly enhanced ADCC-mediated NK degranulation and lysis of SARS-CoV-2 antigen-positive target cells. These data support the notion that Siglec-9 expression is a potent inhibitor of NK cell cytotoxicity. + CD56 dim These findings support a model in which NK cell subpopulations, even when suppressed by Siglec-9 inhibitors, exhibit cytotoxicity against SARS-CoV-2+ cells. However, using Siglec-9-blocking antibodies to alleviate Siglec-9-mediated restrictions on NK cell cytotoxicity can further improve anti-SARS-CoV-2 NK immune surveillance. Collectively, the results presented herein identify a novel glyco-immune checkpoint mechanism that contributes to the ability of SARS-CoV-2+ cells to evade NK immune surveillance and suggest potential strategies to overcome this evasion.

[0223] The data presented here combine patient data with in vivo and in vitro immunological data to identify and characterize a novel targetable glyco-immune checkpoint mechanism during SARS-CoV-2 infection. It is described herein that NK cells from hospitalized patients with SARS-CoV-2 are less able to target SARS-CoV-2 than NK cells from patients with mild SARS-CoV-2 disease and uninfected controls. Siglec-9 against SARS-CoV-2 + CD56 dim Cytotoxicity of NK cell subpopulations is reduced by the Siglec-9 molecule, and blocking Siglec-9 enhances the ability of NK cells to target cells expressing SARS-CoV-2 antigens. + CD56 dimNK cell subpopulations express Siglec-9 - CD56 dim Compared with NK cell subpopulations, they are highly activated, mature, and cytotoxic against SARS-CoV-2 antigen-expressing cells. The results described here point to Siglec-9 / sialoglycan interactions as a novel immunotherapeutic target in the carbohydrate-immune checkpoint mechanism, which may contribute to SARS-CoV-2's ability to evade NK immune surveillance.

[0224] The experimental results are described below.

[0225] NK cells from hospitalized COVID-19 patients degranulate less against target cells expressing the SARS-CoV-2 spike than NK cells from mild COVID-19 patients and uninfected controls To investigate whether severe COVID-19 impairs the anti-SARS-CoV-2 function of NK cells, peripheral blood mononuclear cells (PBMCs) and plasma were collected from 79 patients with three COVID-19 disease states: 1) SARS-CoV-2-negative controls (negative; n = 12), COVID-19 outpatients (mild; n = 26), and hospitalized COVID-19 patients (hospitalized; n = 41) (Table 1). First, we evaluated the anti-SARS-CoV-2-specific direct cytolytic and ADCC activities of NK cells using samples collected from a subset of these patients with sufficient PBMCs (negative; n = 8, mild; n = 12, and hospitalized; n = 21). NK cells have the potential to be multifunctional: they can directly lyse target cells by releasing cytolytic granules; and they can secrete cytokines and chemokines, such as IFN-γ and TNF-α (Cooper MA, et al., 2001, Trends Immunol 22:633-40). The multifunctionality of NK cells has been associated with enhanced antiviral immune responses (Kamya P, et al., 2011, J Virol 85:5949-60). Therefore, direct cytotoxicity and ADCC activity, both mediated by NK degranulation (expression of CD107a) and cytokine production (expression of IFN-γ and TNF-α), were assessed against SARS-CoV-2 spike-expressing 293T target cells by flow cytometry (see Figure 2A for gating strategy). Direct cytotoxicity was calculated by subtracting background NK degranulation / cytokine production from PBMCs cultured alone from that observed when PBMCs were co-cultured with target cells (Figure 1A, left). To assess ADCC, bulk IgG was isolated from the plasma of study participants. IgG from SARS-CoV-2-negative donors was pooled to create a negative antibody pool, and IgG from SARS-CoV-2-positive donors was pooled to create a positive antibody pool. IgG was pooled to ensure consistent quantitative and qualitative characteristics of the antibodies used in the ADCC assay. Consistent levels of SARS-CoV-2-specific antibodies allowed us to examine the ADCC capacity of NK cells from different donors without the potentially confounding effects of different levels or properties of SARS-CoV-2-specific antibodies.NK degranulation and cytokine production due to ADCC were measured by co-culturing PBMCs with target cells in the presence of either a negative or positive antibody pool. ADCC was then assessed by subtracting the NK degranulation and cytokine production in the co-culture with the negative antibody pool from the NK degranulation and cytokine production in the co-culture with the positive antibody pool (after subtracting background NK degranulation and cytokine production) (Figure 1A, right).

[0226] First, we investigated the CD56 expression of SARS-CoV-2 spike-expressing cells in hospitalized COVID-19 patients. dimWe found that direct cytolytic degranulation / cytokine production (Figures 1B-1E, 2B-2D) and ADCC-mediated degranulation (Figures 1F-1I, 2E-2G) of NK cells were lower than those of NK cells from mildly ill or SARS-CoV-2-negative controls. Subanalysis revealed that NK cells from female participants exhibited higher cytotoxicity than those from male participants, suggesting gender differences in NK activity against SARS-CoV-2 spike-expressing targets (Figure 3). We next investigated whether the reduced degranulation / cytokine production observed in NK cells from hospitalized COVID-19 donors resulted in reduced lysis of SARS-CoV-2 spike-expressing target cells. These experiments used PBMCs (as effector cells) from hospitalized or mildly ill COVID-19 patients and SARS-CoV-2 S CHO-K1 cells (as target cells). SARS-CoV-2 S CHO-K1 cells stably express the SARS-CoV-2 spike (S) protein and HaloTag-HiBit protein. When these cells are lysed by ADCC, the intracellular HaloTag-HiBit protein interacts with an extracellular detection reagent to generate a luminescent signal, which can be quantified to measure target cell lysis. As shown in Figure 4A, PBMCs from hospitalized COVID-19 donors exhibited lower ADCC than PBMCs from mild COVID-19 donors, consistent with the NK degranulation / cytokine production data. Finally, we examined NK cytotoxicity in three COVID-19 pathologies against cells (K562 cells) that do not express the SARS-CoV-2 spike protein. NK cells from hospitalized COVID-19 patients were found to degranulate less against K562 cells than NK cells from mild COVID-19 patients and SARS-CoV-2 negative controls (Figures 4B–F). These data suggest that NK cells from hospitalized COVID-19 patients have an overall reduced cytotoxic activity.

[0227] Next, we investigated whether ex vivo NK degranulation correlated with in vivo disease severity markers. The amount of SARS-CoV-2 plasma nucleocapsid (N) antigen in plasma correlates with disease severity (Wang H, et al., 2021, Clin Chem 68:204-213; Yokoyama R, et al., 2021, Front Microbiol 12:791-489; Shan D, et al., 2021, Nat Commun 12:1931). Therefore, we measured N antigen levels in plasma samples using the ultrasensitive Simoa SARS-CoV-2 N protein assay and found a correlation between N antigen levels and NK direct cytolytic and ADCC activities. As shown in Figures 1J–1M, NK degranulation was inversely correlated with SARS-CoV-2 N antigen levels. Collectively, these data suggest that NK degranulation in response to SARS-CoV-2 is suppressed in severe COVID-19. Therefore, developing strategies to enhance anti-SARS-CoV-2 NK cytotoxicity may improve disease outcomes. However, developing such strategies requires a deeper understanding of the factors that determine NK function against SARS-CoV-2.

[0228] Siglec-9 + CD56 dim NK cells bind Siglec-9 to SARS-CoV-2 spike-expressing target cells. - CD56 dim Exhibits greater antibody-mediated cytotoxicity than NK cells To clarify the molecular mechanisms involved in NK cell cytotoxicity against SARS-CoV-2, we investigated the role of Siglecs (Siglec-9 and Siglec-7) expressed on NK cells in establishing direct cytolytic or ADCC activity of NK cells against SARS-CoV-2 spike-expressing target cells. + CD56 dim NK cells or Siglec-9 - CD56 dimThe experimental data in Figure 1 were used after gating on either NK cells. There was no significant difference in the direct cytolytic activity of these two subpopulations, but regardless of the pathology, Siglec-9 expression was significantly higher. + CD56 dim NK cells express Siglec-9 - CD56 dim They showed significantly higher ADCC against target cells than NK cells (Figures 5A-5I). This was consistently observed when evaluating the proportion of cells expressing CD107a, IFN-γ, and TNF-α (Figures 5A-5C), or cells co-expressing CD107a and IFN-γ, CD107a and TNF-α, or IFN-γ and TNF-α (Figures 5D-5F) in each of the two subpopulations. The results were also consistent when the mean fluorescence intensity (MFI) of IFN-γ, IFN-γ, and TNF-α was evaluated in each of the two subpopulations (Figures 5G-5I). Next, Siglec-9 + CD56 dim and Siglec-9 - CD56 dim The direct cytolytic activity or ADCC activity of the subpopulations was examined, measured ex vivo and in vivo with plasma N antigen levels (Figure 5J). The direct cytolytic activity of both subpopulations was negatively correlated with plasma N antigen levels, and Siglec-9 was negatively correlated with plasma N antigen levels. + We show that NK cell subpopulations likely do not play a unique role in direct anti-SARS-CoV-2 cytotoxicity. However, Siglec-9 + The ADCC activity of IgG1 showed a negative correlation with plasma N antigen levels, whereas Siglec-9 - CD56 dim This was not the case in NK cells (Fig. 5J). These data support the idea that Siglec-9 + CD56 dim These findings suggest that NK cells are a subpopulation of NK cells with potentially high anti-SARS-CoV-2 ADCC activity.

[0229] Siglec-7 + CD56 dim NK cells express Siglec-7 against SARS-CoV-2 spike-expressing target cells. - CD56 dim They exhibit higher direct cytotoxicity and ADCC than NK cells. Siglec-7 + CD56 dim NK cells and Siglec-7 - CD56 dim The direct cytotoxic activity and ADCC activity of NK cells were evaluated (Figure 6). + CD56 dim Unlike NK cells, Siglec-7 + CD56 dim NK cells express Siglec-7 against SARS-CoV-2 spike-expressing target cells. - It showed higher direct cytolytic activity than its counterpart, as estimated by the percentage of cells expressing CD107a, TNF-α, and CD107a and TNF-α (Figures 6A-C). + CD56 dim NK cells express Siglec-7 - CD56 dim The SARS-CoV-2-specific ADCC was higher than that of NK cells, which were expressed by cells expressing CD107a, IFN-γ, and TNF-α (Figures 6D-6F), or cells co-expressing CD107a and IFN-γ, CD107a and TNF-α, or IFN-γ and TNF-α. + These data were consistent when examining the MFI of CD107a, IFN-γ, and TNF-α (Figure 7). These cytolytic activities were also associated with Siglec-7. - CD56 dim The activity of the NK cell subpopulation correlated more strongly with a decrease in plasma N antigen levels (Fig. 6J). These data support the conclusion that Siglec-7 + CD56 dim These data are consistent with the high direct cytolytic and ADCC activities of NK cell subpopulations. - This suggests that NK cell subpopulations are dysfunctional during SARS-CoV-2 infection.

[0230] Siglec-9 + CD56 dim NK cells exhibit an activated and mature phenotype in vivo. NK cell subpopulations expressing Siglec-9 or Siglec-7 exhibit higher cytotoxicity against SARS-CoV2-infected cells than Siglec-negative subpopulations. Therefore, we assessed the expression of multiple activating and inhibitory receptors on NK cells from the entire cohort (n=79). Measurements included Siglec-9 and Siglec-7. + , Siglec-9 - , Siglec-7 + , and Siglec-7 - CD56 dim These included the expression of CD16 (FcγRIII; a mediator of ADCC), CD57 (a maturation marker), NKG2C (an activating receptor), and NKG2A (an inhibitory receptor) on NK cells (Fig. 8 ).

[0231] Siglec-9 binds to CD56 dim Because it is expressed only on a subset of NK cells, we used qPCR to validate the specificity of a Siglec-9 antibody (clone K8, Biolegend) in identifying NK cell subpopulations with high levels of Siglec-9 transcripts. The data in Figure 9 show that this antibody identifies cells with higher levels of Siglec-9 transcripts compared to Siglec-9-negative NK cells. Subanalysis also showed that CD56 dim We also found that the expression of Siglec-7 and Siglec-9 on NK cells was gender-dependent, particularly in female participants, where the expression of CD56 dim NK cells expressed higher levels of Siglec-9 than cells from male participants. + cells, higher levels of Siglec-7 + cells, lower levels of Siglec-9 - Siglec-7 - cells, lower levels of Siglec-9 + Siglec-7 - cells, higher levels of Siglec-9 - Siglec-7 + cells and higher levels of Siglec-9+ Siglec-7 + expressing cells (Figure 10).

[0232] Consistent with previous reports (Varchetta S, et al., 2021, Cell Mol Immunol 18:604-612), CD56 in hospitalized COVID-19 patients dim NK cells were found to exhibit a decreased proportion of CD16-expressing cells (Fig. 11A), an increased proportion of CD57-expressing cells (Fig. 11B), and a decreased proportion of Siglec-7-expressing cells (Fig. 11C) compared to controls. + , Siglec-9 - , Siglec-7 + , and Siglec-7 - When we looked at the expression of these markers in cells, we found that Siglec-9 + CD56 dim NK cells express Siglec-9 - CD56 dim Compared to NK cells, they were found to exhibit an activated and mature phenotype, which is due to the Siglec-9 + Cells express Siglec-9 - This is because the expression of activation markers / receptors CD16, CD57, and NKG2C was higher and the expression of the inhibitory receptor NKG2A was lower than that of normal cells (Figure 12A). + CD56 dim The maturation / activation state of cells is determined by Siglec-7 - CD56 dim The effect was not clear compared to NK cells. + CD56 dim Cells express Siglec-7 - CD56 dim Although the expression levels of CD16 and NKG2C were higher in the IL-16+ / IL-2+ / IL-1 ... + CD56 dimThese results suggest that the cells represent an activated and mature NK cell subpopulation, which may explain their high ADCC activity against SARS-CoV-2-spike-expressing target cells.

[0233] Siglec-9 binds CD56 with high ADCC activity against SARS-CoV-2 dim Marks NK cells but not Siglec-7. The data in Figures 5 and 6 are from Siglec-9 + and Siglec-7 + D56 dim These results suggest that both Siglec-9 and Siglec-7 NK cells exhibit high ADCC activity against SARS-CoV-2. However, Siglec-9 and Siglec-7 are not mutually exclusive on NK cells, and there is a population of NK cells that express both Siglec-9 and Siglec-7 (Figure 13A). To investigate whether NK cells expressing Siglec-9 and / or Siglec-7 have higher ADCC activity against SARS-CoV-2 than NK cells expressing only Siglec-9 or Siglec-7, we reanalyzed the data. The in vivo phenotypic data shown in Figure 4 were reanalyzed to identify four possible CD56 dim NK cell subpopulation (Siglec-9 - Siglec-7 - , Siglec-9 - Siglec-7 + , Siglec-9 + Siglec-7 - , and Siglec-9 + Siglec-7 + The expression of CD16, CD57, NKG2C, and NKG2A was examined in each of the 13B to 13E. + These results show that the cells express high levels of CD16, CD57, and NKG2C, and low levels of NKG2A, regardless of Siglec-7 expression. + The cells express high levels of CD16, but not CD57 or NKG2C, regardless of Siglec-9 expression. +The cells express high levels of the inhibitory receptor NKG2A. These data suggest that Siglec-9, but not Siglec-7, marks cells with an activated and mature phenotype.

[0234] To examine the ADCC potential of each of the four NK cell subpopulations, the data in Figures 5 and 6 were reanalyzed. The data in Figures 13F to 13H show that the ADCC potential of CD56 NK cells expressing Siglec-9 was significantly higher than that of the control group. dim These data suggest that NK cells, regardless of Siglec-7 expression, have higher ADCC activity against SARS-CoV-2 compared with cells that do not express Siglec-9. Collectively, these data suggest that Siglec-9-expressing NK cells exhibit an activated and mature phenotype, which may contribute to their high ADCC against SARS-CoV-2 spike-expressing target cells.

[0235] Blocking the interaction of Siglec-9 with a Siglec-9 blocking antibody inhibits CD56 dim Enhanced anti-SARS-CoV-2 ADCC of NK cells. Data suggest that NK cells expressing Siglec-9 exhibit high ADCC activity against SARS-CoV-2. However, the Siglec-9 molecule itself is an inhibitory receptor, which functions as a glyco-immune checkpoint that limits NK cytotoxicity (Adeniji OS, et al., 2021, PLoS Pathog 17:e1010034; Jandus C, et al., 2014, J Clin Invest 124:1810-20; Zhao D, et al., 2018, Front Immunol 9:1124). Therefore, we used our in-house Siglec-9 blocking antibody to block Siglec-9 inhibitory signaling, thereby inhibiting Siglec-9 activity against SARS-CoV-2. +We further examined whether the ADCC activity of NK cells could be enhanced. We examined the effect of Siglec-9 blocking antibodies on ADCC-mediated NK degranulation against SARS-CoV-2 compared to isotype controls (Figures 14A-14E). These experiments used PBMCs from six healthy controls (as effector cells) and SARS-CoV-2 spike-expressing 293T cells (as target cells). Blocking Siglec-9 significantly increased the expression of CD107a. + (Fig. 14A), CD107a + IFN-γ + (Fig. 14B), CD107a + TNF-α (Fig. 14C), TNF-α+ (Fig. 14D), or IFN-γ+TNF-α + (Figure 14E) CD56 dim CD56 as evidenced by an increased proportion of NK cells dim The ADCC ability of NK cells was significantly enhanced.

[0236] Further experiments determined whether the enhanced NK degranulation / cytokine production caused by Siglec-9 blockade would lead to increased lysis of target cells. These experiments used purified NK cells isolated from PBMCs of five healthy donors (as effector cells) and SARS-CoV-2 S CHO-K1 cells (as target cells). As shown in Figure 14F, blocking Siglec-9 enhanced ADCC-mediated lysis of target cells compared to the isotype control. These data suggest that Siglec-9 interaction mediates the cytotoxicity of already highly activated Siglec-9 cells. + These results demonstrate that it indeed limits the ADCC activity of NK cells, suggesting that strategies targeting Siglec-9 may enhance NK cell cytotoxicity against SARS-CoV-2.

[0237] Previous reports have associated severe COVID-19 with altered NK cell profiles (Varchetta S, et al., 2021, Cell Mol Immunol 18:604-612). However, whether these changes lead to impaired anti-SARS-CoV-2 NK cytotoxicity has not been fully investigated. In this report, we performed functional assays to examine both direct cytolysis and ADCC-mediated degranulation of NK cells in hospitalized COVID-19 patients and controls. The data suggest that severe COVID-19 is associated not only with altered NK cell phenotypic profiles but also with impaired cytotoxic and ADCC activities. Although the mechanisms behind these dysfunctions are unknown, severe COVID-19 is associated with a subsequent cytokine storm (Blanco-Melo D, et al., 2020, Cell 181:1036-1045 e9; Guan WJ, et al., 2020, N Engl J Med 382:1708-1720) and a hyperinflammatory state characterized by dysregulated myeloid cell function (Junqueira C, et al., 2022, Nature 606:576-584; Knoll R, et al., 2021, Front Immunol 12:720109). NK cell function can be significantly regulated by the cytokine environment (Brady J, et al., 2010, J Immunol 185:6679-88; Romee R, et al., 2014, Scientifica (Cairo) 2014:205796; Zwirner NW, et al., 2010, Biofactors 36:274-88; Zwirner NW, et al., 2017, Front Immunol 8:25) and interactions with myeloid cells (Knoll R, et al., 2021, Front Immunol 12:720109).Further research is needed to determine whether dysregulated cytokine secretion, such as TGF-β (Barros-Martins J, et al., 2022, Signal Transduct Target Ther 7:32), or myeloid cell dysfunction contributes directly or indirectly to decreased NK function during severe COVID-19. It is also unclear whether decreased NK function is a causal or consequential factor in the progression of COVID-19. To explore this potential relationship, studies using animal models of SARS-CoV-2 infection are needed. However, regardless of whether these functions contribute to disease severity, the likely decreased anti-SARS-CoV-2 cytolytic activity of NK cells during severe COVID-19 suggests the need to develop strategies to enhance NK function during severe SARS-CoV-2 and other similar emerging virus infections, thereby controlling infection and reducing disease severity. These strategies will be particularly important for immunocompromised patients who may require immunotherapeutic approaches to help control viral infections (Abbasi J., 2021, JAMA 326:2250; Goldman JD, et al., 2021, J Immunother Cancer 9; Helleberg M, et al., 2020, J Infect Dis 222:1103-1107; Lee A, et al., 2022, BMJ 376:e068632; Rahav G, et al., 2021, EClinicalMedicine 41:101158; Rubin EJ, et al., 2022, N Engl J Med 386:e71; Rubin R., 2022, JAMA 327:1853-1855).

[0238] Identification of NK cell subpopulations capable of targeting virus-infected cells may be an essential step in developing efficient strategies to enhance NK cytotoxicity against SARS-CoV-2 and other viral infections. In this report, we focused on NK cells expressing Siglec-7 and / or Siglec-9. Siglecs are emerging ITIM-containing, MHC-independent inhibitory receptors that regulate host immune responses by interacting with sialoglycans on the surface of target cells. Siglec-7 is expressed on almost all NK cells and binds α2-8 sialic acid, while Siglec-9 binds CD56. dim It is selectively expressed on a subset of NK cells and binds to α2-3 sialic acid (Adeniji OS, et al., 2021, PLoS Pathog 17:e1010034; Belisle JA, et al., 2010, Mol Cancer 9:118). Siglec-7 accumulates during the severe stages of COVID-19. - CD56 dim A subpopulation of NK cells was identified as a dysfunctional NK cell subpopulation during SARS-CoV-2 infection. This is consistent with previous reports that described reduced Siglec-7 levels as a marker of dysfunctional NK cells during HIV infection (Brunetta E, et al., 2009, Blood 114:3822-30; Varchetta S, et al., 2013, Retrovirology 10:154; Zulu MZ, et al., 2017, AIDS Res Hum Retroviruses 33:1205-1213). In addition to Siglec-7, Siglec-9, which has not previously been implicated in SARS-CoV-2 infection, was also identified. + CD56 dim A subpopulation of NK cells was also identified as a highly cytotoxic NK cell subpopulation, consistent with previous reports that this subpopulation exhibits enhanced antiviral activity during HIV infection (Adeniji OS, et al., 2021, PLoS Pathog 17:e1010034).

[0239] Siglec-9 + CD56dim NK cells have an activated phenotype (high expression of activating receptors and low expression of inhibitory receptors) during cancer (16), HBV infection (Zhao D, et al., 2018, Front Immunol 9:1124), and HIV infection (Adeniji OS, et al., 2021, PLoS Pathog 17:e1010034). Indeed, Siglec-9 + CD56 dim NK cells express Siglec-9 during SARS-CoV-2 infection. - CD56 dim Compared to NK cells, these cells exhibited an activated phenotype with higher levels of activation / maturation receptors and markers and lower expression of the inhibitory receptor NKG2A. Based on these results, these cells also exhibit an activated phenotype in healthy individuals, suggesting that this cell population is naturally activated, has potential cytotoxicity, and can be utilized against multiple viral and non-viral infections.

[0240] Siglec-9 + CD56 dim The highly activated phenotype of NK cells is mediated by Siglec-9 + NK cells express Siglec-9 - These results are consistent with the results of functional analysis showing that Siglec-9 exhibits higher ADCC than NK cells. + This is consistent with the high cytotoxicity of NK cells. However, the Siglec-9 receptor itself does not bind to these naturally highly cytotoxic Siglec-9 receptors. +Siglec-9 is an inhibitory receptor that suppresses the cytotoxic activity of NK cells. Binding of Siglec-9 to α2-3 sialic acid on target cells induces an inhibitory signaling cascade by recruiting the tyrosine phosphatase SHP-1, which prevents phosphorylation-mediated activation of other signaling molecules (Crocker PR, et al., 2007, Nat Rev Immunol 7:255-66; Avril T, et al., 2004, J Immunol 173:6841-9). Indeed, blocking Siglec-9 further enhanced the ability of NK cells to kill SARS-CoV-2 antigen-expressing target cells by ADCC. This result is consistent with the known inhibitory function of the Siglec-9 molecule itself against these originally cytotoxic cells. These data support the role of Siglec-9 in the suppression of NK cell death. + CD56 dim These data support a model in which NK cells are cytotoxic but are suppressed by the inhibitory properties of Siglec-9 receptor signaling (Figure 15, left two panels). Furthermore, these data suggest that blocking the interaction with Siglec-9 may reduce the otherwise highly cytotoxic Siglec-9 receptor signaling. + This suggests that this is a promising strategy to unlock the maximum potential of NK cell subpopulations (Figure 15, right panel).

[0241] In this study, viral infection was shown to inhibit the cytotoxicity of Siglec-9. +We focused on the Siglec-9 / sialic acid axis as a potential carbohydrate-immune checkpoint mechanism for evading immune surveillance by NK cells. We investigated the possibility that Siglec-9 blocking antibodies could enhance anti-SARS-CoV-2 ADCC activity. Indeed, in a proof-of-concept experiment, we found that blocking Siglec-9 using a blocking antibody enhanced the anti-SARS-CoV-2-specific ADCC activity of NK cells in vitro. Over the past few years, several Siglec-blocking antibodies have been tested for their ability to prevent Siglec-mediated inhibition of immune function. Blocking antibodies against Siglec-7 and Siglec-9 enhance antitumor immune activity both in vitro and in vivo (Jandus C, et al., 2014, J Clin Invest 124:1810-20; Hudak JE, et al., 2014, Nat Chem Biol 10:69-75; Beatson R, et al., 2016, Nat Immunol 17:1273-1281; Stanczak MA, et al., 2018, J Clin Invest 128:4912-4923; Ibarlucea-Benitez I, et al., 2021, Proc Natl Acad Sci USA 118; Choi H, Ho M, et al., 2021, Front Oncol 11:778989). Similarly, blocking Siglec-9 interactions enhanced NK cell cytotoxicity against HIV-infected targets (Adeniji OS, et al., 2021, PLoS Pathog 17:e1010034) and reversed NK cell dysfunction during HBV infection (Zhao D, et al., 2018, Front Immunol 9:1124). These blocking antibodies are promising tools for enhancing NK cytotoxicity against virus-infected cells.However, Siglecs are also expressed on other immune cells, including myeloid cells (Kamya P, et al., 2011, J Virol 85:5949-60; Choi H, Ho M, et al., 2021, Front Oncol 11:778989; Schwarz F, et al., 2015, Elife 4), and play an important role as an immune checkpoint against excessive inflammation and autoimmunity (Schwarz F, et al., 2015, Elife 4; Varki A, et al., 2012, Ann NY Acad Sci 1253:16-36). Therefore, monospecific antibodies that block Siglecs may induce nonspecific inflammation. For example, it has recently been shown that Siglec-9 interaction on neutrophils plays an important role in regulating inflammation during COVID-19 (Delaveris CS, et al., 2021, ACS Cent Sci 7:650-657). Therefore, to take advantage of the potential positive effects of Siglec blocking on enhancing anti-SARS-CoV-2 NK immune function while avoiding nonspecific inflammatory side effects, bispecific antibodies that target specific immune cells (NK cells) and / or virus-infected cells may be required.

[0242] This study identified Siglec-9 as an NK cell subpopulation that could be used to develop new immunotherapeutic tools against SARS-CoV-2-infected cells. + CD56 dim This is the first description of NK cells.

[0243] The materials and methods are described below.

[0244] Study cohort characteristics PBMCs and plasma from 67 patients who tested positive for SARS-CoV-2 (PCR) and 12 negative controls were used. The 67 SARS-CoV-2-positive patients were either outpatients (mild symptoms; n = 26) or hospitalized patients (hospitalized; n = 41) (Table 1). Samples from hospitalized patients were collected at the time of admission. [Table 1]

[0245] Direct cytotoxicity assay against SARS-CoV-2 spike-expressing 293T cells. Frozen PBMCs were thawed in complete growth medium (RPMI containing 10% FBS) and allowed to stand overnight. PBMCs (1 × 10 6 ) were cultured in complete growth medium with spike-expressing 293T (S-293T) target cells (1 × 10 5 The cells were co-cultured with 100% BD Biosciences cells at an effector-to-target (E:T) ratio of 10:1 in the presence of GolgiStop (BD Biosciences) and anti-CD107a PE antibody (BD Biosciences). The co-cultured cell mixture was then pelleted by centrifugation at 200 × g for 2 minutes and incubated at 37°C for 16 hours. After incubation, cells were stained for the following surface markers: CD56 (APC-Cy7, Biolegend), CD3 (Alexa-488, BD Biosciences), Siglec-9 (APC, Biolegend), or Siglec-7 (Alexa-700, Biolegend). Cells were washed twice, fixed (using Cytofix / Cytoperm, BD Biosciences), and permeabilized (using Perm / Wash buffer, BD Biosciences). After permeabilization, cells were stained intracellularly for IFN-γ (BV421, BD Biosciences) and TNF-α (PE-Dazzle594, Biolegend). At least 100,000 events were acquired by flow cytometry using a BD Biosciences LSR II flow cytometer. Cytotoxic NK cells were identified by CD3 - and CD56 dim Direct cytotoxicity was calculated by subtracting background NK degranulation / cytokine production in PBMC monocultures from NK degranulation / cytokine production in PBMC cocultures with target cells (Fig. 1A, left).

[0246] ADCC assay against 293T cells expressing the SARS-CoV-2 spike. IgG was isolated from donor plasma using the Pierce Protein G Spin Plate for IgG (Thermo Scientific) kit. Purified IgG was quantified using NanoDrop (A280 absorbance). Equal concentrations of purified IgG from SARS-CoV-2-negative donors were pooled to obtain a negative pool. Equal concentrations of purified IgG from SARS-CoV-2-positive donors were pooled to obtain a positive pool. ADCC assays were performed similarly to the direct cytotoxicity assays, except that target cells were preincubated (15 min) with either the negative or positive pool (10 μg per well) before co-culture with PBMCs from each donor. ADCC activity was then assessed by subtracting the NK degranulation activity in co-cultures with the negative antibody pool from the NK degranulation activity in co-cultures with the positive antibody pool (after subtracting background NK degranulation activity) (Figure 1A, right).

[0247] Target cell lysis of spike-expressing CHO-K1 cells using PBMCs Target cell lysis was performed using Promega's HaloTag-HiBit ADCC kit according to the manufacturer's instructions. Briefly, cryopreserved PBMCs and spike-expressing CHO-K1 cells were thawed and incubated overnight. After incubation, CHO-K1 target cells were incubated with either a SARS-CoV-2-positive or SARS-CoV-2-negative IgG pool at a concentration of 0.5 μg / well. After 15 min, PBMC cells (2.5 × 10 4 Effector cells (2,500 cells) and CHO-K1 target cells (2,500 cells) were cocultured for 5 hours in complete growth medium at an effector-to-target (E:T) ratio of 10:1. After 5 hours, substrate was added, and luminescence was measured after an additional 10 minutes. Specific target cell lysis was calculated by subtracting the luminescence value of each SARS-CoV-2 IgG-negative pool from the luminescence value of the SARS-CoV-2 IgG-positive pool for each donor.

[0248] Direct cytotoxicity assay against K562 cells. Frozen PBMCs were thawed in complete growth medium (RPMI containing 10% FBS) and allowed to stand overnight. PBMCs (1 × 10 6cells) to K562 target cells (2 × 10 5 PBMCs were co-cultured at an E:T ratio of 5:1 in the presence of GolgiStop (BD Biosciences) and anti-CD107a PE antibody (BD Biosciences). The co-cultured cell mixture was pelleted by centrifugation at 200 × g for 2 minutes and incubated at 37°C for 3 hours. After incubation, cells were stained with the same antibodies as above, fixed, permeabilized, and intracellularly stained for IFN-γ and TNF-α as described above. At least 100,000 events were acquired by flow cytometry using a BD Biosciences LSR II flow cytometer. Cytotoxicity was calculated by subtracting background NK degranulation / cytokine production in PBMC monocultures from NK degranulation / cytokine production in PBMC and target cell co-cultures.

[0249] Quantification of SARS-CoV-2 nucleocapsid (N) antigen The amount of SARS-CoV-2 N antigen in plasma was quantified using a single molecule array (Simoa) immunoassay on a Simoa HD-X analyzer (Quanterix) as previously described (Shan D, et al., 2021, Nat Commun 12:1931).

[0250] Siglec-7 + and Siglec-9 + CD56 dim Phenotypic characterization of NK cells Phenotypic characterization of Siglec-7 and Siglec-9 expressing NK cells was performed on cryopreserved PBMCs (n=79) from the study cohort by multiparameter flow cytometry. Briefly, cryopreserved PBMCs were thawed in prewarmed RPMI (RPMI 1640 medium; supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma), 1% penicillin-streptomycin (Lonza), and 2 mM L-glutamine (Sigma) (Mediatech)) and collected by centrifugation. Cells were then incubated with Ca ++ / Mg ++The cells were washed with DPBS-CMF (containing no HCl) and collected by centrifugation. Cells were then stained with the Aqua Live / Dead Cell Stain Kit (Invitrogen) to assess cell viability, washed with DPBS-CMF, and retained for cell surface staining. Next, the cells were incubated with a cocktail of fluorochrome-conjugated anti-human monoclonal antibodies (BD Biosciences' CD3 AF700, CD19 AF700, CD14 AF700, HLA DR APC-H7, CD56 PE-Cy7, CD16 BV605, CD57 FITC, CD38 PE-CF594, NKG2A BB700, NKG2C BV786, and CD161 (BV421)) mixed with Siglec-9 APC and Siglec-7 PE (Biolegend). Cells were washed with FACS buffer (DPBS-CMF + 0.5% BSA + 0.1% sodium azide) and then fixed with 1% paraformaldehyde (PFA, Polysciences) before acquisition on an LSRFortessa SORP flow cytometer (BD Biosciences). Data were analyzed using FlowJo software version 9.9.6 (Treestar Inc.).

[0251] CD56 based on Siglec expression dim Sorting of NK cells and measurement of relative copy number of Siglec-9 mRNA using qPCR. Approximately 10 million primary NK cells were negatively selected from PBMCs isolated from three healthy donors using the Human EasySep NK Isolation Kit (StemCell Technologies) according to the manufacturer's instructions. Cells were then stained for CD56 (APC-Cy7; Biolegend), CD3 (Alexa-488; BD Biosciences), and Siglec-9 (APC; Biolegend). - CD56 dim NK cells were classified into three populations: no Siglec-9 expression, low Siglec-9 expression, and high Siglec-9 expression, using a FACSSymphony S6 SE (FACSAria II) (Fig. 9).

[0252] Sorted cells were lysed, and total RNA was extracted using the RNAeasy Mini Kit (QIAGEN) with on-column DNase treatment (QIAGEN) according to the manufacturer's instructions. cDNA was generated using SuperScript VILO MasterMix (Invitrogen) according to the manufacturer's instructions. The relative copy number of Siglec-9 transcripts was quantified in qPCR reactions containing 4 pmol of each Siglec-9-specific primer and probe (Life Technologies, Assay ID Hs00534924_ml), 10 μL of 2× TaqMan Universal Master Mix (Applied Biosystems), and 5 μL of diluted cDNA. Reactions were performed on a QuantStudio 6 Flex real-time PCR system (Applied Biosystems) using the following cycling conditions: 50°C for 2 minutes, 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Data were normalized using a eukaryotic 18S rRNA endogenous control (Applied Biosystems) as a housekeeping gene. Relative copy numbers were measured using the comparative Ct method (Schmittgen TD, et al., 2008, Nat Protoc 3:1101-8).

[0253] Generation and characterization of human Siglec-9 blocking antibodies. Transgenic H2L2 mice encoding the human immunoglobulin repertoire (Harbor BioMed, Cambridge, MA) were used for immunization (Widjaja I, et al., 2019, Emerg Microbes Infect 8:516-530). Immunization and antibody sequencing were performed similarly to a detailed protocol recently reported using transgenic H2L2 mice (Duty JA, et al., 2022, Med (NY) 3:705-721 e11). Briefly, mice were immunized with 50 μg of DNA encoding human Siglec-9, administered twice at 2-week intervals. Mice then received two booster immunizations at 2-week intervals; the first booster contained Siglec-9 DNA, and the second booster contained 50 μg of purified recombinant human Siglec-9 protein (R&D Systems). The mouse SP2 / 0-Ag14 (SP2 / 0) myeloma cell line was used to generate hybridomas by chemical fusion with spleen cells from immunized mice. After confirming antibody binding using ELISA, mouse spleen cells were used to generate hybridomas, and the antibodies were sequenced as described in detail recently (49, 55).

[0254] For recombinant expression in mammalian cells, the antibody constructs were cloned into the pCDNA3.4 expression vector. A gene construct encoding full-length IgG was designed (GenScript) and expressed by transient production in HEK293 cells in serum-free suspension culture. The reactivity and specificity of the recombinant anti-Siglec-9 antibodies were examined by enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated overnight at 4°C with human recombinant Siglec-9 protein (1 μg / ml) (R&D Systems), human recombinant Siglec-7 protein (1 μg / ml, as a negative control) (R&D Systems), or HIV gp120 protein (1 μg / ml, as a negative control). After washing with PBS and blocking with 3% BSA, purified anti-Siglec-9 antibodies were added at various dilutions and incubated at room temperature for 1 hour. The wells were then washed and incubated with goat anti-mouse secondary antibody, followed by detection with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The reaction was terminated by adding 1 M H2SO4, and the absorbance was measured at 450 nm using an ELISA reader (Figure 16).

[0255] ADCC-mediated NK degranulation assay in the presence of Siglec-9 blocking antibody Cryopreserved PBMCs from six healthy donors were thawed, incubated overnight, and then incubated with an in-house prepared Siglec-9 blocking antibody (0.5 μg / well) for 15 min. S-293T target cells (1 × 10 5 PBMCs (1 × 10 cells / well) were incubated with the positive or negative pool (0.5 μg / well). After 15 min, pretreated PBMCs (1 × 10 cells / well) were added to the positive or negative pool (0.5 μg / well). 6 ) and S-293T cells were co-cultured at an effector-to-target ratio of 10:1, and degranulation was examined as previously described.

[0256] Isolation of human NK cells and target cell lysis in the presence of Siglec-9 blocking antibodies NK cells were isolated from peripheral blood mononuclear cells (PBMCs) collected from five healthy donors by negative selection using the EasySep Human NK Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer's protocol. Target cell lysis was performed using Promega's HaloTag-HiBit ADCC kit according to the manufacturer's instructions. Briefly, isolated NK cells were incubated with Siglec-9 antibody (0.5 μg / well) for 15 minutes. CHO-K1 cells were also preincubated with either the positive or negative pool at 0.5 μg / well for 15 minutes. CHO-K1 cells (2,500 cells) and NK cells (1.25 × 10 cells) were then incubated. 4 (1 donor) were co-cultured for 5 hours at a 5:1 effector-to-target ratio in complete growth medium. After 5 hours, substrate was added, and luminescence was measured 10 minutes later. The luminescence values ​​for each donor in the positive pool were divided by the values ​​obtained from the respective negative pool to obtain the amount of specific target cell lysis.

[0257] statistical analysis For statistical analysis of Figures 1B-I, 2B-G, 4B-F, and 11, we used the Kruskal-Wallis test with Dunn's multiple comparison correction. For statistical analysis of Figures 1J-M, 5J, and 6J, we used Spearman's rank correlation. For statistical analysis of Figures 5A-I, 6A-I, 12 (comparison of cells within each disease group), and Figure 7, we used the Wilcoxon signed-rank test. For comparison of cells between different group conditions in Figure 4, we used the Mann-Whitney U test. We also used the Mann-Whitney U test for statistical analysis of Figures 3, 4A, and 10. For statistical analysis of Figures 13B-H, we used the Friedman test with Dunn's multiple comparison correction. For analysis of Figures 9 and 14, we used a paired t-test. Data were analyzed using Prism 9.0 (GraphPad Software).

[0258] Example 2: Sequence information HB9-4G4 heavy chain (SEQ ID NO: 1) GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGGGTGGGAGCTACAGGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA HB9-4G4 Heavy chain (SEQ ID NO: 2) EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGWELQDYYYYGMDVWGQGTTVTVSS HB9-4G4 Light chain (SEQ ID NO: 3) ATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA HB9-4G4 Light chain (SEQ ID NO: 4) IVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEIK HB9-6A8 heavy chain (SEQ ID NO: 5) CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCAGGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGT CCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGGGACACGGCTGTGTATTACTGTGCGAGAGGTGATTGTAGTGGTGGTAGCTGTCCTTACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA HB9-6A8 heavy chain (SEQ ID NO: 6) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGDCSGGSCPYWYFDLWGRGTLVTVSS HB9-6A8 Light chain (SEQ ID NO: 7) GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA HB9-6A8 Light chain (SEQ ID NO: 8) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK HB9-6F3 Heavy chain (SEQ ID NO: 9) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGAAGCCTCAGGATTCACCTTTAGAAACTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTCGTGGTAGTGGTAGTAGAACATACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGCGCGAAAGATGAGGGGTTCGGGGACTTATTAGCGCACTATGTTATGGATGCCTGGGGTCAAGGAGCTTCAGTCACTGTCTCCTCA HB9-6F3 heavy chain (SEQ ID NO: 10) EVQLLESGGGLVQPGGSLRLSCEASGFTFRNYAMSWVRQAPGKGLEWVSAIRGSGSRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDEGFGDLLAHYVMDAWGQGASVTVSS HB9-6F3 Light chain (SEQ ID NO: 11) GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCA GGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA HB9-6F3 Light chain (SEQ ID NO: 12) EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIK HB9-9B11 heavy chain (SEQ ID NO: 13) GAGGTGCAGTTGTTGGAGTCTGGGGGGGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAACTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTATGAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACGAATATAGCAGTGGCTGGTACCAATTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HB9-9B11 Heavy chain (SEQ ID NO: 14) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWVSAISMSGGSTYYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCAKDEYSSGWYQFDYWGQGTLVTVSS HB9-9B11 Light chain (SEQ ID NO: 15) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA HB9-9B11 Light chain (SEQ ID NO: 16) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK HB9-10B6 heavy chain (SEQ ID NO: 17) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTACATGGTATGATGAATATAATAATAAATACTATG CAGACTCCGTGAAGGCCGCTTCACCATCTCCAGAGACAATTCCAAGAACATGTTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTATGTATTACTGTGCGAGGAACGTATTACGATATTTTGACTGGACCCTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCG HB9-10B6 heavy chain (SEQ ID NO: 18) QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVTWYDEYNKYYADSVKGRFTISRDNSKNMLYLQMNSLRAEDTAMYYCARNVLRYFDWTLDYWGQGTLVTVSS HB9-10B6 Light chain (SEQ ID NO: 19) ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTATCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCCACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA HB9-10B6 Light chain (SEQ ID NO: 20) IVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK HB9-10E3 Heavy chain (SEQ ID NO: 21) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGCCGGTATAGCAGTGGCTGGGGGATGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA HB9-10E3 heavy chain (SEQ ID NO: 22) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGIAVAGGWYFDLWGRGTLVTVSS HB9-10E3 Light chain (SEQ ID NO: 23) ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTATCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGG GCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA HB9-10E3 Light chain (SEQ ID NO: 24) IVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKLEIK HB9-11B10 Heavy Chain (SEQ ID NO: 25) GAGGTGCAGTTGTTGGAGTCTGGGGGGGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAACTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTATGAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACGAATATAGCAGTGGCTGGTACCAATTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HB9-11B10 Heavy Chain (SEQ ID NO: 26) EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWVSAISMSGGSTYYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCAKDEYSSGWYQFDYWGQGTLVTVSS HB9-11B10 Light Chain (SEQ ID NO: 27) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAACAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCATCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCATTTTATTACTGTCAACAGCGTAGTAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA HB9-11B10 Light chain (SEQ ID NO: 28) EIVLTQSPATLSLSPGERATLSCRASQSVNSYLAWYQQKPGQAPRLLIYDASIRATGIPARFSGSGSGTDFTLTISSLEPEDFAFYYCQQRSNWPPTFGQGTKVEIK HB9-1F11 Heavy chain: (SEQ ID NO: 29) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGRIDPNSGGTKYNEKFKSKATLTVDKPSSTAYMQLSSLTSEDSAVYYCARYDYYGSSYFDYWGQGTTVTVSS HB9-1F11 Light chain: (SEQ ID NO: 30) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL HB9-4C10 Heavy chain: (SEQ ID NO: 31) EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGGINPNNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARNDGYRGYAMDYWGQGTSVTVSS HB9-4C10 Light chain: (SEQ ID NO: 32) DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYCCQQYYSNPPLTFGAGTKLELK HB9-5B7 Heavy chain: (SEQ ID NO: 33) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS HB9-5B7 Light chain: (SEQ ID NO: 34) DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGQVPKLLIYAASALQSGVPSRFSGSGSGTDFTLTITSLQPEDVATYYCHKYNSAPWTFGQGTEVEIK HB9-7B8 Heavy chain: (SEQ ID NO: 35) ELQLLESGGGLVQPGGSLRLSCAASGFTFTNYAMNWVRQAPGKGLEWVSAISGSGGRTYYADSVKGRFTISRDNSRNTLFLQMNSLRPEDTAVYYCAKDQTSGTTGYPYFAYWGQGTLVTVSS HB9-7B8 Light chain: (SEQ ID NO: 36) ETVMTQSPATLSVSPGERAILSCRASQSVSSNLVWYQQKPGQAPRLFIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPRTFGQGTKVEIK HB9-14D4 Heavy chain: (SEQ ID NO: 37) QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS HB9-14D4 Light chain: (SEQ ID NO: 38) DIQMTQSPSSLTASVGDRVTITCRASQSISSYVNWYQQKPGKAPKVLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIK HB9-14H12 Heavy chain: (SEQ ID NO: 39) QVQLKESGPGLVAPSQSLSITCTVSGFLLISNGVHWVRQPPGKGLEWLGVIWAGGNTNYNSALMSRVSISKDNSKSQVFLKMKSLQTDDTAMYYCARDFYDYDVFYYAMDYWGQGTSVTVSS HB9-14H12 Light chain: (SEQ ID NO: 40) QAVVTQESALTTSPGETVPLTCRSSTGTVTTSNFANWVQEKPDHLFTGLIGGTNNRAPGLPARFSGSLIGDKAALTITGAQTEDEAIFFCALWYSNHFVFGGGSQLTVL

[0259] HB9-14D4 Heavy chain CDR1 - SYGMH (SEQ ID NO: 41) Heavy chain CDR2 - VIWFDGTKKYYTDSVKG (SEQ ID NO: 42) Heavy chain CDR3 - DRGIGARRGPYYMDV (SEQ ID NO: 43) Light chain CDR1 - RASQSISSYVN (SEQ ID NO: 44) Light chain CDR2 - AASSLQS (SEQ ID NO: 45) Light chain CDR3 - QQYYSTPLT (SEQ ID NO: 46) [Table 2]

[0260] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it will be apparent that those skilled in the art may devise other embodiments and variations of the present invention without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. Therapeutic compositions comprising Siglec-9 inhibitors as adjuvants for enhancing immune responses to target antigens.

2. The therapeutic composition of claim 1, wherein the Siglec-9 inhibitor comprises an anti-Siglec-9 antibody.

3. 3. The therapeutic composition of claim 2, wherein the anti-Siglec-9 antibody comprises an amino acid sequence selected from one or more of the group consisting of: a) one or more variable heavy chain sequences of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, and 39; b) one or more variable light chain sequences of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, and 40; c) a sequence having at least 95% identity to one or more variable heavy chain sequences of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, and 39; d) a sequence having at least 95% identity to one or more variable light chain sequences of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, and 40; e) a fragment comprising at least 80% of the full-length sequence of one or more variable heavy chain sequences of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, and 39; and f) Fragments comprising at least 80% of the full-length sequence of one or more variable light chain sequences of SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, and 40.

4. 10. The therapeutic composition of claim 1, further comprising one or more additional antibodies targeting the antigen.

5. The therapeutic composition of claim 4, wherein the antigen is a viral antigen.

6. The therapeutic composition of claim 4, wherein the antigen is a SARS-CoV-2 antigen.

7. 7. The therapeutic composition of claim 6, wherein the therapeutic composition comprises a bispecific antibody comprising an inhibitory Siglec-9 antibody domain and a SARS-CoV-2 antigen-binding domain.

8. 10. The therapeutic composition of claim 1, comprising one or more inhibitory nucleic acid molecules specific for binding to Siglec-9 or a fragment thereof.

9. 10. The therapeutic composition of claim 1, comprising one or more mRNA molecules encoding a Siglec-9 inhibitor.

10. 10. The therapeutic composition of claim 1, comprising one or more DNA molecules encoding a Siglec-9 inhibitor.

11. 11. The therapeutic composition of claim 10, wherein the DNA molecule comprises one or more nucleotide sequences selected from the group consisting of: a) a nucleotide sequence encoding a variable heavy chain sequence selected from one or more of SEQ ID NOs: 2, 6, 10, 14, 18, 22, and 26; b) a nucleotide sequence encoding a variable light chain sequence selected from one or more of SEQ ID NOs: 4, 8, 12, 16, 20, 24, and 28; c) a nucleotide sequence having at least 95% identity to one or more variable heavy chain sequences of SEQ ID NOs: 2, 6, 10, 14, 18, 22, and 26; d) a nucleotide sequence having at least 95% identity to one or more variable light chain sequences of SEQ ID NOs: 4, 8, 12, 16, 20, 24, and 28; e) a fragment comprising at least 80% of the full-length sequence of one or more variable heavy chain sequences of SEQ ID NOs: 2, 6, 10, 14, 18, 22, and 26; and f) Fragments comprising at least 80% of the full-length sequence of one or more variable light chain sequences of SEQ ID NOs: 4, 8, 12, 16, 20, 24, and 28.

12. 12. The therapeutic composition of claim 10 or 11, further comprising one or more additional nucleic acid molecules comprising a nucleotide sequence encoding an antibody or fragment thereof that targets the antigen.

13. The therapeutic composition of claim 12, wherein the antigen is a viral antigen.

14. The therapeutic composition of claim 13, wherein the antigen is a SARS-CoV-2 antigen.

15. 12. The therapeutic composition of claim 10 or 11, comprising a nucleotide sequence encoding a bispecific antibody comprising an inhibitory Siglec-9 antibody domain and a SARS-CoV-2 antigen-binding domain.

16. A method of enhancing the effectiveness of immunotherapy in a subject in need thereof, comprising administering to said subject a therapeutic composition according to any one of claims 1 to 15.

17. 17. The method of claim 16, wherein the method increases the level of antibody-dependent cellular cytotoxicity (ADCC) activity against the target antigen.

18. A method for preventing or treating a disease or disorder associated with a viral infection in a subject, comprising administering to the subject a therapeutic composition according to any one of claims 1 to 15.

19. 19. The method of claim 18, wherein the viral infection is a SARS-CoV-2 infection.

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