High-affinity siglec-7 / 9 ligands for cancer immunotherapy

Abstract

The present invention provides novel ligand compounds that specifically bind to inhibitory Siglecs (e.g., Siglec 7 and Siglec 9). The novel Sig7 / 9 ligands and related drug conjugates described herein are capable of inhibiting recruitment of Sig7 / 9 by tumor cells to phagocytic and immunological synapses. By promoting Sig7 / 9 sequestration and degradation, the compositions and methods of the invention enhance cancer immune therapy via suppressing Sig7 / 9-mediated inhibition of both innate adaptive immune cell activation and functions.

Description

HIGH-AFFINITY SIGLEC-7 / 9 LIGANDS FOR CANCER IMMUNOTHERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U.S.Provisional Patent Application Numbers 63 / 705,103 (filed October 9, 2024; now pending), 63 / 620,199 (filed January 12, 2024; now pending), and 63 / 603,675 (filed November 29, 2023; now pending). The full disclosures of the priority applications are incorporated herein by reference in their entireties and for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which is submitted in .xml format and is hereby incorporated by reference in its entirety. Said .xml file is named “2220_3PC_Sequence Listing”, was created on November 13, 2024, and is 6 KB bytes in size.BACKGROUND OF THE INVENTION

[0003] Sialic acid-binding Ig-type lectins (Siglecs) are an essential part of the glycan-binding protein family. They are important regulatory receptors with restricted expression patterns on specific immune cells, with major abundance on innate immune cells and minor expression on adaptive cells. To date, 14 human Siglecs have been characterized and are divided into two groups comprised of conserved Siglecs (CD 169, CD22, MAG and Siglec-15) and evolutionary CD33rSiglecs (CD33, Siglec-5, -6, -7, -8, -9, -10, -11, -14 and -16)1. Through interactions with sialoglycans, Siglecs either actively or negatively regulate immune cells in a variety of immune system disorders involving inflammation, infection, immune surveillance and autoimmunity2. Each Siglec recognizes a particular set of sialic acid structures bearing oc2,3-, a2,6- and oc2,8- linked terminal sialic acid residues, due to the distinct extracellular V-set carbohydrate recognition domain (CRD)3. However, the binding affinity of Siglecs to natural sialic acid is very weak with KD at mM scale. Moreover, the endogenous trans and cis counter receptors that carry the Siglec ligands remain unknown, which renders it elusive to understand the mechanism of Siglec functions.

[0004] To harness Siglecs as switches to boost immune cell functions, therapeutic Siglec blocking antibodies have recently been developed and some are already in clinical trials4. These antibodies could be either agonistic or antagonistic, depending on their effects on regulating downstream signaling pathways of Siglecs. In addition, antibody-drug conjugates (ADCs) are also developed to deliver toxins to lymphoma and leukemia cells such as those based on CD22 and CD33 antibodies. However, these Siglec antibodies are unable to reveal the intrinsic Siglec-sialoglycan interactions, underscoring the limited understanding of how Siglec functions in immune cells in a natural context. Thus, it remains a challenge for therapeutically targeting of Siglecs.

[0005] During cancer progression, tumor cells develop several mechanisms to evade killing and turn the immune system into a tumor-promoting environment. PD1-PDL1 interaction has been extensively studied as an important immune checkpoint for T cell immunity against cancer5. This interaction happens at the immunological synapse so that TCR activation can be inhibited by PD1 when engaged with trans PDL1 on cancer cells. More recently, increasing evidence indicates that sialic acids are becoming popular ‘don’t eat me’ signals when aberrantly expressed on cancer cells, that lead to the prevention of effector cell recognition and promote cancer cell immune escape6.

[0006] There is an unmet need in the art for selective and high-affinity sialyl mimetics targeting inhibitory Siglecs that can be used in clinical applications, e.g., cancer immune therapies. The present invention is directed to this and other unmet needs in the art.SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides novel ligands that specifically bind to inhibitory Siglecs, Siglec 7 and / or Siglec (Siglec-7 / 9). In some embodiments, the Siglec-7 / 9 ligand compounds have a structure that fall under Formula I below or a pharmaceutically acceptable salt thereofwherein Ri is any group or atom; R2 is H or acyl; R3 is alkyne, azide or amine; each of Xi,2,3 is independently any atom except H; each of YI,2 is independently any atom except H; and each of Zi,2,3,4 is independently any atom except H. Some specific examples of these Siglec-7 / 9 ligands are:

[0008] In some other embodiments, the Siglec-7 / 9 ligand compounds have a structure that fall under Formula II below:wherein Ri is any group or atom; R2 is H or acyl; R3 is alkyne, azide or amine; each of Xi,2,3 is independently any atom except H; each of YI,2 is independently any atom except H; and each of Zi,2,3,4 is independently any atom except H. Some specific examples of these Siglec-7 / 9 ligands are:

[0009] In some other embodiments, the Siglec-7 / 9 ligand compounds have a10 structure that fall under F ormula III below:wherein Ri is any group or atom; R2 is H or acyl; R3 is alkyne, azide or amine; each of Xi,2,3 is independently any atom except H; each of YI,2 is independently any atom except H; and each of Zi,2,3,4 is independently any atom except H.

[0010] In still some other embodiments, the Siglec-7 / 9 ligand compounds have a structure that fall under Formula IV below:wherein Ri is any group or atom; R2 is H or acyl; R3 is alkyne, azide or amine; each of Xi,2,3 is independently any atom except H; each of ¥1,2 is independently any atom except H; and each of Zi,2,3,4 is independently any atom except H.

[0011] In a related aspect, the invention provides drug conjugates for targeting and degrading cell surface Siglec-7 / 9 molecules. The drug conjugates contain a novel Siglec-7 / 9 ligand compound described herein and a conjugation or fusion partner to which the Siglec-7 / 9 ligand compound is covalently or non-covalently linked. In various embodiments, the conjugation or fusion partner can be, e.g., a protein, an antibody or antibody fragment, a small molecule, a polysaccharide, a lipid or a PEG molecule. Some drug conjugates of the invention are tetramers that contain a Siglec-7 / 9 ligand compound that is fused to a streptavidin protein via biotin. In some preferred embodiments, the fusion partner is capable of directing Siglec-7 / 9 molecules when bound by the ligand compounds to degradation machinery. In some of these embodiments, the fusion partner contains a moiety targeting M6PR or TG2 / LRP-1, and a protein, an antibody or antibody fragment that is fused to the moiety. In some of these embodiments, the moiety targeting M6PR is mannose-6-phosphate (M6P) or IGF2. In some drug conjugates of the invention, the fusion partner contains a carrier molecule (e.g., streptavidin or IgGl Fc domain as exemplified herein) and also a binding motif that can direct the fusion molecule to protein degradation machinery (e.g., M6P, IGF2 or a LSS motif). Some of these drug conjugates contain a Siglec-7 / 9 ligand, a carrier molecule such as streptavidin or an IgGl Fc domain, and mannose-6-phosphate (M6P)or IGF2. In some other embodiments, the fusion partner in the drug conjugates of the invention is an antibody or antibody fragment that recognizes an E3 ligase.

[0012] In another related aspect, the invention provides methods for promoting degradation of inhibitory Siglecs on the surface of immune cells. These methods entail contacting the immune cells with a novel Siglec-7 / 9 ligand compound or drug conjugate described herein. In various embodiments, the immune cells intended for the methods are primary macrophages, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), T lymphocytes or NK cells. In another aspect, the invention provides methods for stimulating and augmenting anti -turn or immune responses against a cancer in a subject. These methods involve administering to the subject a pharmaceutical composition that contains a therapeutically effective amount of a novel Siglec-7 / 9 ligand compound or drug conjugate described herein. In some preferred embodiments, the subject intended for the methods is a human. In some embodiments, the subject is additionally administered with a second anti-tumor agent. In some of these methods, the employed second antitumor agent is an immune checkpoint inhibitor.

[0013] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Application of SuFEx click chemistry for construction ofSiglec ligands. (A) Concept of sulfamide bond-based sialyl ligand construction. (B) Enhanced Siglec binding events are expected to achieve by sulfamide-linked sialyl ligands.

[0015] Figure 2. Synthesis of library of sulfamide-linked Siglec ligands. (A)Synthesis of a2,6 and a2,3 9-NH2-SiaLacNAc-Az. (B) Synthesis of library of SuFExable compounds from amines. (C) Construction of library of sulfamide-linked Siglec ligands. (D) Synthesis of natural a2,6 and a2,3 SiaLacNAc-Az.

[0016] Figure 3. Optimization of high-affinity Siglec-7 ligands. (A) Scheme of optimization of the Siglec-7 hit ligand. (B) Binding activities of optimized analog compounds.

[0017] Figure 4. Characterization of selectivity and avidity of Siglec-7 / 9 ligands. (A) Scheme showing design and preparation of Siglec ligand tetramer. (B) Chemical structures of biotinylated Siglec ligands. (C) Diagram showing the ELISA- like assay for measurement of Siglec-ligand binding affinity. (D) ELISA assay showing the selective binding to Siglec-7 and / or Siglec-9.

[0018] Figure 5. Design of Siglec-7 / 9 degrader by conjugation of mannose 6- phosphate (M6P) to Siglec ligand tetramer. (A) Scheme showing effect of Siglec-7 / 9 depletion on immune cell responses. (B) Design, synthesis and characterization of SA- M6P4. (C) Activity of SA-M6P4 in internalizing biotinylated dye. (D) Depletion of cell surface Siglec-7 and Siglec-9 by M6P-conjugated Siglec ligand tetramers. (E) Degradation of Siglec-7 and Siglec-9 proteomes in macrophages by M6P-conjugated Siglec ligand tetramers. (F) Microscopy analysis showing Siglec-7 and Siglec-9 degradation in macrophages.

[0019] Figure 6. Siglec-7 / 9 degradation synergizes with antibody therapy in macrophage phagocytosis of cancer cells. (A) Cell-surface Siglec-7 and Siglec-9 depletion by Siglec-7 / 9 degrader (Sig7 / 9 e) in human monocyte-derived macrophages (hMDMs). (B) Western blot experiment showing degradation of Siglec-7 and Siglec-9 proteomes in hMDMs. (C) Flow cytometry -based measurement of macrophage phagocytosis of cancer cells. (D) Microscope-based measurement of HT29 phagocytosis. (E) Disruption of phagocytic synapse by Siglec-7 and Siglec-9 recruited to the synapse. (F) Microscopy analysis showing SHP tyrosine phosphatases are recruited by Siglec-7 and Siglec-9 accumulated at phagocytic synapse.

[0020] Figure 7. Siglec-7 / 9 degradation proceeds in vivo and promotes tumor control. Siglec-7 / 9 inhibit both human and mouse T cell activations, we carried out the in vivo Siglec-7 / 9 degradation assay and accessed if this degradation can better control tumor progression. (A) Scheme showing the treatment of B16-GMCSF melanoma tumor with Sig7 / 9 e. (B) Analysis cell surface Siglec-7 / 9 expressions on tumor- infitrating myeloid cells upon treatment with Sig7 / 9 e. (C) Measurement of tumor growth in B16-GMCSF tumor model with administration of Sig7 / 9 e. (D) Measurement of effect of Sig7 / 9t / c treatment on tumor volume in mouse pacreatic cancer model.

[0021] Figure 8. Construction of IGF2-IgGl-Fc Sig ligand conjugate-based degrader. (A) Sig7 / 9 Ligand 8. (B) Sortage mediated coupling of Sig7 / 9 ligand to IGF2Fc fusion. A sortage tag, LPETGG (SEQ ID NO: 1), is added to the Fc domain prior to coupling. (C) Alternative coupling strategy via click chemistry.

[0022] Figure 9. Scheme of an IgGl-Fc Sig ligand conjugate containing a lysosome sorting sequence (LSS) as the targeting agent. Two exemplary LSS molecules (SEQ ID NOs:4 and 5) and the employed sortase tag (SEQ ID NO: 1) are shown.

[0023] Figure 10. Siglec degraders induced efficient Siglec-7 and -9 degradation in U937-derived macrophages. (A, B) Western blot analysis of Siglec-7 and -9 degradation efficacy in WT and R-mutant U937-derived macrophages after treatment with lOnM Ligand 8-modified IGF2-Fc for 24 hours. Siglec-7 and -9 KO U937 cells were used as the control. (C) Analysis of cell-surface Siglec-7 and -9 levels in U937-derived macrophages after treatment with Sig7 / 9L-modified IGF2-Fc for 1 hour at various doses by flow cytometry. (D) Assessment of in vivo Siglec-7 degradation among tumor-infiltrating immune cells in B16-GMCSF tumors in Sig7 / 9+mice by i.v. injection of 200 pg Fc control or Degrader 2, IGF2-Fc-(Sig7 / 9L)i, on day 5 after tumor cell inoculation, followed by flow cytometry analysis after 48 hrs.

[0024] Figure 11. Siglec degradation suppresses tumor growth in a B 16 melanoma model. (A) B16-GMCSF tumor growth in SigEKOn= 10 mice) and Sig7 / 9+mice that were i.v. administrated with (n= 7 mice per group) or Degrader 2 (n= 5 mice per group).DETAILED DESCRIPTIONI. Overview

[0025] Among the Siglec family receptors, Siglec-7 and Siglec-9 (also termed “Siglec-7 / 9” or “ Sig7 / 9” herein) are important regulatory receptors widely expressed on all kind of innate immune cell populations, which can arm Sig7 / 9 profound inhibitory effects in modulation of innate immune responses. Generally, Sig7 / 9 are intrinsically expressed in peripheral NK cells and myeloid phagocytes including monocytes and neutrophils1. Recent studies also show Sig7 / 9 expression is found in mast cells7'8, and Siglec-7 is individually expressed in eosinophils9and platelets10. Interestingly, high-level expression of Sig7 / 9 is observed in tumor- associated macrophages (TAMs)11and myeloid-derived suppressor cells (MDSCs)12in several types of human tumor tissues. The engagement of inhibitory Sig7 / 9 with tumor- associated sialoglycans further upregulate immunosuppressive pathways that can helpcancer cell invasion. However, little is known about how Sig7 / 9 regulate and interfere immune cell activation, especially in tumor microenvironment (TME).

[0026] To understand and decipher the roles of Sig7 / 9 in TME as potential therapeutic targets, the present inventors undertook studies to develop precision sialoglycan tools that can specifically target and block Sig7 / 9 with high-affinity as ‘antibody surrogate’. As detailed below, the present application describes (1) a novel design of Siglec ligands enabled by SuFEx click chemistry; (2) efficient synthesis and construction of library of sulfamide-linked Siglec ligands on live cell surface; (3) a straightforward detection and evaluation of high-affinity and selective sialyl ligands for indicated Siglecs; (4) design and application of Siglec ligand tetramers for labelling specific Siglec-expressing cells; (5) degradation of Sig7 / 9 receptors by decorating Siglec ligand tetramers with lysosome-associated mannose-6-phosphate receptor (M6PR) substrate; (6) synergy between Sig7 / 9 degradation and antibody therapy in macrophage phagocytosis of cancer cells via reduced recruitment of SHP to phagocytic synapse; (7) T cell acquiring Sig7 / 9 receptors from interacting macrophages, and an inhibitory role played by the acquired Sig7 / 9 in T cell activation; (8) mouse T cells’ efficient capturing of Sig7 / 9 molecules from transgenic Sig7 / 9+BMDMs, and decreased T cells activation due to the captured Sig7 / 9; (9) in vivo Sig7 / 9 degradation via intertumoral administration of Sig7 / 9 degrader, and resulting enhancement in controlling tumor growth in B16-GMCSF melanoma model; and (10) synergistic effects from Sig7 / 9 degradation and anti-CTLA4 treatment in controlling MT5 pancreatic tumor progression.

[0027] In accordance with the studies exemplified herein, the invention provides novel high-affinity ligands for specifically targeting inhibitory Sig7 / 9 and multimerized agents or drug conjugates containing the Sig7 / 9-targeting compounds, as well as related therapeutic applications in cancer therapy. As Siglecs are mostly cismarked on immune cell surface, the engagement of trans high-affinity ligands should be able to sequester Siglec receptors from endogenous counter-receptors and further block the Siglecs’ inhibitory functions. The Siglec-7 / 9 high-affinity ligands, multimerized derivatives and drug conjugates described herein provide novel antitumor drugs for treatment of human cancers. In specific applications, the compositions and methods described herein provide novel means for boosting immune cell effector functions, especially macrophage phagocytosis and T cell anti-tumor immunity. Forexample, since a lot of tumor cells coat themselves with high densities of sialic acidrich glycoproteins as self-molecules for immune escape, blocking the trans Siglec- sialoglycan axis could enhance anticancer immune responses. Also, the novel Sig-7 / 9 degrader can be employed to stop and eradicate tumor progression when used in conjunction with classic immune checkpoint antibodies in combination cancer therapies.

[0028] Unless otherwise specified herein, the methods and compositions described herein can all be generated or performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art. See, e.g., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978- 0121821906); U.S. Pat. Nos. 4,965,343, and 5,849,954; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3rded., 2000); Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley -Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998). The following sections provide additional guidance for practicing the compositions and methods of the present invention.

[0029] The following sections provide more detailed guidance for practicing the invention.II. Definitions

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic PressDictionary of Science and Technology, Morris (Ed.), Academic Press (1sted., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3rded., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1sted., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and .4 Dictionary of Biology (Oxford Paperback Reference) , Martin and Hine (Eds.), Oxford University Press (4thed., 2000). Further clarifications of some of these terms as they apply specifically to this invention are provided herein.

[0031] The phrase “a” or “an” entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0032] As used herein, unless specifically indicated otherwise, the word "or" is used in the "inclusive" sense of "and / or" and not the "exclusive" sense of "either / or".

[0033] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0034] The term "antibody" also synonymously called "immunoglobulins"(Ig), or "antigen-binding fragment" refers to polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes. Unless otherwise noted, antibodies or antigen-binding fragments used in the invention can have sequences derived from any vertebrate species. They can be generated using any suitable technology, e.g., hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof. Unless otherwise noted, the term “antibody” as used in the present invention includes intact antibodies, antigen-binding polypeptide fragments and other designer antibodies that are described below or well known in the art (see, e.g., Serafini, J Nucl. Med. 34:533-6, 1993).

[0035] An intact “antibody” typically comprises at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) inter-connected by disulfide bonds. The recognized immunoglobulin genes encoding antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0036] Each heavy chain of an antibody is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region of most IgG isotypes (subclasses) is comprised of three domains, CHI, C H2 and C H3, some IgG isotypes, like IgM or IgE comprise a fourth constant region domain, CH4 Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the first component (Clq) of the classical complement system.

[0037] The VH and VL regions of an antibody can be further subdivided into regions of hypervariability, also termed complementarity determining regions (CDRs), which are interspersed with the more conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The locations of CDR and FR regions and a numbering system have been defined by, e.g., Kabat el al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, U.S. Government Printing Office (1987 and 1991).

[0038] “Binding affinity” is generally expressed in terms of equilibrium association or dissociation constants (KA or KD, respectively), which are in turn reciprocal ratios of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may correspond to different rate constants, so long as the ratio of the rate constants remains the same. The binding affinity of an antibody is usually be expressed as the KD of a monovalent fragment (e.g. a Fab fragment) of the antibody, with KD values in the single-digit nanomolar range or below(subnanomolar or picomolar) being considered as very high and of therapeutic and diagnostic relevance.

[0039] The term “antibody drug conjugate” or “ADC” refers to an antibody to which a therapeutically active substance (e.g., a Sig7 / 9-targeting compound described herein) or an active pharmaceutical ingredient (API) has been conjugated (e.g., covalently coupled), such that the therapeutically active substance or an active pharmaceutical ingredient (API) can be targeted to the binding target of the antibody to exhibit its pharmacologic function. The attachment of a therapeutically active substance or an active pharmaceutical ingredient can be performed in a non-site specific manner (e.g., using standard chemical linkers that couple payloads to lysine or cysteine residues), or preferably the conjugation is performed in a site-specific manner, that allows full control of conjugation site and drug to antibody ratio (DAR) of the ADC to be generated.

[0040] The term "agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” are used interchangeably herein.

[0041] The term "analog" or “derivative” is used herein to refer to a molecule that structurally resembles a reference molecule (e.g., a Sig7 / 9 ligand exemplified herein) but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs to identify variants of known compounds having improved traits is an approach that is well known in pharmaceutical chemistry.

[0042] Antigen presenting cells refer to a type of immune cell that enables aT lymphocyte (T cell) to recognize an antigen and mount an immune response against the antigen. APCs include (but are not limited to) macrophages, dendritic cells, and B lymphocytes (B cells).

[0043] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0044] For polypeptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0045] The term “contacting” has its normal meaning and refers to combining two or more agents (e.g., an antibody and a chemical agent), combining agents and cells, or combining two populations of different cells. Contacting can occur in vitro, e.g., mixing an antibody or ADC and a cell or mixing a population of compounds with a population of cells in a test tube or growth medium. Contacting can also occur in a cell or in situ, e.g., contacting an antibody agent in a cell by coexpression in the cell of recombinant polynucleotides encoding the antibody, or in a celllysate. Contacting can also occur in vivo inside a subject, e.g., by administering an agent to a subject for delivery the agent to a target cell.

[0046] The term "independently" is used herein to indicate that a variable is applied in any one instance without regard to the presence or absence of a variable having that same or a different definition within the same compound. Thus, in a compound in which “R” appears twice and is defined as "independently selected from” means that each instance of that R group is separately identified as one member of the set which follows in the definition of that R group. For example, “each R1and R2is independently selected from carbon and nitrogen" means that both R1and R2can be carbon, both R1and R2can be nitrogen, or R1or R2can be carbon and the other nitrogen or vice versa.

[0047] When any variable occurs more than one time in any moiety or formula depicting and describing compounds employed or claimed in the present invention, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and / or variables are permissible only if such compounds result in stable compounds.

[0048] The term “optional” or “optionally” as used herein means that a subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted” means that the “optionally substituted” moiety may incorporate a hydrogen or a substituent.

[0049] The phrase “optional bond” means that the bond may or may not be present, and that the description includes single, double, or triple bonds. If a substituent is designated to be a "bond" or "absent", the atoms linked to the substituents are then directly connected.

[0050] Administration "in conjunction with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0051] As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specified ingredients.

[0052] As used herein, the term “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein. Representative pharmaceutically acceptable salts include, e.g., alkalimetal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bi sulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3 -hydroxy -2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1- methene-bis-2-hydroxy-3 -naphthoate, einbonate), pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0053] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

[0054] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c, 1970; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443,1970; by the search for similarity method of Pearson and Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI); or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

[0055] The term "subject" or “patient” refers to human and non-human animals (especially non-human mammals). The term "subject" is used herein, for example, in connection with therapeutic methods, to refer to human or non-human subjects. Examples of non-human subjects include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.

[0056] As used herein, T cell inhibitory co-receptors refers to a group of molecules expressed on the surface of T cells that play an inhibitory role in the activation of T cells by antigen-presenting cells (APCs). The activation of naive T cells requires both the stimulation of the T-cell receptor (TCR) by a major histocompatibility complex (MHC)-peptide complex and co-stimulatory signaling by co-stimulatory receptors (e.g., CD28) with their corresponding ligands on antigen-presenting cells (APCs). T cell inhibitory co-receptors negatively regulate TCR driven signals and therefore T-cell activation. Examples of T cell inhibitory co-receptors include CTLA-4 and PD1.

[0057] The terms "treat," "treating," "treatment," and "therapeutically effective" used herein do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment recognized by one of ordinary skill in the art as having a potential benefit or therapeutic effect. In this respect, the inventive method can provide any amount of any level of treatment. Furthermore, the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated.

[0058] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication ofthe attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as "expression vectors".III. Inhibitory Siglec-7 / 9 binding ligands and derivative compounds

[0059] Sialyated glycans, such as tumor-associated sialoglycans, can attenuate immune cell effector functions to promote cancer invasion and progression. This process involves regulatory Siglec receptors expressed on immune cell surface that can recognize the tumor-associated sialoglycans. The traw -sialoglycans expressed on tumor cells can induce inhibitory Siglec recruitment to immunological synapses, resulting in disruption of activating signals and suppression of the immune responses. Targeting the Siglec-sialoglycan axis, also known as glyco-immune checkpoint, could have clinical implications in the regulation of innate immune responses, e.g., in anticancer immunity.

[0060] The invention provides novel compounds that specifically bind to inhibitory Siglecs. In preferred embodiments, the Siglec ligands of the invention are specific for Siglec 7 and / or Siglec 9, and are hence termed Siglec-7 / 9 ligands. Some of the Siglec-7 / 9 binding compounds of the invention are a2,6 SiaLacNAc ligands having a structure shown in Formula I below,whereinRi is any group or atom; R2 is H or acyl; R3 is alkyne, azide or amine; each of Xi,2,3 is independently any atom except H; each of ¥1,2 is independently any atom except H; and each of Z 1,2, 3, 4 is independently any atom except H.

[0061] Some specific examples of compounds encompassed by Formula I are

[0062] As described herein (see, e.g., Example 5), these compounds demonstrated strong binding avidity towards Siglec 7 and / or Siglec 9.

[0063] In some other embodiments, the Siglec-7 / 9 binding compounds of the invention are a2,3 SiaLacNAc ligands having a structure encompassed by Formula II below,whereinRi is any group or atom; R2 is H or acyl; R3 is alkyne, azide or amine; each of Xi,2,3 is independently any atom except H; each of ¥1,2 is independently any atom except H; and each of Z 1,2, 3, 4 is independently any atom except H.

[0064] Some specific examples of compounds encompassed by Formula II are

[0065] As described herein (see, e.g., Example 5), these compounds demonstrated strong binding avidity towards Siglec 7 and / or Siglec 9.

[0066] In some other embodiments, the novel compounds of the invention that specifically bind to inhibitory Siglecs are a2,6 SiaLacNAc ligands having a structure encompassed by Formula III below,whereinRi is any group or atom;R2 is H or acyl;R3 is alkyne, azide or amine;each of XI.2,3 is independently any atom except H; each of YI,2 is independently any atom except H; and each of Z 1,2, 3, 4 is independently any atom except H.

[0067] In still some other embodiments, the novel compounds of the invention that specifically bind to inhibitory Siglecs are a2,3 SiaLacNAc ligands having a structure encompassed by Formula IV below,whereinRi is any group or atom;R2 is H or acyl;R3 is alkyne, azide or amine; each of XI,2,3 is independently any atom except H; each of ¥1,2 is independently any atom except H; and each of Z 1,2, 3, 4 is independently any atom except H.

[0068] In addition to the compounds encompassed by Formulas I-IV, theSiglec ligands of the invention also include pharmaceutically acceptable salts thereof.

[0069] The invention also provides related compounds that are derived from the compounds encompassed by Formulas I-IV. Some derivative compounds of the invention are drug conjugates containing the Siglec ligands. Some of the derivative compounds are multimerized forms of the Siglec ligands described herein (e.g., tetramers). In some embodiments, multimerization of the Siglec ligands can be achieved via biotin-streptavidin mediated conjugation, as exemplified. In some other embodiments, multimerization of the ligands can be achieved via an Fc antibody fragment. Additional drug conjugates of the invention, including various forms of ADCs, are described in more detail below. As demonstrated herein, some Siglec ligand multimers (e.g., tetramer “degraders” described in Examples 9 and 10) or drug conjugates described herein can function as antibody surrogates, and can better target cells with surface expression of the inhibitory Siglecs.IV. Drug conjugates containing novel Sig7 / 9 ligands

[0070] The novel Sig7 / 9-targeting ligands or degraders described herein can be further linked to a conjugation or fusion partner to form drug conjugates. The conjugation partner can be, e.g., a protein, an antibody or antibody fragment, a small molecule, a polysaccharide, a lipid or a PEG molecule. In some drug conjugates of the invention, the conjugation partner to which the Siglec ligand is conjugated to is a carrier moiety (e.g., a protein) that enhances the stability and serum half-life of the compound. The carrier moiety (“carrier” or “carrier molecule”) is typically a large, slowly metabolized macromolecule such as proteins; polysaccharides (such as latex functionalized SEPHAROSE™, agarose, cellulose, cellulose beads and the like); polymeric amino acids (such as polyglutamic acid, polylysine, and the like); amino acid copolymers; and inactive virus particles or attenuated bacteria, such as Salmonella. In various embodiments, the carrier moiety can be a carrier protein, an immunoglobulin, a Fc domain, a PEG molecule or other polymer. In some embodiments, the compounds can be conjugated to a protein or an antibody moiety, which can stabilize the ligand compounds and / or promote degradation of the inhibitory Siglecs (e.g., Siglec-7 and / or Siglec-9) in vivo. In these embodiments, the Siglec ligand is conjugated at the C- terminus or N-terminus of a carrier protein. The connection between the drug conjugate and the carrier moiety can be either covalent or non-covalent via any conventional methods.

[0071] In some embodiments, the drug conjugate contains a Siglec ligand described herein that is tetramerized by biotinylation and fusion to streptavidin. Streptavidin is a tetrameric protein isolated from the bacterium Streptomyces avidinii. It’s well known for its lesser non-specific binding with the biotin (or Vitamin H) molecule. The protein has a molecular weight of 60 kDa. Streptavidin tetramer structure is composed of four monomeric subunits, with each having a high affinity for biotin with a dissociation constant of - 1014mol / 1. Its homologs have been identified in a range of organisms, including bacteria, fungus, chickens, and frogs. The binding of biotin to streptavidin is considered one of the strongest non-covalent bondings. This binding affinity is around 103-106times higher than the antigen-antibody interactions.

[0072] In some embodiments, the drug conjugate of the invention contains aSiglec ligand that is fused to the Fc domain of an IgG molecule. Fusion with an Fc-domain provides a number of beneficial biological and pharmacological properties. For example, the presence of the Fc domain can markedly increase the plasma half-life of the conjugate molecule, which prolongs therapeutic activity, owing to its interaction with the salvage neonatal Fc-receptor, as well as to the slower renal clearance for larger sized molecules. The attached Fc domain also enables the molecules to interact with Fc- receptors (FcRs) found on immune cells, a feature that is particularly important for their use in oncological therapies and vaccines.

[0073] In some embodiments, the fusion partner in the drug conjugates of the invention is capable of directing Siglec-7 / 9 molecules when bound by the ligand compounds to degradation machinery. For example, the fusion partner can contain a targeting moiety that specifically binds to a protein promoting lysosome mediated protein degradation. In some of these embodiments, the fusion partner contains a carrier molecule (e.g., streptavidin or an antibody domain as exemplified herein) and also a binding motif or targeting agent that can target the fusion molecule to protein degradation. In some of these embodiments, the binding motif targeting protein degradation machinery is an agent that binds to a protein that facilitates lysosome mediated internalization and digestion of the inhibitory Siglecs. For example, a Siglec ligand can be fused to IGF2 via an IgGl Fc domain, or tetramerized via streptavidin and fused to mannose-6-phosphate (M6P). By binding to lysosome associated cationindependent M6P receptor (CI-M6PR; aka insulin growth factor 2 receptor, IGF2R) in lysosomes, M6P or IGF2 allows a target protein (e.g., an inhibotory Siglec) recognized by a fusion partner (e.g., a Siglec ligand) to be co-intemalized with CI-M6PR and selectively digested in lysosomes. See, e.g., Yu et al., J. Am. Chem. Soc. 2023, 145, 19107-19119. As exemplified herein, the Siglec ligand can be fused to M6P via the same biotin-streptavidin conjugation scheme to produce a tetramized form of the conjugate. In some other embodiments, the Siglec ligand can be fused to a binding motif targeting protein degradation machinery through an antibody molecule or antibody domain via appropriate linkers. For example, the ligand can be conjugated to IGF2 or a lysosome sorting sequence (LSS) via an IgGl Fc domain as exemplified herein.

[0074] In some other embodiments, the binding motif is an agent that can target protein degradation machinery via another lysosome associated pathway. For example, the binding motif can be an agent that targets lysosomal degradation throughthe TG2 / LRP-1 pathway. See, e.g., Loppinet et al., J. Am. Chem. Soc. 2023, 145, 18705-18710. In these embodiments, the drug conjugates can contain a Siglec-7 / 9 ligand described herein, a carrier molecule, and a ligand of transglutaminase 2 (TG2). Examples of TG2 ligands that may be employed in these drug conjugates include any known high-affinity TG2 substrates or inhibitors, such as Ac-PQLPF-NH2, HB-2-30, HB-3-2, HB-3-3, and HB-3-23 as described in, e.g., Lopinnet et al., Chem. Biol. 2023, 30, 55-68. elO; and Loppinet et al., J. Am. Chem. Soc. 2023, 145, 18705-18710.

[0075] In still some other embodiments, the fusion partner in the drug conjugates of the invention is an antibody or antibody fragment that can target the drug conjugates, upon binding to Siglec-7 / 9, to degradation. For example, the Siglec ligands can be conjugated to an antibody that recruits membrane-bound E3 ligases for the degradation of cell-surface Siglec-7 / 9. These antibody-drug conjugates (ADCs) are suitable for enhanced inhibitory Siglec degradation and use in cancer immunotherapy. These ligands can also be conjugated to antibodies that recruit membrane-bound E3 ligases for the degradation of cell-surface Siglec-7 / 9. In some embodiments, the ADCs of the invention contain a Sig7 / 9 ligand described herein that is conjugated to an antibody targeting the ubiquitin proteasome system (UPS). UPS is essential for maintaining cellular homeostasis by degrading and replacing cellular and extracellular proteins. E3 ubiquitin ligases are an important part of the UPS and regulate the last step of the enzymatic cascade, which also consists of ubiquitin activating enzymes (Els) and ubiquitin conjugating enzymes (E2s). E3 ligases can selectively attach ubiquitin to lysine, serine, threonine or cysteine residues to the specific substrates. Some ADCs of the invention contain an antibody that target a E3 ligase. Depending on the specific tumor or cell type that is desired for targeted Sig7 / 9 inhibition or degradation, the employed antibody can be specific for a number of E3 ligases. As E3 ligases can directly bind to substrates and determine the specificity of ubiquitin proteasome system, there are a large number of E3 ligases but only a few El and E2 ligases in distinct organisms. Based on the difference of structure and function, E3 ligases can be approximately divided into four types: HECT type, U-box type, RING-finger type, and RBR type. See, e.g., Rotin et al., Nat Rev Mol Cell Biol. 2009; 10:398-409.

[0076] The ADCs of the invention can contain an antibody that target any of the E3 ligases known in the art, esp. E3 ligases from human and other mammalian species. These include HECT E3 ligases, U-box E3 ligases, RING-finger E3 ligases,and RBR E3 ligases. HECT (homologous to the E6AP carboxyl terminus) E3 ligases family is one of the largest and earliest studied E3 ligases. They contain a common homologous to E6-associated protein C-terminus (HECT) domain, where the activated E2 ligase can transfer Ub to the active cysteine site before binding to the target substrate. See, e.g., Huibregtse et al., Proc Natl Acad Sci USA 1995; 92:2563-2567. RING E3 ligases are the major type of E3 ligases and characterized by their RING domain. See, e.g., Zheng et al., Annu Rev Biocheni. 2017;86: 129-157; and Freeniont et al.. Cell. 1991;64(3):483-484. There are more than 600 different RING type ligases expressed in human cells (Deshaies et al., Annu Rev Biochem. 2009;78:399-434). U- box E3 ubiquitin ligases are a relatively small family, which is necessary for controlling the quality of post-translational protein in eukaiyotic cells. The C-terminus of U-box E3 ligases contains a conserved U-box domain of about 70 amino acid residues from yeast to humans. See, e.g., Hu et al., Int J Mol Sci. 2018; 19(12). The RBR E3 ligases are a family of RING-HECT hybrid E3 ligases, which are not the same as RING and HECT types. The RBR E3 ligases are specialized by a conserved catalytic region, including a RING1, a central in-between-RINGs (IBR) and a RING2 domain. See, e.g., Aguilera et al., Genetics. 2000; 155: 1231-1244.

[0077] Depending on the specific Siglec ligand and the conjugation partner used in the drug conjugates of the invention, various means known in the art can be used and adapted for linking the Siglec ligand to the conjugation partner. See, e.g., Boutureira, O. & Bernardes, G. J. Chem Rev 115, 2174-2195, 2015; Zhang, Y. et al. Chem Soc Rev 47, 9106-9136, 2018; Dai, X. et al. RSC Advances 9, 4700-4721, 2019; Huang, C. Curr Opin Biotechnol 20, 692-699, 2009; Czajkowsky, D. M. et al. EMBO Mol Med 4, 1015-1028, 2012; and Muller, D. BioDrugs 28, 123-131, 2014. The conjugation between the Siglec ligand and the conjugation partner can be either direct, or indirect via a suitable linker moiety. The conjugation can be either covalent or non- covalent as exemplified herein. In some embodiments, conjugation of a Siglec ligand to the partner moiety can be accomplished via a site-specific linkage. When the conjugation partner is a polypeptide (e.g., an antibody moiety), the Siglec ligand can be fused non-selectively to the polypeptide, e.g., via lysine side chains. Alternatively, the Siglec ligand can be fused site-specifically to the polypeptide, e.g., at its C-terminus or the N-terminus. In various embodiments, the conjugation can be achieved with suitable coupling chemistry that can be readily designed in accordance with the structure of theSiglec ligand. For example, site-specific conjugation of a Siglec ligand to an antibody moiety can be performed with sortase-enzyme mediated antibody conjugation (“SMAC”). In some embodiments, the conjugation can utilize robust thiol-maleimide and transcyclooctene(TCO) / tetrazine(TZ) chemistries. These biorthogonal reactions allow for selective covalent bond formation in buffered aqueous solutions.

[0078] In some embodiments, the Siglec ligand can be conjugated to lysine side-chains of a carrier protein such as an Fc fragment, an antibody or other protein. For example, lysine amide coupling can be used to conjugate the Siglec ligand to an Fc moiety. Amide coupling is a major ADC conjugation method connecting a payload and solvent accessible lysine residues on the antibody using linkers containing activated carboxylic acid esters. See, e.g., Fu et al., Sig. Transduct. Target. Ther. 2022; 7: 93; and Tsuchikama and An, Protein Cell. 2018; 9: 33-46. Amide coupling of an amine and an activated carboxylic acid is one of the most reliable, high-yielding chemical conversions in organic synthesis. In some preferred embodiments, conjugation of a small molecule Siglec ligand to a protein or antibody can be performed via a click chemistry reaction. For example, the antibody can be labeled at exposed lysine residues with tetrazine-NHS, and an acetazolamide based Siglec ligand can be functionalized with transcyclooctene. The labeled antibody can then be subject to a “click” reaction with sialyl ligand-transcyclooctene, generating the Siglec ligand-antibody conjugate.V. Targeting and degrading inhibitory Siglecs in therapeutic applications

[0079] Siglec-7 and Siglec-9 (Sig7 / 9) are important regulatory receptors widely expressed on all kind of innate immune cell populations. They exert inhibitory effects in modulation of innate immune cell functions. Sig7 / 9 are expressed in peripheral NK cells, myeloid phagocytes, and mast cells. Siglec-7 is individually expressed in eosinophils and platelets. High-level expression of Sig7 / 9 is also observed in tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) in several types of human tumor tissues. The engagement of inhibitory Sig7 / 9 with tumor-associated sialoglycans further upregulate immunosuppressive pathways that can help cancer cell invasion.

[0080] The novel Sig7 / 9 ligands and related drug conjugates described herein are capable of inhibiting recruitment of Siglec-7 / 9 by trans tumor sialoglycans and promoting their degradation. In some embodiments, the Siglec ligands and drugconjugates of the invention can be used to degrade inhibitory Siglecs on immune cells such as innate myeloid cells and adaptive T cells. In various embodiments, the immune cells on which Sig7 / 9 can be targeted and degraded by the drug conjugates of the invention include, e.g., primary macrophages, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), T lymphocytes or NK cells. In some embodiments, the invention provides methods for promoting degradation of cell surface inhibitory Siglecs such as Siglec 7 and Siglec 9, and thereby inhibiting tumor cell recruitment of Siglec-7 / 9-expressing immune cells. In these methods, immune cells overexpressing or aberrantly expressing Sig7 / 9 are contacted with a Sig7 / 9 ligand or drug conjugate described herein. Some of the methods are directed to promoting Siglec-7 / 9 degradation in tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) orNK cells. In some of these embodiments, the Siglec-7 / 9 ligand or drug conjugate thereof is administered to a subject (e.g., a human cancer patient) and contacted with the immune cells in vivo. In a related aspect, the invention provides methods for enhancing anti-cancer immunity by suppressing Siglec-7 / 9 mediated inhibition of both innate and adaptive immune cell activation and functions. These therapeutic applications of the invention in cancer treatment entail administering to a subject afflicted with a cancer a therapeutically effective amount of the Siglec-7 / 9 ligand described herein or a drug conjugate containing the Siglec-7 / 9 ligand. In some preferred embodiments, the subject to be treated is a human patient.

[0081] The therapeutic methods of the invention can be employed in cancer immunotherapies whereas enhanced immune response is desired. By promoting Siglec- 7 / 9 degradation and inhibiting tumor cell recruitment of Siglec-7 / 9, the Sig7 / 9 ligands and related drug conjugates provided by the invention can substantially improve clinical efficacy of immunotherapies for various types of cancers that are associated with Sig7 / 9 overexpression or aberrant expression. Examples of cancers that are suitable for the methods of the invention include, but are not limited to, melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renal cell carcinoma, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatocellular carcinoma,nasopharyngeal carcinoma, LGL, ALL, AML, CML, CLL, and other neoplasms known in the art.

[0082] In some embodiments, the Sig7 / 9 ligands and related drug conjugates of the invention can be used with other therapeutic agents in combination therapies. In some of these embodiments, the Siglec ligands or drug conjugates can be used together with immune-checkpoint inhibitor antibodies, e.g., those binding to PD1, PDL1, CTLA4, 0X40, TIM3, GITR, LAG3 and the like. In some other embodiments, they can be used together with cytokines such as interferon a and IL-2a. For example, the therapeutic compositions described herein can be used in conjunction with any of the known antibody drugs targeting checkpoint inhibitors that have been approved by the FDA for treating various types of cancers. These include antibody drugs that target PD- 1, Pembrolizumab (Keytruda), Nivolumab (Opdivo) and Cemiplimab (Libtayo), as well as antibody drugs that target CTLA-4, Ipilimumab and Tremelimumab. Additionally, a number of other known antibodies targeting checkpoint inhibitors have also been extensively characterized and evaluated for clinical utility. These include, e.g., PD1 antibodies Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IB 1308), Tislelizumab (BGB-A317), Toripalimab (JS 001), Dostarlimab (TSR-042, WBP-285), AMP-224 and AMP-514 (MEDI0680). Other than these PD1 and CTLA-4 antibodies, some specific antibodies that block T cell inhibitory co-receptors Tim-3, TIGIT and LAG-3 are also known in the art. See, e.g., Sakuishi et al., J. Exp. Med. 207: 2187- 2194, 2010; Rangachari et al., Nat. Med. 18: 1394-1400, 2012; He et al., Onco. Targets Ther. 11 :7005-7009, 2018; Hung et al., Oncoimmunology 7: el466769, 2018; Solomon et al., Cancer Immunol. Immunother. 67: 1659-67, 2018; Wu et al., Cancer Immunol. Res. &: 1700-13, 2019; Grosso et al., J. Clin. Invest. 117: 3383-92, 2007; Wierz et al., Blood 131 : 1617-21, 2018; and Nguyen et al., Nat. Rev. Immunol. 15: 45-56, 2015. Any of these known antibodies or antigen binding fragments derived therefrom can be used in combination therapies with the novel Sig7 / 9 ligands and related drug conjugates described herein.VI. Pharmaceutical compositions

[0083] For use in the therapeutic methods described herein, the invention also provides pharmaceutical compositions that contain a Sig7 / 9 ligand or a related drug conjugate and a pharmaceutically acceptable carrier. Pharmaceutical compositionscan be prepared from any of the Sig7 / 9 ligands or drug conjugates described herein. The pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier. It can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances which are suitable for administration into a human or veterinary patient (e.g., a physiologically acceptable carrier or a pharmacologically acceptable carrier). The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the use of the active ingredient, e.g., the administration of the active ingredient to a subject. The pharmaceutically acceptable carrier can be co-mingled with one or more of the active components, e.g., a hybrid molecule, and with each other, when more than one pharmaceutically acceptable carrier is present in the composition, in a manner so as not to substantially impair the desired pharmaceutical efficacy. Pharmaceutically acceptable materials typically are capable of administration to a subject, e.g., a patient, without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset. It is, for example, desirable for a composition comprising a pharmaceutically acceptable carrier not to be immunogenic when administered to a human patient for therapeutic purposes.

[0084] Pharmaceutical compositions of the invention can additionally contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt. The compositions can also optionally contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal. Pharmaceutical compositions of the invention can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the antibody of the invention into association with a carrier that constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0085] A composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the inventive composition, which preferably is isotonic with the blood of the recipient. This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also can be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3 -butane diol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed, such as synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found, e.g., in Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20thed., 2000.

[0086] Preparation of pharmaceutical compositions of the invention and their various routes of administration can be carried out in accordance with methods well known in the art. See, e.g., Remington, supra; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The delivery systems useful in the context of the invention include time-released, delayed release, and sustained release delivery systems such that the delivery of the inventive composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. The inventive composition can be used in conjunction with other therapeutic agents or therapies. Such systems can avoid repeated administrations of the inventive composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain compositions of the invention.

[0087] Many types of release delivery systems are available and known to those of ordinary skill in the art. Suitable release delivery systems include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the active composition is contained in a form within a matrix such as those described in U.S. Patents 4,452,775, 4,667,014, 4,748,034, and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such as described in U.S.Patents 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.EXAMPLES

[0088] The following examples are offered to illustrate, but not to limit the present invention.Example 1 Using SuFEx click chemistry for construction of Siglec ligands

[0089] This Example describes construction of Siglec ligands via SuFEx click chemistry (Figure 1). Figure 1 (panel A) illustrates the concept of sulfamide bondbased Siglec ligand construction. We proposed a new Siglec ligand construct by straightforward sulfamidation of primary amine modified a2,6 and a2,3 SiaLacNAc (sialyl-Gaipi-4GlcNAc) on C9 or C5 position of sialic acid scaffold. Using SuFEx click chemistry recently developed by Sharpless and co-workers13, the amine tagged SiaLacNAc can react with a library of iminosulfur oxy difluorides to offer sulfamidebased sialyl mimetics in nearly quantitative yields. The generated crude products can be directly used for construction of cell-surface multivalent Siglec ligands. Figure 1 (panel B) shows that enhanced Siglec binding events are expected to achieve by sulfamidelinked Siglec ligands, because of improved ligand recognition within the V-set binding domain of indicated Siglec.Example 2 Synthesis of library of sulfamide-linked Siglec ligands

[0090] This Example describes the synthesis of a2,6 and a2,3 9-NH2-SiaLacNAc-Az (Figure 2). As shown in Figure 2 (panel A), we started the synthesis from natural sialic acid (NeuAc) by esterification of C-2 carboxylic acid, followed by tosylation of C-9 hydroxyl group. Mild azidation of the resulting tosyl group by NaNs yielded 9-azido modified sialic acid ester (2), that was further reduced by hydrogen- Pd / C under acidic conditions to give the 9-amine substituted sialic acid (3). After neutralization with aqueous NaOH, this product can be directly used for transfer to galactose of LacNAc-Az (2-azidoethyl LacNAc) using a one-pot chemoenzymatic approach. This enzymatic reaction requires recombinant nmCSS (CMP-sialic acid synthetase from N. meningitidis) enzyme and CTP for conversion of 9-NH2 sialic acid(3) to CMP-sialyl format in Tris buffer (pH 8.0), the in situ generated CMP-sialyl intermediate is transferred to SiaLacNAc-Az under catalyzation of specific recombinant bacterial sialyltransferases. When recombinant Pd2,6ST was applied, a2,6 SiaLacNAc sialoside (4) was specifically obtained, while recombinant PmSTl (M144D) exclusively yielded a2,3 SiaLacNAc product (5).

[0091] Figure 2 (panel B) shows the synthesis of library of SuFExable compounds from amines. The library of iminosulfur oxydifluorides was produced quantitatively by conversion of commercially available amine compounds using SOF4 gas in acetonitrile solvent with addition of triethylamine to neutralize HF side product. Figure 2 (panel C) shows the construction of library of sulfamide-linked Siglec ligands. Both a2,6 and a2,3 SiaLacNAc-Az were applied to the SuFEx reaction with a library of iminosulfur oxy difluorides to afford the biocompatible sulfamide-based sialyl derivatives in quantitative yields, which were directly used for the following cellsurface click chemistry without further purification. Figure 2 (panel D) shows the synthesis of natural a2,6 and a2,3 SiaLacNAc-Az. Natural sialic acid modified LacNAc-Az as control was prepared by NeuAc and 2-azidoethyl LacNAc under the above mentioned one-pot chemoenzymatic condition. Recombinant bacterial sialyltransferases Pd2,6ST and PmSTl produced a2,6 and a2,3 SiaLacNAc-Az products (6 and 7) respectively.Example 3 Siglec ligand screening platform

[0092] This Example describes cell-surface CuAAc (Cu^catalyzed azide alkyne cycloaddition) based Siglec ligand screening platform. To achieve a natural and multivalent Siglec-sialyl ligand binding event, we designed a cell-surface construction of Siglec ligands. Because Jurkat cell line does not express human Siglecs, we first prepared the alkynalylated Jurkat cells by metabolic glycoengineering of cells with 50 pM Ac4ManPoc for 3 days culture. During this procedure, Ac4ManPoc is transformed to CMP-SiaNPoc intracellularly, that is further transferred to N or ( -glycans on cellsurface glycoproteins though Golgi transport, resulting in the alkyne tagging on the cell surface. Then the alkynalylated cells were seeded to 96-well microplate (2xl05cells per well) in PBS buffer containing 1% FBS and pre-mixed CuSCh / BTTPS (75 pM / 450 pM, 1 :6). After treatment of above generated SiaLacNAc-Az ligands (200 pM)individually in each well, Na ascorbate (2.5 mM) enables the initiation of cell-surface alkyne reaction with sialyl ligands via ligand-accelerated CuAAc, in which, the livecell biocompatible BTTPS ligand was developed by our group14.

[0093] The click reaction was carried out at room temperature (r.t.) for 15 min and quenched with ImM BCS (bathocuproine disulfonate). During the reaction, cell viability was maintained after washing with PBS. Following this cell-surface CuAAc protocol, a collection of cell-surface Siglec ligands were produced from the library of azide-tagged sulfamide-linked sialyl ligands. The in situ cell-surface constructed sialyl ligands were further probed by fluorophore-conjugated Siglec-Fc chimera proteins by incubation of sialyl-ligand bound cells with pre-mixed Siglec-Fc and fluorescent anti-human IgG Fc, finally followed by FACS analysis of indicated cells for comparison of mean fluorescence intensity (MFI) between experiments with control subsets (6 and 7). The higher MFI indicates a stronger binding event of fluorophore-conjugated Siglec-Fc to cell-surface sialyl ligands.Example 4 Discovery of Siglec-7 ligand hit

[0094] This Example describes identification of hit compounds from Siglec-7 ligand screening. After screening a library of synthetic sulfamide-linked SiaLacNAc-Az ligands via cell-surface CuAAc, we discovered a high-affinity Siglec-7 ligand (<z2,6 benzothiazolyl SiaLacNAc-Az, abbr. <z2,6 BTZ-SLN-Az (8)) which bears a benzothiazole group on the terminal of trisaccharide ligand in an a2,6 sialoside format. In the control experiment, the binding event did not happen in the absence of CuAAc or Siglec-7-Fc, which means the <z2,6 BTZ-SLN-Az ligand constructed on cell surface is indeed capable of binding to Siglec-7. Another control is natural SiaLacNAc (<z2,6 N- SLN-Az, (6)) ligand which showed negligible binding towards Siglec-7. The discovered Siglec-7 ligand 8 also exhibited cross-avidity towards other inhibitory Siglecs, for example, this hit ligand shows moderate binding avidity with Siglec-9 and weak binding activity with hSiglec-3 (hCD33) and Siglec-10. In some experiments, a cross-reactive sialyl ligand is required to target several Siglecs simultaneously.Therefore, this hit is actually very promising for targeting multiple Siglecs in specific experimental and clinical requirements.Example 5 Optimization of high-affinity Siglec-7 ligands

[0095] This Example describes the optimization of high-affinity Siglec-7 ligands (Figure 3). With the BTZ-SLN-Az (8) ligand in hand, we attempted to diversify the benzothiazolyl group to obtain a better avidity in both a2,3 and a2,6 fashions. To realize the Siglec-7 hit ligand optimization, we prepared a small library of benzoheterocycle analogues with SuFExable group by conversion of amine precursors using SOF4 gas. Then these analogues reacted with a2,6 SiaLacNAc-Az (4) or a2,3 SiaLacNAc-Az (5) under 5 mM concentration in DMSO / PBS (v / v, 1 : 1) overnight at 37 °C. The resulting reaction mixture was directly applied to biocompatible cell-surface CuAAc for introduction of sialyl ligand analogues to cell surface at 200 pM, following by FACS analysis (Figure 3, panel A). After screening of benzoheterocycle SiaLacNAc ligands against Siglec-7-Fc, we found both the original BTZ-SLN-Az (8) ligand and the methylated benzothiazolyl SiaLacNac (MBTZ-SLN, 9) ligand equally showed the best binding avidity towards Siglec-7 in a2,6 linkage. While the a2,3 linked both ligands exhibit comparably less avidity towards Siglec-7 with significantly reduced binding ability by half. In addition, benzoxazole SiaLacNAc (BOZ-SLN) ligands including 12 and 13, and quinoxaline SiaLacNAc (quinoxaline- SLN, 18) ligand also exhibited moderate binding with Siglec-7 in a2,6 fashion. The results are shown in Figure 3, panel B. Very interestingly, a close derivative ligand 10 of 8 (only N and S are switched) revealed very weak binding affinity.Example 6 Characterization of selectivity and avidity of Siglec-7 / 9 ligands

[0096] This Example describes the characterization of selectivity and avidity of Siglec-7 / 9 ligands (Figure 4). We obtained two high-affinity Siglec-7 ligands (a2,6 BTZ-SLN-Az (8) and a.2,6 MBTZ-SLN (9)) from the above ligand optimization, and ligand 8 also shows moderate binding avidity towards Siglec-9. Then we investigated the cross-avidity of our best two ligands towards the rest of Siglecs. Considering the development of trans Siglec ligand tools will be more efficient to specifically target Siglec as ‘antibody surrogate’, we designed the model of Siglec Ligand (SigL) tetramer by combining four-equivalent biotinylated SiaLacNAc ligand with each streptavidin (SA), in which SA can be conjugated with a fluorophore or HRP for functional studies. The scheme showing the design and preparation of SigL tetramer is depicted in Figure 4, Panel A. Chemical structures of biotinylated Siglec ligands are shown in Figure 4,Panel B. Figure 4, Panel C, depicts the diagram showing the ELISA-like assay for measurement of Siglec-SigL binding affinity, in which HRP-conjugated SigL-SA tetramer was used. Figure 4, panel D, shows the ELISA assay indicating that ligand 9 is Siglec-7 specific and ligand 8 binds to both Siglec-7 and Siglec-9 with high affinity, besides these best two ligands exhibit negligible cross-avidity towards other Siglecs.Example 7 Siglec-7 / 9L tetramers specifically label Sig7 / 9-expressing cells as an antibody surrogate

[0097] This Example describes specific labeling of Sig7 / 9-expressing cells by Siglec-7 / 9L tetramers. When a fluorescent dye is conjugated to SA, the SigL-SA tetramers should be eligible to stain Siglec-expressing cells as an antibody surrogate. It was found that, like the commercial Siglec-7 antibody (Biolegend clone 6-434), (Sig7L)4-SA-AF647 (8' and 9') tetramers were able to label Sig7-WT U937-derived macrophages but not the Sig7-KO and Sig7-R124A cells. R124 in Siglec-7 is the critical binding site that forms the salt bridge with carboxylic acid from sialic acid ligand. R124 mutation successfully blocked the (Sig7L)4-SA tetramer binding to Siglec-7, which means the (Sig7L)4-SA tetramer labeling is dependent on Siglec-sialic acid recognition. Our discovered Siglec-7 ligands (8' and 9') evidently outcompete the reported ligand (19') that only weakly labels a small population. Same as (Sig7L)4-SA labeling results, (Sig9L)4-SA-AF647 also stained Sig9-WT but not Sig9-KO and Sig9- R120A mutant U937-derived macrophages. The labeling of Sig9-KO and Sig9-R120A mutant cells was not completely blocked because of ‘weak’ Siglec-7 expression on these cells.Example 8 SigL tetramers partially internalize Siglec receptors and cell-surface Siglec expression is recovered upon removal of the SigL-SA tetramers

[0098] This Example describes internalization of cell-surface Siglec receptors by SigL tetramers. As the SigL tetramers are capable of labeling of Siglec- expressing cells as an antibody surrogate, we are interested in interrogation of the fate of Siglec molecules when oligomerized by SigL tetramers. First, flow cytometry analysis showed that U937 macrophage cell-surface Siglec molecules are diminished upon treatment with lOOnM (SigL)4-SA-AF488 at 37°C. This indicates that Sig7L and Sig9L tetramers might partially internalize Siglec-7 and Siglec-9 respectively, and thisinternalization happened in 5 min. Additionally, microscopic imaging was obtained, which showed that the internalized Siglec molecules are mostly colocalized with the early endosome marker (Rab5). Finally, cell surface expression of Siglec-7 and Siglec- 9 was rapidly recovered upon treatment with lOOnM SigL tetramers for 30 min followed by washing to remove SigL tetramers in medium. This could be due to the Siglec recycling from early endosomes to cell surface15.Example 9 Design of Siglec-7 / 9 degrader by conjugation of M6P to SigL tetramer

[0099] This Example describes the design of Siglec-7 / 9 degrader by conjugation of M6P to SigL tetramer (Figure 5). Since Siglec-7 / 9 are inhibitory receptors in regulating immunes cell fuctions, and Siglec-7 / 9 can quickly recyle back to cell surface upon antibody or high-affinity ligands-induced endocytosis, we envision that Siglec-7 / 9 depletion could persistently release the brake of inhibited immune cell effector responses, especially against cancer invasion, because most tumor cells aberrantly and highly upregulate their sialoglycans on the cell surface as the ’don’t eat me’ signal. Recently, a cell-surface protein degradation technolgoy named as ‘LYRAC’16that applies mannose-6-phosphate (M6P)-conjugated antibody to selectively degrade target receptor on the cell surface. When M6P is fused to an antibody, the target protein co-internalizes with the lysosome associated mannosephosphate receptor (CLM6PR) in lysosomes, M6PR returns back to cell surface while internalized target protein is digested in lysosomes. Therefore, we conjugated M6P to SA and assembled the resulting SA-M6P4 with biotinylated SigL to generate a potential Siglec degrader (SigL4-SA-M6P4) (Figure 5, panel A). Our rationale is that when the M6PR recognizes M6P, it would deliver the Siglec molecules bound with the SigL4- SA-M6P4 complex to lysosomes for degradation. Figure 5 (Panel B) shows the design, synthesis and characterization of SA-M6P4. Figure 5 (Panel C) shows that SA-M6P4 internalizes biotinylated dye in an efficient way compared to control (without M6P), and that SA-M6P4 induced dye internalization is completely dependent on M6PR-M6P interaction as co-treatment with either extra amount of M6P substrate or endocytosis inhibitor chloroquine fully blocked the internalization. Figure 5 (Panel D) shows that M6P-conjugated SigL tetramers are able to deplete most of cell surface Siglec-7 and Siglec-9 in U937 macrophages in 1 hour, while the control (without M6P) only removes a small quantity of cell surface Siglecs. Figure 5 (Panel E) shows that bothSiglec-7 and Siglec-9 proteomes in U937 macrophages are mostly degraded upon treatment with M6P-conjugated SigL tetramers for 1 day in comparison with controls, and that the degradation is dependent on Siglec-sialic acid recognition as the Sig7 / 9-R mutations are resistant to degradation. The degradation mechanism is dependent on lysosome degradation pathway as evidenced by blockage of degradation endowed with lysosome inhibitor Bafilomycin Al (BafAl) but not proteasome inhibitor MG132. Figure 5 (Panel F) shows that microscope data confirms Siglec-7 and Siglec-9 degradation in U937 macrophages.Example 10 Siglec-7 / 9 degradation synergizes with antibody therapy in macrophage phagocytosis of cancer cells

[0100] This Example describes that Siglec-7 / 9 degradation synergizes with antibody therapy in macrophage phagocytosis of cancer cells (Figure 6). Since ligand 8 is able to bind both Siglec-7 and Siglec-9 with high affinity, we name 8'4-SA-M6P4 as Siglec-7 / 9 degrader (Sig7 / 9 e) and use this degrader to target and deplete both Siglecs in immune cells. Figure 6 (Panel A) shows that cell-surface Siglec-7 and Siglec-9 are depleted upon treatment with Sig7 / 9t / c in human monocyte-derived macropahges (hMDMs). Figure 6 (Panel B) shows that western blot experiment confirmed both Siglec-7 and Siglec-9 proteomes are degraded in hMDMs. Figure 6 (Panel C) shows flow cytometry -based measurement of macrophage phagocytosis of Sig7 / 9L+cancer cells including breast (MDA-MB-435, MDA-MB-231, T47D and JIMT-1), ovarian (SKOV-3, CAOV-3 and OVCAR-4) and colon (HT29) cancer cell lines. Sig7 / 9 degradation alone in hMDMs does not initiate macrophage phagocytosis of all indicated cancer cells, but Sig7 / 9 degradation significantly synergizes with antibody treatment (anti-CD47 and anti-HER2) for enhancing phagocytosis of cancer cells, Sig7 / 9 degradation in combination with dual anti-CD47 / anti-HER2 treatment further boosted the phagocytosis of MDA-MB-435(HER2++) and SKOV3 cells compared to dual antibody treatment. MDA-MB-453 cell line that does not present Sig7 / 9 ligands has no synergistic effect, which indicates the observed synergy is dependent on Siglec- sialic acid engagement. Figure 6 (Panel D) shows that microscope-based measurement of HT29 phagocytosis corroborates the result obtained from flow cytometry -based assay. Figure 6 (Panel E) shows that both Siglec-7 and Siglec-9 are recruited to phagocytic synapse, leading to synapse disruption compared to Siglec-depleted cases incombination with anti-CD47 treatment. Figure 6 (Panel F) shows that Siglec-7 and Siglec-9 accumulated at the phagocytotic synapse recruit SHP tyrosine phosphatase that dampens the activating phagocytic signaling.Example 11 T cells acquire Siglec-7 / 9 receptors from interacting macrophages and the acquired Siglec molecules impede T cell activation

[0101] This Example describes that T cells acquire Siglec-7 / 9 receptors from interacting macrophages, and that the acquired Siglec molecules impede T cell activation. As macrphages are highly abuandant in many types of human tumor tissues, we wonder whether Siglec-7 / 9 receptors have regulatory effects when macropahges interact with tumor-infitrating T cells. We first found that Siglec-7 / 9 ligands are highly presented on PBMC T cells from healthy donors. Desialyation of T cells almost completely blocked Siglec-9 ligand staining, but only partially reduced Siglec-7 ligand staining, which indicates Siglec-9’ s binding to T cells is dependent on sialic acid recognition, however Siglec-7’ s engagement with T cells is additionally dependent on protein backbone interactions. Then we co-cultured staphylococcal enterotoxin (SEB) superantigen-pulsed hMDMs as the antigen presenting cells (APCs) with PBMC T cells to investigate if Siglec-7 / 9 molecues are able to be recruited at immunelogical synpase. The confocal microscopy imaging shows that Siglec-7 / 9 molecules not only accumulate at the synapse, but are also surprisingly found on T cells. This data indicates that T cells might acquire Siglec-7 / 9 molecules from interacting macrophages via a process known as trygocytosis. Further, flow cytometry analysis of T cells from the above co-culture expeirments confirmed that significant amount of Siglec-7 / 9 receptors appear on T cell surface as compared to the control of only T cells. In comparision with Siglec-9, Siglec- 7 is prone to be acquried by T cells as evidenced by 25% Siglec-7+T cells after coculture with SEB-pulsed macropahges. It was observed that Siglec-7 / 9 transfer to T cells is completely blocked when Siglec-7 / 9 are pre-degraded in macrophages. This indicates that T cells acquire Siglec-7 / 9 molecules from interacting macrophages but not T cell itself. We have substantiated the inhibitory role of Siglec-7 / 9 receptors in macrophage phagocytsis of cancer cells, but whether Siglec-7 / 9 also have an inhibitory effect on T cells is elusive.

[0102] Previously it was reported that overexpression of individual Siglec-7 and Siglec-9 on Jurakt T cells can inhibit TCR signaling thought their intracellularITEM domains.17We accordingly analyzed the activation markers of Jurkat T cells (mock, Siglec-7 and Siglec-9) upon activation with anti-CD3(OKT3) / anti-CD28. We found that both CD69 and PD1 expressions are significantly downregulated in Siglec-7 and Siglec-9 expreesing T cells compared to mock, which corroborates Siglec-7 / 9’s inhibitory role in T cell activation. In the experiment of co-culture of hMDMs with donor-matched PBMC CD8+T cells, we found that Siglec-7 / 9 degradation in hMDMs indeed evidently increased the expression of activation makers (CD69, CD25 and PD1). Moreover, Siglec-7 / 9 degradation also improved T cell proliferation and cytokine secretions including IFNy and TNFa.Example 12 Siglec-7 / 9 transfer onto mouse T cells leading to impeded T cell activation

[0103] This Example describes that Siglec-7 / 9 transferred onto transgenic mouse T cells lead to impeded T cell activation. We further investigated if Siglec-7 / 9 receptors exhibit inhibitory function in mouse T cells. We observed that Siglec-7 / 9 ligands as well as Siglec-E (homolog of Sig7 / 9) ligands are highly presented on WT C57BL / 6J (B6) mouse T cells. We prepared bone marrow-derived macrophages (BMDMs) from Siglec-E-KO (SigE-KO) and Siglec-7 / 9+SigE-KO (Sig7 / 9+) transgenic B6 mice. After co-culcuture of SEM-pulsed BMDMs with WT B6 splenic T cells, >80% and ~8% T cells acquired Siglec-7 and Siglec-9 molecues, respectively. It was found that T cells only or co-cultured with SigE-KO BMDM did not show any Siglec- 7 / 9 expressions. It was also observed that both Siglec-7 and Siglec-9 can be degraded in Sig7 / 9+BMDMs. Co-culture of BMDMs with WT CD8+T cells using anti-CD3 antigen (clone 17A2) showed that Siglec-7 / 9 degradation is able to significantly enhance T cell proliferation and IFNy secretion like the SigE-KO case. Finally, P14 T cell activation study using gp33-pulsed BMDMs confirmed that Siglec-7 / 9 degradation and SigE-KO can evidently improve the T cell activation.Example 13 Siglec-7 / 9 degradation proceeds in vivo and promotes tumor control

[0104] This Example describes that Siglec-7 / 9 degradation can proceed in vivo and promote tumor control (Figure 7). Siglec-7 / 9 inhibit both human and mouse T cell activations, we carried out the in vivo Siglec-7 / 9 degradation assay and accessed if this degradation can better control tumor progression. GMCSF facilites recruitment anddifferentiation of of monocyte-derived macrophages (such as TAMs) and MDSCs to the tumor tissue. In our study, IxlO6B16-GMCSF melanoma tumor cells were inoculated in Sig7 / 9+B6 mice on day -5, Sig7 / 9t / c was intratumorally administrated at 5 pg, lOpg and 15 g doses on day 0. Tumor tissues were harvested at day 1, 2 and 3 for flow cytometry analysis of Sig7 / 9 degradation in CD1 lb+cell populaiton (CD1 lb markers most of the myeloid cells in tumor microenveriment). As shown in Figure 7 (panel A), lOpg of Sig7 / 9t / c is good as 15 pg to reach the maximal degradation in different time courses, and day 2 is the best time point to generate optimal degradation in which ~ 60% Siglec-7 and -70% Siglec-9 were depleted in CD1 lb+cells. Figure 7 (panel B) shows analysis of CD1 lb+cell surface expression of Sig7 / 9 upon introtumoral treatment with lOpg Sig7 / 9t / c. Shown in Figure 7 (panel C), IxlO6B16- GMCSF tumor cells were subcutaneously inoculated in the flanks of SigE-KO and Sig7 / 9+B6 mice. When tumors became palpable, lOpg Sig7 / 9t / c was intratumorally administrated on day 4, 6, 8 and 10. Like the SigE-KO, Sig7 / 9t / c treatment better controlled tumor growth compared to PBS control. In a pacreatic cancer model, 0.4xl06MT5 tumor cells were subcutaneously inoculated in the flanks of SigE-KO and Sig7 / 9+B6 mice (Figure 7, panel D). lOpg Sig7 / 9t / c was intratumorally administrated on day 6, 9, 12, 15 and 18 in the indicated Sig7 / 9+mice, and 200pg anti-CTLA4 (clone 9H10) was intraperitoneally injected to indicated Sig7 / 9+and SigE-KO mice on day 10, 13, 16 and 19. Sig7 / 9 degradation alone and SigE-KO only weakly inhibited tumor growth compared to IgG / PBS control, while anti-CTLA4 alone infirmly controlled tumor grwoth. Intriguingly, Sig7 / 9 degradation or SigE-KO in combiation with anti-CTLA4 treatment significantly inhibited tumor growth and 2 / 5 mice became tumor free in both cases.Example 14 SigL-Fc conjugates for targeted Siglec-7 and -9 degradation

[0105] We further constructed ADCs containing the high affnity Sig7 / 9 ligands and an antibody fusion partner for targeting Siglec-7 and -9 to lysosome for degradation. Instead of using Man-6-P or GalNAc / galactose as the targeting moiety, some of these ADCs used IGF2 as the targeting agent. The reason for this choice is that Man-6-P bind with micromolar affinity to their receptors, such that multiple copies are required. By contrast, IGF2 binds to IGF2R with nanomolar affinity and can be easily used for the construction of genetically encoded fusion proteins.

[0106] To construct the IGF2-IgGl-Fc SigL conjugate-based degrader, we first coupled IGF2 via a (GGGGS; SEQ ID N0:3)n (n=l,2) linker to the N-terminus of a silenced human IgGl Fc mutant to form a fusion protein. See, e.g., Wang et al., Cancer Immunol. Res. 2019;7:2013-24. A N297A mutation is introduced into IgGl Fc to remove N-glycosylation, thereby eliminating Fey receptor (FcyR) binding. A sortase- tag was then installed to the Fc C-terminus, allowing site-specific incorporation of 2 or 4 copies of Siglec-7 / 9 ligands, e.g., Ligand 8 (Figure 8, panel A), via sortase-mediated ligation to form degraders (Figure 8, panel B). Alternatively, the ligand can be coupled to the IGF2-Fc fusion protein via NHS coupling reaction and copper-free click chemistry (Figure 8, panel C).

[0107] Some ADC degraders contain a lysosome sorting sequence (LSS) in lieu of IGF2 as the targeting agent. The degraders are examined to determine whether such a design has better degradation efficacy. An LSS is recognized by components of the clathrin coat to ensure accurate transport of proteins to the lysosome through clathrin-mediated endocytosis. Examples of LSS include YXX0 (0 stands for an amino acid with a bulky hydrophobic side chain) or DDSDEDLLHI (SEQ ID NO:2). See, e.g., Chen et al, J Biol Chem 272, 7003-7012 (1997); and Bonifacino et al., Annu Rev Biochem 72, 395-447 (2003). In these Fc-LSS-SigL fusion degraders, the LSS motif can be conjugated to the Fc C-terminus, and the SigL can be attached to the N- terminus. Additionally, two modifications are introduced into the IgGl Fc: (1) N297A mutation removes N-glycosylation, thereby eliminating Fey receptor (FcyR) binding, and (2) M252Y / S254T / T256E (YTE) triple mutation at the CH2-CH3 interface to enhance FcRn binding and serum half-life. As illustrated in Figure 9, a sortase tag is installed to the Fc N-terminus, allowing the incorporation of 2 copies of the SigL (e.g., Ligand 8) via sortase-mediated ligation to form the degraders.

[0108] Activities of the ADC degraders in Siglec-7 and -9 degradation were examined. Specifically, we observed efficient Siglec-7 and -9 degradation in U937 macrophages upon treatment with IGF2-Fc-based degraders functionalized with 1-3 copies of Ligand 8 (Figure 10, panel A). The observed degradation occurred in a dosedependent manner (Figure 10, panel C). We further found that mutating the critical Arg residues responsible for sialic acid binding, Siglec-7 (R124A) and Siglec-9 (R120A), completely abolished the degradation (Figure 10, panel B).

[0109] We further examined in vivo anti-tumor activities of the IGF2-Fc-SigL degraders. As shown in Figure 11, these ADCs when employed as a single agent were able to effectively control anti-PD-1 resistant B 16 s.c. tumors in Sig7 / 9+mice. It was observed that their therapeutical efficacy was similar to that observed in SigEKOmice.

[0110] Some cited references:1. Duan, S.; Paulson, J. C. Siglecs as Immune Cell Checkpoints in Disease. Annual review of immunology 2020, 38, 365-395.2. Macauley, M. S.; Crocker, P. R.; Paulson, J. C. Siglec-mediated Regulation of Immune Cell Function in Disease. Annual review of immunology 2014, 14, 653-66.3. Bomhofft, K. F.; Goldammer, T.; Rebl, A.; Galuska, S. P. Siglecs: A journey through the evolution of sialic acid-binding immunoglobulin-type lectins. Developmental & Comparative Immunology 2018, 86, 219-231.4. Angata, T.; Nycholat, C. M.; Macauley, M. S. Therapeutic Targeting of Siglecs using Antibody- and Glycan-Based Approaches. Trends in Pharmacological Sciences 2015, 36, 645-660.5. Freeman, G. J.; Long, A. J.; Iwai, Y.; Bourque, K.; Chernova, T.; Nishimura, H.; Fitz, L. J.; Malenkovich, N.; Okazaki, T.; Byrne, M. C.; Horton, H. F.; Fouser, L.; Carter, L.; Ling, V.; Bowman, M. R.; Carreno, B. M.; Collins, M.; Wood, C. R.; Honjo, T. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. Journal of Experimental Medicine 2000, 192, 1027-1034.6. Stanczak, M. A.; Laubli, H. Siglec receptors as new immune checkpoints in cancer. Molecular Aspects of Medicine 2023, 90, 101112.7. Mizrahi, S.; Gibbs, B. F.; Karra, L.; Ben-Zimra, M.; Levi-Schaffer, F. Siglec-7 is an inhibitory receptor on human mast cells and basophils. Journal of Allergy and Clinical Immunology 2014, 134, 230-233.8. Miralda I.; Samanas, N. B.; Seo, A. J.; Foronda, J. S.; Sachen, J.; Hui, Y.; Morrison, S. D, Oskeritzian, C. A.; Piliponsky, A. M. Siglec-9 is an inhibitory receptor on human mast cells in vitro. Journal of Allergy and Clinical Immunology 2023. doi: 10.1016 / j.jaci.2023.04.007.9. Legrand, F.; Landolina, N.; Zaffran, I.; Emeh, R. O.; Chen, E.; Klion, A. D.; Levi- Schaffer, F. Siglec-7 on peripheral blood eosinophils: Surface expression and function. H / / ergy 2019, 74, 1257-1265.10. Nguyen, K. A.; Hamzeh-Cognasse, H.; Palle, S.; Anselme-Bertrand, I.; Arthaud, C. A.; Chavarin, P.; Pozzetto, B.; Garraud, O.; Cognasse, F. Role of Siglec-7 in apoptosis in human platelets. PLoS One 2014, 9, el06239.11. Rodriguez, E.; Boelaars, K.; Brown, K.; Eveline Li, R. J.; Kruijssen, L.; Bruijns, S. C. M.; van Ee, T.; Schetters, S. T. T.; Crommentuijn, M. H. W.; van der Horst, J. C.; van Grieken, N. C. T.; van Vliet, S. J.; Kazemier, G.; Giovannetti, E.; Garcia-Vallejo, J. J.; van Kooyk, Y. Sialic acids in pancreatic cancer cells drive tumour-associatedmacrophage differentiation via the Siglec receptors Siglec-7 and Siglec-9. Nature Communications 2021, 72, 1270.12. Santegoets, K. C. M.; Gielen, P. R.; Bull, C.; Schulte, B. M.; Kers-Rebel, E. D.; Kiisters, B.; Bossman, S. A. J. F. H.; Ter Laan, M.; Wesseling, P.; Adema, G. J. Expression profiling of immune inhibitory Siglecs and their ligands in patients with glioma. Cancer Immunology, Immunotherapy 2019 , 68, 937-949.13. Liu, F.; Wang, H.; Li, S.; Bare, G. A. L.; Chen, X.; Wang, C.; Moses, J. E.; Wu, P.; Sharpless, K. B. Biocompatible SuFEx Click Chemistry: Thionyl Tetrafluoride (SOF4)- Derived Connective Hubs for Bioconjugation to DNA and Proteins. Angewandte Chemie International Edition 2019, 58, 8029-8033.14. Wang, W.; Hong, S.; Tran, A.; Jiang, H.; Triano, R.; Liu, Y.; Chen, X.; Wu, P. Sulfated ligands for the copper(I)-catalyzed azide-alkyne cycloaddition. Chemistry - An Asian Journal 2011, 6, 2796-2802.15. Bonifacino, J. S.; Traub, L. M. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annual Review of Biochemistry 2003, 72, 395-447.16. Banik, S. M.; Pedram, K.; Wisnovsky, S.; Ahn, G.; Riley, N. M.; Bertozzi, C. R. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 2020, 584, 291-297.17. Ikehara, Y.; Ikehara, S. K.; Paulson, J. C. Negative regulation of T cell receptor signaling by Siglec-7 (p70 / AIRM) and Siglec-9. Journal of Biological Chemistry 2004, 279, 43117-43125.18. Ahn, G. et al. LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nature Chemical Biology 17, 937-946 (2021).19. Caianiello, D.F. et al. Bifunctional small molecules that mediate the degradation of extracellular proteins. Nature Chemical Biology 17, 947-953 (2021).20. Brown, J. et al. Structure and functional analysis of the IGF-IEIGF2R interaction. EMB0 J 1, 265-276 (2008).21. Tong, P.Y. & Kornfeld, S. Ligand interactions of the cation-dependent mannose 6- phosphate receptor. Comparison with the cation-independent mannose 6-phosphate receptor. J Biol Chem 264, 7970-7975 (1989).22. Chen, H.J., Yuan, J. & Lobel, P. Systematic mutational analysis of the cationindependent mannose 6-phosphate / insulin-like growth factor II receptor cytoplasmic domain. An acidic cluster containing a key aspartate is important for function in lysosomal enzyme sorting. J Biol Chem 272, 7003-7012 (1997).***

[0111] The invention thus has been disclosed broadly and illustrated in reference to representative embodiments described above. It is understood that various modifications can be made to the present invention without departing from the spirit and scope thereof. It is further noted that all publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entiretyand for all purposes as if each is individually so denoted. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

WHAT IS CLAIMED IS:

1. A Siglec-7 / 9 ligand compound of Formula I below or a pharmaceutically acceptable salt thereofwhereinRi is any group or atom;R2 is H or acyl; R3 is alkyne, azide or amine; each of XI,2,3 is independently any atom except H; each of ¥1,2 is independently any atom except H; and each of Z 1,2, 3, 4 is independently any atom except H.

2. The compound of claim 1, which is3. A Siglec-7 / 9 ligand compound of Formula II below or a pharmaceutically acceptable salt thereofwherein Ri is any group or atom;R2 is H or acyl;R3 is alkyne, azide or amine; each of XI,2,3 is independently any atom except H;each of YI,2 is independently any atom except H; and each of Z 1,2, 3, 4 is independently any atom except H.

4. The compound of claim 3, which is5. A Siglec-7 / 9 ligand compound of Formula III below or a pharmaceutically acceptable salt thereofwhereinRi is any group or atom;R2 is H or acyl;R3 is alkyne, azide or amine; each of XI,2,3 is independently any atom except H; each of ¥1,2 is independently any atom except H; and each of Z 1,2, 3, 4 is independently any atom except H.

6. A Siglec-7 / 9 ligand compound of Formula IV below or a pharmaceutically acceptable salt thereofwhereinRi is any group or atom;R2 is H or acyl;R3 is alkyne, azide or amine;each of XI.2,3 is independently any atom except H; each of YI,2 is independently any atom except H; and each of Z 1,2, 3, 4 is independently any atom except H.

7. A drug conjugate, comprising the Siglec ligand compound of any one of claims 1-6 that is conjugated to a fusion partner.

8. The drug conjugate of claim 7, wherein the fusion partner is a protein, an antibody or antibody fragment, a small molecule, a polysaccharide, a lipid or a PEG molecule.

9. The drug conjugate of claim 7, which is a tetramer comprising the Siglec ligand compound that is fused to a streptavidin protein via biotin.

10. The drug conjugate of claim 9, further comprising mannose-6- phosphate (M6P) that is fused to the streptavidin protein.

11. The drug conjugate of claim 7, wherein the fusion partner comprises a moiety targeting M6PR or TG2 / LRP-1 that is fused to a protein, an antibody or antibody fragment.

12. The drug conjugate of claim 7, wherein the fusion partner comprises mannose-6-phosphate (M6P) that is fused to a protein, an antibody or antibody fragment.

13. The drug conjugate of claim 7, comprising the Siglec ligand compound that is coupled to an antibody that recognizes an E3 ligase.

14. The drug conjugate of claim 7, wherein the fusion partner is an IgGl Fc domain.

15. The drug conjugate of claim 14, further comprising a targeting moiety that is fused to the Fc domain, wherein the targeting moiety specifically binds to a protein that facilitates lysosome mediated protein degradation.

16. The drug conjugate of claim 14, wherein the targeting moiety is IGF2 or a lysosome sorting sequence (LSS).

17. A method for promoting degradation of inhibitory Siglecs on the surface of immune cells, comprising contacting the immune cells with the Siglec-7 / 9 ligand compound of any one of claims 1-6 or a drug conjugate of claim 7.

18. The method of claim 17, wherein the immune cells are primary macrophages, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), T lymphocytes or NK cells.

19. A method for stimulating and augmenting anti-tumor immune responses against a cancer in a subject, comprising administering to the subject a pharmaceutical composition that comprises a therapeutically effective amount of a Siglec-7 / 9 ligand compound of any one of claims 1-6 or a drug conjugate of claim 7.

20. The method of claim 19, wherein the subject is a human.

21. The method of claim 19, further comprising administering to the subject a second anti -turn or agent.

22. The method of claim 21, wherein the second anti -tumor agent is an immune checkpoint inhibitor.

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