EP4637822A2

EP4637822A2 - Siglec 9 inhibitors and methods of use thereof for enhancing immunotherapy efficacy

Info

Publication number: EP4637822A2
Application number: EP23908671.3A
Authority: EP
Inventors: Mohamed ABDEL-MOHSEN
Original assignee: Wistar Institute of Anatomy and Biology
Current assignee: Wistar Institute of Anatomy and Biology
Priority date: 2022-12-23
Filing date: 2023-12-23
Publication date: 2025-10-29
Also published as: JP2025542407A; CN121100001A; WO2024138213A3; AU2023411024A1; WO2024138213A2; KR20250127138A

Abstract

Disclosed herein are compositions comprising a Siglec-9 inhibitor, a fragment thereof, a variant thereof, or a combination thereof, and methods of use thereof for enhancing an immune response. Also disclosed herein is a therapeutic composition comprising a combination of Siglec-9 inhibitor and an anti-SARS-CoV-2 antibody, or a bispecific anti- Siglec-9/anti-SARS-CoV-2 antibodies and methods of use for treating or preventing COVID- 19.

Description

SIGLEC 9 INHIBITORS AND METHODS OF USE THEREOF FOR ENHANCING IMMUNOTHERAPY EFFICACY

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant DK123733 awarded by the National Institutes of Health. The Government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/477,088, filed December 23, 2022 which is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

COVID-19 has rapidly emerged as a global public health crisis joining severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) in a growing number of coronavirus-associated illnesses which have jumped from animals to people. There are at least seven identified coronaviruses that infect humans. Disease symptoms can range from mild flu-like to severe cases with life-threatening pneumonia (Huang et al., 2020, Lancet, 395:497-506). Most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) experience mild symptoms; however, many need hospitalization (Guan WJ, et al., 2020, N Engl J Med 382: 1708-1720; Gandhi RT, et al., 2020, N Engl I Med).

The mechanisms underlying coronavirus disease 2019 (COVID-19) severity are likely multifactorial, and disruption in immunological functions could be one of these mechanisms. Severe COVID-19 has been associated with alterations to the 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, 1 Allergy Clin Immunol 148:996-1006 el8; Maucourant C, et al., 2020, Sci Immunol 5; Osman M, et al., 2020, Blood Adv 4:5035-5039). NK cells are effector innate immune cells that play a central role in anti-viral immunity through direct cytotoxicity and/or antibodydependent cell cytotoxicity (ADCC) (Hammer Q, et al., 2018, Nat Immunol 19:800-808). However, whether severe COVID-19 impairs the anti-SARS-CoV-2 functions of NK cells and what host factors modulate these functions remain unclear.

The cytotoxic potential of NK cells is determined by the balance of opposing signals resulting from multiple activating (such as NKG2C) and inhibitory (such as NKG2A) receptors expressed on the surface of these cells (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 two receptors that belong to a family of emerging glyco-immune checkpoints called Siglecs: Siglec-7 and Siglec-9 (Nicoll G, et al., 1999, J Biol Chem 274:34089-95; Adeniji OS, et al., 2021, PLoS Pathog 17:el010034). Siglecs are sialic-acid-binding, immunoglobulin-like lectins that inhibit immune functions by interacting with sialogylcans (sialic acid-containing glycomic 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-sialogylcan interactions help tumor cells to 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 U S A 111 : 14211-6). Recently, these interactions have been suggested to also help HBV- and SARS-CoV-2- infected cells to evade NK immune surveillance (Adeniji OS, et al., 2021, PLoS Pathog 17:el010034; Zhao D, et al., 2018, Front Immunol 9: 1124). Despite a growing appreciation of Siglecs as glyco-immune negative checkpoints during cancer, their role in helping SARS- CoV-2 evade immune surveillance has never been examined.

Thus there remains a pressing need for novel immunotherapeutic tools to treat or prevent severe disease associated with viral infections, such as COVID-19. This invention addresses this unmet need. SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a therapeutic composition comprising a Siglec-9 inhibitor as an adjuvant to enhance the immune response against 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 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 sequence having at least 95% identity to 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 fragment comprising at least 80% of the full- length sequence of 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 fragment comprising at least 80% of the full-length sequence of 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 therapeutic composition further comprises one or more additional antibody targeting an 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 therapeutic composition comprises a bispecific antibody comprising an inhibitory Siglec-9 antibody domain and a SARS-CoV-2 antigen binding domain.

In one embodiment, the therapeutic composition comprises one or more inhibitory nucleic acid molecule specific for binding to Siglec-9 or a fragment thereof. In one embodiment, the therapeutic composition comprises one or more mRNA molecule encoding the Siglec-9 inhibitor. In one embodiment, the therapeutic composition comprises one or more DNA molecule encoding the Siglec-9 inhibitor. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a 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 nucleotide sequence encoding a 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 nucleotide sequence having at least 95% identity to a 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 nucleotide sequence having at least 95% identity to a 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 a 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 a variable light chain sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28.

In one embodiment, the therapeutic composition further comprises one or more additional nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof targeting an 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 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.

In one embodiment, the invention relates to a method of enhancing immunotherapy efficacy in a subject in need thereof, the method comprising administering to the subject a therapeutic composition comprising a Siglec-9 inhibitor as an adjuvant to enhance the immune response against 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 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 sequence having at least 95% identity to 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 fragment comprising at least 80% of the full- length sequence of 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 fragment comprising at least 80% of the full-length sequence of 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 method further comprises administering one or more additional antibody targeting an 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 invention relates to a method of increasing the level of antibody-dependent cell cytotoxicity (ADCC) activities against a target antigen, the method comprising administering to the subject a therapeutic composition comprising a Siglec-9 inhibitor as an adjuvant to enhance the immune response against 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 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 sequence having at least 95% identity to 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 fragment comprising at least 80% of the full- length sequence of 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 fragment comprising at least 80% of the full-length sequence of 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 method further comprises administering one or more additional antibody targeting an 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 invention relates to a method of 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 against 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 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 sequence having at least 95% identity to 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 fragment comprising at least 80% of the full- length sequence of 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 fragment comprising at least 80% of the full-length sequence of 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 viral infection is SARS-CoV-2 infection.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended 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.

Figure 1A through Figure IM depicts data demonstrating that hospitalized COVID-19 is associated with reduced CD56^dim NK cells degranulation against SARS-CoV- 2 Spike-expressing target cells. Figure 1 A depicts a schematic overview of the experiments to evaluate the direct cytotoxicity and ADCC mediated degranulation of CD56^dim NK cells during different severities of COVID-19 infection. To examine direct cytotoxicity -mediated degranulation, PBMCs from each donor, from three COVID-19 status groups (n=8 SARS- CoV-2 negative, n=12 mild COVID-19, and n=21 hospitalized COVID-19) were cocultured (2) or not (1) with SARS-CoV-2 Spike-expressing 293T target cells (10:1 (E:T) ratio). To examine ADCC-mediated degranulation, identical co-cultures were performed in the presence of the negative (3) or positive (4) antibody pool. Direct cytotoxicity was assessed by subtracting (1) from (2). (10:1 (E:T) ratio). ADCC was assessed by subtracting the results of subtracting (1) from (3) from the results of subtracting (1) from (4). Figure IB depicts a graph demonstrating direct cytotoxicity-mediated degranulation and cytokine production of the CD56^dim NK population assessed as the percentage of CD107a⁺ IFN-y cells. Medians and interquartile ranges (IQR) are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 1C depicts a graph demonstrating direct cytotoxicity-mediated degranulation and cytokine production of the CD56^dun NK population assessed as the percentage of CD107a⁺ TNF-a⁺ cells. Medians and interquartile ranges (IQR) are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure ID depicts a graph demonstrating direct cytotoxicity-mediated degranulation and cytokine production of the CD56^dim NK population assessed as the percentage of IFN-y⁺ cells. Medians and interquartile ranges (IQR) are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure IE depicts a graph demonstrating direct cytotoxicity -mediated degranulation and cytokine production of the CD56^dim NK population assessed as the percentage of IFN-y⁺TNF-a⁺ cells. Medians and interquartile ranges (IQR) are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure IF depicts a graph demonstrating ADCC-mediated degranulation and cytokine production of the CD56^dim NK population assessed as the percentage of CD107a⁺ IFN-y⁺ cells. Median and IQR are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 1G depicts a graph demonstrating ADCC-mediated degranulation and cytokine production of the CD56^dim NK population assessed as the percentage of CD107a⁺ TNF-a⁺ cells. Median and IQR are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 1H depicts a graph demonstrating ADCC-mediated degranulation and cytokine production of the CD56^dim NK population assessed as the percentage of IFN-y cells. Median and IQR are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure II depicts a graph demonstrating ADCC-mediated degranulation and cytokine production of the CD56^dim NK population assessed as the percentage of IFN-y TNF-a⁺ cells. Median and IQR are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 1J depicts a regression graph plotting Spearman's rank-order correlations between plasma N-antigen load and the percentage of CD56^dim NK cells expressing CD107a⁺ during direct cytotoxicity assays; only samples from COVID-19 positive donors were used (n=33). Figure IK depicts a regression graph plotting Spearman's rank-order correlations between plasma N-antigen load and the percentage of CD56^dim NK cells expressing IFN-y during direct cytotoxicity assays; only samples from COVID-19 positive donors were used (n=33). Figure IL depicts a regression graph plotting Spearman's rank-order correlations between plasma N-antigen load and the percentage of CD56^dim NK cells expressing CD107a⁺ IFN-y during direct cytotoxicity assays; only samples from COVID-19 positive donors were used (n=33). Figure IM depicts a regression graph plotting Spearman's rank-order correlations between plasma N-antigen load and the percentage of CD56^dim NK cells expressing IFN-y TNF-a⁺ during direct cytotoxicity assays; only samples from COVID-19 positive donors were used (n=33).

Figure 2 depicts data measuring the mean fluorescence intensity of CD 107a, IFN-y, and TNF-a in CD56^dim NK cells after co-culturing with SARS-CoV-2 Spikeexpressing 293T cells. Figure 2A depicts a gating strategy for experiments in Figure 1, Figure 2, Figure 3, Figure 5F through Figure 5H, and Figure 6A through Figure 6E. Direct cytotoxicity and ADCC were calculated, as mentioned in Figure 1, within each COVID-19 status group (n=8 SARS-CoV-2 negative, n=12 mild COVID-19, and n=21 hospitalized COVID-19). Figure 2B depicts a graph plotting the mean fluorescence intensity (MFI) of CD107a to evaluate direct cytotoxicity. Median and IQR are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 2C depicts a graph plotting the mean fluorescence intensity (MFI) of IFN-y to evaluate direct cytotoxicity. Median and IQR are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 2D depicts a graph plotting the mean fluorescence intensity (MFI) of TNF-a to evaluate direct cytotoxicity. Median and IQR are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 2E depicts a graph plotting the mean fluorescence intensity (MFI) of CD107a to evaluate ADCC. Median and IQR are displayed. Kruskal- Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 2F depicts a graph plotting the mean fluorescence intensity (MFI) of IFN-y to evaluate ADCC. Median and IQR are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 2G depicts a graph plotting the mean fluorescence intensity (MFI) of TNF-a to evaluate ADCC. Median and IQR are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses.

Figure 3 depicts representative data on sex-dependent differences in the cytolytic activities on NK cells against SARS-CoV-2 Spike expressing 293 T cells. The direct cytolytic and ADCC activities of CD56^dim NK cells from SARS-CoV-2 negative controls (n=8; grey dots), mild COVID-19 donors (n=12; orange dots), and hospitalized COVID-19 donors (n=21; maroon dots) were compared based on sex (female; n=l 8, and male; n=23). Figure 3A depicts a graph evaluating direct cytotoxicity calculated as described in Figure 1. Direct cytotoxicity was measured as the percentage of cells expressing CD107a⁺, IFN-y⁺, TNF-a⁺, CD107a⁺IFN-y⁺, CD107a⁺ TNF-a⁺, IFN-y⁺ TNF-a⁺, and the mean fluorescence intensity (MFI) of CD107a, IFN-y, and TNF-a. Median and IQR are displayed. Mann-Whitney U tests were used for statistical analyses. Figure 3B depicts a graph evaluating ADCC calculated as described in Figure 1. ADCC was measured as the percentage of cells expressing CD107a⁺, IFN-y⁺, TNF-a⁺, CD107a⁺IFN-y⁺, CD107a⁺ TNF- a⁺, IFN-y⁺TNF-a⁺, and the mean fluorescence intensity (MFI) of CD107a, IFN-y, and TNF- a. Median and IQR are displayed. Mann-Whitney U tests were used for statistical analyses.

Figure 4A through Figure 4F depicts representative data demonstrating hospitalized COVID- 19 is associated with reduced NK cytotoxicity. Figure 4A depicts representative data from experiments examining the 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). CH0-K1 target cells were incubated with either the positive or negative SARS-CoV-2 antibody pools for 15 min. After 15 min, the cells were co-cultured at 10:1 (E:T) ratio for 5 h. The SARS-CoV-2 S CHO-K1 cells stably express the SARS-CoV-2 Spike (S) protein and a HaloTag-HiBiT protein. When the target cells are lysed by ADCC, the intracellular HaloTag-HiBiT protein interacts with the extracellular detection reagent to generate a luminescence signal that can quantitatively measure the degree of target cell lysis. Luminescence values of each donor for the positive pool were subtracted by the values obtained from the respective negative pool to obtain the target cell lysis values. Median and IQR are displayed. Mann-Whitney U test was used for statistical analyses. Figure 4B through Figure 4F depicts representative data from experiments demonstrating that hospitalized COVID- 19 is associated with reduced CD56^dim NK cells degranulation against K562 target cells. PBMCs from three COVID-19 status groups (n=l 1 SARS-CoV-2 negative, n=9 mild COVID-19, and n=16 hospitalized COVID-19) were cocultured at 5: 1 (E:T) ratio with K562. Direct cytotoxicity was assessed by subtracting the degranulation/cytokine production of PBMCs culture alone from the degranulation/cytokine production of PBMCs co-cultured with target cells. Figure 4B depicts a graph plotting the percentage of CD107a⁺ cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production of the CD56^dim NK population against K562 target cells. Median and IQR are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 4C depicts a graph plotting the percentage of IFN- cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production of the CD56^dim NK population against K562 target cells. Median and IQR are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 4D depicts a graph plotting the percentage of TNF-a⁺ cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production of the CD56^dim NK population against K562 target cells. Median and IQR are displayed. Kruskal- Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 4E depicts a graph plotting the percentage of CD107a⁺ IFN-y cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production of the CD56^dim NK population against K562 target cells. Median and IQR are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 4F depicts a graph plotting the percentage of CD107a⁺TNF-a⁺ cells as an assessment of direct cytotoxicity-mediated degranulation and cytokine production of the CD56^dim NK population against K562 target cells. Median and IQR are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses.

Figure 5A through Figure 51 depicts representative data from experiments demonstrating that the Siglec-9⁺CD56^dimNK sub-population exhibits a higher SARS-CoV-2 specific ADCC than the Siglec-9" CD56^dlinNK sub-population. Figure 5 A depicts a graph plotting ADCC-mediated degranulation and\or cytokine production of the Siglec-9⁺ and Siglec-9' CD56^dimNK cells within each COVID-19 status group as assessed by the percentage of cells expressing CD 107a. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺ and Siglec-9' CD56^dimNK cells within each COVID-19 status group. Figure 5B depicts a graph plotting ADCC-mediated degranulation and\or cytokine production of the Siglec-9⁺ and Siglec-9" CD56^dimNK cells within each COVID-19 status group as assessed by the percentage of cells expressing IFN-y. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺and Siglec-9' CD56^dimNK cells within each COVID-19 status group. Figure 5C depicts a graph plotting ADCC-mediated degranulation and\or cytokine production of the Siglec-9⁺ and Siglec-9' CD56^dimNK cells within each COVID- 19 status group as assessed by the percentage of cells expressing TNF-a. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺and Siglec-9' CD56^dimNK cells within each COVID-19 status group. Figure 5D depicts a graph plotting ADCC-mediated degranulation and\or cytokine production of the Siglec-9 and Siglec-9' CD56^dimNK cells within each COVID-19 status group as assessed by the percentage of cells expressing CD107a and IFN-y. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺and Siglec-9' CD56^dimNK cells within each COVID- 19 status group. Figure 5E depicts a graph plotting ADCC- mediated degranulation and\or cytokine production of the Siglec-9⁺ and Siglec-9" CD56^dim NK cells within each COVID-19 status group as assessed by the percentage of cells expressing CD107a and TNF-a. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺ and Siglec-9" CD56^dimNK cells within each COVID-19 status group. Figure 5F depicts a graph plotting ADCC-mediated degranulation and\or cytokine production of the Siglec-9⁺ and Siglec-9' CD56^dimNK cells within each COVID-19 status group as assessed by the percentage of cells expressing IFN-y and TNF-a. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺and Siglec-9' CD56^dimNK cells within each COVID-19 status group. Figure 5J depicts a Spearman's correlation heat-map showing associations between direct cytotoxicity and ADCC-mediated NK degranulation of the Siglec-9⁺ and Siglec-9' CD56^dimNK cells and plasma N-antigen load. The color of the squares represents the strength of the correlation, with blue representing negative correlations and red representing positive correlations; only samples from COVID-19 positive donors were used (n=33).

Figure 6A through Figure 6J depicts representative data from experiments demonstrating that the Siglec7⁺ CD56^dimNK subpopulation exhibits higher SARS-CoV-2 specific direct cytolytic and ADCC activities than does the Siglec-7' CD56^dimNK subpopulation. Figure 6A through Figure 6C depicts graphs plotting direct cytolytic- mediated degranulation and\or cytokine production of the Siglec-7⁺ and Siglec-7' CD56^dim NK cells within each COVID-19 status group as assessed by the percentage of cells expressing (A) CD107a, (B) TNF-a, and (C) CD107aand TNF-a. Wilcoxon's signed-rank tests were used to compare the Siglec-7⁺ and Siglec-7' CD56^dimNK cells within each disease group. Figure 6 A depicts a graph plotting the percentage of cells expressing CD 107a. Figure 6B depicts a graph plotting the percentage of cells expressing TNF-a. Figure 6C depicts a graph plotting the percentage of cells expressing CD 107a and TNF-a. Figure 6D through Figure 61 depicts graphs plotting ADCC-mediated degranulation and\or cytokine production of the Siglec-7⁺and Siglec-7' CD56^dimNK cells within each COVID-19 status group as assessed by the percentage of cells expressing CD107a, IFN-y, TNF-a, CD107a and IFN-y, CD107a and TNF-a, or IFN-y and TNF-a. Wilcoxon's signed-rank tests were used to compare the Siglec-7⁺ and Siglec-7' CD56^dimNK cells within each disease group. Figure 6D depicts the percentage of cells expressing CD107a. Figure 6E depicts the percentage of cells expressing IFN-y. Figure 6F depicts the percentage of cells expressing TNF-a. Figure 6G depicts the percentage of cells expressing CD107a and IFN-y. Figure 6H depicts the percentage of cells expressing CD107aand TNF-a. Figure 61 depicts the percentage of cells expressing IFN-y and TNF-a. Figure 6J depicts a Spearman's correlation heat-map showing associations between direct cytotoxicity and ADCC-mediated NK degranulation of the Siglec-7⁺ and Siglec-7' CD56^dimNK cells and plasma N-antigen load. The color of the squares represents the strength of the correlation, with blue representing negative correlations and red representing positive correlations; only samples from COVID- 19 positive donors were used (n=33).

Figure 7A through Figure 7C depicts representative data from experiments demonstrating that the Siglec7⁺ CD56^dunNK subpopulation exhibits higher SARS-CoV-2 specific ADCC activities than the Siglec-7' CD56^dimNK subpopulation. The ADCC- mediated degranulation of the Siglec-7⁺ and Siglec-7' CD56^dltnNK cells within each COVID-19 status group (n=8 SARS-CoV-2 negative; n=12 mild COVID-19, and n=21 hospitalized COVID-19) as assessed by the mean fluorescence intensity (MFI) was also evaluated. Figure 7A depicts a graph plotting the MFI for CD 107a. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺ and Siglec-9' CD56^dimNK cells within each disease group. Figure 7B depicts a graph plotting the MFI for IFN-y. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺and Siglec-9' CD56^dimNK cells within each disease group. Figure 7C depicts a graph plotting the MFI for TNF-a. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺and Siglec-9' CD56^dimNK cells within each disease group.

Figure 8 depicts a gating strategy for experiments in Figures 12 and Figures 13A through Figure 13E.

Figure 9A through Figure 9B depicts representative data from experiments demonstrating that Siglec-9 antibody marks Siglec-9⁺ populations with high expression of Siglec-9 mRNA transcripts. To validate the specificity of the Siglec-9 Ab (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, and high Siglec-9 expression. qPCR was then used to measure the relative copy number of Siglec-9 transcripts in the sorted populations. Figure 9A depicts a gating strategy for the sorting experiments. Figure 9B depicts a graph plotting the relative copy number of Siglec-9 transcripts measured by qPCR and normalized using the eukaryotic 18S rRNA endogenous control. Relative copy numbers were determined using the comparative Ct method. Means and standard error of the mean (SEM) are displayed. Paired t-tests were used for statistical analysis.

Figure 10A through Figure 10F depicts representative data from experiments assessing sex-dependent expression of Siglec-7 and Siglec-9 on CD56^dim NK cells. To examine the potential impact of sex on the expression of Siglec-9 and Siglec-7 on CD56^dim NK cells, 79 individuals with three COVID-19 disease states (negative; n=12 (grey dots), mild; n=26 (orange dots), and hospitalized; n=41 (maroon dots), were divided based on sex (female; n=32, and male; n=47). Figure 10A depicts a graph plotting the percentage of Siglec-9⁺ CD56^dim NK cells. Median and IQR are displayed. Mann-Whitney U tests were used to compare the groups. Figure 10B depicts a graph plotting the percentage of Siglec-7⁺ CD56^dim NK cells. Median and IQR are displayed. Mann-Whitney U tests were used to compare the groups. Figure IOC depicts a graph plotting the percentage of Siglec-9' Siglec- 7' CD56^dim NK cells. Median and IQR are displayed. Mann-Whitney U tests were used to compare the groups. Figure 10D depicts a graph plotting the percentage of Siglec-9⁺ Siglec- 7' CD56^dim NK cells. Median and IQR are displayed. Mann-Whitney U tests were used to compare the groups. Figure 10E depicts a graph plotting the percentage of Siglec-9' Siglec- 7⁺ CD56^dim NK cells. Median and IQR are displayed. Mann-Whitney U tests were used to compare the groups. Figure 10F depicts a graph plotting the percentage of Siglec-9⁺ Siglec- 7⁺ CD56^dim NK cells. Median and IQR are displayed. Mann-Whitney U tests were used to compare the groups.

Figure 11 A through Figure 11C depicts representative data from experiments demonstrating that hospitalized COVID- 19 is associated with a decrease in the expression of CD16 and Siglec-7 and an increase in CD57 on CD56^dim NK cells. Figure 11 A depicts a graph plotting the expression of CD16 on CD56^dim NK cells from n=79 (n=12 SARS-CoV-2 negative; n=26 mild COVID-19, and n=41 hospitalized COVID-19). Median and IQR are displayed. Kruskal-Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 1 IB depicts a graph plotting the expression of CD57 on CD56^dim NK cells from n=79 (n=12 SARS-CoV-2 negative; n=26 mild COVID-19, and n=41 hospitalized COVID-19). Median and IQR are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 11C depicts a graph plotting the expression of Siglec-7 on CD56^dim NK cells from n=79 (n=12 SARS- CoV-2 negative; n=26 mild COVID-19, and n=41 hospitalized COVID-19). Median and IQR are displayed. Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses.

Figure 12A through Figure 12B depicts representative data from experiments demonstrating that Siglec-9 marks activated and mature CD56^dimNK subpopulation in vivo. Figure 12A depicts graphs plotting comparisons of the expression of CD16 (FcyRIII; mediator of ADCC), CD57 (maturation marker), NKG2C (activating receptor), and NKG2A (inhibitory receptor) on Siglec-9⁺ and Siglec-9' CD56^dim NK cells obtained from 79 individuals with three COVID-19 disease states (negative; n=12, mild; n=26, and hospitalized; n=41). Siglec-9⁺ cells exhibit higher levels of CD16, CD57, NKG2C, and lower levels of NKG2A compared to Siglec-9' cells. Wilcoxon's signed-rank tests were used to compare the Siglec-9⁺and Siglec-9' CD56^dimNK cells within each COVID-19 state group. Mann-Whitney U tests were used to compare the disease state groups. Figure 12B depicts graphs plotting comparisons of the expression of CD 16, CD57, NKG2C, and NKG2A on the Siglec-7⁺ and Siglec-7' CD56^dun NK cells. Siglec-7⁺ cells exhibit higher levels of CD 16, NKG2C, and NKG2A than Siglec-7' cells. No differences were observed in the expression of CD57 between the Siglec-7⁺ and Siglec-7' cells. As in panel A, Wilcoxon's signed-rank tests were used to compare the Siglec-7⁺ and Siglec-7' CD56^dimNK cells within each COVID-19 status group, and Mann-Whitney U tests were used to compare the status groups.

Figure 13A through Figure 13H depicts representative data from experiments demonstrating that Siglec-9, but not Siglec-7, marks CD56^dim NK cells with high ADCC activity against SARS-CoV-2. Figure 13 A depicts 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 the CD56^dun NK cells from all donors (n=79). Figure 13B depicts a graph plotting the in vivo expression of CD 16 on the indicated cell sub-populations (n=79). Median and IQR are displayed. Friedman tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 13C depicts a graph plotting the in vivo expression of CD57 on the indicated cell sub-populations (n=79). Median and IQR are displayed. Friedman tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 13D depicts a graph plotting the in vivo expression of NKG2C on the indicated cell sub-populations (n=79). Median and IQR are displayed. Friedman tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 13E depicts a graph plotting the in vivo expression of NKG2A on the indicated cell sub-populations (n=79). Median and IQR are displayed. Friedman tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 13F depicts a graph plotting the ADCC-mediated degranulation of the indicated NK cell subpopulations (n=41) as assessed by the percentage of cells expressing CD 107a. Median and IQR are displayed. Friedman tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 13G depicts a graph plotting the ADCC-mediated degranulation of the indicated NK cell subpopulations (n=41) as assessed by the MFI of CD 107a. Median and IQR are displayed. Friedman tests with Dunn’s multiple comparisons correction were used for statistical analyses. Figure 13H depicts a graph plotting the ADCC- mediated degranulation of the indicated NK cell subpopulations (n=41) as assessed by the percentage of cells expressing CD107aand IFN-y. Median and IQR are displayed. Friedman tests with Dunn’s multiple comparisons correction were used for statistical analyses.

Figure 14A through Figure 14F depicts representative data from experiments demonstrating that blocking Siglec-9 interactions, using a Siglec-9 blocking antibody, enhances the anti-SARS-CoV-2 ADCC of CD56^dim NK cells. Figure 14A through Figure 14E depicts representative data from experiments assessing the impact of the Siglec-9 blocking antibody, compared to an isotype control, on the ADCC-mediated NK degranulation and\or cytokine production against SARS-CoV-2. PBMCs from six healthy controls were used as effector cells, and SARS-CoV-2 Spike-expressing 293T cells were used as target cells. The cells were co-cultured at 10: 1 (E:T) ratio for 12 h. Figure 14A depicts a graph plotting NK degranulation and\or cytokine production as assessed by the percentage of cells expressing CD 107a. Paired t-tests were used for statistical analysis. Figure 14B depicts a graph plotting NK degranulation and\or cytokine production as assessed by the percentage of cells expressing CD 107a and IFN-y. Paired t-tests were used for statistical analysis. Figure 14C depicts a graph plotting NK degranulation and\or cytokine production as assessed by the percentage of cells expressing CD107aand TNF-a. Paired t-tests were used for statistical analysis. Figure 14D depicts a graph plotting NEC degranulation and\or cytokine production as assessed by the percentage of cells expressing TNF-a. Paired t-tests were used for statistical analysis. Figure 14E depicts a graph plotting NK degranulation and\or cytokine production as assessed by the percentage of cells expressing IFN-y and TNF-a. Paired t-tests were used for statistical analysis. Figure 14F depicts a graph plotting representative data from experiments assessing the impact of the Siglec-9 blocking antibody, compared to an isotype control, on the ADCC-mediated lysis of SARS-CoV-2 target cells. Purified NK cells isolated from the PBMCs of five healthy donors were used as effector cells, and the SARS-CoV-2 S CH0-K1 cells were used as target cells. The cells were co-cultured at 5: 1 E:T ratio for 5 h. The SARS-CoV-2 S CHO- K1 cells stably express the SARS-CoV-2 Spike (S) protein and a HaloTag-HiBiT protein. When the target cells are lysed by ADCC, the intracellular HaloTag-HiBiT protein interacts with the extracellular detection reagent to generate a luminescence signal that can quantitatively measure the degree of target cell lysis. Paired t-test was used for statistical analysis.

Figure 15 depicts a model of how a Siglec-9 blocking antibody increases the cytotoxicity of Siglec-9⁺ NK cells. Left panel: Siglec-9' cells have low cytotoxicity. Middle panel: The Siglec-9⁺ CD56^dim NK subset has high ADCC activity, possibly due to elevated expression of CD16 (FcyRIII; a mediator of ADCC activity), CD57 (maturation marker), and NKG2C (activating receptor), and to reduced expression of the inhibitory receptor NKG2A, compared to the Siglec-9' CD56^dim NK cells. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains the cytolytic ability of these highly cytotoxic Siglec-9⁺ CD56^dun NK cells by binding to Sialic acid on the surface of target cells. Right panel: Blocking the inhibitory receptor, Siglec-9, using a blocking antibody can unleash a higher ADCC potential of the Siglec-9⁺ CD56^dim sub-population.

Figure 16 depicts representative data from experiments on Siglec-9 blocking antibody characterization. The binding of different dilutions of the recombinantly expressed anti-Siglec-9 antibody to: 1) recombinant Siglec-9 protein, 2) recombinant Siglec-7 protein (as a negative control), and 3) HIV-1 -gp 120 protein (as a negative control) was determined by ELISA. Each point represents the OD value (mean ± SEM).

DETAILED DESCRIPTION

In some embodiments, the present invention includes comprising a Siglec-9 inhibitor for use in increasing the cytolytic and antibody-dependent cell cytotoxicity (ADCC) activities of natural killer (NK) cells in response to the presence of a target antigen. In some embodiments, the invention relates to compositions comprising at least one anti- Siglec-9 antibody comprising a domain specific for binding to Siglec-9.

In some embodiments, the Siglec-9 inhibitor of the invention is administered in combination with one or more additional therapeutic agent. In some embodiments, the additional thereapeutic agent is a therapeutic antibody. In some embodiments, the Siglec-9 inhibitor of the invention functions as an adjuvant to increase the cytotoxicity of NK cells against the antigen targeted by the therapeutic antibody.

In one embodiment, the therapeutic antibody targets a viaral antigen. In one embodiment, the viral antigen is a SARS-CoV-2 antigen. The antigen may be a SARS-CoV- 2 viral antigen, or fragment thereof, or variant thereof. The SARS-CoV-2 antigen can be from a factor that allows the virus to replicate, infect or survive. In some embodiments, the SARS-CoV-2 antigen can be a spike antigen or a fragment thereof.

In one embodiment, the invention provides compositions comprising one or more bispecific antibody comprising an anti-Siglec-9 domain for inhibiting Siglec-9 activity in a NK cell and a target antigen binding domain specific for binding to a target antigen. In one embodiment, the target antigen is a viaral antigen. In one embodiment, the viral antigen is a SARS-CoV-2 antigen. The antigen may be a SARS-CoV-2 viral antigen, or fragment thereof, or variant thereof. The SARS-CoV-2 antigen can be from a factor that allows the virus to replicate, infect or survive. In some embodiments, the SARS-CoV-2 antigen can be a spike antigen or a fragment thereof.

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

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in 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 not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

“Antibody” may mean an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, 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 the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

“Antibody fragment” or “fragment of an antibody” as used interchangeably herein refers to a portion of an intact antibody comprising the antigen-binding site or variable region. The portion does not include the constant heavy chain domains (i.e. CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. 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 containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

“Antigen” refers to proteins that have the ability to generate an immune response in a host. An antigen may be recognized and bound by an antibody. An antigen may originate from within the body or from the external environment.

“Coding sequence” or “encoding nucleic acid” as used herein may mean refers to the nucleic acid (RNA or DNA molecule) that comprise a nucleotide sequence which encodes an antibody as set forth herein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to whom the nucleic acid is administered. The coding sequence may further include sequences that encode signal peptides.

“Complement” or “complementary” as used herein may mean a nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

“Endogenous antibody” as used herein may refer to an antibody that is generated in a subject that is administered an effective dose of an antigen for induction of a humoral immune response.

“Fragment” may mean a polypeptide fragment of an antibody that is function, i.e., can bind to desired target and have the same intended effect as a full length antibody. A fragment of an antibody may be 100% identical to the full length except missing at least one amino acid from the N and/or C terminal, in each case with or without signal peptides and/or a methionine at position 1. Fragments 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, 99% or more percent of the length of the particular full length antibody, excluding any heterologous signal peptide added. The fragment may comprise a fragment of a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antibody and additionally comprise an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise an N terminal methionine and/or a signal peptide such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The N terminal methionine and/or signal peptide may be linked to a fragment of an antibody.

A fragment of a nucleic acid sequence that encodes an antibody may be 100% identical to the full length except missing at least one nucleotide from the 5' and/or 3' end, in each case with or without sequences encoding signal peptides and/or a methionine at position 1. Fragments 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, 99% or more percent of the length of the particular full length coding sequence, excluding any heterologous signal peptide added. The fragment may comprise a fragment that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antibody and additionally optionally comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity. Fragments may further comprise coding sequences for an N terminal methionine and/or a signal peptide such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The coding sequence encoding the N terminal methionine and/or signal peptide may be linked to a fragment of coding sequence.

“Genetic construct” as used herein refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes 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 capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed. “Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of 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 may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

“Impedance” as used herein may be used when discussing the feedback mechanism and can be converted to a current value according to Ohm's law, thus enabling comparisons with the preset current.

“Immune response” as used herein may mean the activation of a host’s immune system, e.g., that of a mammal, in response to the introduction of one or more nucleic acids and/or peptides. The immune response can be in the form of a cellular or humoral response, or both.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

“Operably linked” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

A “peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

“Promoter” as used herein may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and the CMV IE promoter.

“Signal peptide” and “leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein set forth herein. Signal peptides/leader sequences typically direct localization of a protein. Signal peptides/leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides/leader sequences are linked at the N terminus of the protein.

“Stringent hybridization conditions” as used herein may mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., about 10-50 nucleotides) and at least about 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C.

“Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc) and a human). In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.

“Substantially complementary” as used herein may mean that a first 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 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, or that the two sequences hybridize under stringent hybridization conditions.

“Substantially identical” as used herein may mean that a first and second sequence are 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% over a region of 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 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

“Synthetic antibody” as used herein refers to an antibody that is encoded by the recombinant nucleic acid sequence described herein and is generated in a subject.

“Treatment” or “treating,” as used herein can mean protecting of a subject from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering an antibody of the present invention to a subject prior to onset of the disease. Suppressing the disease involves administering a antibody of the present invention to a subject after induction of the disease but before its clinical appearance. Repressing the disease involves administering an antibody of the present invention to a subject after clinical appearance of the disease.

“Variant” used herein with respect to a nucleic acid may mean (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto. “Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

A variant may be a nucleic acid sequence that is substantially identical over the full length of the full 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 full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino 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 full length of the amino acid sequence or a fragment thereof.

“Vector” as used herein may mean a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome.

For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

Compositions

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

In various embodiments, the invention provides compositions and methods for inhibiting Siglec-9 to increase the cytolytic and antibody-dependent cell cytotoxicity (ADCC) activities of natural killer cells in response to the presence of a target antigen (e.g., a SARS-CoV-2 antigen.)

In various embodiments, the present invention includes compositions and methods of treating a viral infection in a subject. In various embodiments, the composition for treating a viral infection comprises an inhibitor of Siglec-9. In one embodiment, the inhibitor of the invention decreases the amount of Siglec-9 polypeptide, the amount of Siglec-9 mRNA, the amount of Siglec-9 activity, or a combination thereof. It will be understood by one skilled in the art, based upon the disclosure provided herein, that a decrease in the level of Siglec-9 encompasses the decrease in the expression, including transcription, translation, or both. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that a decrease in the level of Siglec-9 includes a decrease in the activity of Siglec-9. Thus, decrease in the level or activity of Siglec-9 includes, but is not limited to, decreasing the amount of polypeptide of Siglec-9, and decreasing transcription, translation, or both, of a nucleic acid encoding Siglec-9; and it also includes decreasing any activity of Siglec-9 as well.

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

One skilled in the art will appreciate, based on the disclosure provided herein, that one way to decrease the mRNA and/or protein levels of Siglec-9 in a cell is by reducing or inhibiting expression of the nucleic acid encoding Siglec-9. Thus, the protein level of Siglec-9 in a cell can also be decreased using a molecule or compound that inhibits or reduces gene expression such as, for example, siRNA, an antisense molecule or a ribozyme. However, the invention should not be limited to these examples.

In one embodiment, siRNA is used to decrease the level of Siglec-9. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. 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); and 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 a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the present invention also includes methods of decreasing levels of Siglec-9 at the protein level using RNAi technology.

In other related aspects, the invention includes an isolated nucleic acid encoding an inhibitor, wherein an inhibitor such as an siRNA or antisense molecule, inhibits Siglec-9, a derivative thereof, a regulator thereof, or a downstream effector, operably linked to a nucleic acid comprising a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and as described elsewhere herein. In another aspect of the invention, Siglec-9 or a regulator thereof, can be inhibited by way of inactivating and/or sequestering one or more of Siglec-9, or a regulator thereof. As such, inhibiting the effects of Siglec-9 can be accomplished by using a transdominant negative mutant.

In another aspect, the 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. The incorporation of a desired polynucleotide into a vector and the choice of vectors is well-known in the art as described in, for example, Sambrook et al., supra.

The siRNA or antisense polynucleotide can be cloned into a number of 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. In order to assess the expression of the siRNA or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

In one embodiment of the invention, an antisense nucleic acid sequence which is expressed by a plasmid vector is used to inhibit Siglec-9. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of Siglec-9.

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

The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

Alternatively, antisense molecules of the invention may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243). Compositions and methods for the synthesis and expression of antisense nucleic acids are as described elsewhere herein.

Ribozymes and their use for inhibiting gene expression are also well 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., International Publication No. WO 92/07065; Altman et al., U.S. Patent No. 5,168,053). Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences encoding these RNAs, molecules can be engineered to recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of this approach is the fact that ribozymes are sequence-specific.

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

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

When the inhibitor of the invention is a small molecule, a small molecule antagonist may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.

Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.

In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores.

In another aspect of the invention, Siglec-9 can be inhibited by way of inactivating and/or sequestering Siglec-9. As such, inhibiting the effects of Siglec-9 can be accomplished by using a transdominant negative mutant. Alternatively an antibody specific for Siglec-9 (e.g., an antagonist to Siglec-9) may be used. In one embodiment, the antagonist is a protein and/or compound having the desirable property of interacting with a binding partner of Siglec-9 and thereby competing with the corresponding protein. In another embodiment, the antagonist is a protein and/or compound having the desirable property of interacting with Siglec-9 and thereby sequestering Siglec-9.

As will be understood by one skilled in the art, any antibody that can recognize and bind to an antigen of interest is useful in the present invention. Methods of making and using antibodies are well known in the art. For example, polyclonal antibodies useful in the present invention are generated by immunizing rabbits according to standard immunological techniques well-known in the art (see, e.g., Harlow et al., 1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY). Such techniques include immunizing an animal with a chimeric protein comprising a portion of another protein such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and/or a moiety such that the antigenic protein of interest is rendered immunogenic (e.g., an antigen of interest conjugated with keyhole limpet hemocyanin, KLH) and a portion comprising the respective antigenic protein amino acid residues. The chimeric proteins are produced by cloning the appropriate nucleic acids encoding the marker protein into a plasmid vector suitable for this purpose, such as but not limited to, pMAL-2 or pCMX.

However, the invention should not be construed as being limited solely to methods and compositions including these antibodies or to these portions of the antigens. Rather, the invention should be construed to include other antibodies, as that term is defined elsewhere herein, to antigens, or portions thereof. Further, the present invention should be construed to encompass antibodies, inter alia, bind to the specific antigens of interest, and they are able to bind the antigen present on Western blots, in solution in enzyme linked immunoassays, in fluorescence activated cells sorting (FACS) assays, in magnetic affinity cell sorting (MACS) assays, and in immunofluorescence microscopy of a cell transiently transfected with a nucleic acid encoding at least a portion of the antigenic protein, for example.

One skilled in the art would appreciate, based upon the disclosure provided herein, that the antibody can specifically bind with any portion of the antigen and the full- length protein can be used to generate antibodies specific therefor. 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 the protein to produce an antibody that specifically binds with a specific antigen. That is, the invention includes immunizing an animal using an immunogenic portion, or antigenic determinant, of the antigen.

Siglec-9 antibody

In various embodiments, the inhibitor of Siglec-9 comprises an antibody, a fragment thereof, or a variant thereof specific for binding to Siglec-9. As used herein, the term "antibody" or "immunoglobulin" refers to proteins (including glycoproteins) of the immunoglobulin (Ig) superfamily of proteins. An antibody or immunoglobulin (Ig) molecule may be tetrameric, comprising two identical light chain polypeptides and two identical heavy chain polypeptides. The two heavy chains are linked together 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.

An anti-Siglec-9 antibody, or antigen-binding fragment thereof, includes, but is not limited to a polyclonal antibody, a monoclonal fusion proteins, antibodies or fragments thereof , chimerized or chimeric fusion proteins, antibodies or fragments thereo , humanized fusion proteins, antibodies or fragments thereof , deimmunized humfusion proteins, antibodies or fragments thereof , fully humfusion proteins, antibodies or fragments thereof , single chain antibody, single chain Fv fragment (scFv), Fv, Fd fragment, Fab fragment, Fab' fragment, F(ab')2 fragment, diabody or antigen- binding fragment thereof, minibody or antigen-binding fragment thereof, triabody or antigen- binding fragment 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 antibody, or antigenbinding fragment thereof, identified with a repetitive backbone array (e.g. repetitive antigen display).

The immune system produces several different classes of Ig molecules (isotypes), including IgA, IgD, IgE, IgG, and IgM, each distinguished by the particular class of heavy chain polypeptide present: alpha (a) found in IgA, delta (8) found in IgD, epsilon (s) found in IgE, gamma (y) found in IgG, and mu (p) found in IgM. There are at least five different y heavy chain polypeptides (isotypes) found in IgG. In contrast, there are only two light chain polypeptide isotypes, referred to as kappa (K) and lambda (X) chains. The distinctive characteristics of antibody isotypes are defined by sequences of the constant domains of the heavy chain.

An IgG molecule comprises two light chains (either K or X form) and two heavy chains (y form) bound together by disulfide bonds. The K and X forms of IgG light chain each contain a domain of relatively variable amino acid sequences, called the variable region (variously referred to as a "VL-," "VK-," or " "Vx-region") and a domain of relatively conserved amino acid sequences, called the constant region (Ct-region). Similarly, each IgG heavy chain contains a variable region (Vu-region) and one or more conserved regions: a complete IgG heavy chain contains three constant domains ("Cui-," " CH2-," and " Cu3- regions") and a hinge region. Within each VL- or Vu-region, hypervariable regions, also known as complementarity-determining regions ("CDR"), are interspersed between relatively conserved framework regions ("FR"). Generally, the variable region of a light or heavy chain polypeptide 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 that IgG molecule binds. Each IgG molecule is dimeric, able to bind two antigen molecules. Cleavage of a dimeric IgG with the protease papain produces two identical antigen-binding fragments ("Fab"') and an "Fc" fragment or Fc domain, so named because it is readily crystallized.

As used throughout the present disclosure, the term "antibody" further refers to a whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule that is generated by any one of a variety of methods that are known in the art and described herein. The term "antibody" includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a deimmunized human antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody.

As used herein, the term "epitope" refers to the site on a protein that is bound by an antibody. "Overlapping epitopes" include at least one (e.g., two, three, four, five, or six) common amino acid residue(s).

In one embodiment, the antibody of the invention specifically binds to a a SARS-CoV-2 antigen. As used herein, the terms "specific binding" or "specifically binds" refer to two molecules forming a complex that is relatively stable under physiologic conditions. Typically, binding is considered specific when the association constant (K_a) is higher than 10⁶ M-l . Thus, an antibody can specifically bind to a target with a Ka of at least (or greater than) 10⁶ (e.g., at least or greater than 10⁷, 10⁸, 10⁹, IO¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ or higher) M’¹.

Methods for determining whether an antibody binds to an antigen and/or the affinity for an antibody to 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 a variety of techniques such as, but not limited to, Western blot, dot blot, surface plasmon resonance method (e.g., BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.), or enzyme-linked immunosorbent assays (ELISA). See, e.g., Harlow and Lane (1988) "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Benny K. C. Lo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Borrebaek (1992) "Antibody Engineering, A Practical Guide," W.H. 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.

Immunoassays which can be used to analyze immunospecific binding and cross-reactivity of the antibodies include, but are not limited to, competitive and noncompetitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art.

Antibodies can also be assayed using any surface plasmon resonance (SPR)- based assays known in the art for characterizing the kinetic parameters of the interaction of the antibody with its target or epitope. Any SPR instrument commercially available including, but not limited to, BIAcore Instruments (Biacore AB; Uppsala, Sweden); lAsys 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) can be used in the methods described herein. See, e.g., 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; and Rich et ai. (2000) Curr Opin Biotechnol 11:54-61.

The antibodies and fragments thereof can be, in some embodiments, "chimeric." Chimeric antibodies and antigen-binding fragments thereof comprise portions from two or more different species (e.g., mouse and human). Chimeric antibodies can be produced with mouse variable regions of desired specificity spliced onto human constant domain gene segments (see, for example, U.S. Patent No. 4,816,567). In this manner, nonhuman antibodies can be modified to make them more suitable for human clinical application (e.g., methods for treating or preventing a complement associated disorder in a human subject).

The monoclonal antibodies of the present disclosure include "humanized" forms of the non-human (e.g., mouse) antibodies. Humanized or CDR-grafted mAbs are particularly useful as therapeutic agents for humans because they are not cleared from the circulation as rapidly as mouse antibodies and do not typically provoke an adverse immune reaction. Methods of preparing humanized antibodies are generally well known in the art. For example, humanization can be essentially performed following the method of Winter and co-workers (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. Also see, e.g., Staelens et al. (2006) Mol Immunol 43:1243-1257. In some embodiments, humanized forms of non-human (e.g., mouse) antibodies are human antibodies (recipient antibody) in which hypervariable (CDR) region residues of the recipient antibody are replaced by hypervariable region residues from a non- human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and binding capacity. In some instances, framework region residues of the human immunoglobulin are also replaced by corresponding non-human residues (so called "back mutations"). In addition, phage display libraries can be used to vary amino acids at chosen positions within the antibody sequence. The properties of a humanized antibody are also affected by the choice of the human framework. Furthermore, humanized and chimerized antibodies can be modified to comprise residues that are not found in the recipient antibody or in the donor antibody in order to further improve antibody properties, such as, for example, affinity or effector function.

Fully human antibodies are also provided in the disclosure. The term "human antibody" includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. Human antibodies can include 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 or human antibodies may be derived from transgenic mice carrying human antibody genes (carrying the variable (V), diversity (D), joining (J), and constant (C) exons) or from human cells. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, 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 mice strains can be engineered to contain gene sequences from unrearranged human immunoglobulin genes. The human sequences may code for both the heavy and light chains of human antibodies and would function correctly in the mice, undergoing rearrangement to provide a wide antibody repertoire similar to that in humans. The transgenic mice can be immunized with the target protein (to create a diverse array of specific antibodies and their encoding RNA. Nucleic acids encoding the antibody chain components of such antibodies may then be cloned from the animal into a display vector. Typically, separate populations of nucleic acids encoding heavy and light chain sequences are cloned, and the separate populations then recombined on insertion into the vector, such that any given copy of the vector receives a random combination of a heavy and a light chain. The vector is designed to express antibody chains so that they can be 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 a phage coat protein from the outer surface of the phage. Thereafter, display packages can be screened for display of antibodies binding to a target.

Thus, in some embodiments, the disclosure provides, e.g., humanized, deimmunized or primatized antibodies comprising one or more of the complementarity determining regions (CDRs) of the mouse monoclonal antibodies described herein, which retain the ability (e.g., at least 50, 60, 70, 80, 90, or 100%, or even greater than 100%) of the mouse monoclonal antibody counterpart to bind to its antigen. In addition, human antibodies can be derived 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 created which use randomized combinations of synthetic human antibody V-regions. By selection on antigen fully human antibodies can be made in which the V- regions are very human-like in nature. 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 which are incorporated herein by reference in their entirety.

For the generation of human antibodies, also see 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 hereby incorporated by reference in their entirety. Human antibodies are further discussed and delineated in U.S. Patent Nos.: 5,939,598; 6,673,986; 6,1 14,598; 6,075, 181; 6, 162,963; 6,150,584; 6,713,610; and 6,657, 103 as well as U.S. Patent Application Publication Nos. 2003- 0229905 Al, 2004-0010810 Al, US 2004- 0093622 Al, 2006-0040363 Al, 2005-0054055 Al, 2005-0076395 Al, and 2005-0287630 Al . See also International Publication Nos. WO 94/02602, WO 96/34096, and WO 98/24893, and European Patent No. EP 0 463 151 Bl. The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, 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) are formed into a construct for insertion into an animal. This approach is described in, e.g., 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; 5,612,205; 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 hereby incorporated by reference. See also European Patent No. 0 546 073 Bl, International Patent Publication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884, the disclosures of each of which are hereby incorporated by reference in their entirety. See further 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: 1 17; 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. I. Immunol. 10: 2998-3005, the disclosures of each of which are hereby incorporated by reference in their entirety.

Methods for making antibodies are within the purview of those skilled in the art. Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy -chain/light-chain pairs, where the two heavy chain/light-chain pairs have different specificities (Milstein and Cuello (1983) Nature 305:537- 539). Antibody variable domains with the desired binding specificities (antibodyantigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably with an immunoglobulin heavy-chain constant domain, including 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 are co-transfected into a suitable host organism. For further details of illustrative currently known methods for generating bispecific antibodies see, e.g., Suresh et al. (1986) Methods in Enzymology 121 :210; PCT Publication No. WO 96/27011 ; Brennan et al. (1985) Science 229:81 ; Shabby 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 hetero-conjugate antibodies. Hetero-conjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross- linking techniques.

Various techniques for making and isolating antibody fragments directly from recombinant cell culture have also been described. 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 the Fos and Jun proteins may be linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers may be reduced at the hinge region to form monomers and then re- oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy- chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen- binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibodies can be "linear antibodies" as described in, e.g., Zapata et al. (1995) Protein Eng. 8(10): 1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH- CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies (e.g., trispecific antibodies) are also contemplated and described in, e.g., Tutt et al. (1991) J Immunol 147:60.

The disclosure also embraces variant forms of multi-specific antibodies such as the dual variable domain immunoglobulin (DVD-lg) molecules described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297. The DVD-lg molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CHI and Fc region. Methods for making DVD-lg molecules from two parent antibodies are further described in, e.g., PCT Publication Nos. WO 08/024188 and WO 07/024715.

The disclosure also provides camelid or dromedary antibodies (e.g., antibodies derived from Camelus bactrianus, Calelus dromaderius, or lama paccos). Such antibodies, unlike the typical two-chain (fragment) or four-chain (whole antibody) antibodies from most mammals, generally lack light chains. See U.S. patent 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.

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, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be "humanized" to thereby further reduce the potential immunogenicity of the antibody.

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 NO: 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 NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40, or a fragment or variant thereof.

In some embodiments, a variant of an amino acid sequence as described herein comprises 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 higher identity over a specified region when compared to a defined amino acid sequence. In some embodiments, a variant of an amino acid sequence as described herein comprises 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 higher identity over the full length of variable heavy chain having an amino acid sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, or 39, or variable light chain having an amino acid sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, or 40.

In some embodiments, the present disclosure also provides antibodies, or antigen-binding fragments thereof, which are variants of a peptide, protein or antibody described herein. In some embodiments, such a variant peptide, protein or antibody maintains the binding or inhibitory ability of the parent peptide, protein or antibody. Methods to prepare variants of known proteins, peptides or antibodies are known in the art. In some embodiments, such a variant comprises at least a single amino acid substitution, deletion, insertion, or other modification. In some embodiments, fusion proteins, antibodies or fragments thereof described herein comprises 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, fusion proteins, antibodies or fragments thereof described herein does not contain an amino acid modification in a CDR. In some embodiments, fusion proteins, antibodies or fragments thereof described herein does 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 modifications in a CDR.

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

In some embodiments, the antibodies described herein comprise an altered or mutated sequence that leads to altered stability or half-life compared to parent antibodies. This includes, for example, an increased stability or half- life for higher affinity or longer clearance time in vitro or in vivo, or a decreased stability or half-life for lower affinity or quicker removal. Additionally, the antibodies described herein may 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, an altered glycosylation pattern (e.g., the addition of one or more sugar components, the loss of one or more sugar components, or a change in composition of one or more sugar components.

In some embodiments, the antibodies described herein comprise reduced (e.g. or no) effector function. Altered effector functions include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc- receptors, and pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody containing an altered constant region as compared to the activity of the unaltered form of the constant region. In particular embodiments, modulation includes situations in which an activity is abolished or completely absent.

Antibodies with altered or no effector functions may be generated by engineering or producing antibodies with variant constant, Fc, or heavy chain regions; recombinant DNA technology and/or cell culture and expression conditions may be used to produce antibodies with altered function and/or activity. For example, recombinant DNA technology may be used to engineer one or more amino acid substitutions, deletions, or insertions in regions (such as, for example, Fc or constant regions) that affect antibody function including effector functions. Alternatively, changes in post- translational modifications, such as, e.g., glycosylation patterns, may be achieved by manipulating the cell culture and expression conditions by which the antibody is produced. Suitable methods for introducing one or more substitutions, additions, or deletions into an Fc region of an antibody are well known in the art and include, e.g., standard DNA mutagenesis techniques as described in, e.g., Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual, 2nd Edition," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; 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.

In some embodiments, the antibody may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“FR”) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N- terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding site, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment, which comprises both antigen-binding sites. Accordingly, the antibody can be the Fab or F(ab’)2. The Fab can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can include the VH region and the CHI region. The light chain of the Fab can include the VL region and CL region.

The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and CL region.

The antibody can be a polyclonal or 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. The humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

The antibody can be a bispecific antibody as described below in more detail. The antibody can be a bifunctional antibody as also described below in more detail.

As described above, the antibody can be generated in the subject upon administration of the composition to the subject. The antibody may have a half-life within the subject. In some embodiments, the antibody may be modified to extend or shorten its half-life within the subject. Such modifications are described below in more detail.

The antibody can be defucosylated as described in more detail below.

As described above, the inhibitor of the invention can comprise an anti- Siglec-9 antibody, a fragment thereof, a variant thereof, or a combination thereof. In some embodiments, the anti-Siglec-9 antibody inhibits Siglec-9 signaling. Therefore, in some embodiments, the antibody of the invention comprises an inhibitory Siglec-9 binding domain.

The inhibitory Siglec-9 binding domain may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“FR”) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding domain, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment, which comprises both antigen-binding sites. Accordingly, the target antigen binding domain of the BiTE can be the Fab or F(ab’)2. The Fab can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can include the VH region and the CHI region. The light chain of the Fab can include the VL region and CL region. The anti-Siglec-9 antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CHI region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and CL region.

The anti-Siglec-9 antibody can be a polyclonal or 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. The humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

ScFv Antibody

In one embodiment, the inhibitor of the invention is a ScFv antibody fragment. In one embodiment, ScFv relates to a Fab fragment without the of CHI and CL regions. Thus, in one embodiment, the ScFv relates to a Fab fragment comprising the VH and VL. In one embodiment, the ScFv comprises a linker between VH and VL. In one embodiment, the inhibitor of the invention is an ScFv-Fc. In one embodiment, the ScFv-Fc comprises the VH, VL and the CH2 and CH3 regions. In one embodiment, the ScFv-Fc comprises a linker between VH and VL. In one embodiment, the ScFv of the invention has modified expression, stability, half-life, antigen binding, heavy chain - light chain pairing, tissue penetration or a combination thereof as compared to a parental antibody.

In one embodiment, the ScFv of the invention has at least 1.1 fold, at least 1.2 fold, 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 least5.5 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 leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher expression than the parental antibody.

In one embodiment, the ScFv of the invention has at least 1 . 1 fold, at least

1.2 fold, 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 least5.5 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 leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher antigen binding than the parental antibody.

In one embodiment, the ScFv of the invention has at least 1 . 1 fold, at least

1.2 fold, 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 leasts .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 leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold longer half-life than the parental antibody.

In one embodiment, the ScFv of the invention has at least 1 . 1 fold, at least

1.2 fold, 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 least5.5 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 leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold higher stability than the parental antibody.

In one embodiment, the ScFv of the invention has at least 1 . 1 fold, at least

1.2 fold, 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 least5.5 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 leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold greater tissue penetration than the parental antibody.

In one embodiment, the ScFv of the invention has at least 1.1 fold, at least 1.2 fold, 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 least5.5 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 leastlO fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold or greater than 50 fold greater heavy chain - light chain pairing than the parental antibody.

Bispecific Antibody

In some embodiments, the inhibitor of the invention comprises 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 a natural killer cell in proximity to a target cell expressing a target antigen (e.g., a viral antigen.) Therefore, 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 a viral antigen. In some embodiments, the target antigen binding domain is specific for binding a SARS-CoV- 2 viral antigen.

The target antigen binding domain of the NKE may comprise an antibody , a fragment thereof, a variant thereof, or a combination thereof. The target antigen binding domain of the NKE may comprise a heavy chain and a light chain complementarity determining region (“CDR”) set, respectively interposed between a heavy chain and a light chain framework (“FR”) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other. The CDR set may contain three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3,” respectively. An antigen-binding domain, therefore, may include six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment, which comprises both antigen-binding sites. Accordingly, the target antigen binding domain of the BiTE can be the Fab or F(ab’)2. The Fab can include the heavy chain polypeptide and the light chain polypeptide. The heavy chain polypeptide of the Fab can include the VH region and the CHI region. The light chain of the Fab can include the VL region and CL region.

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

The target antigen binding domain of the NKE can be a polyclonal or 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. The humanized antibody can be an antibody from a non-human species that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.

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.

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

A bispecific antibody molecule according to the invention may have two binding sites of any desired specificity.

In one embodiment, the synthetic antibody (e.g., NKE) is directed to Siglec-9 and one or more additional antigen or fragment or variant thereof. The antigen can be a nucleic acid sequence, an amino acid sequence, a polysaccharide or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The polysaccharide can be a nucleic acid encoded polysaccharide.

In one embodiment, a synthetic bispecific NKE of the invention targets two or more antigens. In one embodiment, at least one antigen targeted by a 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.

Bifunctional Antibody

In one embodiment, the inhibitory of the invention is a bifunctional antibody, a fragment thereof, a variant thereof, or a combination thereof. The bifunctional antibody can inhibit Siglec-9 as described elsewhere herein. The bifunctional antibody can also be modified to impart an additional functionality to the antibody beyond recognition of and binding to the antigen. Such a modification can include, but is not limited to, coupling to factor H or a fragment thereof. Factor H is a soluble regulator of complement activation and thus, may contribute to an immune response via complement-mediated lysis (CML).

Extension of Antibody Half-Life

The antibody of the invention may be modified to extend or shorten the halflife of the antibody in the subject. The modification may extend or shorten the half-life of the antibody in the serum of the subject.

The modification may be present in a constant region of the antibody. The modification may be one or more amino acid substitutions in a constant region of the antibody that extend the half-life of the antibody as compared to a half-life of an antibody not containing the 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 as compared to a half-life of an antibody not containing the one or more amino acid substitutions.

In some embodiments, the one or more amino acid substitutions in the constant region may include replacing 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 glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.

In other embodiments, the one or more amino acid substitutions in the constant region may include replacing a methionine residue in the CH2 domain with a tyrosine residue, a serine residue in the CH2 domain with a threonine residue, a threonine residue in the CH2 domain with a glutamate residue, or any combination thereof, thereby extending the half-life of the antibody.

Defucosylation

The antibody of the invention may be a defucosylated antibody or a non- fucosylated antibody, a fragment thereof, a variant thereof, or a combination thereof. Fucosylation includes the addition of the sugar fucose to a molecule, for example, the attachment of fucose to N-glycans, O-glycans and glycolipids. Accordingly, in a defucosylated antibody, fucose is not attached to the carbohydrate chains of the constant region. In turn, this lack of fucosylation may improve FcyRIIIa binding and antibody directed cellular cytotoxic (ADCC) activity by the antibody as compared to the fucosylated antibody. Therefore, in some embodiments, the non-fucosylated antibody may exhibit increased ADCC activity as compared to the fucosylated antibody.

The antibody may be modified so as to prevent or inhibit fucosylation of the antibody. In some embodiments, such a modified antibody may exhibit increased ADCC activity as compared to the unmodified antibody. The modification may be in the heavy chain, light chain, or a combination thereof. The modification may 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.

Nucleic Acid Molecules

Provided herein are polynucleotides that encode the Siglec-9 inhibitors, anti- Siglec-9 antibodies, or NKE antibodies, or fragments thereof, of the invention. In some embodiments, the polynucleotide also comprises a sequence encoding a signal peptide operably linked at the 5' end of the encoding sequence. In some embodiments, the polynucleotide also comprises a sequence encoding a linker sequence.

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

In some embodiments, a variant of a nucleotide sequence as described herein comprises 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 higher identity over a specified region when compared to a defined nucleotide sequence. In some embodiments, a variant of a nucleotide sequence as described herein comprises 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 higher identity over the full length of a nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26, encoding a variable heavy chain, or a nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28, encoding a variable light chain.

In some embodiments, a fragment of a nucleotide sequence as described herein comprises 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 a defined nucleotide sequence. In some embodiments, a fragment of a nucleotide sequence as described herein comprises 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 a nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26, encoding a variable heavy chain, or a nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28, encoding a variable light chain.

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

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

In some embodiments, a variant of a nucleotide sequence as described herein comprises 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 higher identity over a specified region when compared to a defined nucleotide sequence. In some embodiments, a variant of a nucleotide sequence as described herein comprises 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 higher identity over the full length of a nucleotide sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26, encoding a variable heavy chain, or a nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28, encoding a variable light chain.

In some embodiments, a fragment of a nucleotide sequence as described herein comprises 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 a defined nucleotide sequence. In some embodiments, a fragment of a nucleotide sequence as described herein comprises 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 SEQ ID NO: 2, 6, 10, 14, 18, 22, or 26, encoding a variable heavy chain, or a nucleotide sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, or 28, encoding a variable light chain.

The isolated nucleic acid may comprise any type of nucleic acid, including, but not limited to DNA, cDNA, and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding a protein inhibitor or functional fragment thereof. In one embodiment, the composition comprises an isolated RNA molecule encoding a Siglec-9 inhibitor, an anti-Siglec-9 antibody, a NKE, or a functional fragment thereof.

The nucleic acid molecules of the present invention can be modified to improve stability. Modifications can be added to enhance stability, functionality, and/or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the invention. For example, in order to enhance the stability, the 3 ’-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2’- deoxythymidine is tolerated and does not affect function of the molecule.

In one embodiment of the present invention the nucleic acid molecule may contain at least one modified nucleotide analogue. For example, the ends may be stabilized by incorporating modified nucleotide analogues.

Non-limiting examples of nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may 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, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e g., 8-bromo guanosine; deaza nucleotides, e g., 7-deaza-adenosine; 0- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. The above modifications may be combined.

In some instances, the nucleic acid molecule comprises at least one of the following chemical modifications: 2’-H, 2’-0-methyl, or 2’-0H modification of one or more nucleotides. In some embodiments, a nucleic acid molecule of the invention can have enhanced resistance to nucleases. For increased nuclease resistance, a nucleic acid molecule, can include, for example, 2’ -modified ribose units and/or phosphorothioate linkages. For example, the 2’ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. For increased nuclease resistance the nucleic acid molecules of the invention can include 2’-0-methyl, 2’-fluorine, 2’-O-methoxyethyl, 2’-0- aminopropyl, 2’-amino, and/or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 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, can also increase binding affinity to a target.

In one embodiment, the nucleic acid molecule includes a 2’ -modified nucleotide, e.g., a 2’-deoxy, 2 ’-deoxy-2’ -fluoro, 2’-0-methyl, 2’-O-methoxyethyl (2’-0- MOE), 2’-O-aminopropyl (2’-0-AP), 2’-O-dimethylaminoethyl (2’-0-DMA0E), 2’-0- dimethylaminopropyl (2’-0-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-0-DMAE0E), or 2’-O-N-methylacetamido (2’-0-NMA). In one embodiment, the nucleic acid molecule includes at least one 2’-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2’-0-methyl modification.

Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, for example as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termed modified RNAs, are typically the result of a post- transcriptional modification and are within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, for example different from that which occurs in the human body. While they are referred to as “modified RNAs” they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.

Modifications of the nucleic acid of the invention may be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.

The present invention also includes a vector in which the isolated nucleic acid of the present invention is inserted. The art is replete with suitable vectors that are useful in the present invention.

Therefore, in another aspect, the invention relates to a vector, comprising the nucleotide sequence of the invention or the construct of the invention. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In some embodiments, the vector of the invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available. In some embodiments, the expression of synthetic nucleic acids encoding a protein is typically achieved by operably linking a nucleic acid encoding the protein or portions thereof to a promoter and incorporating the construct into an expression vector. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

The recombinant nucleic acid sequence construct can include one or more transcription termination regions. The transcription termination region can be downstream of the coding sequence to provide for efficient termination. The transcription termination region can be obtained from the same gene as the promoter described above or can be obtained from one or more different genes.

The recombinant nucleic acid sequence construct can include one or more initiation codons. The initiation codon can be located upstream of the coding sequence. The initiation codon can be in frame with the coding sequence. The initiation codon can be associated with one or more signals required for efficient translation initiation, for example, but not limited to, a ribosome binding site.

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

The recombinant nucleic acid sequence construct can include one or more polyadenylation signals. The polyadenylation signal can include one or more signals required for efficient polyadenylation of the transcript. The polyadenylation signal can be positioned downstream of the coding sequence. The polyadenylation signal may be a SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, or human 0- globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA). The recombinant nucleic acid sequence 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, for example, but not limited to, an IgG signal peptide and an IgE signal peptide.

The vectors of the present invention may also be used for nucleic acid immunization, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.

The isolated nucleic acid of the invention can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the vector may be provided to a cell in the form of a viral vector. Viral vectortechnology is well 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 in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193. By way of illustration, the vector in which the nucleic acid sequence is introduced can be a plasmid, which is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a tet-on inducible vector for expression in eukaryote cells.

The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells.

In one embodiment, the recombinant expression vectors may also contain nucleic acid molecules, which encode a peptide or protein of invention, described elsewhere herein.

A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

For example, vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

In some embodiments, the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance 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 and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.

A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2012). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

The recombinant expression vectors may also contain a selectable marker gene, which 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, P-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin, such as IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

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 driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor -la (EF-la). However, other constitutive promoter sequences may 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, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.

In order to assess the expression of a protein inhibitor, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

Physical methods for introducing a peptide or protein into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

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

Chemical means for introducing a peptide or protein into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or lipid nanoparticle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

Host cells

Also provided are host cells (such as isolated cells, transient cell lines, and stable cell lines) for expressing the molecule described herein. The host cell may be prokaryotic or eukaryotes. Exemplary prokaryote 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), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans. and various Pseudomonas species. One suitable prokaryotic host cell isE. coli BL21 (Stratagene), which is deficient in the OmpT and Lon proteases, which may interfere with isolation of intact recombinant proteins, and useful with T7 promoter-driven vectors, such as the pET vectors. Another suitable prokaryote is E. coli W3110 (ATCC No. 27325). When expressed by prokaryotes the peptides typically contain an N-terminal methionine or a formyl methionine and are not glycosylated. In the case of fusion proteins, the N-terminal methionine or formyl methionine resides on the amino terminus of the fusion protein or the signal sequence of the fusion protein. These examples are, of course, intended to be illustrative rather than limiting. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for fusion-protein-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC No. 16,045), K. wickeramii (ATCC No. 24,178), K. waltii (ATCC No. 56,500), K. drosophilarum (ATCC No. 36,906; Van den Berg et al., Bio/Technology, 8: 135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259- 5263 (1979)); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A. 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 A. niger (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methyl otrophs, 269 (1982). Host cells also include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.

Examples of useful mammalian host cell lines include, but are not limited to, HeLa, Chinese hamster ovary (CHO), COS-7, L cells, C127, 3T3, BHK, CHL-1, NSO, HEK293, WI38, BHK, Cl 27 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 RPMI 7032 melanoma cells, a readily available human cell line. Cells suitable for use in this invention are commercially available from the ATCC. Substrates

In one embodiment, the present invention provides a scaffold, substrate, or device comprising a bispecific immune cell engager, fragment thereof, or nucleic acid molecule encoding the same. For example, in some embodiments, the present invention provides a tissue engineering scaffold, including but not limited to, a hydrogel, electrospun scaffold, polymeric matrix, or the like, comprising the modulator. In certain embodiments, a bispecific immune cell engager, fragment thereof, or nucleic acid molecule encoding the same, may be coated along the surface of the scaffold, substrate, or device. In certain embodiments, the bispecific immune cell engager, fragment thereof, or nucleic acid molecule encoding the same is encapsulated within the scaffold, substrate, or device.

Delivery Vehicles

In one embodiment, the present invention provides a composition comprising a delivery vehicle comprising a bispecific anti-SARS-CoV-2 immune cell engaging antibody, fragment thereof, or nucleic acid molecule encoding the same, as described herein. In one embodiment, the nucleic acid molecule encoding the bispecific anti-SARS-CoV-2 immune cell engaging antibody comprises an mRNA molecule.

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 a bispecific anti-SARS-CoV-2 immune cell engaging antibody of the invention or a fragment thereof. In one embodiment, the nucleic acid molecule encoding the bispecific anti-SARS-CoV-2 immune cell engaging antibody comprises an mRNA molecule. In one embodiment, the mRNA encoding the bispecific anti-SARS-CoV-2 immune cell engaging 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 engaging antibody encodes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8. In some embodiments, the delivery vehicle provides for controlled release, delayed release, or continual release of its loaded cargo. In some embodiments, the delivery vehicle comprises a targeting moiety that targets the delivery vehicle to a treatment site.

In certain instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 min of in vivo injection of the encoding mRNA. In certain embodiments, using mRNA rather than the protein also has many advantages. Halflives of proteins in the circulation are often short, thus protein treatment would need frequent dosing, while mRNA provides a template for continuous protein production for several days. Purification of proteins is problematic and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

In order to confirm the presence of the mRNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Northern blotting and RT- PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunogenic means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

Combination with Additional Agent(s)

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

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

Treatment Methods

The present invention provides methods of inhibiting Siglec-9 activity in a natural killer cell of a target subject. In some embodiments, the 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 inhibitor of the invention. In some emboidments, the composition comprising one or more Siglec-9 inhibitor further comprises one or more adjuvants, one or more additional therapeutic agents, or a combination thereof. In some emboidments, the composition comprising one or more Siglec-9 inhibitor functions as an adjuvant to increase the efficacy of an immune response against a target antigen. In some emboidments, the composition comprising one or more Siglec-9 inhibitor comprises an immunotherapy for SARS-CoV-2 infection or COVID- 19.

In some embodiments, the method provides or enhances immunity in the target subject to an infection, or a disease, or disorder associated with an infectious agent. The present invention thus provides a method of treating or preventing the infection, or a disease, or disorder associated with an infectious agent. For example, the method may be used to treat or prevent a viral infection, bacterial infection, fungal infection, or a parasitic infection, depending upon the type of antigen of the administered composition. Exemplary antigens and associated infections, diseases, and tumors are described elsewhere herein. In one embodiment, the composition is administered to a target subject having a SARS-CoV-2 infection or COVID-19. In one embodiment, the composition is administered to a subject at risk for developing a SARS-CoV-2 infection or COVID-19.

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

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

In one embodiment, the compositions of the 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 antibody. In one embodiment, the method comprises administering an LNP composition comprising a nucleic acid molecule encoding one or more bispecific anti-Siglec-9 antibody.

In certain embodiments, the method comprises administering to subject a combination of a Siglec-9 inhibitor of the invention and one or more additional therapeutic agent. In some embodiments the additional therapeutic agent is an additional agent for treatment of a target pathogen, and adjuvant, or a combination thereof.

Administration of the compositions of the invention in a method of treatment can be achieved in a number of different ways, using methods known in the art. In one embodiment, the method of the invention comprises systemic administration of the subject, including for example enteral or parenteral administration. In certain embodiments, the method comprises intradermal delivery of the composition. In another embodiment, the method comprises intravenous delivery of the composition. In some embodiments, the method comprises intramuscular delivery of the composition. In one embodiment, the method comprises subcutaneous delivery of the composition. In one embodiment, the method comprises inhalation of the composition. In one embodiment, the method comprises intranasal delivery of the composition.

It will be appreciated that the composition of the invention may be administered to a subject either alone, or in conjunction with another agent.

The therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions comprising a Siglec-9 inhibitor of the invention, an adjuvant, or a combination thereof, described herein to practice the methods of the invention. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from 0.001 ng/kg/day to 100 mg/kg/day. In one embodiment, the invention envisions administration of a dose which results in a concentration of the compound of the present invention from lOnM to 10 pM in a mammal.

Typically, dosages which may be administered in a method of the invention to a mammal, preferably a human, range in amount from 0.01 pg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. Preferably, the dosage of the compound will vary from about 0.1 pg to about 10 mg per kilogram of body weight of the mammal. More preferably, the dosage will vary from about 1 pg to about 1 mg per kilogram of body weight of the mammal.

The composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

In certain embodiments, administration of a Siglec-9 inhibitor or antibody of the present invention, or a nucleic acid molecule encoding the same, may be performed by single administration or boosted by multiple administrations. Examples

The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example l.~ Blocking Siglec-9 interactions increase NK cytotoxicity against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters the immunological profiles of natural killer (NK) cells, effector cells that play an important role in controlling viral infections. However, whether anti-SARS-CoV-2 NK functions are impaired during severe coronavirus disease 2019 (COVID-19) and what host factors modulate these functions remain unclear. The results presented herein show that NK cells from hospitalized COVID-19 patients had significantly less direct cytolytic and antibody-dependent cell cytotoxicity (ADCC) activities against SARS-CoV-2-antigen- expressing cells than did NK cells from mild COVID-19 patients or negative controls. The lower NK cytotoxicity was associated with higher plasma levels of SARS-CoV-2 nucleocapsid-antigen. Detailed phenotypic and functional analyses showed thatNK cells expressing the gly co-immune checkpoint Siglec-9 elicited higher SARS-CoV-2-specific ADCC than Siglec-9' NK cells. Consistently, Siglec-9⁺ NK cells expressed an activated and mature phenotype with higher expression of CD16 (FcyRIII; mediator of ADCC), CD57 (maturation marker), and NKG2C (activating receeptor), along with lower expression of the inhibitory receptor NKG2A, than Siglec-9' CD56^dim NK cells. These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic against SARS-CoV-2. However, the Siglec-9 molecule itself is an inhibitory receptor that is established to restrain NK cytotoxicity during cancer and other viral infections. Indeed, blocking Siglec-9 using an anti-Siglec-9 antibody significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells. These data support a model in which the Siglec-9⁺ CD56^dim NK subpopulation is cytotoxic against SARS-CoV-2+ cells even while being restrained by the inhibitory effects of Siglec-9. However, alleviating the Siglec-9-mediated restriction on NK cytotoxicity using Siglec-9 blocking antibodies can further improve the anti-SAR-CoV-2 NK immune surveillance. Taken together, the results presented herein identify a novel glyco-immune checkpoint mechanism that contributes to the ability of SARS-CoV-2+ cells to evade NK immunosurveillance and suggest a potential strategy to overcome this evasion.

The data presented herein combines patient data with in vivo and in vitro immunological data to identify and characterize a targetable novel glyco-immune checkpoint mechanism during SARS-CoV-2 infection. It is described herein that NK cells from SARS-CoV-2 hospitalized patients are less able to target SARS-CoV-2 than are NK cells from SARS-CoV-2 mild patients or uninfected controls. The cytotoxicity of the Siglec- 9⁺ CD56^dim NK subpopulation against SARS-CoV-2 is decreased by the Siglec-9 molecule and blocking Siglec-9 enhances the ability of NK cells to target cells expressing SARS- CoV-2 antigens. The Siglec-9⁺ CD56^dimNK subpopulation is highly activated, mature, and cytotoxic against SARS-CoV-2 antigen-expressing cells compared to the Siglec-9' CD56^dim NK subpopulation. The herein described results depict a new immunotherapeutic target in the glyco-immune checkpoint mechanism, Siglec-9/sialoglycan interactions, that may contribute to the ability of SARS-CoV-2 to evade NK immune surveillance.

The results of the experiments are now described.

NK cells from hospitalized COVID-19 patients degranulate less than NK cells from mild COVID- 19 patients or uninfected controls against SARS-CoV-2-Spike-expressing target cells

To examine whether severe COVID-19 impairs the anti-SARS-CoV-2 functions of NK cells, peripheral blood mononuclear cells (PBMCs) and plasma were collected from 79 individuals with three COVID-19 disease states: 1) SARS-CoV-2 negative control (negative; n=12), COVID-19 outpatients (mild; n=26), and COVID-19 inpatients (hospitalized; n=41) (Table 1). First, using samples from a subset of these individuals with sufficient PBMCs (n=8 negative, n=12 mild, and n=21 hospitalized), the anti -SARS-CoV-2 specific direct cytolytic and ADCC activities of NK cells was assessed. NK cells could be polyfunctional: directly lyse target cells by releasing cytolytic granules; and secrete cytokines and chemokines, such as IFN-y and TNF-a (Cooper MA, et al., 2001, Trends Immunol 22:633-40). The polyfunctionality of NK cells has been associated with enhanced anti-viral immune responses (Kamya P, et al., 2011, J Virol 85:5949-60).

Therefore, direct cytotoxicity and ADCC by both NK degranulation (expression of CD 107a) and cytokine production (the expression of IFN-y and TNF-a) was assessed against SARS- CoV-2 Spike-expressing 293T target cells measured by flow cytometry (a gating strategy is in Figure 2A). Direct cytotoxicity was calculated by subtracting the background NK degranulation/cytokine production of PBMCs cultured alone from the NK degranulation/cytokine production when PBMCs were co-cultured with target cells (Figure 1A, left). For assessing ADCC, bulk IgGs were isolated from the plasma of the study participants. The IgGs from the SARS-CoV-2 negative donors were pooled to create a negative antibody pool, and the IgGs from the SARS-CoV-2 positive donors were pooled to create a positive antibody pool. IgGs were pooled to ensure that the quantitative and qualitative features of the antibodies used in the ADCC assays are constant. Having constant levels of SARS-CoV-2 specific antibodies would allow examining the ADCC capacity of NK cells from different donors without the potential confounding effects of different levels or qualities of SARS-CoV-2 specific antibodies. NK degranulation/cytokine production by ADCC was measured by co-culturing PBMCs and target cells in the presence of the negative antibody pool or the positive antibody pool. ADCC was then assessed by subtracting the percent NK degranulation/cytokine production of the co-culture with the negative antibody pool from the percent NK degranulation/cytokine production of the coculture with the positive antibody pool (after subtracting the background NK degranulation/cytokine production) (Figure 1A, right).

It was first found that the direct cytolytic-mediated degranulation/cytokine production (Figure IB-Figure IE, Figure 2B-Figure 2D) and ADCC-mediated degranulation (Figure IF-Figure II), Figure 2E-Figure 2G) against SARS-CoV-2 Spike-expressing cells of CD56dim NK cells from hospitalized COVID-19 patients were lower than those of the NK cells from the mild group or the SARS-CoV-2 negative controls. In a sub-analysis, it was found that the cytotoxic activities of NK cells from the female participants were higher than those from the male participants, suggesting a sex-dependent difference in NK activities against SARS-CoV-2 Spike-expressing targets (Figure 3). Next, it was examined whether the reduced degranulation/cytokine production observed from the NK cells of hospitalized COVID- 19 donors results in decreased lysis of SARS-CoV-2 Spike-expressing target cells. For these experiments, PBMCs from hospitalized or mild COVID-19 patients (as effector cells) and SARS-CoV-2 S CH0-K1 cells (as target cells) were used. The SARS-CoV-2 S CH0-K1 cells stably express the SARS-CoV-2 Spike (S) protein and a HaloTag-HiBiT protein; when these cells are lysed by ADCC, the intracellular HaloTag-HiBiT protein interacts with an extracellular detection reagent to generate a luminescence signal that can be quantified to measure target cell lysis. As shown in Figure 4A, PBMCs from hospitalized COVID- 19 donors exhibit lower ADCC than PBMCs from mild COVID- 19 donors, consistent with the NK degranulation/cytokine production data. Finally, the cytotoxicity of NK cells from the three COVID-19 disease states was examined against cells not expressing SARS-CoV-2 Spike protein (K562 cells). It was found thatNK cells from hospitalized COVID- 19 patients degranulate less against K562 cells than did NK cells from the mild group or the SARS-CoV-2 negative controls (Figure 4B-Figure 4F). These data suggest that these NK cells from hospitalized COVID-19 patients have a general impairment of cytotoxic activities.

Next, it was examined whether the NK degranulation measured ex vivo correlated with markers of disease severity in vivo. Plasma SARS-CoV-2 plasma nucleocapsid (N) antigen load correlatecorrelates with disease severity (Wang H, et al., 2021, Clin Chem 68:204-213; Yokoyama R, et al., 2021, Front Microbiol 12:791489; Shan D, et al., 2021, Nat Commun 12: 1931). Therefore the N-antigen load was measured in the plasma samples using the ultra-sensitive Simoa SARS-CoV-2 N-protein assay and correlated the N-antigen load with NK direct cytolytic and ADCC activities. As shown in Figure 11-Figure IM, NK degranulation correlated inversely with SARS-CoV-2 N-antigen load. Together, these data suggest that NK degranulation against SARS-CoV-2 is compromised during severe COVID- 19. Therefore, developing strategies to enhance the anti-SARS-CoV-2 NK cytotoxicity may improve disease outcomes. However, developing such strategies requires a better understanding of the factors determining NK functions against SARS-CoV-2.

Siglec-9⁺ CD56^dun NK cells exhibit higher antibody-mediated cytotoxicity against SARS- CoV-2-Spike-expressing target cells than do Siglec-9' CD56^dun NK cells.

To determine the molecular mechanisms involved in NK cytotoxicity against SARS-CoV-2 the Siglecs expressed on NK cells, Siglec-9 and Siglec-7 were examined for their role in establishing either the direct cytolytic activity or the ADCC activity of NK cells against SARS-CoV-2 Spike-expressing target cells. This used data from experiments in Figure 1 after gating on either Siglec-9⁺ CD56^dim NK cells or Siglec-9' CD56^dim NK cells. There was no observable difference in the direct cytolytic activity of these two subpopulations; however, the Siglec-9⁺CD56^dim NK cells exhibited significantly higher ADCC against target cells than the Siglec-9' CD56^dim NK cells, irrespective of disease state (Figure 5A-Figure 51). This was consistently observed when evaluating the percentage of cells expressing CD107a, IFN-y, and TNF-a (Figure 5A-Figure 5C) or co-expressing CD107a and IFN-y, CD107aand TNF-a, or IFN-y and TNF-a (Figure 5D-Figure 5F) on each of the two sub-populations. It was also consistent when the mean fluorescence intensity (MFI) of IFN107a, IFN-y, and TNF-a was evaluated on each of the two sub-populations (Figure 5G- Figure 51). Next, the associations between the direct cytolytic or ADCC activity of the Siglec-9⁺ CD56^dim and the Siglec-9' CD56^dim subpopulations were examined, measured ex vivo, and the plasma N-antigen load, measured in vivo (Figure 5 J). The direct cytolytic activity of both subpopulations correlated negatively with the plasma N-antigen load, indicating that the Siglec-9⁺ NK subpopulation is likely not playing a unique role in anti- SARS-CoV-2 direct cytotoxicity. However, the ADCC ability of the Siglec-9⁺ but not the Siglec-9' CD56^dim NK cells correlated negatively with the plasma N-antigen load (Figure 5J). These data suggest that Siglec-9⁺ CD56^dim NK cells are a subpopulation ofNK cells with potentially high anti-SARS-CoV-2 ADCC activity. Siglec-7⁺ CD56^dun NK cells exhibit higher direct cytotoxicity and ADCC towards SARS- CoV-2-Spike-expressing target cells than do Siglec-7' CD56^dun NK cells.

The direct cytolytic and ADCC activities of the Siglec-7⁺ CD56^dim NK and Siglec-7' CD56^dim NK cells were examined (Figure 6). Unlike Siglec-9⁺CD56^dim NK, Siglec-7⁺ CD56^dun NK cells exhibited higher direct cytolytic activity against SARS-CoV-2- Spike-expressing target cells than their Siglec-7' counterparts, as estimated by the percentage of cells expressing CD 107a, TNF-a, and CD 107a and TNF-a (Figure 6A-Figure 6C). The Siglec-7⁺ CD56^dim NK cells exhibited higher SARS-CoV-2 specific ADCC than did the Siglec-7' CD56^dim NK cells, as estimated by the percentage of cells expressing CD107a, IFN-y, and TNF-a (Figure 6D-Figure 6F), or co-expressing CD107a and IFN-y, CD107a and TNF-a, or IFN-y and TNF-a⁺ (Figure 6G-Figure 61). These data were also consistent when examining the MFI of CD 107a, IFN-y, and TNF-a (Figure 7). These cytolytic activities correlated more strongly with a lower plasma N-antigen load compared with the activities of the Siglec-7' CD56^dim NK subpopulation (Figure 6J). These data are consistent with the high direct cytolytic and ADCC activities of the Siglec-7⁺ CD56^dim NK subpopulation. These data suggest that the Siglec-7' NK subpopulation is dysfunctional during SARS-CoV-2 infection.

Siglec-9⁺ CD56^dun NK cells exhibit an activated and mature phenotype in vivo.

The NK subpopulations that express Siglec-9 or Siglec-7 exhibit higher cytotoxicity against SARS-Cov2 infected cells than their Siglec-negative counterparts. Thus, the expression of several activating and inhibitory receptors on NK cells from the entire cohort (n=79) was evaulated. Measurements included the expression of CD 16 (FcyRIII; mediator of ADCC), CD57 (maturation marker), NKG2C (activating receptor), and NKG2A (inhibitory receptor) on Siglec-9⁺, Siglec-9', Siglec-7⁺, and Siglec-7' CD56^dim NK cells (Figure 8).

Given that Siglec-9 is expressed only on a subset of CD56dim NK cells, qPCR was used to validate the specificity of the Siglec-9 Ab (clone K8; Biolegend) in identifying NK subpopulation with high levels of Siglec-9 transcripts. Data in Figure 9 show that this Ab identifies cells with higher levels of Siglec-9 transcripts compared to Siglec-9 negative NK cells. In a sub-analysis, it was also found that Siglec-7 and Siglec-9 expression on CD56dim NK cells is sex-dependent. In particular, CD56dim NK cells from the female participants express higher levels of Siglec-9+, higher levels of Siglec-7+, lower levels of Siglec-9- Siglec7-, lower levels of Siglec-9+ Siglec-7-, higher levels of Siglec-9- Siglec-7+, and higher levels of Siglec-9+ Siglec-7+ cells than cells from the male participants (Figure 10).

Consistent with previous reports (Varchetta S, et al., 2021, Cell Mol Immunol 18:604-612), it was found that CD56dim NK cells from hospitalized COVID-19 patients, compared to controls, show a reduction in the percentage of cells expressing CD16 (Figure 11A), an increase in the percentage of cells expressing CD57 (Figure 1 IB), and a reduction in the percentage of cells expressing Siglec-7 (Figure 11C). Focusing on the expression of these markers on Siglec-9+, Siglec-9-, Siglec-7+, and Siglec-7- cells, it was found that the Siglec-9+ CD56dim NK cells had an activated and mature phenotype, compared to Siglec-9- CD56dim NK cells, as the Siglec-9+ cells had higher expression of activation markers/receptors CD 16, CD57, and NKG2C, along with lower expression of the inhibitory receptor NKG2A than the Siglec-9- cells (Figure 12A). The maturation/activation status of the Siglec-7+ CD56dim cells, compared to the Siglec-7- CD56dim NK cells, was less clear. Siglec-7+ CD56dim cells had higher levels of CD 16 and NKG2C than Siglec-7- CD56dim cells, but no difference in the expression of CD57, and higher levels of inhibitor marker NKG2A (Figure 12B). These data suggest that Siglec-9+ CD56dim cells are an activated and mature NK subpopulation, which might explain their higher ADCC activity against SARS-CoV-2-Spike expressing target cells.

Siglec-9, but not Siglec-7, marks CD56^dim NK cells with high ADCC activity against SARS- CoV-2,

Data in Figures 5 and 6 suggest that both the Siglec-9⁺ and Siglec-7⁺ CD56^dim NK cells exhibit high ADCC activity against SARS-CoV-2. However, Siglec-9 and Siglec-7 are not mutually exclusively expressed on NK cells, and thus there is a population of NK cells that express both Siglec-9 and Siglec-7 (Figure 13A). Data was reanalyzed to investigate whether NK cells expressing Siglec-9 and/or Siglec-7 have higher ADCC activity against SARS-CoV-2 than do NK cells expressing only Siglec-9 or -7. The in vivo phenotype data presented in Figure 4 was reanalyzed to examine the expression of CD 16, CD57, NKG2C, and NKG2A on each of the four possible CD56^dimNK subpopulations: Siglec-9' Siglec-7', Siglec-9' Siglec-7⁺, Siglec-9⁺ Siglec-7', and Siglec-9⁺ Siglec-7⁺. Data in Figure 13B-Figure 13E show that the Siglec-9⁺ cells, regardless of Siglec-7 expression, express high levels of CD16, CD57, NKG2C, and low levels of NKG2A. On the other hand, the Siglec-7⁺ cells, regardless of Siglec-9 expression, express high levels of CD16 but not CD57 or NKG2C. Furthermore, the Siglec-7⁺ cells express high levels of the inhibitory receptor NKG2A. These data further suggest that Siglec-9, but not Siglec-7, marks cells with activated and mature phenotypes.

To examine the ADCC potential of each of the four NK subpopulations the data presented in Figures 5 and 6 was reanalyzed. Data in Figure 13F-Figure 13H suggest that the CD56^dimNK cells that express Siglec-9, regardless of their Siglec-7 expression, have higher ADCC ability against SARS-CoV-2 compared to cells that do not express Siglec-9. Together, these data suggest that NK cells expressing Siglec-9 exhibit an activated and mature phenotype that may contribute to their high ADCC towards SARS-CoV-2-Spike expressing target cells.

Blocking Siglec-9 interactions with Siglec-9 blocking antibody, enhances the anti-SARS- CoV-2 ADCC of CD56^dun NK cells.

The 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 gly co-immune checkpoint to restrict NK cytotoxicity (Adeniji OS, et al., 2021, PLoS Pathog 17:el010034; Jandus C, et al., 2014, J Clin Invest 124:1810- 20; Zhao D, et al., 2018, Front Immunol 9: 1124). Therefore an in house Siglec-9 blocking antibody was used to determine if blocking the inhibitory signaling of Siglec-9 further enhanced the ADCC activity of Siglec-9+ NK cells against SARS-CoV-2. The impact of the Siglec-9 blocking antibody, compared to an isotype control, on the ADCC-mediated NK degranulation against SARS-CoV-2 (Figure 14A-Figure 14E) was examined. These experminents included PBMCs from six healthy controls (as effector cells) and 293T cells expressing SARS-CoV-2 Spike (as target cells). Blocking Siglec-9 significantly enhanced the ADCC potential of CD56^dimNK cells as shown by the increased percentages of CD107a⁺ (Figure 14A), CD107a⁺IFN-Y⁺ (Figure 14B), CD107a⁺TNF-a (Figure 14C), TNF- a⁺ (Figure 14D), or IFN-y TNF-a⁺ (Figure 14E) CD56^dim NK cells.

Further experiments determined whether the enhanced NK degranulation/cytokine production caused by blocking Siglec-9 resulted in increased lysis of target cells. These experiments used purified NK cells isolated from the PBMCs of five healthy donors (as effector cells) and SARS-CoV-2 S CH0-K1 cells (as target cells). As shown in Figure 14F, blocking Siglec-9 enhanced the ADCC-mediated lysis of target cells compared to the isotype control. These data show that the Siglec-9 interactions indeed restrain the ADCC activity of the already highly activated and cytotoxic Siglec-9⁺ NK cells and suggest that strategies to target Siglec-9 may enhance NK cytotoxicity against SARS- CoV-2.

Previous reports have associated severe COVID-19 with alterations to the profiles of NK cells (Varchetta S, et al., 2021, Cell Mol Immunol 18:604-612). However, whether these alterations result in a dysfunctional anti-SARS-CoV-2 NK cytotoxicity is not fully studied. In this report, functional assays were performed to examine both the direct cytolytic and ADCC-mediated degranulation of NK cells from hospitalized COVID-19 patients and controls. This data suggest that severe COVID-19 is not only associated with alterations to the phenotypic profiles of NK cells but also with a reduction in their cytolytic and ADCC activities. The mechanisms underlying these dysfunctions are unclear; however, severe COVID-19 is associated with a state of hyper-inflammation, characterized by an ensuing 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 dysregulated myeloid cell functions (lunqueira C, et al., 2022, Nature 606:576-584; Knoll R, et al., 2021, Front Immunol 12:720109). NK cell functions can be significantly modulated by the cytokine milieu (Brady J, et al., 2010, J Immunol 185:6679-88; Romee R, et al., 2014, Scientifica (Cairo) 2014:205796; ZwirnerNW, 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). Whether deregulated cytokine secretion, such as TGF-0 (Barros- Martins J, et al., 2022, Signal Transduct Target Ther 7:32), and myeloid cell dysfunction directly and/or indirectly contribute to the diminished NK functions during severe COVID- 19 warrants further investigations. Also unknown is the causative versus consequential effects of the diminished NK functions and COVID-19 severity. Studies in animal models of SARS-CoV-2 infection will be needed to explore this potential link. However, knowing that the anti-SARS-CoV-2 cytolytic activities of NK cells are likely compromised during severe COVID- 19, regardless of the contribution of these functions to the disease severity, suggests the need to develop strategies to enhance NK functions during severe SARS-CoV-2 infections, and other similar emerging viruses, to control the infection and decrease disease severity. These strategies could be particularly important in immunocompromised individuals who might need an immunotherapeutic approach 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).

Identifying NK subpopulations capable of targeting virally-infected cells could be an essential step in developing efficient strategies to enhance NK cytotoxicity against SARS-CoV-2 and other viral infections. In this report, focus was on NK cells expressing Siglec-7 and/or Siglec-9. Siglecs are emerging ITIM-containing, MHC- independent inhibitory receptors that control host immune responses by interacting with sialoglycans on the surface of target cells. Siglec-7 is expressed on almost all NK cells and binds to a2-8 Sialic acid, whereas Siglec-9 is selectively expressed on a subset of CD56^dim NK cells and binds to a2-3 Sialic acid (Adeniji OS, et al., 2021, PLoS Pathog 17:el010034; Belisle JA, et al., 2010, Mol Cancer 9: 118). The Siglec-7’ CD56^dim NK subpopulation was identified, which is being accumulated during severe COVID-19, as a dysfunctional NK subpopulation during SARS-CoV-2 infection. This is consistent with previous reports describing decreased levels of Siglec-7 as a marker for 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, the Siglec-9⁺ CD56^dim NK subpopulation, which has never been implicated during SARS-CoV-2 infection, was also identified as a highly cytotoxic NK subpopulation. This is also consistent with previous reports that this NK subpopulation exhibits high anti-viral activity during HIV infection (Adeniji OS, et al., 2021, PLoS Pathog 17:el010034).

The Siglec-9⁺ CD56^dim NK cells have an activated phenotype (higher expression of activating receptors and lower 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:el010034). Indeed, it was found that the Siglec- 9⁺ CD56^dim NK exhibits an activated phenotype with higher levels of activating/maturation receptors and markers and lower expression of the inhibitory receptor NKG2A, compared to Siglec-9' CD56^dim NK cells, during SARS-CoV-2 infection. Based on these results, these cells have an activated phenotype even in healthy controls, suggesting that this population of cells is naturally activated with potential cytotoxic capacity and can be exploited against several viral and non-viral infections.

The highly activated phenotype of the Siglec-9⁺ CD56^dim NK cells is consistent with the functional analysis demonstrating that the Siglec-9⁺ NK cells exhibit higher ADCC than Siglec-9' NK cells. These results are consistent with the highly cytotoxic nature of Siglec-9⁺ NK cells. However, the Siglec-9 receptor itself is an inhibitory receptor that restrains the cytolytic ability of these otherwise highly cytotoxic Siglec-9⁺ NK cells. The binding of Siglec-9 to a2-3 Sialic acid on target cells induces an inhibitory signal transduction cascade by recruiting the tyrosine phosphatase SHP-1, which counteracts the phosphorylation-mediated activation of other signaling molecules (Crocker PR, et al., 2007, Nat Rev Immunol 7:255-66; Avril T, et al., 2004, 1 Immunol 173:6841-9). Indeed, blocking Siglec-9 further enhanced the ability of NK cells to kill target cells expressing SARS-CoV-2 antigen by ADCC. This result is consistent with the known inhibitory function of the Siglec- 9 molecule itself on these otherwise cytotoxic cells. These data support a model in which Siglec-9⁺ CD56^dun NK cells are cytotoxic but are restrained by the inhibitory nature of Siglec-9 receptor signaling (Figure 15, left two panels). Furthermore, these data suggest that blocking Siglec-9 interactions is a promising strategy to unleash the full potential of the Sigec-9⁺ NK subpopulation that is otherwise highly cytotoxic (Figure 15, right panel). In this study, it was highlighted that the Siglec-9/Sialic acid axis as a glyco- immune checkpoint mechanism that viral infections may exploit to evade immune surveillance by the cytotoxic Siglec-9⁺ NK cells. The potential of Siglec-9 blocking antibody to enhance the anti-SARS-CoV-2 ADCC activity was examined. Indeed, in proof- of-concept experiments, it was found that blocking Siglec-9, using a blocking antibody, enhanced the anti-SARS-CoV-2 specific ADCC activity of NK cells in vitro. In the last few years, several Siglec blocking antibodies have been tested for their ability to prevent Siglec- mediated inhibition of immune functions. Blocking antibodies against Siglec-7 and Siglec-9 enhances anti-tumor 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, 1 Clin Invest 128:4912-4923; Ibarlucea-Benitez I, et al., 2021, Proc Natl Acad Sci U S A 118; Choi H, Ho M, et al., 2021, Front Oncol 11 :778989). Similarly, blocking Siglec-9 interactions enhanced NK cytotoxicity against HIV-infected targets (Adeniji OS, et al., 2021, PLoS Pathog 17:el 010034) and reversed the dysfunctionality of NK cells during HBV infection (Zhao D, et al., 2018, Front Immunol 9:1124). These blocking antibodies are promising tools to enhance NK cytotoxicity against virally infected cells. However, mono-specific antibodies blocking Siglecs may possibly induce non-specific inflammation as Siglecs are 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 immune checkpoints against hyper-inflammation and autoimmunity (Schwarz F, et al., 2015, Elife 4; Varki A, et al., 2012, Ann N Y Acad Sci 1253: 16-36). For instance, it was recently shown that Siglec-9 interactions on neutrophils play an important role in modulating inflammation during COVID-19 (Delaveris CS, et al., 2021, ACS Cent Sci 7:650-657). Therefore, likely bispecific antibodies that target the blocking antibody to specific immune cells (NK cells) and/or virally infected cells will be needed to utilize the potential positive effects of the Siglec blockade on enhancing anti- SARS-CoV-2 NK immune functions while avoiding any potential non-specific inflammatory side-effects. This study is the first to describe Siglec-9⁺ CD56^dim NK cells as an NK subpopulation that can be exploited to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells.

The Materials and Methods are now Described

Characteristics of the study cohort

PBMCs and plasma was used from 67 individuals who tested positive for SARS-CoV-2 (by PCR) and 12 negative controls. The 67 SARS-CoV-2 positive individuals were either outpatients (mild; n=26) or inpatients (hospitalized; n=41) (Table 1). Samples from hospitalized patients were collected when patients were admitted to the hospital.

Table 1. Demographic and clinical characteristics of the study cohort.

Direct cytotoxicity assay against SARS-CoV-2 Spike-expressing 293 T cells. Frozen PBMCs were thawed in complete growth media (RPMI with 10%

FBS) and rested overnight. PBMCs (1 X 10⁶) were then co-cultured with Spike-expressing- 293T (S-293T) target cells (1 X 10⁵) at 10:1 effector-to-target (E: T) ratio in a complete growth medium in the presence of GolgiStop (BD Biosciences) and anti-CD107a PE antibody (BD Biosciences). The co-cultured cell mixture was then pelleted at 200x for 2 minutes and incubated at 37°C for 16 hours. Upon 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 the BD Biosciences’ Cytofix/Cytoperm), and permeabilized (BD Biosciences’ Perm/Wash buffer). Following permeabilization, cells were intracellularly stained for IFN-gamma (BV421; BD Biosciences) and TNF-alpha (PE-Dazzle 594; Biolegend). At least 100,000 events were acquired by flow cytometry on a BD Biosciences LSR II Flow Cytometer. Cytolytic NK cells were gated as CD3" and CD56^dim (Figure 2A). Direct cytotoxicity was calculated by subtracting the background NK degranulation/cytokine production of the PBMCs alone culture from the NK degranulation/cytokine production of the co-cultures of the PBMCs and target cells (Figure 1A, left).

ADCC assay against SARS-CoV-2 Spike-expressing 293 T cells.

IgG was isolated from the plasma of the donors using the Pierce Protein G Spin Plate for IgG (Thermo Scientific) kit. Purified IgG was quantified using NanoDrop (absorbance at A280). Purified IgGs from the SARS-CoV-2 negative donors were pooled in equal concentrations to obtain a negative pool. Purified IgGs from SARS-CoV-2 positive donors were pooled in equal concentrations to obtain a positive pool. The ADCC assay was performed identically to the direct cytotoxicity assay but with the target cells pre-incubated (for 15 minutes) with the negative or positive pools (at lOpg per well) before co-culturing them with the PBMCs from each donor. ADCC was then assessed by subtracting the NK degranulation of the co-culture with the negative antibody pool from NK degranulation of the co-culture with the positive antibody pool (after subtracting background NK degranulation) (Figure 1A, right).

Target cell lysis of Spike expressing CHO-K1 using PBMCs.

Target cell lysis was performed using the Promega HaloTag-HiBit ADCC kit, following the manufacturer's instructions. Briefly, cryopreserved PBMCs and spikeexpressing CHO-K1 cells were thawed, rested overnight. Upon resting, CHO-K1 target cells were incubated with either the SARS-CoV-2 positive or SARS-CoV-2 negative IgG pools at 0.5pg/well concentration. After 15 min, PBMC cells (2.5 X 10⁴) and CHO-K1(2,500) target cells were co-cultured at 10:1 effector-to-target (E:T) ratio, in complete growth media for 5 hours. After 5 hours, the substrate was added, and luminescence was measured after an additional 10 min. Luminescence values of each donor for the positive SARS-CoV-2 IgG pool were subtracted by the values obtained from the respective negative SARS-CoV-2 IgG pool to obtain the specific target cell lysis.

Direct cytotoxicity assay against K562 cells.

Frozen PBMCs were thawed in complete growth media (RPMI with 10 % FBS) and rested overnight. PBMCs (1 X 10⁶) were then co-cultured with K562 target cells (2 X 10⁵) at 5: 1 (E:T) ratio in a complete growth medium in the presence of GolgiStop (BD Biosciences) and anti-CD107a PE antibody (BD Biosciences). The co-cultured cell mixture was then pelleted at 200x for 2 minutes and incubated at 37°C for 3 hours. Upon incubation, cells were stained with the same antibodies described above, fixed, permeabilized, and intracellularly stained for IFN-gamma and TNF-alpha, as described above. At least 100,000 events were acquired by flow cytometry on a BD Biosciences LSR II Flow Cytometer. Cytotoxicity was calculated by subtracting the background NK degranulation/cytokine production of the PBMCs alone culture from the NK degranulation/cytokine production of the co-cultures of the PBMCs and target cells.

SARS-CoV-2 nucleocapsid (N) antigen quantification

The SARS-CoV-2 N-antigen plasma load was quantified using a Single Molecular Array (Simoa) immunoassay on the Simoa HD-X analyzer (Quanterix), as previously described (Shan D, et al., 2021, Nat Commun 12: 1931).

Phenotypic characterization of Siglec-7⁺ and Siglec-9⁺ CD56^dim NK cells

Phenotypic characterization of NK cells expressing Siglec-7 and Siglec-9 was performed on cryopreserved PBMC (n=79) from the study cohort by multiparameter flow cytometry. In brief, cryopreserved PBMC were thawed in pre-warmed RPMI (RPMI 1640 medium; (Mediatech) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Sigma), 1% penicillin-streptomycin (Lonza), and 2 mM L-glutamine (Sigma) and collected by centrifugation. Cells were then washed in DPBS without Ca⁺⁺\Mg⁺⁺ (DPBS- CMF) and collected by centrifugation. Next, the cells were stained with Aqua Live/Dead cell stain kit (Invitrogen) to assess the viability of the cells, washed in DPBS-CMF, and held for cell surface staining. Cells were then incubated with a cocktail of fluorochrome conjugated anti-human monoclonal antibodies: CD3 AF700, CD19 AF700, CD14 AF700, HLA DR APC-H7, CD56 PE-Cy7, CD 16 BV605, CD57 FITC, CD38 PE-CF594, NKG2A BB700, NKG2C BV786, and CD161 (BV421) from BD Biosciences along with Siglec-9 APC and Siglec-7 PE from Biolegend. Cells were washed in FACS buffer (DPBS-CMF + 0.5% BSA + 0.1% Na Azide) and then fixed in 1% paraformaldehyde (PF A, Polysciences) before acquiring on LSRFortessa SORP flow cytometer (BD Biosciences). Data were analyzed using FlowJo Software version 9.9.6 (Treestar Inc).

Sorting CD56^dim NK cells based on their Siglec expressing and measuring 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 Human EasySep NK Isolation Kit as per the manufacturer's instructions (StemCell Technologies). Cells were then stained for CD56 (APC-Cy7; Biolegend), CD3 (Alexa-488; BD Biosciences), Siglec-9 (APC; Biolegend). CD3' CD56^dim NK cells were sorted into three populations with no, low, and high Siglec-9 expression using the FACSymphony S6 SE (FACSAriall) (Figure 9).

Sorted cells were lysed, and total RNA was extracted from them using the RNAeasy mini kit (QIAGEN) with on-column DNase treatment (QIAGEN) according to the manufacturer’s instructions. cDNA was generated using the SuperScript VILO MasterMix (Invitrogen) according to the manufacturer’s instructions. The relative copy number of Siglec-9 transcripts was quantified in a qPCR reaction containing 4 pmol of each Siglec-9 specific primer and probe (Life technologies, assay ID Hs00534924_ml), 10 uL of 2x TaqMan Universal Master Mix (Applied Biosystems), and 5pL of diluted cDNA. Reactions were performed in a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) using the following cycling conditions: 50°C for 2 min, 95° C for 10 min, followed by 45 cycles of 95° C for 15 s, and 60° C for 1 min. Data were normalized using the eukaryotic 18S rRNA endogenous control (Applied Biosystems) as a housekeeping gene. Relative copy numbers were determined using the comparative Ct method (Schmittgen TD, et al., 2008, Nat Protoc 3: 1101-8).

Generation and characterization of human Siglec-9 blocking antibody.

Transgenic H2L2 mice (Harbor BioMed, Cambridge, MA) that encode the human immunoglobulin repertoire were used for immunization (Widjaja I, et al., 2019, Emerg Microbes Infect 8:516-530). Immunization and antibody sequencing were performed similarly to the detailed protocol described recently using the transgenic H2L2 mice (Duty JA, et al., 2022, Med (N Y) 3:705-721 el l). Briefly, mice were immunized with 50pg of DNA encoding human Siglec-9 two times at 2-week intervals. Mice then received two booster injections at two-week intervals, the first booster contained Siglec-9 DNA and the second booster contained 50pg of purified recombinant human Siglec-9 protein (R&D Systems). Murine SP2/0-Agl4 (SP2/0) myeloma cell lines were used to generate hybridomas by the chemical fusion of splenocytes from immunized mice. After antibody binding confirmation using ELISA, mouse splenocytes were used to generate hybridomas and sequence antibodies as described, in detail, recently (49, 55).

For recombinant expression in mammalian cells, antibody constructs were cloned into the pCDNA3.4 expression vector. Gene constructs encoding full-length IgG were designed (GenScript), and transient production in suspension HEK293 cells was performed in serum-free suspension culture to express the full-size antibodies. The reactivity and specificity of the recombinant anti-Siglec-9 antibody were examined by enzyme-linked immunosorbent assay (ELISA). The ELISA plates were coated with human recombinant Siglec-9 (Ipg/ml) protein (R&D Systems), human recombinant Siglec-7 (Ipg/ml; as a negative control) protein (R&D Systems), or HIV gpl20 protein (Ipg/ml; as a negative control) overnight at 4°C. After being washed with PBS and blocked by 3% BSA, the purified anti-Siglec-9 antibody was added at different dilutions and incubated for 1 hour at room temperature. The wells were then washed and detected by 3, 3', 5, 5'- Tetramethylbenzidine (TMB) substrate after incubation with goat anti-mouse secondary antibody. The reaction was stopped by the addition of 1 M H2SO4, and the absorbance was measured at 450 nm by an ELISA reader (Figure 16).

ADCC-mediated NK degranulation assay in the presence of Siglec-9 blocking antibody.

Cryopreserved PBMCs from six healthy donors were thawed, rested overnight, and incubated with an in-house Siglec-9 blocking antibody at 0.5pg/well for 15 minutes. S-293T target cells (1 X 10⁵) were incubated with either the positive or negative pool at 0.5pg/well concentration. After 15 min, pre-treated PBMCs (1 X 10⁶) and S-293T cells were co-cultured at 10:1 effector-to-target ratio, and degranulation was examined as previously described.

Human NK cell isolation and target cell lysis in the presence of Siglec-9 blocking antibody.

NK cells were isolated by negative selection from peripheral blood mononuclear cells (PBMC) obtained from five healthy donors using the EasySep Human NK Cell Isolation Kit (STEMCELL Technologies) following the manufacturer’s protocol. Target cell lysis was performed using the Promega HaloTag-HiBit ADCC kit, following the manufacturer's instructions. Briefly, isolated NK cells were incubated with the Siglec-9 antibody (at 0.5pg /well) for 15 minutes. CHO-K1 cells were also pre-incubated with either the positive or negative pool at 0.5pg per well for 15 minutes. CHO-K1 (2,500) and NK cells (1.25 X 10⁴) were then co-cultured at 5:1 effector-to-target ratio in complete growth media for 5 hours. After 5 hours, the substrate was added, and luminescence was measured after 10 min. Luminescence values of each donor for the positive pool were divided by the values obtained from the respective negative pool to obtain the specific target cell lysis.

Statistical analysis.

Kruskal -Wallis tests with Dunn’s multiple comparisons correction were used for statistical analyses in Figure 1B-I, Figure 2B-Figure 2G, Figure 4B-Figure 4F, and Figure 11. Spearman's rank-order correlations were used for statistical analyses in Figure 1J- Figure IM, Figure 5J, and Figure 6J. Wilcoxon's signed-rank tests were used for statistical analyses in Figure 5A- Figure 51, Figure 6A- Figure 61, and Figure 12 (to compare the cells within each disease group), and Figure 7. Mann-Whitney U tests were used to compare cells between the different group states in Figure 4 and were also used for statistical analyses in Figure 3, Figure 4A, and Figure 10. Friedman tests with Dunn's multiple comparisons correction were used for statistical analyses in Figure 13B- Figure 13H. Paired T-tests were used in analyses in Figure 9 and Figure 14. Data were analyzed using Prism 9.0 (GraphPad Software)

Example 2: Sequence Information

HB9-4G4

Heavy chain

(SEQ ID NO:1)

GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTT

AGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGG

TCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAA

CTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCT

CAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGAAAACCG

AGGACACAGCCGTGTATTACTGTACCACAGGGTGGGAGCTACAGGACTACTACT

ACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

HB9-4G4

Heavy chain

(SEQ ID NO:2)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKT

DGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGWELQDYYYY

GMDVWGQGTTVTVSS

HB9-4G4

Light chain

(SEQ ID NO:3)

ATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCA

CCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCA

GAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACT

GGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCA

TCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAA

CTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

HB9-4G4

Light chain

(SEQ ID NO:4)

IVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIP

ARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEIK

HB9-6A8

Heavy chain (SEQ ID N0:5)

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTG

TCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGAT

CCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGG

AAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAC

GTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCT

GTGTATTACTGTGCGAGAGGTGATTGTAGTGGTGGTAGCTGTCCTTACTGGTACT

TCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA

HB9-6A8

Heavy chain

(SEQ ID N0:6)

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGST

NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGDCSGGSCPYWYFDLW GRGTLVTVSS

HB9-6A8

Light chain

(SEQ ID N0:7)

GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGG

CCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTA

TTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTG GGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCA CAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTA

CTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGA AATCAAA

HB9-6A8

Light chain

(SEQ ID NO: 8)

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSN

RASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK

HB9-6F3

Heavy chain

(SEQ ID NO:9)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG

AGACTCTCCTGTGAAGCCTCAGGATTCACCTTTAGAAACTATGCCATGAGCTGGG

TCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTCGTGGTAGTG

GTAGTAGAACATACTACGCAGACTCTGTGAAGGGCCGGTTCACCATCTCCAGAG

ACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACA CGGCCGTGTATTACTGCGCGAAAGATGAGGGGTTCGGGGACTTATTAGCGCACT

ATGTTATGGATGCCTGGGGTCAAGGAGCTTCAGTCACTGTCTCCTCA

HB9-6F3

Heavy chain

(SEQ ID NO: 10)

EVQLLESGGGLVQPGGSLRLSCEASGFTFRNYAMSWVRQAPGKGLEWVSAIRGSGS

RTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDEGFGDLLAHYVM

DAWGQGASVTVSS

HB9-6F3

Light chain

(SEQ ID NO: 11)

GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGA

GCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACC

AGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGG

CCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCT

CACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTAT

AATAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

HB9-6F3

Light chain

(SEQ ID NO: 12)

EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGI

PARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIK

HB9-9B11

Heavy chain

(SEQ ID NO: 13)

GAGGTGCAGTTGTTGGAGTCTGGGGGGGGCTTGGTACAGCCTGGGGGGTCCCTG

AGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAACTATGCCATGAACTGGG

TCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTATGAGTG

GTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAG

ACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACA

CGGCCGTATATTACTGTGCGAAAGACGAATATAGCAGTGGCTGGTACCAATTTGA

CTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

HB9-9B11

Heavy chain (SEQ ID NO: 14)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWVSAISMSGG

STYYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCAKDEYSSGWYQFDYW GQGTLVTVSS

HB9-9B11

Light chain

(SEQ ID NO: 15)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAG

TCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCA

GCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAA

AGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA

CCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTA

CAGTACCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

HB9-9B11

Light chain

(SEQ ID NO: 16)

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV

PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK

HB9-10B6

Heavy chain

(SEQ ID NO: 17)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTG

AGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGG

TCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTACATGGTATGATG

AATATAATAAATACTATGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGA

CAATTCCAAGAACATGTTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACAC

GGCTATGTATTACTGTGCGAGGAACGTATTACGATATTTTGACTGGACCCTTGAC

TACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCG

HB9-10B6

Heavy chain

(SEQ ID NO: 18)

QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVTWYD

EYNKYYADSVKGRFTISRDNSKNMLYLQMNSLRAEDTAMYYCARNVLRYFDWTLD

YWGQGTLVTVSS

HB9-10B6 Light chain

(SEQ ID NO: 19)

ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCA

CCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTATCAACA

GAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACT

GGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCA

TCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAA

CTGGCCTCCCACTTTTGGCCAGGGGACCAAGGTGGAGATCAAA

HB9-10B6

Light chain

(SEQ ID NO:20)

IVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP

ARFSGSGSGTOFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

HB9-10E3

Heavy chain

(SEQ ID NO:21)

GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG

AGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGG

TCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTG

GTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAG

ACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACA

CGGCCGTATATTACTGTGCGAAAGCCGGTATAGCAGTGGCTGGGGGATGGTACTT

CGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA

HB9-10E3

Heavy chain

(SEQ ID NO:22)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG

STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGIAVAGGWYFDL WGRGTLVTVSS

HB9-10E3

Light chain

(SEQ ID NO:23)

ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCA

CCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTATCAACA

GAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACT

GGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCA TCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAA

CTGGCCTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

HB9-10E3

Light chain

(SEQ ID NO:24)

IVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIP

ARFSGSGSGTOFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKLEIK

HB9-11B10

Heavy Chain

(SEQ ID NO:25)

GAGGTGCAGTTGTTGGAGTCTGGGGGGGGCTTGGTACAGCCTGGGGGGTCCCTG

AGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAACTATGCCATGAACTGGG

TCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTATGAGTG

GTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAG

ACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACA

CGGCCGTATATTACTGTGCGAAAGACGAATATAGCAGTGGCTGGTACCAATTTGA

CTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

HB9-11B10

Heavy Chain

(SEQ ID NO:26)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWVSAISMSGG

STYYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCAKDEYSSGWYQFDYW GQGTLVTVSS

HB9-11B10

Light chain

(SEQ ID NO:27)

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAG

CCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAACAGCTACTTAGCCTGGTACCA

ACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCATCAGGGCC

ACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCA

CCATCAGCAGCCTAGAGCCTGAAGATTTTGCATTTTATTACTGTCAACAGCGTAG

TAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

HB9-11B10

Light chain (SEQ ID NO:28)

EIVLTQSPATLSLSPGERATLSCRASQSVNSYLAWYQQKPGQAPRLLIYDASIRATGIP

ARFSGSGSGTOFTLTISSLEPEDFAFYYCQQRSNWPPTFGQGTKVEIK

HB9-1F11

Heavy Chain:

(SEQ ID NO:29)

QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGRIDPNS

GGTKYNEKFKSKATLTVDKPSSTAYMQLSSLTSEDSAVYYCARYDYYGSSYFDYW GQGTTVTVSS

HB9-1F11

Light chain:

(SEQ ID NO:30)

QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRA

PGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL

HB9-4C10

Heavy Chain:

(SEQ ID N0:31)

EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGGINPNNG

GTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARNDGYRGYAMDYW GQGTSVTVSS

HB9-4C10

Light chain:

(SEQ ID NO:32)

DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYW

ASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYCCQQYYSNPPLTFGAGTKLELK

HB9-5B7

Heavy Chain:

(SEQ ID NO:33)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS

GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW GQGTLVTVSS HB9-5B7

Light chain:

(SEQ ID NO:34)

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGQVPKLLIYAASALQSG

VPSRFSGSGSGTDFTLTITSLQPEDVATYYCHKYNSAPWTFGQGTEVEIK

HB9-7B8

Heavy chain:

(SEQ ID NO:35)

ELQLLESGGGLVQPGGSLRLSCAASGFTFTNYAMNWVRQAPGKGLEWVSAISGSGG

RTYYADSVKGRFTISRDNSRNTLFLQMNSLRPEDTAVYYCAKDQTSGTTGYPYFAY WGQGTLVTVSS

HB9-7B8

Light chain:

(SEQ ID NO:36)

ETVMTQSPATLSVSPGERAILSCRASQSVSSNLVWYQQKPGQAPRLFIYGASTRATGI

PARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPRTFGQGTKVEIK

HB9-14D4

Heavy chain:

(SEQ ID NO:37)

QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDG

TKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYM DVWGKGTTVTVSS

HB9-14D4

Light chain:

(SEQ ID NO:38)

DIQMTQSPSSLTASVGDRVTITCRASQSISSYVNWYQQKPGKAPKVLIFAASSLQSGV

PSRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIK

HB9-14H12

Heavy chain:

(SEQ ID NO:39) QVQLKESGPGLVAPSQSLSITCTVSGFLLISNGVHWVRQPPGKGLEWLGVIWAGGNT

NYNSALMSRVSISKDNSKSQVFLKMKSLQTDDTAMYYCARDFYDYDVFYYAMDY WGQGTSVTVSS

HB9-14H12

Light chain:

(SEQ ID NO:40)

QAVVTQESALTTSPGETVPLTCRSSTGTVTTSNFANWVQEKPDHLFTGLIGGTNNRA

PGLPARFSGSLIGDKAALTITGAQTEDEAIFFCALWYSNHFVFGGGSQLTVL

HB9-14D4

Heavy Chain CDR1 - SYGMH (SEQ ID N0:41)

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: SEQUENCES

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art 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

CLAIMS What is claimed is:

1. A therapeutic composition comprising a Siglec-9 inhibitor as an adjuvant to enhance the immune response against a target antigen.

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

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) a variable heavy chain sequence of one or more of SEQ ID NOs 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, and 39; b) a variable light chain sequence of one or more 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 a variable heavy chain sequence of one or more 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 a variable light chain sequence of one or more 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 a variable heavy chain sequence of one or more of SEQ ID NOs 1, 5, 9, 13, 17, 21, 25, 29, 31, 33, 35, 37, and 39; and

1) a fragment comprising at least 80% of the full-length sequence of a variable light chain sequence of one or more of SEQ ID NOs 3, 7, 11, 15, 19, 23, 27, 30, 32, 34, 36, 38, and 40.

4. The therapeutic composition of claim 1, further comprising one or more additional antibody targeting an 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. 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. The therapeutic composition of claim 1, comprising one or more inhibitory nucleic acid molecule specific for binding to Siglec-9 or a fragment thereof.

9. The therapeutic composition of claim 1, comprising one or more mRNA molecule encoding the Siglec-9 inhibitor.

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

11. The therapeutic composition of claim 10, wherein the DNA molecule comprises one or more nucleotide sequence 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 a variable heavy chain sequence of one or more of SEQ ID NOs 2, 6, 10, 14, 18, 22, and 26; d) a nucleotide sequence having at least 95% identity to a variable light chain sequence of one or more 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 a variable heavy chain sequence of one or more of SEQ ID NOs 2, 6, 10, 14, 18, 22, and 26; and

1) a fragment comprising at least 80% of the full-length sequence of a variable light chain sequence of one or more of SEQ ID NOs 4, 8, 12, 16, 20, 24, and

12. The therapeutic composition of claim 10 or 11, further comprising one or more additional nucleic acid molecule comprising a nucleotide sequence encoding an antibody or fragment thereof targeting an 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. 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 immunotherapy efficacy in a subject in need thereof, the method comprising administering to the subject a therapeutic composition of any one of claims 1-15.

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

18. A method of 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 of any one of claims 1-15.

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