Anti-siglec-6 antibody formulations

Abstract

The present disclosure provides pharmaceutical compositions (e.g., liquid formulations) comprising a monoclonal antibody that binds to a human Siglec-6 for subcutaneous administration, as well as articles of manufacture related thereto.

Description

ANTI-SIGLEC-6 ANTIBODY FORMULATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 623,153, filed on January 19, 2024, the disclosure of which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (701712002240seqlist.xml; Size: 150,615 bytes; and Date of Creation: January 14, 2025) is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to pharmaceutical compositions (e.g., liquid formulations) comprising a monoclonal antibody that binds to a human Siglec-6 for subcutaneous administration, as well as articles of manufacture related thereto.BACKGROUND

[0004] Siglecs (sialic acid-binding immunoglobulin-like lectins) are single-pass transmembrane cell surface proteins found predominantly on leukocytes and that are characterized by their specificity for sialic acids attached to cell-surface glycoconjugates. The Siglec family contains at least 15 members that are found in mammals (Pillai et al., Annu Rev Immunol., 2012, 30:357-392). These members include sialoadhesion (Siglec-1), CD22 (Siglec- 2), CD33 (Siglec-3), myelin associated glycoprotein (Siglec-4), Siglec-5, OBBP1 (Siglec-6), AIRMI (Siglec-7), SAF-2 (Siglec-8), and CD329 (Siglec-9).

[0005] Siglec-6 (also known as CD327) is an inhibitory receptor that is selectively expressed on mast cells, e.g., human tissue mast cells, HMC-1 cells, and CD34+ derived human mast cells. Mast cells are considered pathogenic drivers of numerous autoimmune and inflammatory diseases, including but not limited to food allergy, mast cell activation syndrome, mastocytosis, IPF, COPD, and others. See, e.g., Yu, Y. et al. (2018) Front. Immunol. 9:2138; Yokoi, H. et al. (2006) Allergy 61 :769-76; W02005124358; and US PG Pub. Nos. US20060269556A1 and US20080267973 Al . Engagement of Siglec-6 with antibody is thought to inhibit IgE-mediated mast cell activation.

[0006] Monoclonal antibodies (mAbs) have become essential therapeutic agents in treating various diseases. Traditional mAb formulations often have lower concentrations, requiring large volumes for therapeutic doses. This can lead to challenges in administration, especially for subcutaneous injections, and can impact patient compliance and comfort.

[0007] There is a growing need for high concentration mAb formulations to reduce the volume of administration while maintaining or enhancing the drug's stability and potency. There remains a need for improved formulations for anti-Siglec-6 antibodies that allow for significantly higher antibody concentrations (e.g., up to 150mg / mL) than conventional mAb formulations while also providing stability, solubility, and potency for the anti-Siglec-6 antibody.

[0008] All references cited herein, including patent applications, patent publications, and scientific literature, are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.BRIEF SUMMARY

[0009] To meet this and other needs, the present disclosure provides, inter alia, formulations (e.g., liquid formulations for subcutaneous administration) comprising a monoclonal antibody that binds to Siglec-6 (i.e., an anti-Siglec-6 antibody), and kits or articles of manufacture related thereto. These formulations are based at least in part on the demonstrated herein of an anti- Siglec-6 antibody formulation that allows for exceptionally antibody high concentration, reaching up to 200 mg / mL, without significantly changing appearance or viscosity. The formulations is designed to improve the solubility and provide adequate stability for an anti- Siglec-6 antibody. A combination of stabilizing agents and buffer systems that allow for high antibody concentration without the common issues of aggregation, precipitation, or denaturation often encountered in high-concentration formulations. The formulation also includes a pH- adjusted solvent system that is optimized for the anti-Siglec-6 antibody and is biologically compatible. Without wishing to be bound to theory, it is thought that this high-concentration formulation is advantageous for subcutaneous administration, allowing for reduced injection volumes and enhanced patient comfort without compromising the therapeutic potency of the anti-Siglec-6 antibody.

[0010] Accordingly, certain aspects of the present disclosure relate to pharmaceutical compositions (e.g., liquid formulations) comprising a monoclonal antibody that binds to a human Siglec-6, wherein the antibody is in a concentration of about 50 mg / mL to about 200 mg / mL;and (b) histidine in a concentration of about 5 mM to about 100 mM; and wherein the pH of the liquid formulation is between 5.7 and 6.5.

[0011] In some embodiments according to any of the embodiments described herein, the antibody comprises: (1) a heavy chain comprising a heavy chain variable (VH) region comprising: an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 168; an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 169; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 7; and (2) a light chain comprising a light chain variable (VL) region comprising: an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 161, and the VL region comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, the heavy chain further comprises a human IgGFc region. In some embodiments, the human IgG Fc region is a human IgGl Fc region. In some embodiments, the human IgGl Fc region is non-fucosylated. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 165 or 166, and the light chain comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, at least one or two of the heavy chains of the antibody is / are non-fucosylated.

[0012] In some embodiments according to any of the embodiments described herein, the histidine is in a concentration of about 15 mM to about 25 mM. In some embodiments, the histidine is in a concentration of 20 mM. In some embodiments, the histidine comprises L- histidine and / or L-histidine hydrochloride. In some embodiments, the histidine comprises 9 mM L-histidine and 11 mM L-histidine hydrochloride monohydrate. In some embodiments, the formulation further comprises a disaccharide in a concentration of about 90 mM to about 250 mM. In some embodiments, the formulation comprises the disaccharide in a concentration of 100 mM to 248 mM or 160 mM to 248 mM. In some embodiments, the disaccharide is sucrose. In some embodiments, the formulation comprises sucrose in a concentration of 170 mM. In some embodiments, the formulation further comprises polysorbate 80. In some embodiments, the polysorbate 80 is in a concentration of about 0.025% to about 0.075% (w / v). In some embodiments, the formulation comprises polysorbate 80 in a concentration of 0.05% (w / v). In some embodiments, the formulation does not comprise sodium chloride. In some embodiments, the pH of the liquid formulation is between 5.8 and 6.2. In some embodiments, the pH of theliquid formulation is 6.0. In some embodiments, the antibody is in a concentration of 135 mg / mL to 200 mg / mL. In some embodiments, the antibody is in a concentration of 135 mg / mL to 165 mg / mL. In some embodiments, the antibody is in a concentration of 150 mg / mL. In some embodiments, the formulation further comprises arginine, proline, or glutamate. In some embodiments, the formulation comprises arginine hydrochloride in a concentration of about 1 mM to about 100 mM. In some embodiments, the formulation comprises L-proline in a concentration of about 1 mM to about 200 mM. In some embodiments, the formulation comprises L-glutamate in a concentration of about 1 mM to about 100 mM. In some embodiments, the formulation has an osmolality of between about 260 mOsm / kg and about 435 mOsm / kg. In some embodiments, the formulation has an osmolality of about 290 mOsm / kg.

[0013] In some embodiments according to any of the embodiments described herein, the formulation comprises the antibody that binds to a human Siglec-6 in a concentration of 150 mg / mL; 20 mM L-histidine and / or L-histidine hydrochloride; 170 mM sucrose; and 0.05% polysorbate 80 (w / v); wherein the pH of the liquid formulation is 6.0. In some embodiments, the formulation comprises the antibody that binds to a human Siglec-6 in a concentration of 150 mg / mL; 20 mM L-histidine and / or L-histidine hydrochloride; 248 mM sucrose; and 0.05% polysorbate 80 (w / v); wherein the pH of the liquid formulation is 6.0. In some embodiments according to any of the embodiments described herein, the formulation comprises the antibody that binds to a human Siglec-6 in a concentration of 150 mg / mL; 20 mM L-histidine and / or L- histidine hydrochloride; 160 mM sucrose; and 0.05% polysorbate 80 (w / v); wherein the pH of the liquid formulation is 5.8.

[0014] Other aspects of the present disclosure relate to articles of manufacture or kits comprising a container enclosing the formulation according to any one of the embodiments described herein. In some embodiments, the container is a glass vial. In some embodiments, the articles of manufacture or kits further comprise instructions for administering the formulation subcutaneously.

[0015] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to one of skill in the art. These and other embodiments of the present disclosure are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows a flow chart outlining the workflow for formulation development of an anti-Siglec-6 antibody.

[0017] FIG. 2 shows that sample pH increased with increasing concentration of anti-Siglec-6 antibody during ultra-filtration.

[0018] FIG. 3 shows the appearance of surfactant screening solutions before and after physical stress was applied. Solutions comprising no surfactant, 0.05% (w / v) polysorbate 80 (PS80), or 0.05% (w / v) polysorbate 20 (PS20) were tested, as indicated.

[0019] FIG. 4 shows the surfactant screening results before and after physical stress.

[0020] FIG. 5 shows the appearance of excipient solutions at time T=0.

[0021] FIG. 6 shows a table quantifying the pH, osmolality, protein concentration, viscosity, differential scanning calorimetry (DSC), appearance, and sub-visible particles (SVP) results of the samples at time T=0 for excipient testing.

[0022] FIG. 7 shows a table quantifying size-exclusion high performance liquid chromatography (SE-HPLC) and cation-exchange high performance liquid chromatography (CEX-HPLC) results of the samples at time T=0 for excipient testing.

[0023] FIG. 8 shows the appearance of excipient solutions after 2 weeks at 5 °C.

[0024] FIG. 9 shows stability results of the samples after 2 weeks at 5 °C, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0025] FIG. 10 shows the appearance of excipient solutions after 2 weeks at 25°C / 60% relative humidity (RH).

[0026] FIG. 11 shows stability results of the samples after 2 weeks at 25°C / 60% RH, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0027] FIG. 12 shows the appearance of excipient solutions after 2 weeks at 40°C / 75% RH.

[0028] FIG. 13 shows stability results of the samples after 2 weeks at 40°C / 75% RH, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0029] FIG. 14 shows the appearance of excipient solutions after 4 weeks at 5 °C.

[0030] FIG. 15 shows stability results of the samples after 4 weeks at 5 °C, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0031] FIG. 16 shows the appearance of excipient solutions after 4 weeks at 25°C.

[0032] FIG. 17 shows stability results of the samples after 4 weeks at 25°C, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0033] FIG. 18 shows the appearance of excipient solutions after 4 weeks at 40°C.

[0034] FIG. 19 shows stability results of the samples after 4 weeks at 40°C, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0035] FIG. 20 shows the appearance of excipient solutions after 8 weeks at 5 °C.

[0036] FIG. 21 shows stability results of the samples after 8 weeks at 5 °C, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0037] FIG. 22 shows the appearance of excipient solutions after 8 weeks at 25°C.

[0038] FIG. 23 shows stability results of the samples after 8 weeks at 25°C, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0039] FIG. 24 shows the appearance of excipient solutions after 8 weeks at 40°C.

[0040] FIG. 25 shows stability results of the samples after 8 weeks at 40°C, quantifying protein concentration, appearance, SVP, SE-HPLC, and CEX-HPLC.

[0041] FIG. 26 shows light obscuration particle count (LPC) test comparison results using liquid particle counter (EUAC) among formulations F01, F02, F03 and F09.

[0042] FIG. 27 shows SE-HPLC comparison results among formulations F01, F02, F08 and F12.

[0043] FIG. 28 shows CEX-HPLC (% acidic) comparison results between formulations F01, F02, F03 and Fl 2.

[0044] FIG. 29 shows the appearance of excipient solutions after 5x freeze-thaw.

[0045] FIG. 30 shows stability results of the samples after 5x freeze-thaw, quantifying protein concentration, appearance, SVP and SE-HPLC.

[0046] FIG. 31 shows the appearance of excipient solutions after physical stress.

[0047] FIG. 32 shows stability results of the samples after physical stress, quantifying protein concentration, appearance, SVP and SE-HPLC.

[0048] FIG. 33 shows the percent of samples in the main peak from SE-HPLC for formulations F01, F02, Fl 1 and F12 at 5°C, 25°C and 40°C, and at T=0, after 2 weeks, 4 weeks and 8 weeks.

[0049] FIG. 34 shows the percent of samples in the main peak from CEX-HPLC for formulations F01, F02, Fl 1 and F12 at 5°C, 25°C and 40°C, and at T=0, after 2 weeks, 4 weeks and 8 weeks.

[0050] FIG. 35 shows the measured sub-ambient glass transition temperature (Tg’) onset as compared to the weight fraction of protein.

[0051] FIG. 36 shows the sequence of the anti-Siglec-6 antibody heavy and light chains, with the potential aspartate isomerization residues underlined. Sequences shown correspond to SEQ ID Nos: 166 (upper) and 167 (lower), respectively.

[0052] FIG. 37 shows the appearance of the anti-Siglec-6 antibody before concentration (20 mg / mL), after concentration (220 mg / mL), and after dilution in the indicated formulation buffer.

[0053] FIG. 38 shows the overlaid chromatograms of anti-Siglec-6 antibody before and after concentrating using SE-HPLC.DETAILED DESCRIPTIONI. Definitions

[0054] It is to be understood that the present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0055] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0056] It is understood that aspects and embodiments of the present disclosure include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0057] The term “antibody” includes polyclonal antibodies, monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with poly epitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, andsingle-chain molecules), as well as antibody fragments (e.g., Fab, F(ab')2, and Fv). The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

[0058] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the oc and y chains and four CH domains for p and s isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0059] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated oc, 8, s, y and p, respectively. The y and oc classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in the present disclosure. Common allotypic variants in human populations are those designated by the letters a, f, n, z.

[0060] An “isolated” antibody is one that has been identified, separated and / or recovered from a component of its production environment (e.g., naturally or recomb inantly). In some embodiments, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody is prepared by at least one purification step.

[0061] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or posttranslation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. In some embodiments, monoclonal antibodies have a C-terminal cleavage at the heavy chain and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the C- terminus of heavy chain and / or light chain. In some embodiments, the C-terminal cleavage removes a C-terminal lysine from the heavy chain. In some embodiments, monoclonal antibodies have an N-terminal cleavage at the heavy chain and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the N-terminus of heavy chain and / or light chain. In some embodiments, monoclonal antibodies are highly specific, being directed against a single antigenic site. In some embodiments, monoclonal antibodies are highly specific, being directed against multiple antigenic sites (such as a bispecific antibody or a multispecific antibody). The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies tobe used in accordance with the present disclosure may be made by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, phage-display technologies, and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0062] The term “naked antibody” refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0063] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0064] An “antibody fragment” comprises a portion of an intact antibody, the antigen binding and / or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0065] Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen -bin ding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0066] The Fc fragment comprises the carboxy -terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0067] ‘ ‘Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0068] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a review of the sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0069] ‘ ‘Functional fragments” of the antibodies of the present disclosure comprise a portion of an intact antibody, generally including the antigen binding or variable region of the intact antibody or the Fv region of an antibody which retains or has modified FcR binding capability. Examples of antibody fragments include linear antibody, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[0070] The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with an antigen of interest. As used herein, “humanized antibody” is used as a subset of “chimeric antibodies.”

[0071] ‘ ‘Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. In some embodiments, the number of these amino acid substitutions in the FR are no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 :105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, humanized antibodies are directed against a single antigenic site. In some embodiments, humanized antibodies are directed against multiple antigenic sites. An alternative humanization method is described in U.S. Pat. No. 7,981,843 and U.S. Patent Application Publication No. 2006 / 0134098.

[0072] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites.

[0073] The term “hypervariable region,” “HVR,” or “HV,” when used herein refers to the regions of an antibody-variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu etal. Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman etal., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0074] A number of HVR delineations are in use and are encompassed herein. The HVRs that are Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institute of Health, Bethesda, MD (1991)). Chothia HVRs refer instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.Loop Kabat Chothia ContactLI L24-L34 L26-L34 L30-L36L2 L50-L56 L50-L56 L46-L55L3 L89-L97 L91-L96 L89-L96Hl H31-H35B H26-H32 H30-H35B (Kabat Numbering)Hl H31-H35 H26-H32 H30-H35 (Chothia Numbering)H2 H50-H65 H53-H56 H47-H58H3 H95-H102 H95-H102 H93-H101

[0075] Unless otherwise indicated, the variable-domain residues (HVR residues and framework region residues) are numbered according to Kabat etal., supra.

[0076] “Framework” or “FR” residues are those variable-domain residues other than the HVR residues as herein defined.

[0077] The expression “variable-domain residue-numbering as in Kabat” or “amino-acid- position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy -chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0078] An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0079] ‘ ‘Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

[0080] An antibody that “binds to”, “specifically binds to” or is “specific for” a particular a polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In some embodiments, binding of an anti-Siglec-6 antibody described herein (e.g., an antibody that binds to human Siglec-6) to an unrelated non-Siglec-6 polypeptide is less than about 10% of the antibody binding to Siglec-6 as measured by methods known in the art (e.g., enzyme-linked immunosorbent assay (ELISA)). In some embodiments, an antibody that binds to a Siglec-6 (e.g., an antibody that binds to human Siglec-6) has a dissociation constant (Kd) of < IpM, < 100 nM, < 10 nM, < 2 nM, < 1 nM, < 0.7 nM, <0 .6 nM, < 0.5 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 1 O'8M or less, e.g. from 10'8M to 10'13M,e.g., from 10'9M to IO3M), about 250pM or less, about lOOpM or less, about lOpM or less, or about IpM.

[0081] The term “anti-Siglec-6 antibody” or “an antibody that binds to human Siglec-6” refers to an antibody that binds to a polypeptide or an epitope of human Siglec-6 without substantially binding to any other polypeptide or epitope of an unrelated non-Siglec-6 polypeptide.

[0082] The term “Siglec-6” as used herein refers to a human Siglec-6 protein. The term also includes naturally occurring variants of Siglec-6, including splice variants or allelic variants. Siglec-6, the sialic acid binding Ig-like lectin 6, is also known as CD327, CD33L, OBBP1, CD33L1, CD33L2, and CDW327. In some embodiments, a human Siglec-6 protein is any protein or polypeptide expressed by a human SIGLEC6 gene. An exemplary human SIGLEC6 gene is described by NCBI Ref. Seq. Gene ID No. 946. Amino acid sequences of exemplary human Siglec-6 proteins and domains thereof are described herein. For example, in some embodiments, a human Siglec-6 protein comprises an extracellular domain (ECD) comprising the amino acid sequenceQERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEEV QEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRVM ALTHRPNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTITP RPQDHSTNLTCQVTFPGAGVTMERTIQLNVSYAPQKVAISIFQGNSAAFKILQNTSSLPV LEGQALRLLCDADGNPPAHLSWFQGFPALNATPISNTGVLELPQVGSAEEGDFTCRAQH PLGSLQISLSLFVHWKPEGRAGGV (SEQ ID NO:1).

[0083] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell- mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0084] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for killing of the targetcell by this mechanism. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and I<inet, 4ww. Rev. Immunol. 9: 457-92 (1991). In some embodiments, an anti-Siglec-6 antibody (e.g., an antibody that binds to human Siglec-6) described herein enhances ADCC. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998). Other Fc variants that alter ADCC activity and other antibody properties include those disclosed by Ghetie et al., Nat Biotech. 15:637-40, 1997; Duncan et al, Nature 332:563-564, 1988; Lund et al., J. Immunol 147:2657-2662, 1991; Lund et al, Mol Immunol 29:53-59, 1992; Alegre et al, Transplantation 57:1537-1543, 1994; Hutchins et al., Proc Natl. Acad Sci USA 92:11980-11984, 1995; Jefferis et al, Immunol Lett. 44: 111-117, 1995; Lund et al., FASEB J9: 115-119, 1995; Jefferis et al, Immunol Lett 54: 101-104, 1996; Lund et al, J Immunol 157:4963-4969, 1996; Armour et al., Eur J Immunol 29:2613-2624, 1999; Idusogie et al, J Immunol 164:4178-4184, 200; Reddy et al, J Immunol 164:1925-1933, 2000; Xu et al., Cell Immunol 200:16-26, 2000; Idusogie et al, J Immunol 166:2571-2575, 2001; Shields et al., J Biol Chem 276:6591-6604, 2001; Jefferis et al, Immunol Lett 82:57-65. 2002; Presta et al., Biochem Soc Trans 30:487-490, 2002; Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005-4010, 2006; U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056; 6,821,505; 6,277,375; 7,335,742; and 7,317,091.

[0085] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. Suitable native-sequence Fc regions for use in the antibodies of the present disclosure include human IgGl, IgG2, IgG3 and IgG4. A single amino acid substitution (S228P according to Kabat numbering; designatedIgG4Pro) may be introduced to abolish the heterogeneity observed in recombinant IgG4 antibody. See Angal, S. et al. (1993) Mol Immunol 30, 105-108.

[0086] ‘ ‘Non-fucosylated” or “fucose-deficient” antibody refers to a glycosylation antibody variant comprising an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, an antibody with reduced fucose or lacking fucose has improved ADCC function. Non-fucosylated or fucose-deficient antibodies have reduced fucose relative to the amount of fucose on the same antibody produced in a cell line. In some embodiments, a non-fucosylated or fucose-deficient antibody composition contemplated herein is a composition wherein less than about 50% of the N-linked glycans attached to the Fc region of the antibodies in the composition comprise fucose.

[0087] The terms "fucosylation" or “fucosylated” refers to the presence of fucose residues within the oligosaccharides attached to the peptide backbone of an antibody. Specifically, a fucosylated antibody comprises a (l,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or both of the N-linked oligosaccharides attached to the antibody Fc region, e.g. at position Asn 297 of the human IgGl Fc domain (EU numbering of Fc region residues). Asn297 may also be located about + 3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in immunoglobulins.

[0088] The "degree of fucosylation" is the percentage of fucosylated oligosaccharides relative to all oligosaccharides identified by methods known in the art e.g., in an N-glycosidase F treated antibody composition assessed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). In a composition of a "fully fucosylated antibody" essentially all oligosaccharides comprise fucose residues, i.e. are fucosylated. In some embodiments, a composition of a fully fucosylated antibody has a degree of fucosylation of at least about 90%. Accordingly, an individual antibody in such a composition typically comprises fucose residues in each of the two N-linked oligosaccharides in the Fc region. Conversely, in a composition of a "fully non-fucosylated" antibody essentially none of the oligosaccharides are fucosylated, and an individual antibody in such a composition does not contain fucose residues in either of the two N-linked oligosaccharides in the Fc region. In some embodiments, a composition of a fully non- fucosylated antibody has a degree of fucosylation of less than about 10%. In a composition of a "partially fucosylated antibody" only part of the oligosaccharides comprise fucose. An individualantibody in such a composition can comprise fucose residues in none, one or both of the N- linked oligosaccharides in the Fc region, provided that the composition does not comprise essentially all individual antibodies that lack fucose residues in the N-linked oligosaccharides in the Fc region, nor essentially all individual antibodies that contain fucose residues in both of the N- linked oligosaccharides in the Fc region. In one embodiment, a composition of a partially fucosylated antibody has a degree of fucosylation of about 10% to about 80% (e.g., about 50% to about 80%, about 60% to about 80%, or about 70% to about 80%).

[0089] “Binding affinity” as used herein refers to the strength of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In some embodiments, the binding affinity of an antibody for a Siglec-6 polypeptide or sub-domain thereof (e.g., the ECD, Domain 1, Domain 2, or Domain 3, e.g., as described herein) can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0090] “Binding avidity” as used herein refers to the binding strength of multiple binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).

[0091] An “isolated” nucleic acid molecule encoding the antibodies herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. In some embodiments, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies herein existing naturally in cells.

[0092] The term “pharmaceutical formulation” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to an individual to which the formulation would be administered. Such formulations are sterile.

[0093] “ Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0094] As used herein, the term “treatment” or “treating” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. An individual is successfully “treated”, for example, if one or more symptoms associated with a disease (e.g., viral infection) are mitigated or eliminated. For example, an individual is successfully “treated” if treatment results in increasing the quality of life of those suffering from a disease, decreasing the dose of other medications required for treating the disease, reducing the frequency of recurrence of the disease, lessening severity of the disease, delaying the development or progression of the disease, and / or prolonging survival of individuals.

[0095] As used herein, “in conjunction with” or “in combination with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” or “in combination with” refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0096] As used herein, the term “prevention” or “preventing” includes providing prophylaxis with respect to occurrence or recurrence of a disease in an individual. An individual may be predisposed to a disease, susceptible to a disease, or at risk of developing a disease, but has not yet been diagnosed with the disease.

[0097] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired or indicated effect, including a therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and theability of the antibody to elicit a desired response in the individual. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at the dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in individuals prior to or at the earlier stage of disease, the prophylactically effective amount can be less than the therapeutically effective amount.

[0098] “ Chronic” administration refers to administration of the medicament(s) in a continuous as opposed to acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0099] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0100] As used herein, an “individual” or a “subject” is a mammal. A “mammal” for purposes of treatment includes humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is a human.II. Compositions

[0101] In some aspects, also provided herein are compositions (e.g., pharmaceutical compositions, such as liquid formulations) comprising any of the anti-Siglec-6 antibodies described herein (e.g., an antibody that binds to Siglec-6). Exemplary anti-Siglec-6 antibodies contemplated for use are provided infra. In some embodiments, the composition is for subcutaneous administration. In some embodiments, provided herein is a liquid formulation comprising: (a) a monoclonal antibody that binds to a human Siglec-6, wherein the antibody is in a concentration of about 50 mg / mL to about 200 mg / mL; and (b) histidine in a concentration of about 5 mM to about 100 mM; wherein the pH of the liquid formulation is between 5.7 and 6.5.

[0102] In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable (VH) region comprising: an HVR-H1 comprising the amino acid sequence of SEQ ID liNO:168; an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 169; an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 7; and a light chain comprising a light chain variable (VL) region comprising: an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8; an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 161, and the VL region comprises the amino acid sequence of SEQ ID NO: 162.

[0103] In some embodiments, the heavy chain further comprises a human IgGFc region, e.g., a human IgGl Fc region. In some embodiments, the antibody comprises a Fc region and N- glycoside-linked carbohydrate chains linked to the Fc region, wherein less than about 50% of the N-gly coside-linked carbohydrate chains contain a fucose residue. In some embodiments, at least one or two of the heavy chains of the antibody is non-fucosylated. In some embodiments, substantially none of the N-gly coside-linked carbohydrate chains contain a fucose residue.

[0104] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 165 or 166, and the light chain comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, one or both of the antibody Fc regions or heavy chains do not have a C- terminal lysine. As is known in the art, the C-terminal lysine of some antibody heavy chain species may be cleaved off in some fraction of molecules. In some embodiments, a formulation of the present disclosure comprises an anti-Siglec-6 antibody that comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 165 or 166 and a light chain that comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, a formulation of the present disclosure comprises an anti-Siglec-6 antibody that comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 165 and a light chain that comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, a formulation of the present disclosure comprises an anti-Siglec-6 antibody that comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 166 and a light chain that comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, a formulation of the present disclosure comprises a mixture of anti-Siglec-6 antibody species, wherein each species comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 165 or 166 and a light chain that comprises the amino acid sequence of SEQ ID NO: 167.

[0105] Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wikiins, Pub., Gennaro Ed., Philadelphia, Pa. 2000). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA and / or non-ionic surfactants. Exemplary formulations are described herein. Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall.

[0106] The instant application demonstrates formulations suitable for an anti-Siglec-8 antibody at high concentrations sufficient for subcutaneous administration without instability or aggregation. In some embodiments, the antibody is in a concentration of about 50 mg / mL to about 200 mg / mL, about 60 mg / mL to about 200 mg / mL, about 70 mg / mL to about 200 mg / mL, about 80 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 125 mg / mL to about 200 mg / mL, about 135 mg / mL to about 200 mg / mL, about 135 mg / mL to about 175 mg / mL, about 135 mg / mL to about 165 mg / mL, about 50 mg / mL to about 150 mg / mL, about 60 mg / mL to about 150 mg / mL, about 70 mg / mL to about 150 mg / mL, about 80 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, about 125 mg / mL to about 150 mg / mL, or about 135 mg / mL to about 150 mg / mL. In some embodiments, the antibody concentration is any concentration within a range having an upper limit (in mg / mL) of about any of: 200, 190, 180, 170, 165, 160, 150, 140, 135, 130, 120, 110, or 100; and an independently selected lower limit (in mg / mL) of about any of: 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 140, or 150; wherein the upper limit is greater than the lower limit. In some embodiments, the antibody is in a concentration of about any one of 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 165 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, or 200 mg / mL. In some embodiments, the antibody is in a concentration of 150 mg / mL.

[0107] Buffers can be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. In some embodiments, the formulationcomprises histidine and / or a salt thereof, e.g., L-histidine and / or L-histidine hydrochloride. In some embodiments, the formulation comprises histidine (e.g., L-histidine and / or a salt thereof, including L-histidine and / or L-histidine hydrochloride) in a concentration of about 5 mM to about 100 mM, about 10 mM to about 100 mM, about 15 mM to about 100 mM, about 5 mM to about 95 mM, about 5 mM to about 90 mM, about 10 mM to about 90 mM, about 10 mM to about 80 mM, about 10 mM to about 75 mM, about 10 mM to about 50 mM, about 10 mM to about 60 mM, about 15 mM to about 75 mM, about 15 mM to about 65 mM, about 15 mM to about 60 mM, about 15 mM to about 50 mM, about 15 mM to about 40 mM, about 15 mM to about 30 mM, about 10 mM to about 40 mM, about 10 mM to about 30 mM, or about 15 mM to about 25 mM. In some embodiments, the formulation comprises histidine (e.g., L-histidine and / or a salt thereof, including L-histidine and / or L-histidine hydrochloride) in a concentration within a range having an upper limit (in mM) of about any of: 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, or 10; and an independently selected lower limit (in mM) of about any of: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90; wherein the upper limit is greater than the lower limit. In some embodiments, the formulation comprises histidine (e.g., L-histidine and / or a salt thereof, including L-histidine and / or L-histidine hydrochloride) in a concentration within a range having an upper limit (in mM) of about any of: 50, 45, 40, 35, 30, or 25; and an independently selected lower limit (in mM) of about any of: 5, 10, 15, or 20; wherein the upper limit is greater than the lower limit. In some embodiments, the formulation comprises histidine (e.g., L-histidine and / or a salt thereof, including L-histidine and / or L-histidine hydrochloride) in a concentration of any one of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0108] In some embodiments, the formulation comprises L-histidine and L-histidine hydrochloride (e.g., L-histidine hydrochloride monohydrate) at a total concentration of about 5 mM to about 100 mM. n some embodiments, the formulation comprises L-histidine and L- histidine hydrochloride (e.g., L-histidine hydrochloride monohydrate) at a total concentration of 20 mM. For example, in some embodiments, the formulation comprises 9 mM L-histidine and 11 mM L-histidine hydrochloride monohydrate, 5 mM L-histidine and 15 mM L-histidine hydrochloride monohydrate, 10 mM L-histidine and 10 mM L-histidine hydrochloride monohydrate, or 15 mM L-histidine and 5 mM L-histidine hydrochloride monohydrate.

[0109] In some embodiments, the formulation comprises a disaccharide. Disaccharides used in biologic formulations can include, without limitation, trehalose (e.g., trehalose dihydrate), sucrose, lactose, and maltose. In some embodiments, the formulation comprises a disaccharide in a concentration of about 90 mM to about 250 mM, about 100 mM to about 250 mM, about 150 mM to about 250 mM, about 90 mM to about 200 mM, about 90 mM to about 150 mM, about 100 mM to about 200 mM, or about 100 mM to about 150 mM. In some embodiments, the formulation comprises a disaccharide in a concentration of 100 mM to 248 mM, 120 mM to 248 mM, 140 mM to 248 mM, 160 mM to 248 mM, 100 mM to 225 mM, 120 mM to 225 mM, 140 mM to 225 mM, or 160 mM to 225 mM. In some embodiments, the formulation comprises a disaccharide in a concentration of 100 mM, 120 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 220 mM, 240 mM, 248 mM, or 250 mM.

[0110] In some embodiments, the formulation comprises sucrose. In some embodiments, the formulation comprises sucrose in a concentration of about 90 mM to about 250 mM, about 100 mM to about 250 mM, about 150 mM to about 250 mM, about 90 mM to about 200 mM, about 90 mM to about 150 mM, about 100 mM to about 200 mM, or about 100 mM to about 150 mM. In some embodiments, the formulation comprises sucrose in a concentration of 100 mM to 248 mM, 120 mM to 248 mM, 140 mM to 248 mM, 160 mM to 248 mM, 100 mM to 225 mM, 120 mM to 225 mM, 140 mM to 225 mM, or 160 mM to 225 mM. In some embodiments, the formulation comprises sucrose in a concentration of 100 mM, 120 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 220 mM, 240 mM, 248 mM, or 250 mM.[OHl] Non-ionic surfactants or detergents (also known as “wetting agents”) can be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active antibody. Non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0112] In some embodiments, the formulation further comprises polysorbate 80. In some embodiments, the formulation further comprises polysorbate 80 in a concentration (w / v) of about 0.025% to about 0.075% (w / v), about 0.035% to about 0.075% (w / v), about 0.045% to about 0.075% (w / v), about 0.025% to about 0.065% (w / v), about 0.025% to about 0.055% (w / v), about 0.035% to about 0.065% (w / v), or about 0.045% to about 0.055% (w / v). In some embodiments, the formulation further comprises polysorbate 80 in a concentration (w / v) of any one of 0.025%, 0.030%, 0.035%, 0.040%, 0.045%, 0.050%, 0.055%, 0.060%, 0.065%, 0.070%, or 0.075%.

[0113] In some embodiments, the formulation does not comprise sodium chloride. Without wishing to be bound to theory, it is thought that sodium chloride may be unsuitable as a tonicity modifier for the anti-Siglec-6 antibody at high concentrations.

[0114] In some embodiments, the formulation further comprises arginine, proline, or glutamate. In some embodiments, the formulation comprises arginine or a salt thereof (e.g., arginine hydrochloride) in a concentration of about 1 mM to about 100 mM or up to 100 mM. In some embodiments, the formulation comprises proline or a salt thereof (e.g., L-proline) in a concentration of about 1 mM to about 200 mM or up to 200 mM. In some embodiments, the formulation comprises glutamate or a salt thereof (e.g., L-glutamate) in a concentration of about 1 mM to about 100 mM or up to 100 mM.

[0115] In some embodiments, the pH of the formulation is between 5.7 and 6.5, between 5.8 and 6.5, between 5.7 and 6.3, between 5.7 and 6.2, between 5.8 and 6.2, or between 6.0 and 6.5. In some embodiments, the pH of the formulation is any one of 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

[0116] In some embodiments, the formulation has an osmolality of between about 260 mOsm / kg and about 435 mOsm / kg, about 260 mOsm / kg and about 400 mOsm / kg, about 260 mOsm / kg and about 375 mOsm / kg, about 260 mOsm / kg and about 350 mOsm / kg, about 260 mOsm / kg and about 325 mOsm / kg, about 260 mOsm / kg and about 300 mOsm / kg, about 275 mOsm / kg and about 435 mOsm / kg, about 275 mOsm / kg and about 400 mOsm / kg, about 275 mOsm / kg and about 375 mOsm / kg, about 275 mOsm / kg and about 350 mOsm / kg, about 275 mOsm / kg and about 325 mOsm / kg, or about 275 mOsm / kg and about 300 mOsm / kg. In some embodiments, the formulation has an osmolality of 260 mOsm / kg, 270 mOsm / kg, 280 mOsm / kg, 290 mOsm / kg, 300 mOsm / kg, 310 mOsm / kg, 320 mOsm / kg, 330 mOsm / kg, 340 mOsm / kg, 350mOsm / kg, 360 mOsm / kg, 370 mOsm / kg, 380 mOsm / kg, 390 mOsm / kg, 400 mOsm / kg, 410 mOsm / kg, 420 mOsm / kg, 430 mOsm / kg, or 435 mOsm / kg.

[0117] In some embodiments, the formulation comprises the anti-Siglec-6 antibody in a concentration of about 50 mg / mL to about 200 mg / mL, histidine and / or a salt thereof (e.g., L- histidine and L-histidine hydrochloride) in a concentration of about 5 mM to about 100 mM, sucrose in a concentration of about 90 mM to about 250 mM, and polysorbate 80 in a concentration of about 0.025% to about 0.075% (w / v), wherein the pH of the formulation is between 5.7 and 6.3.

[0118] In some embodiments, the formulation comprises the anti-Siglec-6 antibody in a concentration of about 135 mg / mL to about 165 mg / mL, histidine and / or a salt thereof (e.g., L- histidine and L-histidine hydrochloride) in a concentration of about 15 mM to about 25 mM, sucrose in a concentration of about 150 mM to about 250 mM, and polysorbate 80 in a concentration of about 0.035% to about 0.065% (w / v), wherein the pH of the formulation is between 5.8 and 6.2.

[0119] In some embodiments, the formulation comprises the anti-Siglec-6 antibody in a concentration of 150 mg / mL, histidine and / or a salt thereof (e.g., L-histidine and L-histidine hydrochloride) in a concentration of 20 mM, sucrose in a concentration of 170 mM, and polysorbate 80 in a concentration of 0.050% (w / v), wherein the pH of the formulation is 6.0.

[0120] In some embodiments, the formulation comprises the anti-Siglec-6 antibody in a concentration of 150 mg / mL, histidine and / or a salt thereof (e.g., L-histidine and L-histidine hydrochloride) in a concentration of 20 mM, sucrose in a concentration of 160 mM, and polysorbate 80 in a concentration of 0.050% (w / v), wherein the pH of the formulation is 6.0.

[0121] In some embodiments, the formulation comprises the anti-Siglec-6 antibody in a concentration of 150 mg / mL, histidine and / or a salt thereof (e.g., L-histidine and L-histidine hydrochloride) in a concentration of 20 mM, sucrose in a concentration of 248 mM, and polysorbate 80 in a concentration of 0.050% (w / v), wherein the pH of the formulation is 6.0.

[0122] In some embodiments, the formulation comprises the anti-Siglec-6 antibody in a concentration of 150 mg / mL, histidine and / or a salt thereof (e.g., L-histidine and L-histidine hydrochloride) in a concentration of 20 mM, sucrose in a concentration of 160 mM, and polysorbate 80 in a concentration of 0.050% (w / v), wherein the pH of the formulation is 5.8.

[0123] In some embodiments, the formulation comprises the anti-Siglec-6 antibody in a concentration of 150 mg / mL, histidine and / or a salt thereof (e.g., L-histidine and L-histidine hydrochloride) in a concentration of 20 mM, sucrose in a concentration of 160 mM, and polysorbate 80 in a concentration of 0.050% (w / v), wherein the pH of the formulation is 6.5.

[0124] In order for the formulations to be used for in vivo administration, they must be sterile. The formulation may be rendered sterile by filtration through sterile filtration membranes. The therapeutic compositions herein generally are placed into a container having a sterile access port, for example, a vial having a stopper pierceable by a hypodermic injection needle, a pre-filled syringe, or auto injector.

[0125] The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., by subcutaneous injection.

[0126] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active compounds are suitably present in combination in amounts that are effective for the purpose intended.

[0127] Antibodies described herein that bind to human Siglec-6 can be used either alone or in combination with other agents in the methods described herein. Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the present disclosure can occur prior to, simultaneously, and / or following, administration of the one or more additional therapeutic agents. In some embodiments, administration of an anti-Siglec-6 antibody described herein and administration of one or more additional therapeutic agents occur within about one month, about two months, about three months, about four months, about five months or about six months of each other. In some embodiments, administration of an anti-Siglec-6 antibody described herein and administration of one or more additional therapeutic agents occur within about one week, about two weeks or about three weeks of each other. In some embodiments, administration of an anti- Siglec-6 antibody described herein and administration of one or more additional therapeutic agents occur within about one day, about two days, about three days, about four days, about five days, or about six days of each other.

[0128] The formulations of the present disclosure can be administered to treat or prevent a variety of indications, e.g., for treatment of a disease or condition characterized by increased activity and / or number of mast cells (e.g., mast cells expressing Siglec-6). Exemplary diseases or conditions characterized by increased activity and / or number of mast cells (e.g., mast cells expressing Siglec-6), i.e., mast cell-mediated disorders or conditions, are described infra. In some embodiments, the individual to be treated with a formulation of the present disclosure has or has been diagnosed with a mast cell-mediated disorder or condition. In some embodiments, the subject is at risk of developing the mast cell-mediated disorder or condition. Exemplary mast cell-mediated disorders and conditions are known in the art and described herein. For example, mast cell-mediated disorders or conditions can include, but are not limited to, mastocytosis (e.g., indolent systemic mastocytosis, ISM; or aggressive systemic mastocytosis, ASM), mast cell leukemia, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, bone fracture, Alzheimer’s disease, chronic neuropathic pain, hyperalgesia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), graft vs. host disease (GVH), colitis (e.g., microscopic colitis or ulcerative colitis), hereditary alpha tryptasemia, neurofibroma, Kounis syndrome, urticaria (e.g., chronic spontaneous urticaria or an inducible urticaria), atopic dermatitis, contact dermatitis, angioedema, prurigo nodularis, cholangitis, psoriasis, irritable bowel syndrome (IBS), functional dyspepsia, asthma (e.g., eosinophilic or non-eosinophilic asthma), allergy (e.g., food allergy or pseudo allergy), keloid, chronic rhino sinusitis (e.g., with or without nasal polyps), aspirin exacerbated respiratory disease (AERD), chronic obstructive pulmonary disease (COPD), bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitital cystitis, hidradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn’s disease, endometriosis, interstitial cystitis, headache, migraine, rheumatoid arthritis, gastroesophageal reflux disease, viral infection, achalasia, postural tachycardia syndrome, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), complex regional pain syndrome, or Ehlers-Danlos syndrome. In some embodiments, the subject has or has been diagnosed with mastocytosis (e.g., indolent systemic mastocytosis, ISM; or aggressive systemic mastocytosis, ASM), mast cell leukemia, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, bone fracture, Alzheimer’s disease, chronic neuropathic pain, hyperalgesia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), graft vs. host disease (GVH), colitis(e.g., microscopic colitis or ulcerative colitis), hereditary alpha tryptasemia, neurofibroma, Kounis syndrome, urticaria (e.g., chronic spontaneous urticaria or an inducible urticaria), atopic dermatitis, contact dermatitis, angioedema, prurigo nodularis, cholangitis, psoriasis, irritable bowel syndrome (IBS), functional dyspepsia, asthma (e.g., eosinophilic or non-eosinophilic asthma), allergy (e.g., food allergy or pseudo allergy), keloid, chronic rhinosinusitis (e.g., with or without nasal polyps), aspirin exacerbated respiratory disease (AERD), chronic obstructive pulmonary disease (COPD), bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitital cystitis, hidradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn’s disease, endometriosis, interstitial cystitis, headache, migraine, rheumatoid arthritis, gastroesophageal reflux disease, viral infection, achalasia, postural tachycardia syndrome, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), complex regional pain syndrome, or Ehlers-Danlos syndrome.

[0129] In some embodiments according to any of the embodiments described herein, the administration results in a depletion or reduction of mast cells, e.g., comparing the mast cell population number in a sample (e.g., a tissue sample or a biological fluid sample) from a subject after treatment with the antibody to the mast cell population number in a sample from a subject before treatment with the antibody. In some embodiments, the depletion or reduction of mast cells is measured by comparing the mast cell population number in a sample (e.g., a tissue sample or a biological fluid sample) from a subject after treatment with the antibody to a reference number or sample, e.g., the mast cell population number in a sample from another subject without the antibody treatment, or average mast cell population number in samples from subjects without the antibody treatment. In some embodiments, depletion of reduction of mast cells is due to ADCC and / or ADCP.

[0130] In some embodiments according to any of the embodiments described herein, the administration results in a decreased level of an inflammatory mediator or mast cell product in a sample obtained from the subject, e.g., as compared to a level of the inflammatory mediator or mast cell product in a reference number or sample. For example, level of an inflammatory mediator or mast cell product in a sample from the individual obtained after treatment with the antibody or composition can be compared to: a level of the inflammatory mediator or mast cell product in a sample obtained from the subject prior to treatment with the antibody or composition, a level of the inflammatory mediator or mast cell product in a sample obtainedfrom a subject not treated with the antibody or composition, average level of the inflammatory mediator or mast cell product in samples obtained from subject(s) not treated with the antibody or composition, or a reference or normal lab value for level of the inflammatory mediator or mast cell product in a corresponding type of sample. Exemplary inflammatory mediators include, but are not limited to, proteases (e.g., pan-tryptase, active or beta-tryptase, chymase, CPA3, heparin, etc.), leukotrienes (e.g., leukotriene C4 or B4, platelet activating factor, prostaglandin D2 or E2, etc.), amines (e.g., histamine, serotonin, dopamine, polyamines, etc.), growth factors (e.g., SCF, GM-CSFG-CSF, FGF, EGF, NGF, VEGF, PDGF, etc ), cytokines (e.g, TNF, IL-lb, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, IL-18, IL-31, IL-36, etc.), chemokines (e.g., CCL2, CCL3, CCL4, CCL5, CCL11, CCL12, CCL13, CCL24, CCL26 ,CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, etc.), exosomes, and extracellular traps. In some embodiments, the inflammatory mediator or mast cell product is one or more of: active tryptase, CCL2, IL-13, IL-8, CCL4, IL-6, histamine, chymase, CPA3, a prostaglandin (e.g., prostaglandin D2 or E2), a leukotriene (e.g., leukotriene C4 or B4), or a mast cell marker (e.g., CD63, CD107a, CD203c, IgE, or MRGPRX2). In some embodiments, the inflammatory mediator or mast cell product is a substance secreted by mast cells, and the sample is a solid sample (e.g., a biopsy sample), or a liquid sample (e.g., a serum, plasma, or urine sample). In some embodiments, the inflammatory mediator or mast cell product is a substance or marker expressed but not secreted by mast cells, and the sample is a solid sample (e.g., a biopsy sample).

[0131] In some embodiments according to any of the embodiments described herein, the administration results in a decreased level of a mast cell marker in a sample (e.g., a biopsy sample, such as a biopsy sample comprising one or more mast cells) obtained from the subject, e.g., as compared to a level of the mast cell marker in a reference number or sample. In some embodiments, level of a mast cell marker in a sample from the individual obtained after treatment with the antibody or composition can be compared to: a level of the mast cell marker in a sample obtained from the subject prior to treatment with the antibody or composition, a level of the mast cell marker in a sample obtained from a subject not treated with the antibody or composition, average level of the mast cell marker in samples obtained from subject(s) not treated with the antibody or composition, or a reference or normal lab value for level of the mast cell marker in a corresponding type of sample. Exemplary mast cell markers are known in the art and include without limitation CD63, CD107a, CD203c, IgE, and MRGPRX2.

[0132] In some embodiments according to any of the embodiments described herein, the administration results in a decrease in one or more symptoms in the individual. For example, a level, amount, or presence of one or more symptoms in the individual after treatment can be compared to a level, amount, or presence of one or more symptoms in the individual at a baseline, e.g., prior to treatment. In some embodiments, the one or more symptoms can include without limitation nausea, cramping, constipation, abdominal pain, bloating, vomiting, diarrhea, fatigue, eye pain, light sensitivity, redness, discharge, runny nose, headache, dizziness, brain fog, itching, flushing, sweating, hives, hypotension, shortness of breath, bone pain, joint pain, weight loss, osteoporosis, angioedema, chest pain, anxiety, depression, rapid heartbeat, bronchoconstriction, and general pain.Antibodies

[0133] In some embodiments, the anti-Siglec-6 antibody is a humanized antibody that binds to Domain 1 of an extracellular domain of human Siglec-6, e.g., comprising the amino acid sequence QERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEEV QEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRVM ALTHR (SEQ ID NO:2).

[0134] In some embodiments, the anti-Siglec-6 antibody binds to Domain 2 of an extracellular domain of human Siglec-6, e.g., comprising the amino acid sequence PNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTITPRPQDH STNLTCQVTFPGAGVTMERTIQLNVSYA (SEQ ID NO:3). In some embodiments, the antibody is a humanized or human antibody.

[0135] In some embodiments, the anti-Siglec-6 antibody binds to Domain 3 of an extracellular domain of human Siglec-6, e.g., comprising the amino acid sequence PQKVAISIFQGNSAAFKILQNTSSLPVLEGQALRLLCDADGNPPAHLSWFQGFPALNATPI SNTGVLELPQVGSAEEGDFTCRAQHPLGSLQISLSLFVHWKPEGRAGGV (SEQ ID NO:4). In some embodiments, the antibody is a humanized or human antibody.

[0136] In some embodiments, the anti-Siglec-6 antibody comprises 1, 2, 3, 4, 5, or all 6 HVR sequences of a single anti-Siglec-6 antibody as set forth in Table 2. In some embodiments, the anti-Siglec-6 antibody comprises a VH region comprising 1, 2, or all 3 HVR sequences of a VHregion of a single anti-Siglec-6 antibody as set forth in Table 2. In some embodiments, the anti- Siglec-6 antibody comprises a VL region comprising 1, 2, or all 3 HVR sequences of a VL region of a single anti-Siglec-6 antibody as set forth in Table 2.Table 2. Anti-Siglec-6 antibody HVR sequences (HVRs shown according to Kabat definition unless otherwise specified).

[0137] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 89, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:90, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:91; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:92, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:93, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 94.

[0138] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 5, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:7; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, anHVR-L2 comprising the amino acid sequence of SEQ ID NO:9, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

[0139] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 83, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:84, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:85; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:86, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:87, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 88.

[0140] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11 , an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

[0141] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 77, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:78, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:80, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:81, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 82.

[0142] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:20, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:21, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:22.

[0143] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:71, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:72, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:73; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:75, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:76.

[0144] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 53, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:55; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:56, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:57, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 58.

[0145] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:31 ; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:32, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:33, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0146] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:23, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:24, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:25; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:26, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:27, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:28.

[0147] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:35, an HVR-H2 comprising the amino acid sequence ofSEQ ID NO:36, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:37; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:40.

[0148] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:41, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:42, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:43; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:44, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:45, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:46.

[0149] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:47, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:48, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:49; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:50, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:51, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 52.

[0150] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 59, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:60, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:61 ; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:62, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:63, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 64.

[0151] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:65, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:66, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:67; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ IDNO:68, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:69, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:70.

[0152] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:135, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:136, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:137; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 138, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 139, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 140.

[0153] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:141, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 142, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 143; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 144, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 145, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 146.

[0154] In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region; wherein the VH region comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 147, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 149; and wherein the VL region comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 150, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 152.

[0155] In some embodiments, an anti-Siglec-6 antibody provided herein competes for binding to human Siglec-6 (e.g., an ECD or sub-domain thereof of a human Siglec-6 protein) with a reference antibody, e.g., an anti-Siglec-6 antibody of the present disclosure. In some embodiments, an anti-Siglec-6 antibody provided herein competes for binding to human Siglec-6 (e.g., an ECD or sub-domain thereof of a human Siglec-6 protein) with one or more of the following anti-Siglec-6 antibodies described herein: AK04, AK05, AK02, AK14, AK11, AK15, AK13, AK12, AKIO, AK09, AK08, AK07, AK06, AK03, AK01, AK16, AK17, and AK18. In some embodiments, an anti-Siglec-6 antibody provided herein competes for binding to Domain 1of human Siglec-6 with one or more of the following anti-Siglec-6 antibodies described herein: AK04, AK05, AK02, AK07, AK06, AK03, and AK01. In some embodiments, an anti-Siglec-6 antibody provided herein competes for binding to Domain 2 of human Siglec-6 with one or more of the following anti-Siglec-6 antibodies described herein: AKIO and AK11. In some embodiments, an anti-Siglec-6 antibody provided herein competes for binding to Domain 3 of human Siglec-6 with one or more of the following anti-Siglec-6 antibodies described herein: AK09, AK08, AK12, AK13, AK14, and AK15. In some embodiments, the antibody competes for binding to human Siglec-6 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:5, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:6, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:7 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:8, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:9, and an HVR- L3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody competes for binding to human Siglec-6 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:11, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody competes for binding to human Siglec-6 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:20, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:21, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody competes for binding to human Siglec-6 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:29, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:30, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:31 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:32, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:33, and an HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 34. In some embodiments, the antibody competes for binding to human Siglec-6 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO:23, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:24, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:25 and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:26, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:27, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, the antibody competes for binding to human Siglec-6 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 135, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 137, and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 138, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 139, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, the antibody competes for binding to human Siglec-6 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 141, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 142, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 143, and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 144, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 145, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 146. In some embodiments, the antibody competes for binding to human Siglec-6 with a reference antibody that comprises a VH region comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 147, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 148, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 149, and a VL region comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 150, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 152.

[0156] In some embodiments, an anti-Siglec-6 antibody described herein binds to an extracellular domain (ECD) of a human Siglec-6 protein. In some embodiments, the Siglec-6 ECD comprises the amino acid sequence QERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEEV QEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRVMALTHRPNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTITP RPQDHSTNLTCQVTFPGAGVTMERTIQLNVSYAPQKVAISIFQGNSAAFKILQNTSSLPV LEGQALRLLCDADGNPPAHLSWFQGFPALNATPISNTGVLELPQVGSAEEGDFTCRAQH PLGSLQISLSLFVHWKPEGRAGGV (SEQ ID NO:1).

[0157] In some embodiments, an anti-Siglec-6 antibody described herein binds to Domain 1, Domain 2, or Domain 3 of a human Siglec-6 protein (e.g., an ECD of a human Siglec-6 protein). In some embodiments, an anti-Siglec-6 antibody described herein binds to Domain 1 of a human Siglec-6 protein (e.g., an ECD of a human Siglec-6 protein). In some embodiments, Domain 1 comprises the amino acid sequence QERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEEV QEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRVM ALTHR (SEQ ID NO:2). In some embodiments, an anti-Siglec-6 antibody described herein binds to Domain 2 of a human Siglec-6 protein (e.g., an ECD of a human Siglec-6 protein). In some embodiments, Domain 2 comprises the amino acid sequence PNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTITPRPQDH STNLTCQVTFPGAGVTMERTIQLNVSYA (SEQ ID NO:3). In some embodiments, an anti- Siglec-6 antibody described herein binds to Domain 3 of a human Siglec-6 protein (e.g., an ECD of a human Siglec-6 protein). In some embodiments, Domain 3 comprises the amino acid sequencePQKVAISIFQGNSAAFKILQNTSSLPVLEGQALRLLCDADGNPPAHLSWFQGFPALNATPI SNTGVLELPQVGSAEEGDFTCRAQHPLGSLQISLSLFVHWKPEGRAGGV (SEQ ID NO:4).

[0158] In some embodiments, an anti-Siglec-6 antibody provided herein binds the same epitope on human Siglec-6 (e.g., an ECD or sub-domain thereof of a human Siglec-6 protein) as an anti-Siglec-6 antibody of the present disclosure. In some embodiments, an anti-Siglec-6 antibody provided herein binds the same epitope on human Siglec-6 Domain 1, 2, or 3 as an anti- Siglec-6 antibody of the present disclosure. In some embodiments, an anti-Siglec-6 antibody provided herein binds the same epitope as AK05. In some embodiments, an anti-Siglec-6 antibody provided herein binds the following amino acids on Siglec-6: 29, 30, 34, 38, 63, 64, 68, 74, 76, 99, 100, 103, 104, 106, and 114 (numbering according to the Siglec-6 ECD as shown in SEQ ID NO: 1). In some embodiments, an anti-Siglec-6 antibody provided herein binds the same epitope as AK04. In some embodiments, an anti-Siglec-6 antibody provided herein binds thefollowing amino acids on Siglec-6: 26, 29, 30, 52, 64, 74, 75, 79, 98, 100, 104, 106, and 107 (numbering according to the Siglec-6 ECD as shown in SEQ ID NO:1). Exemplary assays for epitope mapping are known in the art and exemplified herein. For example, epitope mapping can be performed using cross-linking mass spectrometry (XL-MS), e.g. , as exemplified herein. Other assays include without limitation X-ray crystallography and alanine scanning mutagenesis.Table 5. Variable domain sequences.

[0159] Many definitions for CDR or HVR sequences of an antibody variable domain are known in the art and may be used to describe an antibody of the present disclosure, e.g., by CDR / HVR sequences. In some embodiments, antibody CDR / HVR sequences are defined as in Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institute of Health, Bethesda, MD (1991)). In some embodiments, antibody CDR / HVR sequences are defined as in Chothia (see, e.g., Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). For example, the humanized variable domain sequences of AK19, AK20, and AK21 listed in Table 5 above have their CDR sequences according to Chothiadefinition underlined. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises all 3 CDR sequences according to Chothia of AK19 as shown in Table 5 and a VL domain that comprises all 3 CDR sequences according to Chothia of AK19 as shown in Table 5. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises all 3 CDR sequences according to Chothia of AK20 as shown in Table 5 and a VL domain that comprises all 3 CDR sequences according to Chothia of AK20 as shown in Table 5. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises all 3 CDR sequences according to Chothia of AK21 as shown in Table 5 and a VL domain that comprises all 3 CDR sequences according to Chothia of AK21 as shown in Table 5.

[0160] Table 6 shows the CDR sequences of anti-Siglec-6 antibodies according to Kabat with the Chothia CDRs underlined. As shown in Table 6, the Kabat and Chothia definitions for CDRs CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are identical, whereas the Chothia CDR-H1 is amino acids 1-7 of the corresponding Kabat CDR, and the Chothia CDR-H2 is amino acids 3-7 or 3-8 of the corresponding Kabat CDR. Therefore, in some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:5, an HVR-H2 comprising amino acids 3-7 of SEQ ID NO:6, an HVR- H3 comprising the amino acid sequence of SEQ ID NO: 7; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:9, and an HVR-L3 comprising the amino acid sequence of SEQ ID NOTO. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO: 11, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO: 12, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO: 17, an HVR- H2 comprising amino acids 3-8 of SEQ ID NO: 18, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20, an HVR-L2 comprising the amino acid sequence of SEQ IDN0:21, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:23, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:24, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:25; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:26, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:27, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:28. In some embodiments, an anti- Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:29, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:30, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:31 ; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:32, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:33, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:34. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:35, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:36, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:37; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:40. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:41, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:42, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:43; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:44, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:45, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:46. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO: 135, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:136, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 138, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 139, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 140. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises aVH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:47, an HVR- H2 comprising amino acids 3-8 of SEQ ID NO:48, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:49; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 50, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:51, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:53, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO: 54, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:55; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:56, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:57, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, an anti- Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:59, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:60, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:61 ; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:62, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:63, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:64. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:65, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:66, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:67; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:70. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:141, an HVR-H2 comprising ammo acids 3-8 of SEQ ID NO:142, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 143; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 144, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 145, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 146. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:71, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:72, an HVR-H3 comprising theamino acid sequence of SEQ ID NO:73; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:74, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO: 77, an HVR- H2 comprising amino acids 3-8 of SEQ ID NO:78, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:79; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 80, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:81, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 82. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO: 147, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO: 148, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 149; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 150, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 151, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 152. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR- H1 comprising amino acids 1-7 of SEQ ID NO: 83, an HVR-H2 comprising amino acids 3-8 of SEQ ID NO:84, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:85; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO:86, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 87, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:88. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises an HVR-H1 comprising amino acids 1-7 of SEQ ID NO:89, an HVR-H2 comprising amino acids 3-7 of SEQ ID NO:90, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:91; and a VL domain that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 92, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 93, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:94. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain that comprises all three CDR sequences of a single anti- Siglec-6 antibody according to Chothia as shown in Table 6 and a VL domain that comprises all three CDR sequences of the same anti-Siglec-6 antibody according to Chothia as shown in Table 6.Table 6. CDRs as defined by Kabat, with Chothia CDRs underlined.

[0161] In some embodiments, antibody CDR / HVR sequences are defined as in IMGT (see, e.g., Lefranc, M.P. (1999) The Immunologist 7:132-136). In some embodiments, CDR / HVR sequences of a single antibody are defined as by mixing two or more definitions, e.g., Kabat, Chothia, and / or IMGT.

[0162] In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 105 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 106. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK15 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK15 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 107 and / or a VLdomain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 108. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK14 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK14 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 109 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 110. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK13 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK13 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 111 and / or a VL domain comprising 1 , 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:112. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK12 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK12 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 113 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 114. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK11 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK11 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 115 and / or a VL domain comprising 1 , 2, orall 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 116. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AKIO as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AKIO as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 117 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 118. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK09 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK09 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 119 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 120. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK08 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK08 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 121 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 122. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK07 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK07 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 123 and / or a VL domain comprising 1 , 2, or all 3 CDR or HVR sequences presentin the amino acid sequence of SEQ ID NO: 124. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK06 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK06 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 125 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 126. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK05 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK05 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 127 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 128. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK04 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK04 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 129 and / or a VL domain comprising 1 , 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO:130. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK03 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK03 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 131 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence ofSEQ ID NO: 132. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK02 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK02 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 133 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 134. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK01 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK01 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 153 and / or a VL domain comprising 1 , 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 154. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK16 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK16 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 155 and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 156. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK17 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK17 as described herein (see, e.g., Table 5). In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 157 and / or a VL domain comprising 1 , 2, or all 3 CDR or HVR sequences present in the amino acid sequence of SEQ ID NO: 158. In someembodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VH domain sequence of AK18 as described herein (see, e.g., Table 5) and / or a VL domain comprising 1, 2, or all 3 CDR or HVR sequences present in the VL domain sequence of AK18 as described herein (see, e.g., Table 5). In some embodiments according to any of the embodiments described herein, the antibody is a humanized antibody.

[0163] In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH region that comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 168, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 169, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 7; and / or a VL region that comprises an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:9, and an HVR-L3 comprising the amino acid sequence of SEQ ID NOTO. In some embodiments, the antibody is a humanized antibody.

[0164] In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH region that comprises the amino acid sequence of SEQ ID NO: 159; and a VL region that comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH region that comprises the amino acid sequence of SEQ ID NO: 161; and a VL region that comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a VH region that comprises the amino acid sequence of SEQ ID NO: 163 ; and a VL region that comprises the amino acid sequence of SEQ ID NO: 164.

[0165] In some embodiments, an anti-Siglec-6 antibody of the present disclosure binds to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 inhibits activation of the mast cell. Assays for assessing mast cell activation are known in the art and exemplified herein. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 inhibits CD63 expression, e.g., of a human mast cell. In some aspects, an anti-Siglec-6 antibody described herein inhibits one or more mast cell-mediated activities. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 inhibits expression and / or release (e.g., by a human mast cell) of one or more of the following: IL-6, IL-8, IL-13, CCL2, CCL4, histamine, chymase, and tryptase (e.g., activetryptase). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 inhibits expression and / or release (e.g., by a human mast cell) of one or more of the following inflammatory mediators: proteases (e.g., pan-tryptase, active or beta-tryptase, chymase, CPA3, heparin, etc.), leukotrienes (e.g., leukotriene C4 or B4, platelet activating factor, prostaglandin D2 or E2, etc.), amines (e.g., histamine, serotonin, dopamine, polyamines, etc.), growth factors (e.g, SCF, GM-CSFG-CSF, FGF, EGF, NGF, VEGF, PDGF, etc ), cytokines (e.g, TNF, IL-lb, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17, IL-18, IL-31, IL-36, etc ), chemokines (e.g, CCL2, CCL3, CCL4, CCL5, CCL11, CCL12, CCL13, CCL24, CCL26 ,CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, etc ), exosomes, and extracellular traps. For example, total and active tryptase as well as histamine, N-methyl histamine, and 11 -beta-prostaglandin F2 can be measured in blood or urine to assess the reduction in mast cells. See, e.g., U.S. Patent Application Publication No. US 20110293631 for an exemplary mast cell activity assay.

[0166] In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to reduced levels of Siglec-6 on the cell surface (i.e., of the human mast cell). Binding of the antibody can lead to reduced levels of Siglec-6 on the cell membrane, e.g., through Siglec-6 internalization, endocytosis, shedding, cleaving, etc. Assays for assessing levels of Siglec-6 surface expression are known in the art and exemplified herein. In some embodiments, Siglec-6 surface expression is measured by flow cytometry, e.g., using an anti-Siglec-6 antibody with a detectable (e.g., fluorescent) tag. For example, as demonstrated herein, mast cells can be contacted with an anti-Siglec-6 antibody, and surface expression can be measured by flow cytometry with a fluorescent-tagged anti-Siglec-6 antibody that binds to a different epitope on Siglec-6 than the test antibody. Reduced fluorescence in the presence of the test antibody can indicate a reduced level of Siglec-6 surface expression. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to reduced levels of Siglec-6 surface expression regardless of the presence / absence of an antibody Fc region, or regardless of the ability of the antibody Fc region to bind an Fc receptor (e.g., expressed on an effector cell). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to an expression level Siglec-6 on the cell membrane that is reduced by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, e.g., as compared to surface expression of Siglec-6 in the absence of an antibody, or in the absence of an antibody that does not bind to Siglec-6.

[0167] In other embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell does not lead to reduced levels of Siglec-6 on the cell membrane (i.e., of the human mast cell). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell does not lead to reduced levels of Siglec-6 on the cell membrane regardless of the presence / absence of an antibody Fc region, or regardless of the ability of the antibody Fc region to bind an Fc receptor (e.g., expressed on an effector cell).

[0168] In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to reduced levels of Siglec-6 on the cell membrane (i.e., of the human mast cell) in the presence of a cell (e.g., an effector cell) expressing an Fc receptor. In some embodiments, the antibody comprises an Fc region (e.g., an active Fc region). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec-6 dependent upon interaction between the antibody Fc region (e.g., an active Fc region) and an Fc receptor or an effector cell, e.g., that expresses an Fc receptor. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec- 6 only when the antibody is a full-length antibody comprising an Fc region (e.g., an active Fc region), e.g., in the presence of an Fc receptor or an effector cell (e.g., expressing an Fc receptor). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface- expressed Siglec-6 when the antibody is an antibody fragment, e.g., lacking an Fc region. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec- 6 when the antibody is a full-length antibody comprising an Fc region that does not bind an Fc receptor (e.g., an inactive or dead Fc region lacking effector function). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to a reduced level of surface-expressed Siglec-6 when theantibody is a full-length antibody comprising an Fc region (e.g., an active Fc region), e.g., in the presence of an Fc receptor or an effector cell (e.g., expressing an Fc receptor), but not when the antibody is an antibody fragment lacking an Fc region or comprising an Fc region that does not bind an Fc receptor (e.g., an inactive or dead Fc region). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell leads to an expression level Siglec-6 on the cell membrane that is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% when the antibody comprises an active Fc region, e.g., as compared to surface expression of Siglec-6 using an antibody that does not comprise an Fc region, or does not comprise an active Fc region.

[0169] In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces dimerization and / or internalization of Siglec-6. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces dimerization and internalization of Siglec-6. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces endocytosis of Siglec-6. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces shedding of Siglec-6 (e.g., the Siglec-6 ECD). In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces cleaving of Siglec-6 (e.g., the Siglec-6 ECD).

[0170] In some embodiments, an anti-Siglec-6 antibody described herein depletes mast cells expressing human Siglec-6 in vitro and / or in vivo, e.g., in the presence of effector cells. In some embodiments, binding of the antibody to the extracellular domain of human Siglec-6 when expressed on a surface of a human mast cell induces ADCC and / or ADCP activity, e.g., in the presence of effector cells in vitro and / or in vivo. In some other aspects, an anti-Siglec-6 antibody described herein kills mast cells expressing Siglec-6 by ADCC activity in vitro and / or in vivo. In some other aspects, an anti-Siglec-6 antibody described herein induces phagocytosis of mast cells expressing Siglec-6 by ADCP activity in vitro and / or in vivo. In some embodiments, a composition comprises non-fucosylated (i.e., afucosylated) anti-Siglec-6 antibodies. In someembodiments, a composition comprising non-fucosylated anti-Siglec-6 antibodies described herein enhances ADCC activity as compared to a composition comprising partially fucosylated anti-Siglec-6 antibodies.

[0171] Assays for assessing ADCC activity are well known in the art and described herein. In an exemplary assay, to measure ADCC activity, effector cells and target cells are used, e.g., at a specific ratio (e.g., 1 :50 target cells: effector cells) in the presence of an antibody to be evaluated. Examples of effector cells include natural killer (NK) cells, large granular lymphocytes (LGL), lymphokine-activated killer (LAK) cells and PBMC comprising NK and LGL, or leukocytes having Pc receptors on the cell surfaces, such as neutrophils, eosinophils and macrophages. The target cell is any cell which expresses on the cell surface antigens that antibodies to be evaluated can recognize. An example of such a target cell is a mast cell which expresses Siglec-6 on the cell surface. Target cells are labeled with a reagent that enables detection of cytolysis.Examples of reagents for labeling include a radio-active substance such as sodium chromate (Na251CrC>4) and carboxyfluorescein succinimidyl ester. See, e.g., Immunology, 14, 181 (1968); J. Immunol. Methods, 172, 227 (1994); and J. Immunol. Methods, 184, 29 (1995). The number of remaining target cells can then be assessed (e.g., by flow cytometry) after co-incubation and normalized (e.g., to number of remaining target cells co-incubated with effector cells treated with an antibody that does not bind to Siglec-6 or treated with no antibody).

[0172] Assays for assessing ADCP activity are well known in the art and described herein.For example, target cells can be co-cultured with macrophages (e.g., human monocyte-derived macrophages), monocytes, or PBMCs at a specific ratio (e.g., 1 :50 target cells: macrophages) in the presence of an antibody to be evaluated. The number of remaining target cells can then be assessed (e.g., by flow cytometry) after co-incubation and normalized (e.g., to number of remaining target cells co-incubated with macrophages treated with an antibody that does not bind to Siglec-6, or treated with no antibody).

[0173] In one aspect, an anti-Siglec-6 antibody described herein is a monoclonal antibody. In one aspect, an anti-Siglec-6 antibody described herein is an antibody fragment (including antigen-binding fragment), e.g., a Fab, Fab'-SH, Fv, scFv, or (Fab '^fragment. In one aspect, an anti-Siglec-6 antibody described herein is a chimeric, humanized, or human antibody. In one aspect, any of the anti-Siglec-6 antibodies described herein are purified.

[0174] An anti-Siglec-6 antibody described herein may comprise any suitable framework variable domain sequence, provided that the antibody retains the ability to bind human Siglec-6. As used herein, heavy chain framework regions are designated "HC-FR1-FR4," and light chain framework regions are designated "LC-FR1-FR4."

[0175] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated oc, 8, s, y and p, respectively. The y and oc classes are further divided into subclasses e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in some of the embodiments herein. Common allotypic variants in human populations are those designated by the letters a,f,n,z or combinations thereof.

[0176] In any of the embodiments herein, the antibody may comprise a heavy chain Fc region, e.g., a human Fc region or human IgG Fc region. In further embodiments, the human IgG Fc region comprises a human IgGl or IgG4 Fc region. In some embodiments, the human IgG4 Fc region comprises the amino acid substitution S228P, wherein the amino acid residues are numbered according to the EU index as in Kabat. In some embodiments, the human Fc region comprises one or more mutation(s) that reduce effector function.

[0177] In some embodiments, the human IgGl Fc region comprises one or more mutation(s) that reduce effector function. In some embodiments, the human IgGl Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) L234 and / or L235; (b) A327, A330, and / or P331; (c) E233, L234, L235, and / or G236; (d) E233, L234, and / or L235; (e) E233, L234, L235, G236, A327, A330, and / or P331; (f) E233, L234, L235, A327, A330, and / or P331; (g) N297; (h) L242, N297, and / or K334; (i) A287, N297, and / or L306; (j) R292, N297, and / or V302; (k) N297, V323, and / or 1332; (1) V259, N297, and / or L306; (m) L234, L235, K322, M252, S254, and / or T256; or (n) L234, L235, and / or P329. In some embodiments, the antibody comprises a human IgGl Fc region with one or more of the following mutation(s), numbering based on EU index: (a) L234A and / or L235A; (b) A327G, A330S, and / or P331S; (c) E233P, L234V, L235A, and / or G236del; (d) E233P, L234V, and / or L235A; (e) E233P, L234V, L235A, G236del, A327G, A330S, and / or P331S; (f) E233P, L234V, L235A, A327G, A33 OS, and / or P33 IS; (g) N297A; (h) N297G; (i) N297Q; (j) L242C, N297C, and / or K334C; (k) A287C, N297G, and / or L306C; (1) R292C, N297G, and / or V302C; (m)N297G, V323C, and / or 1332C; (n) V259C, N297G, and / or L306C; (o) L234F, L235Q, K322Q, M252Y, S254T, and / or T256E; (p) L234A, L235A, and / or P329G; or (q) L234A, L235Q, and K322Q. See, e.g., Schlothauer, T. et al. (2016) Protein Eng. Des. Sei. 29:457-466; Armour, K.L. et al. (2003) Mol. Immunol. 40:585-593; Jacobsen, F.W. et al. (2017) J. Biol. Chem. 292:1865- 1875; and Borrok, M.J. et al. (2017) J. Pharm. Sci. 106: 1008-1017. In some embodiments, the antibody heavy chain comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:101 or 102.

[0178] In some embodiments, the anti-Siglec-6 antibody comprises an Fc region that has (i.e., is capable of inducing) one or more effector functions (e.g., in the presence of appropriate effector cell(s)). In some embodiments, the Fc region is an “active” Fc region such as a human IgGl Fc region (e.g., a wild-type human IgGl Fc region). In some embodiments, the Fc region has one or more effector functions including but not limited to Cl q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (e.g., antibody-dependent cell-mediated phagocytosis or ADCP); down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation. In some embodiments, the antibody comprises an Fc region capable of inducing ADCC and / or ADCP activity, e.g., in the presence of one or more effector cells (such as those expressing an appropriate Fc receptor that binds the antibody Fc region).

[0179] In some embodiments, the human IgGl Fc region comprises one or more mutation(s) that increase or enhance effector function. In some embodiments, the human IgGl Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) F243, R292, Y300, V305, and / or P396; (b) S239 and / or 1332; (c) S239, 1332, and / or A330; (d) S298, E333, and / or K334; (e) G236, S239, and / or 1332; (f) K326 and / or E333; (g) S267, H268, and / or S324; or (h) E345, E430, and / or S440. In some embodiments, the human IgGl Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) F243L, R292P, Y300L, V305I, and / or P396L; (b) S239D and / or I332E; (c) S239D, I332E, and / or A330L; (d) S298A, E333A, and / or K334A; (e) G236A, S239D, and / or I332E; (f) K326W and / or E333S; (g) S267E, H268F, and / or S324T; or (h) E345R, E430G, and / or S440Y. See, e.g., Stavenhagen, J.B. et al. (2007) Cancer Res. 67:8882-8890; Lazar, GA. et al. (2006) Proc. Natl. Acad. Sci. USA 103:4005-4010; Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591- 6604; Richards, J.O. etal. (2008) Afo / . Cancer Ther. 7:2517-2527; Idusogie, E.E. et al. (2001) J.Immunol. 166:2571-2575; Moore, G.L. et al. (2010) MAbs 2:181-189; and Diebolder, C.A. et al. (2014) Science 343:1260-1263.

[0180] In some embodiments, the human IgG2 Fc region comprises one or more mutation(s) that reduce effector function. In some embodiments, the human IgG2 Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) A330 and / or P331; (b) V234, G237, P238, H268, V309, A330, and / or P331; or (c) V234, G237, H268, V309, A330, P331, C232, C233, S267, L328, M252, S254, and / or T256. In some embodiments, the human IgG2 Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) A330S and / or P33 IS; (b) V234A, G237A, P238S, H268A, V309L, A330S, and / or P33 IS; or (c) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, and / or T256E. See, e.g., Armour, K.L. et al. (2QQ3)Mol. Immunol. 40:585-593 and US PG Pub. Nos. 20170204193 and 20170240631.

[0181] In some embodiments, the human IgG4 Fc region comprises one or more mutation(s) that reduce effector function. In some embodiments, the human IgG4 Fc region comprises a substitution or deletion at one or more of the following position(s), numbering based on EU index: (a) E233, F234, L235, and / or G236; (b) E233, F234, and / or L235; or (c) S228 and / or L235. In some embodiments, the human IgG4 Fc region comprises one or more of the following mutation(s), numbering based on EU index: (a) E233P, F234V, L235A, and / or G236del; (b) E233P, F234V, and / or L235A; (c) S228P and / or L235E; or (d) S228P and / or L235A. See, e.g., Schlothauer, T. et al. (2016) Protein Eng. Des. Sei. 29:457-466; and Armour, K.L. et al. (2003) Mol. Immunol. 40:585-593. In some embodiments, the antibody heavy chain comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 103.

[0182] In some embodiments, the anti-Siglec-6 antibody of the present disclosure comprises an antibody heavy chain comprising a heavy chain constant region that comprises an amino acid sequence shown in Table 3.Table 3. Antibody heavy chain constant region sequences.

[0183] In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises an antibody light chain comprising a light chain constant (CL) domain, e.g., a human kappa or lambda CL domain. In some embodiments, the CL domain is a human kappa CL domain. In some embodiments, the CL domain comprises the amino acid sequence ofRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 104).

[0184] In some embodiments, an anti-Siglec-6 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence ofQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVSWIRQPPGKGLEWIGVIWHDGSTSYH PSLKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCASDGYSGTFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 165) orQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVSWIRQPPGKGLEWIGVIWHDGSTSYHPSLKSRVTISRDTSKNQVSLKLSSVTAADTAVYYCASDGYSGTFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 166) and / or a light chain comprising the amino acid sequence ofQIVVTQSPATLSL SPGERATL SCTA SS SVS SS YLHWYQQKPGQ APRLLIYST SILA SGIPAR FSGSGSGTDFTLTISSLQPEDFAVYYCHQYHRSPYTFGQGTKLEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 167). In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 165 or 166 and a light chain comprising the amino acid sequence of SEQ ID NO: 167.

[0185] In one aspect, the present disclosure provides anti-Siglec-6 antibodies with reduced or eliminated fucosylation, e.g., as described infra. For example, in some embodiments, at least one or two of the heavy chains of the antibody is non-fucosylated. In some embodiments, the Fc region is non-fucosylated. In some embodiments, the antibody comprises a non-fucosylated human IgGl Fc region. Exemplary assays for measuring antibody fucosylation, as well as methods and cell lines for producing antibodies with altered, reduced, or eliminated fucosylation, are provided herein.

[0186] In one aspect, polynucleotides encoding anti-Siglec-6 antibodies are provided. In certain embodiments, vectors comprising polynucleotides encoding anti-Siglec-6 antibodies are provided. In certain embodiments, host cells comprising such vectors are provided. In another aspect of the invention, compositions comprising anti-Siglec-6 antibodies or polynucleotides encoding anti-Siglec-6 antibodies are provided. In certain embodiments, a composition of the present disclosure is a pharmaceutical formulation for the treatment of a mast cell-mediated disorder, such as those enumerated herein.1. Antibody Affinity 0187] In some embodiments, the anti-Siglec-6 antibody binds to the ECD of human Siglec-6 with an equilibrium dissociation constant (KD) of about 250pM or less, about 225pM or less, about 200pM or less, about 175pM or less, about 150pM or less, about 125pM or less, about lOOpM or less, about 90pM or less, about 80pM or less, about 70pM or less, about 60pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 20pM or less, about lOpM or less, or about IpM. In some embodiments, the anti-Siglec-6 antibody binds to Domain 1 of the ECD of human Siglec-6 with an equilibrium dissociation constant (KD) of about 250pM or less, about 225pM or less, about 200pM or less, about 175pM or less, about 150pM or less, about 125pM or less, about lOOpM or less, about 90pM or less, about 80pM or less, about 70pM or less, about 60pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 20pM or less, about lOpM or less, or about IpM. In some embodiments, the anti-Siglec-6 antibody binds to Domain 2 of the ECD of human Siglec-6 with an equilibrium dissociation constant (KD) of about 250pM or less, about 225pM or less, about 200pM or less, about 175pM or less, about 150pM or less, about 125pM or less, about lOOpM or less, about 90pM or less, about 80pM or less, about 70pM or less, about 60pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 20pM or less, about lOpM or less, or about IpM. In some embodiments, the anti-Siglec-6 antibody binds to Domain 3 of the ECD of human Siglec-6 with an equilibrium dissociation constant (KD) of about 250pM or less, about 225pM or less, about 200pM or less, about 175pM or less, about 150pM or less, about 125pM or less, about lOOpM or less, about 90pM or less, about 80pM or less, about 70pM or less, about 60pM or less, about 50pM or less, about 40pM or less, about 30pM or less, about 20pM or less, about lOpM or less, or about IpM.

[0188] Exemplary assays for determining binding affinity of an antibody for human Siglec-6, its ECD, or a sub-domain thereof are known in the art and exemplified herein. In one embodiment, the binding affinity of the anti-Siglec-6 antibody can be determined by a surface plasmon resonance assay. For example, the Kd or Kd value can be measured by using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore® Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier'sinstructions. Capture antibodies (e.g., anti-human-Fc) are diluted with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 30 pl / minute and further immobilized with an anti- Siglec-6 antibody. For kinetics measurements, two-fold serial dilutions of dimeric Siglec-6 are injected in PBS with 0.05% Tween 20 (PBST) at 25° C at a flow rate of approximately 25 pl / min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to- one Langmuir binding model (BIAcore® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881.

[0189] In another embodiment, biolayer interferometry may be used to determine the affinity of anti-Siglec-6 antibodies against Siglec-6. In an exemplary assay, Siglec-6-Fc tagged protein is immobilized onto anti-human capture sensors, and incubated with increasing concentrations of mouse, chimeric, or humanized anti-Siglec-6 Fab fragments to obtain affinity measurements using an instrument such as, for example, the Octet Red 384 System (ForteBio).

[0190] The binding affinity of the anti-Siglec-6 antibody can, for example, also be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980) using standard techniques well known in the relevant art. See also Scatchard, G., Ann. N.Y. Acad. Sci. 51 :660 (1947).2. Competition Assays

[0191] Competition assays can be used to determine whether two antibodies bind the same epitope by recognizing identical or sterically overlapping epitopes or one antibody competitively inhibits binding of another antibody to the antigen. These assays are known in the art.Typically, antigen or antigen expressing cells is immobilized on a multi-well plate and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured. Common labels for such competition assays are radioactive labels or enzyme labels. In some embodiments, an anti-Siglec-6 antibody described herein competes with a reference antibody described herein for binding to a Siglec-6 polypeptide or an ECD or domain thereof, e.g., expressed on the cell surface of a cell (e.g., a mast cell).III. Antibody Preparation

[0192] The antibody described herein is prepared using techniques available in the art for generating antibodies, exemplary methods of which are described in more detail in the following sections.1. Antibody Fragments

[0193] The present invention encompasses antibody fragments. Antibody fragments may be generated by traditional means, such as enzymatic digestion, or by recombinant techniques. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9: 129-134.

[0194] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can nowbe produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from / / coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from / / coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv). See WO 93 / 16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibodies may be monospecific or bispecific.2. Humanized Antibodies

[0195] The present disclosure encompasses humanized antibodies. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter (Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0196] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. According to the so-called “best- fit” method, the sequence of the variable domain of a rodent (e.g., mouse) antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151 :2623.

[0197] It is further generally desirable that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those, skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.3 Human A ntibodies

[0198] Human anti-Siglec-6 antibodies of the invention can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s). Alternatively, human monoclonal anti-Siglec-6 antibodies of the invention can be made by the hybridoma method. Human myeloma and mousehuman heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol., 147: 86 (1991).

[0199] It is possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy -chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

[0200] Gene shuffling can also be used to derive human antibodies from non-human (e.g., rodent) antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called “epitope imprinting”, either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain / human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain / human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, i.e., the epitope governs the choice of the human chain partner. When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see PCT WO 93 / 06213 published Apr. 1, 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non- human origin.4. Multispecific Antibodies

[0201] Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. Bispecific antibodies may refer to antibodies that have binding specificities for two different antigens, or two different epitopes on the same antigen. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is for Siglec-6 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of Siglec-6. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express Siglec-6.Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies).

[0202] Methods for making bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305: 537 (1983), WO 93 / 08829 published May 13, 1993, and Traunecker et al., EMBO J., 10: 3655 (1991). For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121 :210 (1986). Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.5. Single-Domain Antibodies

[0203] In some embodiments, an antibody of the invention is a single-domain antibody. A single-domain antibody is a single polyeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. Incertain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 Bl). In one embodiment, a singledomain antibody consists of all or a portion of the heavy chain variable domain of an antibody.6 Antibody Variants

[0204] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made.

[0205] A useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed immunoglobulins are screened for the desired activity.

[0206] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variantsof the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0207] In certain embodiments, an antibody of the invention is altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5 -hydroxy lysine may also be used.

[0208] Addition or deletion of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) is created or removed. The alteration may also be made by the addition, deletion, or substitution of one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0209] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies with a mature carbohydrate structure that lacks fucose attached to an Fc region of the antibody are described in US Pat Appl No US 2003 / 0157108 (Presta, L.). See also US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisecting N- acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody are referenced in WO 2003 / 011878, Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody are reported in WO 1997 / 30087, Patel et al. See, also, WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.) concerning antibodies with altered carbohydrate attached to the Fc region thereof. See also US 2005 / 0123546 (Umana et al.) on antigen-binding molecules with modified glycosylation.

[0210] In certain embodiments, a glycosylation variant comprises an Fc region, wherein a carbohydrate structure attached to the Fc region lacks fucose or has reduced fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or moreamino acid substitutions therein which further improve ADCC, for example, substitutions at positions 298, 333, and / or 334 of the Fc region (Eu numbering of residues). Examples of publications related to “defucosylated” or “fucose-deficient” antibodies include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; W02005 / 053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003 / 0157108 Al, Presta, L; and WO 2004 / 056312 Al, Adams et al., especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)), and cells overexpressing pi,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi p-mannosidase II (Mann).

[0211] Antibodies are contemplated herein that have reduced fucose relative to the amount of fucose on the same antibody produced in a wild-type CHO cell. For example, the antibody has a lower amount of fucose than it would otherwise have if produced by native CHO cells (e.g. , a CHO cell that produce a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene). In certain embodiments, an anti-Siglec-6 antibody provided herein is one wherein less than about 50%, 40%, 30%, 20%, 10%, 5% or 1% of the N-linked glycans thereon comprise fucose. In certain embodiments, an anti-Siglec-6 antibody provided herein is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the antibody is completely without fucose, or has no fucose or is non-fucosylated or is afucosylated. The amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. In some embodiments, at least one or two of the heavy chains of the antibody is non-fucosylated.

[0212] In one embodiment, the antibody is altered to improve its serum half-life. To increase the serum half-life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule (US 2003 / 0190311, U.S. Pat. No. 6,821,505; U.S. Pat. No. 6,165,745; U.S. Pat. No. 5,624,821; U.S. Pat. No. 5,648,260; U.S. Pat. No. 6,165,745; U.S. Pat. No. 5,834,597).

[0213] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. Sites of interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions.” If such substitutions result in a desirable change in biological activity, then more substantial changes, denominated “exemplary substitutions” in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.Table 1.

[0214] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or c) the bulk of the side chain. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):(1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M)(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q)(3) acidic: Asp (D), Glu (E)(4) basic: Lys (K), Arg (R), His (H)

[0215] Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties:(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro,(6) aromatic: Trp, Tyr, Phe.

[0216] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites.

[0217] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have modified (e.g., improved) biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display.Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are displayed from filamentous phage particles as fusions to at least part of a phage coat protein (e.g., the gene III product of Ml 3) packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). In order to identify candidate hypervariable region sites for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigenantibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to techniques known in the art, including those elaborated herein. Once such variants are generated, the panel of variants is subjected to screening using techniques known in the art, including those described herein, and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0218] Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.

[0219] It may be desirable to introduce one or more amino acid modifications in an Fc region of antibodies of the present disclosure, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions including that of a hinge cysteine. In some embodiments, the Fc region variant comprises a human IgGl, IgG2, or IgG4 Fc region. Exemplary Fc region variants are provided herein.

[0220] In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, an antibody of the invention may comprise one or more alterations as compared to the wild type counterpart antibody, e.g. in the Fc region. These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can bemade in the Fc region that would result in altered (i.e., either improved or diminished) Clq binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in WO99 / 51642. See also Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94 / 29351 concerning other examples of Fc region variants. WO00 / 42072 (Presta) and WO 2004 / 056312 (Lowman) describe antibody variants with improved or diminished binding to FcRs. The content of these patent publications are specifically incorporated herein by reference. See, also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described in U.S. Pat. No. 6,194,551B1, WO99 / 51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).7. Vectors, Host Cells, and Recombinant Methods

[0221] For recombinant production of an antibody of the invention, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species.Generating Antibodies Using Prokarvotic Host Cells: a) Vector Construction 0222] Polynucleotide sequences encoding polypeptide components of the antibody of the invention can be obtained using standard recombinant techniques. Desired polynucleotidesequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence. 0223] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes-encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237. 0224] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as XGEM.TM.- 11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

[0225] The expression vector of the invention may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter thatinitiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.

[0226] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and / or expression of the target genes. In some embodiments, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter. 0227] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the 0- galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites. 0228] In one aspect of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.[0229 [ In another aspect, the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and heavychains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., the E. coli trxB-strains) provide cytoplasm conditions that are favorable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0230] Antibodies of the invention can also be produced by using an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and properly assembled antibodies of the invention. Such modulation is accomplished at least in part by simultaneously modulating translational strengths for the polypeptide components.

[0231] One technique for modulating translational strength is disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of the translational initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thereby providing a convenient means by which to adjust this factor for the desired expression level of the specific chain. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes which can alter the amino acid sequence. In certain embodiments, changes in the nucleotide sequence are silent. Alterations in the TIR can include, for example, alterations in the number or spacing of Shine-Dalgamo sequences, along with alterations in the signal sequence. One method for generating mutant signal sequences is the generation of a “codon bank” at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity in making the bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4: 151-158. 0232] In one embodiment, a set of vectors is generated with a range of TIR strengths for each cistron therein. This limited set provides a comparison of expression levels of each chain as well as the yield of the desired antibody products under various TIR strength combinations. TIR strengths can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Based on the translational strength comparison, the desired individual TIRs are selected to be combined in the expression vector constructs of the invention.

[0233] Prokaryotic host cells suitable for expressing antibodies of the invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gramnegative cells are used. In one embodiment, E. coli cells are used as hosts for the invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537) andE. coli RV308(ATCC 31,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above- mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture. b) Antibody Production

[0234] Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. 0235] Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers. Another method for transformation employs polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0236] Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene. 0237] Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thiogly collate, dithioerythritol and dithiothreitol. 0238] The prokaryotic host cells are cultured at suitable temperatures. In certain embodiments, for E. coli growth, growth temperatures range from about 20° C. to about 39° C.; from about 25° C. to about 37° C.; or about 30° C. The pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. In certain embodiments, for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0. 0239] If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter. In one aspect of the invention, PhoA promoters are used for controlling transcription of the polypeptides. Accordingly, the transformed host cells are cultured in a phosphate-limiting medium for induction. In certain embodiments, the phosphate-limiting medium is the C.R.A.P. medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263: 133-147). A variety of other inducers may be used, according to the vector construct employed, as is known in the art.

[0240] In one embodiment, the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolatedand identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[0241] In one aspect of the invention, antibody production is conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, and in certain embodiments, about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose. Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters. 0242] In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12- 50 hours, although longer or shorter induction time may be used. 0243] To improve the production yield and quality of the polypeptides of the invention, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis, trans-isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. The chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199- 210.

[0244] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present invention. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP,Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).(0245 In one embodiment, E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention. c) Antibody Purification

[0246] In one embodiment, the antibody protein produced herein is further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art can be employed. The following procedures are exemplary of suitable purification procedures: fractionation on immuno affinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

[0247] In one aspect, Protein A immobilized on a solid phase is used for immuno affinity purification of the antibody products of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureas which binds with a high affinity to the Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein A is immobilized can be a column comprising a glass or silica surface, or a controlled pore glass column or a silicic acid column. In some applications, the column is coated with a reagent, such as glycerol, to possibly prevent nonspecific adherence of contaminants.

[0248] As the first step of purification, a preparation derived from the cell culture as described above can be applied onto a Protein A immobilized solid phase to allow specific binding of the antibody of interest to Protein A. The solid phase would then be washed to remove contaminants non-specifically bound to the solid phase. Finally the antibody of interest is recovered from the solid phase by elution.Generating Antibodies Using Eukaryotic Host Cells:[0249 [ A vector for use in a eukaryotic host cell generally includes one or more of the following non-limiting components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.a) Signal Sequence Component 0250] A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for such a precursor region is ligated in reading frame to DNA encoding the antibody. b) Origin of Replication

[0251] Generally, an origin of replication component is not needed for mammalian expression vectors. For example, the SV40 origin may typically be used only because it contains the early promoter. c) Selection Gene Component

[0252] Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, where relevant, or (c) supply critical nutrients not available from complex media.

[0253] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0254] Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0255] For example, in some embodiments, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, an appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

[0256] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199. Host cells may include NSO, CHOK1, CHOK1SV or derivatives, including cell lines deficient in glutamine synthetase (GS). Methods for the use of GS as a selectable marker for mammalian cells are described in U.S. Pat. No. 5,122,464 and U.S. Pat. No. 5,891,693. d) Promoter Component

[0257] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding a polypeptide of interest (e.g., an antibody). Promoter sequences are known for eukaryotes. For example, virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. In certain embodiments, any or all of these sequences may be suitably inserted into eukaryotic expression vectors. 0258] Transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.

[0259] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindHI E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describing expression of human -interferon cDNA in mouse cells under the control of a thymidine kinase promoterfrom herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter. e) Enhancer Element Component 0260] Transcription of DNA encoding an antibody of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the human cytomegalovirus early promoter enhancer, the mouse cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) describing enhancer elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody polypeptide-en coding sequence, but is generally located at a site 5' from the promoter. f) Transcription Termination Component

[0261] Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector disclosed therein. g) Selection and Transformation of Host Cells

[0262] Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including insect or vertebrate host cells. Propagation of insect or vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful insect cell lines are Sf-9 and Sf-21 of Spodoptera frugiperda, DS2 cells of Drosophila melanogaster, or High Five cells (BTI-TN-5B1-4) of Trichopulsia ni. See, e.g., Frenzel, A. et al. (2013) Front. Immunol. 4:217. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; CHOK1 cells, CHOK1 SV cells or derivatives and a human hepatoma line (Hep G2). 0263] Host cells are transformed with the above-described-expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. h) Culturing the Host Cells 0264] The host cells used to produce an antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's L10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Bames et al., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENT AMY CIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. i) Purification of Antibody

[0265] When using recombinant techniques, the antibody can be produced intracellularly, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, may be removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems may be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants. 0266] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a convenient technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human yl, y2, or y4 heavy chains (Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein Gis recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached may be agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. 0267] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to further purification, for example, by low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, performed at low salt concentrations (e.g., from about 0-0.25M salt).[0268 [ In general, various methodologies for preparing antibodies for use in research, testing, and clinical use are well-established in the art, consistent with the above-describedmethodologies and / or as deemed appropriate by one skilled in the art for a particular antibody of interest.Production of non-fucosylated antibodies 0269] Provided herein are methods for preparing antibodies with a reduced degree of fucosylation. For example, methods contemplated herein include, but are not limited to, use of cell lines deficient in protein fucosylation (e.g., Led 3 CHO cells, alpha- 1,6-fucosyltransferase gene knockout CHO cells, cells overexpressing pi,4-N-acetylglucosaminyltransferase III and further overexpressing Golgi p-mannosidase II, etc.), and addition of a fucose analog(s) in a cell culture medium used for the production of the antibodies. See Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003 / 0157108 Al, Presta, L; WO 2004 / 056312 Al; Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); and US Pat. No. 8,574,907. Additional techniques for reducing the fucose content of antibodies include Glymaxx technology described in U.S. Patent Application Publication No. 2012 / 0214975. Additional techniques for reducing the fucose content of antibodies also include the addition of one or more glycosidase inhibitors in a cell culture medium used for the production of the antibodies.Glycosidase inhibitors include a-glucosidase I, a-glucosidase II, and a-mannosidase I. In some embodiments, the glycosidase inhibitor is an inhibitor of a-mannosidase I (e.g., kifunensine).

[0270] As used herein, “core fucosylation” refers to addition of fucose (“fucosylation”) to N- acetylglucosamine (“GlcNAc”) at the reducing terminal of an N-linked glycan. Also provided are antibodies produced by such methods and compositions thereof.

[0271] In some embodiments, fucosylation of complex N-glycoside-linked sugar chains bound to the Fc region (or domain) is reduced. As used herein, a “complex N-glycoside-linked sugar chain” is typically bound to asparagine 297 (according to the number of Kabat), although a complex N-gly coside linked sugar chain can also be linked to other asparagine residues. A “complex N-glycoside-linked sugar chain” excludes a high mannose type of sugar chain, in which only mannose is incorporated at the non-reducing terminal of the core structure, but includes 1) a complex type, in which the non-reducing terminal side of the core structure has one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the non-reducing terminal side of Gal-GlcNAc optionally has a sialic acid, bisecting N- acetylglucosamine or the like; or 2) a hybrid type, in which the non-reducing terminal side of thecore structure has both branches of the high mannose N-glycoside-linked sugar chain and complex N-glycoside-linked sugar chain.

[0272] In some embodiments, the “complex N-glycoside-linked sugar chain” includes a complex type in which the non-reducing terminal side of the core structure has zero, one or more branches of galactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and the nonreducing terminal side of Gal-GlcNAc optionally further has a structure such as a sialic acid, bisecting N-acetylglucosamine or the like.

[0273] According to the present methods, typically only a minor amount of fucose is incorporated into the complex N-glycoside-linked sugar chain(s). For example, in various embodiments, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the antibody has core fucosylation by fucose in a composition. In some embodiments, substantially none (i.e., less than about 0.5%) of the antibody has core fucosylation by fucose in a composition. In some embodiments, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, or more than about 99% of the antibody is nonfucosylated in a composition. 0274] In some embodiments, provided herein is an antibody wherein substantially none (i.e., less than about 0.5%) of the N-glycoside-linked carbohydrate chains contain a fucose residue. In some embodiments, provided herein is an antibody wherein at least one or two of the heavy chains of the antibody is non-fucosylated.

[0275] As described above, a variety of mammalian host-expression vector systems can be utilized to express an antibody. In some embodiments, the culture media is not supplemented with fucose. In some embodiments, an effective amount of a fucose analog is added to the culture media. In this context, an “effective amount” refers to an amount of the analog that is sufficient to decrease fucose incorporation into a complex N-glycoside-linked sugar chain of an antibody by at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50%. In some embodiments, antibodies produced by the instant methods comprise at least about 10%, at least about 20%, at least about 30%, at least about 40% or at least about 50%non-core fucosylated protein (e.g., lacking core fucosylation), as compared with antibodies produced from the host cells cultured in the absence of a fucose analog.

[0276] The content (e.g., the ratio) of sugar chains in which fucose is not bound to N- acetylglucosamine in the reducing end of the sugar chain versus sugar chains in which fucose is bound to N-acetylglucosamine in the reducing end of the sugar chain can be determined, for example, as described in the Examples. Other methods include hydrazinolysis or enzyme digestion (see, e.g., Biochemical Experimentation Methods 23: Method for Studying Glycoprotein Sugar Chain (Japan Scientific Societies Press), edited by Reiko Takahashi (1989)), fluorescence labeling or radioisotope labeling of the released sugar chain and then separating the labeled sugar chain by chromatography. Also, the compositions of the released sugar chains can be determined by analyzing the chains by the HPAEC-PAD method (see, e.g., J. Liq Chromatogr. 6:1557 (1983)). (See generally U.S. Patent Application Publication No. 2004 / 0110282.).IV. Articles of Manufacture or Kits

[0277] In another aspect, an article of manufacture or kit is provided which comprises an anti- Siglec-6 antibody formulation of the present disclosure.

[0278] The article of manufacture or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (such as single or dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container holds the formulation.

[0279] The article of manufacture or kit may further comprise a label or a package insert, which is on or associated with the container, may indicate directions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous or other modes of administration, e.g., for treating or preventing a mast cell-mediated disorder in an individual. The container holding the formulation may be a single-use vial or a multi-use vial, which allows for repeat administrations of the reconstituted formulation. The article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may further include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0280] The article of manufacture or kit may further comprise instructions for use of the antibody or formulation in the methods of the present disclosure. Thus, in certain embodiments, the article of manufacture or kit comprises instructions for the use of an anti-Siglec-6 antibody formulation in methods for treating a disease or condition characterized by increased activity and / or number of mast cells (e.g., mast cells expressing Siglec-6), inhibiting activation of mast cells (e.g., mast cells expressing Siglec-6), and / or depleting mast cells (e.g., mast cells expressing Siglec-6), e.g., in a subject in need thereof. In certain embodiments, the individual is a human. In some embodiments, the individual has or is at risk for developing a mast cell- mediated disorder or condition. In some embodiments, the subject has or has been diagnosed with mastocytosis (e.g., indolent systemic mastocytosis, ISM; or aggressive systemic mastocytosis, ASM), mast cell leukemia, mast cell activation syndrome, gastroparesis, osteoporosis, osteopenia, renal osteodystrophy, bone fracture, Alzheimer’s disease, chronic neuropathic pain, hyperalgesia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), graft vs. host disease (GVH), colitis (e.g., microscopic colitis or ulcerative colitis), hereditary alpha tryptasemia, neurofibroma, Kounis syndrome, urticaria (e.g., chronic spontaneous urticaria or an inducible urticaria), atopic dermatitis, contact dermatitis, angioedema, prurigo nodularis, cholangitis, psoriasis, irritable bowel syndrome (IBS), functional dyspepsia, asthma (e.g., eosinophilic or non-eosinophilic asthma), allergy (e.g., food allergy or pseudo allergy), keloid, chronic rhinosinusitis (e.g., with or without nasal polyps), aspirin exacerbated respiratory disease (AERD), chronic obstructive pulmonary disease (COPD), bullous pemphigoid, idiopathic pulmonary fibrosis, systemic sclerosis, interstitital cystitis, hi dradenitis suppurativa, alopecia areata, vitiligo, mast cell gastrointestinal disease, Crohn’s disease, endometriosis, interstitial cystitis, headache, migraine, rheumatoid arthritis, gastroesophageal reflux disease, viral infection, achalasia, postural tachycardia syndrome, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), complex regional pain syndrome, or Ehlers-Danlos syndrome.

[0281] In a specific embodiment, the present invention provides kits for a single doseadministration unit. Such kits comprise a container of an aqueous formulation of therapeutic antibody, including both single or multi-chambered pre-filled syringes. Exemplary pre-filled syringes are available from Vetter GmbH, Ravensburg, Germany.

[0282] The article of manufacture or kit herein optionally further comprises a container comprising a second medicament, wherein the anti-Siglec-6 antibody is a first medicament, and which article or kit further comprises instructions on the label or package insert for treating the subject with the second medicament, in an effective amount.

[0283] In another embodiment, provided herein is an article of manufacture or kit comprising the formulations described herein for administration in a pre-filled syringe or an auto-injector device. An auto-injector can be described as an injection device that upon activation, will deliver its contents without additional necessary action from the patient or administrator. They are particularly suited for self-medication of therapeutic formulations when the delivery rate must be constant, and the time of delivery is greater than a few moments.

[0284] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.EXAMPLES

[0285] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.Example 1: Ultrafiltration / diafiltration (UFDF) Assessment: pH shift

[0286] Siglec-6 (also known as CD327) is an inhibitory receptor that is selectively expressed on mast cells. Mast cells are considered pathogenic drivers of numerous autoimmune and inflammatory diseases, including but not limited to food allergy, mast cell activation syndrome, mastocytosis, IPF, COPD, and others. See, e.g., Yu, Y. et al. (2018) Front. Immunol. 9:2138;Yokoi, H. et al. (2006) Allergy 61:769-76; US PG Pub. Nos. US20060269556A1 and US20080267973A1; and International Pub. No. W02023044390.

[0287] Monoclonal antibodies (mAbs) have become essential therapeutic agents in treating various diseases. Traditional mAb formulations often have lower concentrations, requiring large volumes for therapeutic doses. This can lead to challenges in administration, especially for subcutaneous injections, and can impact patient compliance and comfort. Monoclonal antibodies are known to display complicated solution properties at high concentrations such as over lOOmg / mL, including high viscosity, low stability, and high aggregation propensity (see, e.g., Wang, S. et al. (2015) Mol Pharm 12(12):4478-4487). Denaturation and aggregation are two major manifestations of physical instability exhibited by antibodies under various conditions (see, e.g., Wang, W. et al. (2007) J Pharm Sci. 96(l):l-26; Kheddo, P. et al. (2014) Int J Pharm. 473 (1 -2): 126-133 ; and Kheddo, P. et al. (2016) Toxicol In Vitro 33:88-98). When high- concentration liquid formulations are desired (e.g., for subcutaneous delivery, such as because of injection volume restrictions), issues such as phase separation, induced viscosity, opalescence, and self-association have been observed from molecular crowding effects, potentially leading to significant challenges in processing, formulation, and delivery, as well as product quality and patient comfort / convenience (see, e.g., Muralidhara, B.K. and Wong, M. (2020) Drug Discov Today 25(3): 574-581 ).

[0288] The following Examples describe experiments aimed at identifying the appropriate formulation conditions for subcutaneous administration of an anti-Siglec-6 antibody. The formulation development of the anti-Siglec-6 antibody covers surfactant screening, ultrafiltration / diafiltration (UF / DF) assessment, UF feasibility (UFF) and excipient screening studies. The target anti-Siglec-6 antibody concentration was set to 150 mg / mL, and the antibody comprised a heavy chain that comprises the sequence of SEQ ID NO: 166 and a light chain that comprises the sequence of SEQ ID NO: 167. An overview of the formulation workflow is shown in FIG. 1Materials and Methods

[0289] Size-exclusion high performance liquid chromatography (SE-HPLC) was performed for testing size-exclusion profiles and purity of anti-Siglec-6 antibody sample proteins using HPLC (Waters®, e2695) in accordance with standard protocol. Standard protocol was also used toanalyze size-exclusion chromatography (SEC). Equation 1 was used for calculation of %area of the high molecular weight species (%HMWS), monomer and low molecular weight species (%LMWS). A shoulder peak toward %LMWS peak has been observed and was manually defined considering the relative retention time observed during development to consistently evaluate the relative changes in the sample set.Equation 1. Formula for content calculationSHMWS peak area [pV * seel%HMWS = - - - - -1X 100%Total area [pV * sec]SMonomer peak area [nV * sec]%Monomer = - - - - - - x 100%Total area [pV * sec] SLMWS peak area [pV * sec] %LMWS = - - - - - - - -1x 100%Total area [pV * sec]

[0290] Sub-visible particles (SVP) testing was performed using light obstruction particle count (LPC) and HORIZON. Samples for the excipient screening were tested using LPC (Beckman Coulter, HIAC 9703+) according to standard protocol. Sample volume for each sample was 0.2 mL. The overall HIAC data were converted to particles per container, based upon a container volume of 10 mL.

[0291] Other screening samples were tested using HORIZON (Halo labs, HORIZON) according to standard protocol. HORIZON system is a high throughput screening (HTS) system using backgrounded membrane imaging for sub-visible particles measurement.

[0292] Before sample preparation, a background image of the membrane was collected. Then 30 pL of each sample was loaded on the membrane. These samples were then filtered through using vacuum pressure capturing the particles onto the membrane and then membrane is reimaged. The “background image” was precisely aligned with the “measure image” and then subtracted on a pixel-by -pixel basis so that the background texture is eliminated, and particles were revealed and quantified.

[0293] Differential scanning calorimetry (DSC) (Malvern, MicroCai PEAQ-DSC Automated) was used for measuring Tonset and Tmfor anti-Siglec-6 antibody thermal stability during excipient screening step according to standard protocol.

[0294] The absorption of heat that occurs when a protein unfolds causes a temperature difference (AT) between the cells, resulting in a thermal gradient across the Peltier units. This sets up a voltage, which is converted into power and is used to control the Peltier to return AT (the temperature differential) to 0°C. Alternatively, the cells could be allowed to reach thermal equilibrium passively through conduction. The data from cells are transformed to graph heat capacity and integrated to obtain Tmand Tonset values of the sample.

[0295] Charge variants were monitored during formulation development by cation-exchange chromatography (CEX-HPLC). The CEX-HPLC method was developed for the purpose of comparing the chemical stability of the anti-Siglec-6 antibody in different compositions during development.

[0296] The method employed a Thermo mabPac™ SCX-10 CEX-HPLC column, 5 pm, 4x150 mm (#085198) with Mobile phase A (MP A) composed of sodium phosphate, sodium chloride, pH 5.8, 2.1 mS and Mobile phase B (MP B) composed of sodium phosphate, sodium chloride, pH 8.0, 3.75 mS. The method employed a column temperature of 35°C and a flow rate of 0.4 mL / min and a 40 pg injection load. The elution gradient is summarized in Table A.Table A. CEX-HPLC Gradient

[0297] Protein Concentration (A280) testing for low concentration samples (samples <50 mg / mL) was performed using UV / VIS spectrophotometer (Nanodrop™ One, Thermo Scientific) according to standard protocol. For anti-Siglec-6 antibody, applied extinction coefficient (EC) value is 1.455 AU (mg / mL)'1. Protein Concentration (A280) testing for high concentration samples (samples greater than 50 mg / mL) was performed using a slope spectrometer (SoloVPE®, C-Technologies) according to standard protocol. For anti-Siglec-6 antibody, applied extinction coefficient (EC) value is 1.455 AU (mg / mL)4.

[0298] pH was measured using pH meter (Mettler Toledo, S220) according to standard protocol.

[0299] Osmolality was measured using osmometer (Advanced Instrument, Osmol®) according to standard protocol.

[0300] Color and clarity test for appearance was performed through automatic handler (Unchained labs, JUNIOR). For visible particles testing, manual visual inspection was performed according to standard protocol.

[0301] Viscosity testing was performed using viscometer (Rheosense, m-VROC®) according to standard protocol.

[0302] Glass Transition Temperature (Tg) testing was measured using DSC (Mettler Toledo, DSC 3+) according to standard protocol.

[0303] Target ranges of the methods that were used for formulation development are summarized in Table B.Table B. Summary of Product Quality Targets1:>The drug product (DP) container was filled with a volume 1.2 mL. However, for this analysis per container = 10 mL.

[0304] Before formulation development started, initial target product information was provided as described in Table C.Table C. Initial Target Product ProfileResults

[0305] The goal of doing a preliminary UFDF assessment was to gain an understanding of the extent of the pH shift during the UFDF process due to Gibbs-Donnan and volume exclusion effects. Based upon the literature (see, e.g., Bolton GR. et al. (2011) Biotechnol Prog 27(1): 140- 152), it was expected that processing a protein to concentrations >50 mg / mL in a histidine-based buffer would result in an increase in pH of the product as the protein was concentrated.Therefore, prior to producing the material supply run (MSR) for any subsequent studies, an assessment of the extent of pH shift was conducted for anti-Siglec-6 antibody in concentrating to -240 mg / mL and determining how much the pH changes upon reaching the target concentration. Based upon prior experience, it was anticipated that the pH might shift 0.3 pH units in going from -20 mg / mL to -240 mg / mL. Therefore, the diafiltration buffer pH was targeted to pH 5.7 as an initial estimation. Therefore, if the initial estimation of 0.3 pH unit change was correct, the final pH should be around pH 6.0. Briefly, on small scale, 195 mL of the starting material at -17 mg / mL, was diafiltered into 20 mM Histidine, pH 5.7 as shown in Table D.Table D. Diafiltration ResultsDV = Diavolumes; ND = Not determined

[0306] This material was then concentrated to a target concentration of -240 mg / mL and the resulting pH was measured during the concentration process. As shown in FIG. 2 and Table E, there was a consistent increase in product pH with increasing protein concentration.Table E. Product pH with Increasing Concentration during Ultrafiltration

[0307] Based upon these findings, it was determined that upon reaching a targeted UF2 concentration -200 mg / mL, the pH of the product using a 20mM L-histidine / L-histidine-HCl based diafiltration buffer at a pH of 5.7 was near the target pH of 6.0.Example 2: UF Feasibility

[0308] The purpose of the ultrafiltraton (UF) feasibility (UFF) study was to evaluate the feasibility of processing the anti-Siglec-6 antibody material prior to generating samples for formulation screening. This study enables an understanding of the potential concentration that can be achieved for the purposes of formulation development.

[0309] The goal was to obtain a preliminary view of the molecule's stability in the base buffer system (3 -day hold, ambient laboratory conditions). Summarized in Table F, there was anincrease in the %HMWS (with a corresponding decrease in the monomer content) during the ultrafiltration / diafiltration (UFDF) process. The starting concentration of the starting material (MMX) was 16.86 mg / mL and concentrated 1.3-fold (UFF 1.3x material). There were only slight differences between the product at lx concentration and 1.3x concentration as determined by SE- HPLC. After the 3 -day hold at ambient conditions, the lx material did not show significant changes and the 1.3x material did show an increase in %HMWS, as shown in Table F.

[0310] The protein concentration and corresponding viscosity was measured for the UFF samples. As shown in Table G, as the concentration changed from 150.0 mg / mL to 195.0 mg / mL, there was an observable sizable change in the solution viscosity from 5.0 cP to 11.9 cP.Table F. Stability of UF Feasibility Samples - SE-HPLC ResultsTable G. Product pH with Increasing Concentration’* Viscosity measurements were performed at 25°C.Shear rates between 1431 / second and 3864 / second were employed for the 1.3x.Shear rates between 2288 / second and 3864 / second were employed for the lx.Example 3: Surfactant Screening

[0311] Surfactants are essential in high concentration monoclonal antibody formulations, as they effectively mitigate issues such as aggregation, surface adsorption, and ensuring stabilityand therapeutic efficacy. The purpose of the surfactant screening study was to evaluate the impact of physical stress (agitation) on the physical stability of the anti-Siglec-6 antibody. The goals of the study were to determine a need for a surfactant and to preferentially select between polysorbate 20 (PS20) and polysorbate 80 (PS80) for the anti-Siglec-6 antibody.

[0312] The material supply run (MSR) material was provided at a concentration of 50 mg / mL in 20 mM Histidine buffer, pH 6.0. The MSR material was spiked with stock solutions (5% w / v) of polysorbate (both polysorbate 20 and polysorbate 80) prepared in 20 mM Histidine buffer, pH 6.0 to a target concentration of 0.05% w / v.

[0313] A physical stress (PS) protocol was applied to all samples for the study. The PS test was performed to predict whether protein products would be affected by physical stresses due to gas-liquid interface phenomena leading to high aggregate concentrations. PS testing was performed by mechanically vibrating the product at 3,000 rpm for 4 hours on a multi -vortex meter. After the physical stress, protein content (A280), the sample appearance, SE-HPLC and sub-visible particles (HORIZON) were analyzed.

[0314] As can be observed in the results from FIG. 3 and FIG. 4, the sample formulated without any surfactant was severely degraded due to the physical stress. For the samples containing either surfactant, there were only very slight differences between the physical stress sample and the control samples in terms of color, visible particles, or SE-HPLC (FIG. 4). Closer examination of the results for clarity and sub-visible particles, the physical stress sample containing polysorbate 20 exhibited a slightly higher opacity (larger NTU) value as well as significantly larger number of sub-visible particles. Therefore, polysorbate 80 was determined to be more favorable as compared to polysorbate 20 for the anti-Siglec-6 antibody. Based upon these results, the use of polysorbate 80 at a concentration of 0.05% (w / v) was recommended for future studies.Example 4: Excipient ScreeningFormulation Matrix Design

[0315] Excipient screening in formulation design is important to identify and evaluate additives that are suitable for subcutaneous injection, maintains or improves antibody stability and efficacy, and manufacturability of the drug product, while ensuring safety and regulatory compliance. Excipients are added to a formulation to stabilize the protein, control or target drugdelivery, aid in manufacture, and / or provide tonicity or other qualities to minimize pain upon injection. Different excipients can interact with proteins in a formulation in different ways, such as preferential hydration, electrostatic and cation-pi interactions, hydrogen bonding, and dispersive forces, in different states of the formulation (e.g., liquids, frozen liquids, gels, freeze dried solids, etc.); see, e.g., Kamerzell, T.J. et al. (2011) Adv Drug Deliv Rev. 63(13): 1118-1159.

[0316] A strategy was devised to streamline formulation development activities. The revised development is summarized here.

[0317] The base buffer system was fixed at using a 20 mM L-histidine / L-histidine-HCl buffer, pH 6.0. Therefore, with a fixed buffer system and pH, the only variable to be screened were various excipients. The excipients evaluated are common excipients employed for biopharmaceutical development (see, e.g., Ionova, Y. and Wilson, L. (2020) PLoS One 15(6):e0235076 and Rao, V.A. et al. (2020) Pharm Res. 37(10):200).

[0318] The MSR was prepared at a concentration of 50 mg / mL in 20 mM Histidine buffer, pH 5.7. The MSR was further concentrated using a tangential flow filtration system. The material was concentrated to a UF concentration of 208 mg / mL. The UF2 material was then spiked with concentrated excipient solutions to achieve the final compositions indicated below. Formulations tested included the following:• F01 - 150 mg / mL anti-Siglec-6, 20 mM histidine buffer, 248 mM sucrose, 0.05% (w / v) polysorbate 80, pH 6.0• F02 - 150 mg / mL anti-Siglec-6, 20 mM histidine buffer, 160 mM sucrose, 0.05% (w / v) polysorbate 80, pH 6.0• Fll - 150 mg / mL anti-Siglec-6, 20 mM histidine buffer, 160 mM sucrose, 0.05% (w / v) polysorbate 80, pH 5.8• F12 - 150 mg / mL anti-Siglec-6, 20 mM histidine buffer, 160 mM sucrose, 0.05% (w / v) polysorbate 80, pH 6.5

[0319] In addition, formulations F03-F10 were used to test various common excipients (e.g., sodium chloride, arginine, glycine, proline, and glutamate) in addition to 20 mM histidine buffer, 0.05% (w / v) polysorbate 80, and 150 mg / mL antibody at pH 6.0.

[0320] In addition to Formulations F01 - F10, two additional formulations were included to evaluate the stability above and below the target pH of 6.0. However, since the diafiltrationbuffer was fixed, the best way in which to prepare samples that were above and below the target pH was to pre-adjust the spike solutions in order to adjust the pH of the final formulation.

[0321] In order to achieve a pH below the target pH of 6.0 (Fl 1) and above the pH of 6.0 (Fl 2) using the diafiltered material in 20 mM Histidine pH 6.0, separate spike solutions of 20 mM Histidine, 1.5 M Sucrose, pH 2.0 and 20 mM Histidine, 1.5 M Sucrose at pH 10.5 were prepared prior to addition to the UFDF material. These spike solutions were prepared by weighing an appropriate amount of sucrose and dissolving into either 20 mM Histidine-HCl (as for the spike solution for Fl 1) or dissolving into 20 mM L-Histidine (as for the spike solution for F12). Minor adjustments were made with either 1 M Hydrochloric acid or with 1 M sodium hydroxide to achieve the target pH values for the spike solutions of pH 2 (spike solution for Fl 1) and pH 10.5 (spike solution for Fl 2). These spike solutions were added to the UFDF material to achieve the resulting compositions for Fl 1 and Fl 2.

[0322] After the preparation of the desired compositions, the formulations were sterile filtered through 0.22 pm filters and aseptically filled in a bio safety cabinet (BSC) using sterile vials and stoppers. The 2R vials were filled with 1.2 mL (as the lowest filled volume for an extractable volume of 1 mL from a 2R vial) of each formulation. Vials were then placed on stability at 5 ± 3°C, 25 ± 2°C / 60% relative humidity (RH), and 40 ± 2°C / 75% RH and tested at time 0 (T=0), after 2 weeks, after 4 weeks, and after 8 weeks.

[0323] In addition to thermal stress, the formulations were exposed to physical stress and freeze-thaw (FT) stress. FT test was performed to predict the product stability when the product is frozen and thawed repeatedly. FT study was performed under the following conditions: Freeze at -70 ± 10°C for at least 20 hours and thaw under ambient conditions for 4 ± 0.5 hours (5 cycles, 5x). The conditions for physical stress were as described above.Release (RE) Test (T=0) Results

[0324] The release test (T=0) results are summarized in FIGs. 5-7. A general summary of results are as follows.

[0325] pH / Protein concentration: All measured pH values were within ± 0.1 pH units of target pH. The protein concentrations were at 150.0 mg / mL (± ~5%).

[0326] Osmolality: The measured osmolality of the formulations spanned a broad osmolality range of 262-431 mOsm / kg*water.

[0327] Appearance: All formulations exhibited a single liquid phase with no observable visible particles. However, various formulations did exhibit some opalescence. Formulations exhibiting the greatest opalescence contain an electrolyte excipient (F03, F04, F05, F06, F08). It is worth noting that formulations containing sodium chloride & L-arginine HC1 were visually similar degrees of opalescence, whereas the formulation containing the combination of arginine / glutamate (F08) was less opalescent than F03-F06.

[0328] Thermal stability (DSC): The formulations exhibited differences in the Tmas measured by DSC. Interpretation of the results suggested that formulations containing either sodium chloride or L-arginine HC1, only, were slightly lower in Tmcompared to the other excipient formulations. When comparing the effect of pH, there was a notable change in Tmfor Fl 1 (pH = 5.8) compared to F12 (pH 6.5), where F12 has the highest overall Tmof all of the evaluated formulations.

[0329] Viscosity: All protein concentrations were at 150 mg / mL (± ~5%) and the viscosities all are between 5.5 - 6.5 cP. Some potentially favorable excipients for viscosity are glycine, L- arginine-HCl, proline, and sodium chloride as they are generally regarded as useful viscosity modifiers. However, despite their addition, it seems that all formulations have acceptable viscosities for mobile fluids and parenteral delivery.

[0330] Sub-visible particles: All formulations pass the reference standard as tested by HIAC and were generally low; even compared to 10 mL per container.

[0331] SE-HPLC: All of the formulations at T=0 exhibited >96% main peak.

[0332] CEX-HPLC: All of the formulations at T=0 had % main peak around 43%.Stability Results After T=2 weeks at 5°C, 25°C / 60%> RH, and 40°C / 75% RH

[0333] The results for the 2-week analyses are shown in FIGs. 8-13.

[0334] Protein concentration / Appearance: All formulations remained at the target concentration. There were no significant changes in the appearance at any of the temperatures. There were no observed visible particles except for F04 at 25 °C and 40°C.

[0335] Sub-visible particles: There are some changes in the sub-visible particles after 2-week stability. F03, F04 and F05 showed increasing amounts of sub-visible particles (>10 pm). Nevertheless, all formulations pass the reference standard at all of the temperatures.

[0336] SE-HPLC: The SE-HPLC is able to highlight observable changes in the %HMWS and %LMWS upon storage, where select formulations exhibit greater amounts of %HMWSformation on stability. For example, F03 and F12 exhibited greater increases in %HMWS. These trends are followed throughout the course of the study and summarized at the end of the study.

[0337] CEX-HPLC: The CEX-HPLC method is able to discern some differences between the formulations, in particular, at 40°C. For example, at 40°C at the 2-week timepoint, F03-F06 showed smaller increases in acidic species. These and other trends were followed throughout the course of the study and summarized at the end of the study.Stability Results After T=4 weeks at 5°C, 25°C, and 40°C

[0338] The results for the 4-week analyses are summarized in FIGs. 14-19.

[0339] Protein concentration / Appearance: All of the formulations remained at the target concentration. There were no significant changes in the appearance at any of the temperatures and no visible particles.

[0340] Sub-visible particles: Even though there were some changes in the sub-visible particles after 4-week stability, all formulations pass the reference standard at all of the temperatures.

[0341] SE-HPLC: There are some formulations that exhibit greater changes on stability as determined by SE-HPLC. For example, the formulations containing sodium chloride (F03 and F04) showed a greater formation of HMWS. Additionally, Fl 2 (the higher pH sample) also showed a greater amount of HMWS formation. On the other hand, it appears that F03 has the least amount of HMWS formation after 4 weeks at 5°C, 25°C and 40°C. Stability trends observed by SE-HPLC are summarized at the end of the study.

[0342] CEX-HPLC: The CEX-HPLC method was able to discern some differences between the formulations. In particular, F03-F06 all showed slightly more favorable behavior with regards to changes in acidic peaks. Each of the formulations were followed throughout the course of the study and summarized at the end of the study.Stability Results After T=8 weeks at 5°C, 25°C, and 40°C

[0343] The results from the 8-week analyses are summarized in FIGs. 20-25.

[0344] pH / Protein concentration / Appearance: All the measured pH values were within ± 0.1 pH units of target pH. All the formulations remained at the target concentration. There were no significant changes in the appearance at any of the temperatures and no visible particles.

[0345] Sub-visible particles: All formulations successfully met the requirements of reference standard across all temperature conditions, as shown in FIG. 26. Notably, there were variations in the counts of sub-visible particles following an 8-week stability period. When comparing thesub-visible particle counts of formulations F01 and F02 with formulation F03, which showed a significant change in these counts, and formulation F09, known for its lower particle counts, it is observed that the trends in F01 and F02 were akin to those seen in F09.

[0346] SE-HPLC: The SE-HPLC method was effective in detecting variations in high molecular weight species (HMWS) and low molecular weight species (LMWS) during storage. It was observed that certain formulations demonstrated a higher formation of HMWS during stability studies. These changes were monitored and are discussed in detail below. Specifically, formulation Fl 2 showed significant reductions in the main peak, indicating a notable loss, whereas formulation F08 exhibited minimal changes. However, when these formulations are compared with sucrose-based formulations at pH 6.0 (F01 and F02), the observed trends are strikingly similar. Thus, despite observable alterations in the SE-HPLC profiles, particularly at increased temperatures, the sucrose-based formulations displayed a degradation pattern that is closely aligned with those formulations exhibiting the least change, as illustrated in FIG. 27.

[0347] CEX-HPLC: The CEX-HPLC method discerned some differences between the formulations and were followed throughout the course of the study. Mathematically, formulation Fl 2 exhibited the most increase in acidic peaks, while formulation F03 showed minimal change. However, upon comparing these formulations with those containing sucrose at pH 6.0 (F01 and F02), the observed patterns were quite similar. Thus, although variations in the CEX-HPLC profiles were noted, particularly at higher temperatures, the sucrose-containing formulations demonstrated a degradation trend akin to that of the formulations with the least observable changes, as illustrated in FIG. 28.5x Freeze-Thaw (FT) Test Results

[0348] Freeze-thaw is an important consideration in the development and handling of pharmaceutical products. Formulations were subjected to 5x freeze-thaw and evaluated for visual appearance, SVP and SE-HPLC. The results from the FT analyses are summarized in FIG. 29 and FIG. 30.

[0349] Protein concentration / Appearance / Sub-visible particles: All formulations remained at the target concentration and there were no observable visible particles. With regards to the sub- visible particles, F03, F04, F06, and F08 have higher sub-visible particles (> 10 pm). Despite these observed differences in the sub-visible particle counts, all formulations passed the reference standard requirements after 5x freeze-thaw.

[0350] SE-HPLC: Only F03, F04 and Fl 2 showed a slightly greater amount of %HMWS as compared to the other formulations after 5x freeze-thaw.Physical Stress (PS) Test Results

[0351] Physical stress (agitation) is an important factor in pharmaceutical development as it can impact protein stability, increase aggregation risk, and affect product efficacy and safety under various handling and transportation conditions. Formulations were mechanically vibrated for 4 hours and evaluated for visual appearance, SVP, and SE-HPLC. The results of the PS analyses are summarized in FIG. 31 and FIG. 32.

[0352] Protein concentration / Appearance / Sub-visible particles: All of the formulations remained at the target concentration. There were no significant changes in the appearance or visible particles after physical stress. All formulations pass the reference standard requirements after physical stress. However, F03, F04 and F08 had slightly elevated sub-visible particle counts (> 10 pm) as compared to the other formulations after the physical stress.

[0353] SE-HPLC: F03, F09, F10 and F12 showed a slightly greater amount of HMWS as compared the other formulations after physical stress.Summary of Excipient (EXC) Screening, Results

[0354] The goal of the study was to perform formulation excipient screen and evaluate the physical and chemical stability of the anti-Siglec-6 antibody at 150 mg / mL by exposing each of the candidate formulations to thermal stress (5 °C, 25 °C, and 40°C) for 8 weeks, a 5x freeze-thaw cycling stress, and physical (agitation) stress.

[0355] The freeze-thaw and physical stress conditions did not significantly impact the stability of any of the formulations. This likely suggests that the excipients that were evaluated did not adversely impact the interfacial stability. Additionally, the presence of the 0.05 % (w / v) PS80 appears adequate to stabilize the molecule against interfacial stress in the presence of each of the other formulation components.

[0356] The thermal stress study outcomes identified certain compositions as less advantageous due to their physical and / or chemical stability. The aim was to assess the behavior of each formulation by minimizing the changes observed in the stability assays, as detailed in Table B. For instance, among the pH 6.0 formulations, F03 (100 mM sodium chloride) showed the most significant increase in %HMWS during stability testing. Without wishing to be bound to theory, it is thought that sodium chloride may be unsuitable as a tonicity modifier for the anti-Siglec-6antibody at high concentrations. The data also indicates that minor pH variations, such as -0.2 pH units (Fl 1) and +0.5 pH units (F12), could affect the stability of the anti-Siglec-6 antibody. It seems that a pH of 6.5 (F12) is less favorable for thermal stability than a pH of 6.0 (F02). However, the profdes of pH 6 (F02) and a slightly lower pH of 5.8 (Fl 1) appeared to be similar. In terms of %HMWS formation, the SE-HPLC results showed that formulations F05, F06, F08, F09, F10 and Fl 1 had positive results. Formulations F04, F08, and F09 were advantageous for %LMWS formation. For increases in acidic species based on CEX-HPLC, formulations F03, F04, F05 and F06 were considered more advantageous.

[0357] An 8-week examination revealed that after storing at 25°C, formulation F03 showed the largest increase in the number of particles equal to or larger than 10 pm. Formulations F04, F05 and F06 also showed considerable changes in the count of similar-sized particles at 25°C. It's important to note that all conditions adhered to the reference standard specification for stability after this 8-week period.

[0358] Upon a comprehensive review of all the data, it was observed that many of the excipients evaluated at pH 6.0 showed a high degree of similarity. Specifically, F01 (248 mM sucrose) and F02 (160 mM sucrose) demonstrated similar stability profiles, as depicted in FIG. 33 and FIG. 34. These figures also illustrate the trend of behavior slightly above and below the target pH. Despite minor differences between the slightly varied pH levels, the overall stability profile appears to be highly similar. Even with the noticeable differences in accelerated stability, both F01 and F02 (sucrose-based formulations at pH 6.0) showed similar trends in stability attributes such as sub-visible particles, SE-HPLC, and CEX-HPLC. This makes a sucrose-based formulation at pH 6.0 a favorable choice. Additionally, both F01 and F02 also have acceptable viscosities of 6.46 cP and 6.08 cP, respectively.

[0359] However, there is a significant difference in the osmolality of these two formulations, with F01 having an osmolality of 431 mOsm / kg and F02 having an osmolality of 285 mOsm / kg. Given the similar properties and stability behavior of these two formulations (as shown in FIG. 33 and FIG. 34), the final sucrose concentration was adjusted to bring the final osmolality (or tonicity of the solution) into a more suitable range. Consequently, the final selected formulation was a solution containing 170 mM Sucrose, 0.05 % (w / v) PS80, 20 mM Histidine buffer, pH 6.0, and had an osmolality of 299 mOsm / Kg for a solution containing a target concentration of 150 mg / mL anti-Siglec-6 antibody. This formulation is thought to be suitable for subcutaneousinjections with viscosities less than 6-7 (or less than 8 cP) centipoise (cP) for anti-Siglec-6 concentrations up to 150 mg / mL, and less than 12 cP for anti-Siglec-6 concentrations from 170- 200 mg / mL; that are clear to slightly opalescent, colorless to slightly brownish-yellow, well- controlled from the particle formation (sub-visible particles), and essentially-free of visible particulates.Example 5: Determination of Tg' for Anti-Siglec-6 Antibody Formulations

[0360] The target range of the sub-ambient glass transition temperature (Tg') was not specified. The formulation recommendation for the anti-Siglec-6 antibody is a solution containing 150 mg / mL anti-Siglec-6 antibody, 170 mM sucrose, 0.05% (w / v) polysorbate 80, 20mM L-histidine / histidine-HCl, at pH 6.0. The Tg' for the final formulation was measured to provide supporting evidence for potential drug substance storage temperature. However, the glass transition of the anti-Siglec-6 antibody final drug substance (DS) was not clearly defined. It is believed that it is difficult to discern the Tg' because of the large weight fraction of protein in the formulation. To obtain some experimental evidence of the sub-ambient glass transition temperature, a number of dilutions were made into the formulation buffer to reduce the weight fraction of the protein and determine that relative solution’s Tg'. Using these experimental results, an estimation of the Tg' was determined, by extrapolation of the experimental data in FIG. 35 and Table H, to be -22°C (-21.97°C).Table H. Tg' Result for Diluted Final Formulation of Anti-Siglec-6 AntibodyND = Not determinedConclusion

[0361] Based on the results of Examples 1-5, the final formulation recommendation for anti- Siglec-6 antibody was determined as listed in Table I. The intended storage temperature is 5 ± 3°C.Table I. Final Formulation1:>170 mM sucrose is 58.2 mg / mLExample 6: Sequence Liability Analysis

[0362] As shown in FIG. 36, there is potential for aspartate isomerization in the CDRs of the anti-Siglec-6 antibody heavy chain (SEQ ID NO: 166). Isomerization in the CDRs can impact antibody stability, activity, manufacturability, and batch comparability. In order to analyze this, a study under pro-isomerization conditions was performed to assess whether potency loss occurs. As shown in Table K, no potency loss was observed for the anti-Siglec-6 antibody.Table K. Anti-Siglec-6 Antibody Potency Evaluation Under Pro-Isomerization ConditionsExample 7 : Aggregation AnalysisThe target anti-Siglec-6 antibody concentration was set to 150 mg / mL. The purpose of the aggregation analysis was to establish the feasibility of concentrating anti-Siglec-6 antibody to ahigh concentration. The anti-Siglec-6 antibody starting material (20 mg / mL in 20mM Histidine pH 6.0) was able to be concentrated to 220 mg / mL without difficulty. Excipient was then added, yielding anti-Siglec-6 antibody at 139 mg / mL in 20mM Histidine, 8.5% sucrose, 0.05% polysorbate 80 (PS80) pH 6.0. Upon visual inspection, the protein did not show signs of aggregation either at 220 mg / mL or upon addition of the excipient at 139 mg / mL, demonstrating anti-Siglec-6 antibody is stable at high concentrations, as shown in FIG. 37. SE-HPLC of the starting material (20 mg / mL) and concentrated product (139 mg / mL) resulted in similar chromatography profiles as shown in FIG. 38, further indicating anti-Siglec-6 antibody did not aggregate at high concentrations.

Claims

CLAIMSWhat is claimed is:

1. A liquid formulation comprising: (a) a monoclonal antibody that binds to a human Siglec-6, wherein the antibody is in a concentration of about 50 mg / mL to about 200 mg / mL; and (b) histidine in a concentration of about 5 mM to about 100 mM; wherein the pH of the liquid formulation is between 5.7 and 6.5; and wherein the antibody comprises: (1) a heavy chain comprising a heavy chain variable (VH) region comprising: an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 168; an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 169; an HVR-H3 comprising the amino acid sequence of SEQ ID NO:7; and (2) a light chain comprising a light chain variable (VL) region comprising: an HVR-L1 comprising the amino acid sequence of SEQ ID NO:8; an HVR-L2 comprising the amino acid sequence of SEQ ID NO:9; and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10.

2. The formulation of claim 1, wherein the histidine is in a concentration of about 15 mM to about 25 mM.

3. The formulation of claim 2, comprising histidine in a concentration of 20 mM.

4. The formulation of any one of claims 1-3, wherein the histidine comprises L-histidine and / or L-histidine hydrochloride.

5. The formulation of claim 4, wherein the histidine comprises 9 mM L-histidine and 11 mM L-histidine hydrochloride monohydrate.

6. The formulation of any one of claims 1-5, further comprising a disaccharide in a concentration of about 90 mM to about 250 mM.

7. The formulation of claim 6, comprising the disaccharide in a concentration of 100 mM to 248 mM.

8. The formulation of claim 6, comprising the disaccharide in a concentration of 160 mM to 248 mM.

9. The formulation of any one of claims 6-8, wherein the disaccharide is sucrose.

10. The formulation of claim 9, comprising sucrose in a concentration of 170 mM.

11. The formulation of any one of claims 1-10, further comprising polysorbate 80.

12. The formulation of claim 11, wherein the polysorbate 80 is in a concentration of about 0.025% to about 0.075% (w / v).

13. The formulation of claim 12, comprising polysorbate 80 in a concentration of 0.05% (w / v).

14. The formulation of any one of claims 1-13, wherein the formulation does not comprise sodium chloride.

15. The formulation of any one of claims 1-14, wherein the pH of the liquid formulation is between 5.7 and 6.3.

16. The formulation of claim 15, wherein the pH of the liquid formulation is between 5.8 and 6.2.

17. The formulation of claim 15, wherein the pH of the liquid formulation is 6.0.

18. The formulation of any one of claims 1-17, wherein the antibody is in a concentration of 135 mg / mL to 200 mg / mL.

19. The formulation of claim 18, wherein the antibody is in a concentration of 135 mg / mL to 165 mg / mL.

20. The formulation of claim 18, wherein the antibody is in a concentration of 150 mg / mL.

21. The formulation of any one of claims 1-20, further comprising arginine, proline, or glutamate.

22. The formulation of claim 21, comprising arginine hydrochloride in a concentration of about 1 mM to about 100 mM.

23. The formulation of claim 21, comprising L-proline in a concentration of about 1 mM to about 200 mM.

24. The formulation of claim 21, comprising L-glutamate in a concentration of about 1 mM to about 100 mM.

25. The formulation of any one of claims 1-24, wherein the formulation has an osmolality of between about 260 mOsm / kg and about 435 mOsm / kg.

26. The formulation of claim 25, wherein the formulation has an osmolality of about 290 mOsm / kg.

27. The formulation of claim 1, comprising:(a) the antibody that binds to a human Siglec-6 in a concentration of 150 mg / mL;(b) 20 mM L-histidine and / or L-histidine hydrochloride;(c) 170 mM sucrose; and(d) 0.05% polysorbate 80 (w / v); wherein the pH of the liquid formulation is 6.0.

28. The formulation of claim 1, comprising:(a) the antibody that binds to a human Siglec-6 in a concentration of 150 mg / mL;(b) 20 mM L-histidine and / or L-histidine hydrochloride;(c) 248 mM sucrose; and(d) 0.05% polysorbate 80 (w / v); wherein the pH of the liquid formulation is 6.0.

29. The formulation of claim 1, comprising:(a) the antibody that binds to a human Siglec-6 in a concentration of 150 mg / mL;(b) 20 mM L-histidine and / or L-histidine hydrochloride;(c) 160 mM sucrose; and(d) 0.05% polysorbate 80 (w / v); wherein the pH of the liquid formulation is 5.8.

30. The formulation of any one of claims 1-29, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 161, and the VL region comprises the amino acid sequence of SEQ ID NO: 162.

31. The formulation of any one of claims 1-30, wherein the heavy chain further comprises a human IgG Fc region.

32. The formulation of claim 31 , wherein the human IgG Fc region is a human IgGl Fc region.

33. The formulation of claim 32, wherein the human IgGl Fc region is non-fucosylated.

34. The formulation of any one of claims 1-29, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 165 or 166, and the light chain comprises the amino acid sequence of SEQ ID NO: 167.

35. The formulation of claim 33, wherein at least one or two of the heavy chains of the antibody is / are non-fucosylated.

36. An article of manufacture comprising a container enclosing the formulation of any one of claims 1-35.

37. The article of manufacture of claim 36, wherein the container is a glass vial.

38. The article of manufacture of claim 36 or claim 37, further comprising instructions for administering the formulation subcutaneously.

Images