Crystals of Anti-human TAU monoclonal antibody

Abstract

Crystals of an antibody that specifically binds to human tau protein phosphorylated at serine 413 are provided, as well as methods of producing such antibody crystals, compositions comprising the crystals, and uses of such compositions for treating a disease, e.g., Alzheimer's disease. Crystals suitable for X-ray diffraction are also provided, and the inventors herein have used such crystals to solve the three-dimensional structure of the antibody to 2.76 Å resolution.

Description

25845 CRYSTALS OF ANTI-HUMAN TAU MONOCLONAL ANTIBODY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 569,937 filed March 26, 2024, the entire contents of which are incorporated by reference herein. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (25845-WO-PCT_SL.xml; Size: 14,488 bytes; and Date of Creation: April 9, 2024 are herein incorporated by reference in their entirety. FIELD OF THE INVENTION

[0003] The disclosed invention relates to crystalline forms of an antibody that specifically binds to human tau protein phosphorylated at serine 413, methods of producing such crystalline forms, compositions comprising these crystalline forms, and uses of such compositions for treating a disease, e.g., Alzheimer’s disease. BACKGROUND OF THE INVENTION

[0004] Therapeutic antibodies are traditionally prepared in lyophilized form or in solution. Lyophilized forms generally exhibit enhanced long-term stability, but require reconstitution prior to use, making them less than ideal for self-administration. Solution formulations do not require reconstitution but may suffer from reduced stability and typically require cold storage prior to use. Both lyophilized and solution formulations may fail to provide sufficiently high concentrations to allow for high dose delivery by subcutaneous administration; the maximum volume for subcutaneous administration is quite low (only about 1.2 ml; see Yang, M. X. et al., Proc. Nat. Acad. Sci. USA 100:6934-6939 (2003)). Subcutaneous administration may be method of choice for administration of many therapeutic antibodies, in part because it enables self- administration. Also, high concentration solution formulations of antibodies, even when achievable, can be prone to the antibody dropping out of solution, or the solution being too viscous to be delivered in a narrow-gauge needle required for subcutaneous administration, particularly for self-administration.

[0005] One approach for achieving high concentration antibody formulations is to prepare the formulation with antibody crystals. See, e.g., Yang et al., 2003, supra; U.S. Pat. No.7,833,525, and WO 2012 / 135035. Antibody crystal formulations are desirable also because of the improvedstability of protein crystals in solution at room temperature, lower viscosity of high antibody concentration solutions, and the ability to manipulate crystallization conditions to produce crystals having different morphologies, and consequently different release properties (see, e.g., Yang et al., 2003, supra and Basu, S. K., et al., Expert Opin. Biol. Thera.4:301-317 (2004)).

[0006] However, unlike small molecules, only a few reports of methods for preparing crystalline protein suspensions exist. One such report relates to crystalline insulin suspensions which have been used as sustained release preparations for over fifty years (Brange, J. and Volund, A., Adv Drug Deliv Rev, 1999, 35(2-3):307-335). More recently, crystalline protein suspensions have been proposed for the delivery of interferon (US 6,004,549) and monoclonal antibodies (Yang, et al., 2003, supra). There are also potential opportunities for using crystalline suspensions in non-injectable delivery systems such local or systemic pulmonary delivery. While the ability to produce crystalline suspensions of proteins present opportunities for overcoming difficulties encountered in developing methods of delivering therapeutic protein, unlike large- scale crystallization-based purification of small organic molecules, which are common, only a few examples of large-scale crystallization in purification of proteins are available. An exception is the use of a crystallization step in the manufacture of interferon alpha-2b (IFN-α2b) where a temperature induction method is used in the purification process on a multigram scale. The process can be readily performed at the gram to kilogram range. There are several advantages of using protein crystallization for purification in a manufacturing process. Harvesting of crystals removes small molecule impurities present in the mother liquor or wash. The ability of a protein preparation to crystallize is a confirmation of the purity of the protein.

[0007] Among proteins, antibodies are known to be especially difficult to crystallize due to the flexibility of the multiple chains present in the antibody. Although, there have been numerous reports of crystallization of intact antibodies over the last 30 years, only six structures of such antibodies have been deposited in the RCSB Protein databank. In contrast, over 800 structures of apo Fab (fragment antigen binding) or Fab-antigen complexes have been deposited. As with most soluble proteins, intact antibodies can be screened for their crystallizability in sparse matrix screens using classical liquid or vapor diffusion crystallization methods (Yang et al., Proc. Nat. Acad. Sci. USA 2003, supra).

[0008] Methods to crystallize antibodies were first reported in WO 2002 / 072636, in which crystallization of three commercially available monoclonal antibodies, namely, rituximab, trastuzumab, and infliximab were described. Subsequent reports of antibody crystallization include those for the preparation of crystals of anti-IL-13 mAbs (WO 2005 / 121177), anti-TNF alpha mAbs (WO 2008 / 057240), anti-sclerostin mAbs (WO 2012 / 135035), anti-IL-23 mAbs(WO 2014 / 004436), and anti-PD-1 antibodies (WO 2016 / 137850, WO 2018 / 204368, and WO 2020 / 092233). These reports notwithstanding, it is generally agreed in the art of protein crystallization that identifying suitable crystallization conditions for a particular antibody is still an empirical exercise, and that there is no general rule that can be applied to a particular antibody of interest to reliably predict what crystallization conditions will produce crystals of that antibody.

[0009] Turning now to tau protein, WO 2021 / 262791 describes high affinity antibodies targeting human tau protein phosphorylated at serine 413. Tau is a brain-specific microtubule- associated protein enriched in axons. Its major function is to promote microtubule assembly and maintain microtubule structure. Phosphorylation decreases the ability of tau to bind microtubules. While tau is phosphorylated in healthy brains, it is hyperphosphorylated in tauopathies, which are a group of progressive neurodegenerative disorders that are pathologically defined by the presence of tau protein aggregates in the brain. More than 26 different tauopathies have been identified. Sexton, C et al., Alzheimers Dement, 2022 May; 18(5):988-1007. In some tauopathies, such as PiD (Pick's disease), PSP (Progressive supranuclear palsy), CBD (corticobasal degeneration) and AGD (Argyrophilic grain disease), tau is the major and prominent component of the pathology. In other tauopathies such as Alzheimer’s disease (AD) and repetitive brain injury in chronic traumatic encephalopathy (CTE), tau aggregation is understood to be a response to other pathological proteins or events. For example, in AD, tau pathology is thought to be driven or accelerated by amyloid beta (Aβ)

[0010] In AD, brains of patients having the disease are characterized by the presence of two classes of microscopic abnormal structures: extracellular amyloid plaques and intraneuronal neurofibrillary tangles (NFTs). Miao J et al., Frontiers in Aging Neuroscience, March 2019, vol. 11, Article 34. These structures comprise highly insoluble and densely packed filaments which are produced from soluble building blocks, amyloid-β (Aβ) peptides for plaques and tau for neurofibrillary tangles. In one study for investigating human tauopathies, the authors used transgenic mice dominantly expressing human tau in adult age. The mice exhibited abnormal tau phosphorylation, synapse loss, memory impairment at six months, neurofibrillary tangle formation, and neuronal loss at 24 months, and it was found that in these mice, tau was phosphorylated at serine 413 early and at a high level. Umeda et al., Annals of Clinical and Translational Neurology 2015; 2(3): 241–255. Further, an anti-pSer413 antibody was shown to significantly improve memory. This cognitive improvement was found to parallel reduction in the levels of tau hyperphosphorylation, tau oligomer accumulation, synapse loss, tangle formation, and neuronal loss. These findings suggested that tau, phosphorylated at serine 413(pSer413 tau), is a promising target for intervention in the treatment of tauopathy, and antibodies targeting tau protein phosphorylated at serine 413 (anti-pSer413 tau antibodies) may be therapeutically effective for treating AD.

[0011] Given that compositions of antibody crystals provide advantages including improved stability of the antibody in liquid solutions at room temperature and lower viscosity of high concentration antibody solutions, a need exists for methods to prepare crystalline forms of anti- pSer413 tau antibodies. Such crystalline forms may be useful for preparing improved pharmaceutical compositions comprising anti-pSer413 tau antibody for treating AD. SUMMARY OF THE INVENTION

[0012] In part, the present disclosure provides methods for producing crystals and crystalline suspensions of a high affinity antibody targeting human tau protein phosphorylated at serine 413. This antibody is described in WO 2021 / 262791 and has a light chain amino acid sequence of SEQ ID NO: 73 and a heavy chain amino acid sequence of SEQ ID NO: 74 (represented herein, respectively, as SEQ ID NO: 3 and SEQ ID NO: 8). This high affinity anti-pSer413 tau antibody is referred to hereinafter as HA anti-pSer413 tau mAb.

[0013] Accordingly, the invention disclosed herein provides a crystalline form of HA anti- pSer413tau mAb and methods of producing the same.

[0014] The invention also provides suspensions of the crystalline forms of HA anti-pSer413tau mAb, e.g., as crystalline slurries.

[0015] The invention also provides methods of preparing crystalline forms of HA anti- pSer413tau mAb.

[0016] Further, the invention provides pharmaceutical compositions comprising suspensions of the crystalline forms of HA anti-pSer413tau mAb.

[0017] The crystalline forms of HA anti-pSer413tau mAb of the invention may be used to facilitate purification, storage, and therapeutic administration of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a photomicrograph of crystals in an HA anti-pSer413tau mAb crystalline suspension, obtained by vapor diffusion at 22 °C using a precipitant solution of 1.2 M ammonium sulphate, 200 mM sodium chloride, and 100 mM sodium acetate, pH 4.6 (Example 1, Hit No.1). The photomicrograph was taken at 100× magnification after 1 week.

[0019] Figure 2A is a photomicrograph of a crystal in an HA anti-pSer413tau mAb crystalline suspension, obtained by vapor diffusion at 22 °C using a precipitant solution of 1.2 Mammonium sulphate and 100 mM sodium citrate pH 4.8 (Example 2). The photomicrograph was taken at 100× magnification after 1 week.

[0020] Figure 2B is a photomicrograph of crystals in an HA anti-pSer413tau mAb crystalline suspension, obtained by vapor diffusion at 22 °C using a precipitant solution of 1.3 M ammonium sulphate and 100 mM sodium citrate pH 4.9 (Example 2). The photomicrograph was taken at 100× magnification after 1 week.

[0021] Figure 3 is a photomicrograph of crystals in a HA anti-pSer413tau mAb crystalline suspension, obtained by vapor diffusion at 22 °C using a precipitant solution of 2% PEG 4000 and 1.8 M ammonium citrate dibasic pH 4.8 (Example 3). The photomicrograph was taken at 100× magnification after 1 week.

[0022] Figure 4 is a photomicrograph of crystals in a HA anti-pSer413tau mAb crystalline suspension, obtained by vapor diffusion using seed crystals (Example 4). Specifically, 260 nl of 50 mM ammonium sulphate,100 mM sodium acetate pH 4.5, 34% w / v PEG 4000 (precipitant / reservoir solution) was added to 100 nl of HA anti-pSer413tau mAb (61 mg / ml), and to this mixture, 40 nl of seed crystals stock was added. The photomicrograph was taken at 100× magnification after 1 week. DETAILED DESCRIPTION OF THE INVENTION

[0023] The crystalline suspensions of a high affinity antibody targeting tau protein phosphorylated at serine 413 and pharmaceutical compositions thereof address the need for high concentration solution formulations of the antibody for treating tauopathies, e.g., AD. Definitions

[0024] So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

[0026] As used herein, “about,” when used to modify a numerically defined parameter (e.g., the concentration of a component in a solution) means that the parameter may vary by as much as 10% above or below the stated numerical value for that parameter. For example, a composition comprising about 200 mg / ml of a specified antibody may have between 180 mg / ml and 220mg / ml of the antibody. Similarly, a temperature of about 30 °C means any temperature between 27° C. and 33° C.

[0027] “Administration” and “treatment,” as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. “Administration” and “treatment” can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. “Administration” and “treatment” also include in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell.

[0028] The term “patient” (alternatively referred to as “subject” or “individual” herein) refers to a mammal (e.g., rat, mouse, dog, cat, rabbit) capable of being treated with the formulations of the invention, most preferably a human. In some embodiments, the patient is an adult patient. Those “in need of treatment” include those patients that may benefit from treatment with the formulations of the invention, e.g., a patient suffering from AD.

[0029] As used herein the term “antibody” refers to a tetramer that includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain defines a constant region primarily responsible for effector function. The variable regions of each light / heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are identical.

[0030] Typically, the variable regions of each of the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C- terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 (i.e., CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5thed.; NIH Publ. No.91-3242 (1991); Kabat (1978) Adv. Prot. Chem.32:1-75; Kabat, et al., (1977) J. Biol.25845 Chem.252:6609-6616; Chothia, et al., (1987) J Mol. Biol.196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0031] Ammonium dihydrogen phosphate (NH4H2PO4) or ADP as used herein is synonymous with ammonium phosphate monobasic, mono-ammonium phosphate and prim-ammonium phosphate.

[0032] Ammonium hydrogen phosphate ((NH4)2HPO4) or AHP as used herein is synonymous with ammonium phosphate dibasic, diammonium hydrogen phosphate and diammonium hydrogen phosphate.

[0033] “Concentration”, when used with reference to a crystalline antibody suspension of the invention, refers to the amount of antibody present in a given macroscopic unit volume of solution. The term concentration is used in its customary sense despite the inherent heterogeneity of the suspension, as compared to a traditional solution. The concentration of antibody in a crystalline suspension is equal to the concentration of an equivalent sample in which the antibody is not in crystalline form.

[0034] In this disclosure, terms such as “comprises,” “comprised,” “comprising,” “contains,” “containing” and the like mean “includes,” “included,” “including” and the like. Such terms refer to the inclusion of particular ingredients or set of ingredients without excluding any other ingredients.

[0035] Terms such as “consisting essentially of” and “consists essentially of” allow for the inclusion of additional ingredients or steps that do not detract from the novel or basic characteristics of the disclosure, i.e., they exclude additional unrecited ingredients or steps that detract from the novel or basic characteristics of the disclosure. As a non-limiting example, a pharmaceutical composition that consists essentially of antibody crystals and a specific pharmaceutically acceptable excipient may also include one or more other excipients that do not materially affect the properties of the pharmaceutical composition.

[0036] The terms “consists of” and “consisting of” are closed ended. Accordingly, these terms refer to the inclusion of a particular ingredient or set of ingredients and the exclusion of all other ingredients.

[0037] As used herein, an “antibody solution” may be used to refer to a solution of an anti-tau antibody that is used to generate the crystalline antibody of the invention. “Precipitant solution” refers to a second solution that is mixed with the antibody solution, typically at a 1:1 volume ratio (i.e., equal volumes of the two solutions are mixed) to create a “crystallization solution” or “crystallization mixture” from which antibodies crystals grow. The concentrations of the antibody and precipitant solutions are provided herein for a 1:1 mixture, for convenience, but oneof skill in the art would recognize that the volume ratio used to make the mixture can be changed, and thus so can the concentrations of the solutions making up the mixture. Such modifications are within the scope of the invention if they generate the same crystallization conditions (i.e., the same crystallization solution) as the mixtures described herein.

[0038] A “precipitant” is a compound that decreases the solubility of a polypeptide, such as an antibody, in a concentrated solution. In batch crystallization methods the precipitant is included in the “precipitant solution,” and in bulk dialysis methods the precipitant is included in the “dialysis solution.” Precipitants induce crystallization by forming an energetically unfavorable precipitant-depleted layer around the polypeptide molecules. To minimize the relative amount of this depletion layer, the polypeptides form associations and, ultimately, crystals. This process is explained in Weber (1991) Advances in Protein Chemistry 41:1, which is incorporated herein by reference. Various precipitants are known in the art and include ammonium sulphate, ammonium di-hydrogen phosphate, ethanol, isopropanol, propylene glycol, 3-ethyl-2, 4 pentanediol; and many of the polyglycols, such as polyethylene glycol (e.g., PEG 4000 and PEG 10000). In addition to precipitants, other materials are sometimes added to the polypeptide precipitant solution. These include buffers, such as Tris or HEPES, to adjust the pH of the solution (and hence surface charge on the peptide) and salts, such as ammonium mono-hydrogen phosphate, sodium sulphate, sodium chloride, lithium chloride and sodium citrate, to reduce the solubility of the polypeptide.

[0039] When used with reference to a crystalline antibody suspension of the invention, “concentration” refers to the amount of antibody (in this case anti-tau antibody) present in a given macroscopic unit volume of solution. The term concentration is used in its customary sense despite the inherent heterogeneity of the suspension, as compared to a traditional solution. The concentration of antibody in a crystalline suspension is equal to the concentration of an equivalent sample in which the antibody was not in the crystalline form.

[0040] 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 that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. 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 to be used in accordance with the invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

[0041] As used herein, “HA anti-pSer413 tau mAb” refers to a monoclonal antibody that binds specifically to a human tau protein epitope comprising phosphorylated serine 413 (anti-pSer413 tau). The human tau protein has the amino acid sequence of SEQ ID NO: 1 and the monoclonal antibody has a light chain having the amino acid sequence of SEQ ID NO: 3 and a heavy chain having the amino acid sequence of SEQ ID NO: 8.

[0042] As used herein, “HA anti-pSer413 tau mAb crystal” or “crystalline HA anti-pSer413 tau mAb” refers to a crystal containing the antibody arranged in a lattice structure that repeats periodically in three dimensions. In contrast, a solid, amorphous form of the antibody, e.g., such as produced by lyophilizing an antibody dissolved in a solution, does not display the optical properties such as refractive index and birefringence that are typical of a crystalline antibody form.

[0043] As used herein, and regarding crystallization methods based on dialysis, “dialysis solution” refers to the solution against which a solution of HA anti-pSer413 tau mAb (the “antibody solution”) is dialyzed to drive formation of the crystalline antibody of the invention.

[0044] “Retentate” refers to the antibody solution after dialysis, which may include crystals of the antibody, which are harvested. The antibody solution / retentate are on one side of the dialysis membrane, and the dialysis solution is on the opposite side.

[0045] The terms “micron” and “micrometer” are used interchangeably herein, and each means 1 / 1000000th of a meter. Tau protein

[0046] Tau is a protein encoded by the MAPT gene, which is located on chromosome 17 (17q21) in the human genome. Tau is one of the microtubule-binding proteins abundantly expressed in the central nervous system. It has been found to be a major constituent protein in the paired helical filaments and straight filaments forming neurofibrillary tangles (NFT) in AD.25845

[0047] The MAPT gene encoding tau has been identified as consisting of 14 exons and it can be expressed as multiple protein isoforms via alternative splicing (Martin et al., 2011, Neurochem. Int.58, 458–471). The tau protein comprises an N-terminal acidic domain containing 0-2 repeats of a 29 amino acids sequence (N) encoded by exon 2 and exon 3 depending on alternative splicing of these exons (referred to as 0N, 1N, and 2N), an intermediate domain rich in proline, and a C-terminal microtubule-binding domain (encoded by exons 9 to 12) containing 3 (3R) or 4 (4R) repetitive sequences (R) that contribute to microtubule binding (Barbier P. et al., 2019, Front. Aging Neurosci.11:204). Therefore, human tau has six representative isoforms: 3R0N (352 amino acids), 3R1N (381 amino acids), 3R2N (410 amino acids), 4R0N (383 amino acids), 4R1N (412 amino acids), and 4R2N (441 amino acids), depending on the number of 29 amino acid repetitive sequences (N) and microtubule-binding repetitive sequences (R) that it contains. The difference between the 3R and 4R isoforms results from whether exon 10 is removed via alternative splicing (3R) or present (4R). In this disclosure, tau is used to refer to the longest isoform 4R2N, which has the amino acid sequence of SEQ ID NO: 1 shown below in Table 1.

[0048] Tau is phosphorylated at many serine and threonine residues. One of these serine residues is Ser 413 (isoform 4R2N). Tau phosphorylated at Ser 413 is referred to herein as “pSer413 tau.” As previously shown in WO 2013 / 180238, administration of antibodies that participate in specific antigen-antibody reactions with pSer413 tau to transgenic mice which develop cognitive function impairment while maturing, resulted in restoration of cognitive functions to almost the same level as that of the control group. Interestingly, administration of similar concentration of a monoclonal antibody against a tau protein having a phosphorylated amino acid residue at the position corresponding to Ser 396, which has a stronger affinity to an equivalent antigen than the above antibody, did not result in sufficient improvement in cognitive functions. WO 2018 / 154390 describes humanized anti-phosphorylated tau antibodies exhibiting high binding affinity to tau phosphorylated at Ser413 while having significantly reduced antigenicity to the human body for use as a therapeutic or prophylactic agent for cognitive disorders such as tauopathy in a human subject. The ‘390 application also discloses amino acid modifications within the CDRs as compared to the murine CDRs that reduce deamidation leading to increased stability. General Methods

[0049] Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 19892ndEdition, 20013rdEdition) Molecular Cloning, A Laboratory Manual, Cold25845 Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; and Wu (1993) Recombinant DNA, Vol.217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol.1), cloning in mammalian cells and yeast (Vol.2), glycoconjugates and protein expression (Vol.3), and bioinformatics (Vol.4).

[0050] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described in Coligan, et al. (2000) Current Protocols in Protein Science, Vol.1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins are also described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol.2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol.3, John Wiley and Sons, Inc., NY, NY, pp.16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp.45-89; and Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp.384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described in Coligan, et al. (2001) Current Protocols in Immunology, Vol.1, John Wiley and Sons, Inc., New York; and Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques for characterizing ligand / receptor interactions are also available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol.4, John Wiley, Inc., New York).

[0051] Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Shepherd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp.139-243; Carpenter, et al. (2000) J. Immunol.165:6205; He, et al. (1998) J. Immunol.160:1029; Tang et al. (1999) J. Biol. Chem.274:27371-27378; Baca et al. (1997) J. Biol. Chem.272:10678-10684; Chothia et al. (1989) Nature 342:877-883; and Foote and Winter (1992) J. Mol. Biol.224:487-499; U.S. Pat. No.6,329,511).

[0052] An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol.14:309- 314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146- 156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage25845 Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; and de Bruin et al. (1999) Nature Biotechnol.17:397-399).

[0053] Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nded.; Wiley-Liss, Hoboken, NJ; and Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0054] Standard methods of histology of the immune system are described (see, e.g., Muller- Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; and Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0055] Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available (see, e.g., GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren, et al. (2002) Comput. Methods Programs Biomed.68:177-181; von Heijne (1983) Eur. J. Biochem.133:17-21; and von Heijne (1986) Nucleic Acids Res.14:4683-4690. HA anti-pSer413 tau mAb

[0056] As disclosed in WO 2021 / 262791A1, HA anti-pSer413 tau antibody was generated by yeast display-based affinity maturation of the humanized antibody Ta1505-hIgG4-S228P described in WO 2018 / 154390. Affinity maturation led to mutations in CDR2 of the heavy chain variable region (VH) and in CDR1 and CDR3 of the light chain variable region (VL) of Ta1505- hIgG4-S228P. An additional mutation (K54E or K54D) was introduced in VH for lowering the isoelectric point of antibody while maintaining or improving its affinity. HA anti-pSer413 tau mAb has IgG1 heavy chain and kappa light chain. Additional changes were made to the heavy chain constant region to improve half-life of the antibody (LALA and YTE mutations). The amino acid sequences of the heavy and light chain (with and without signal peptide) as well the25845 variable regions of the heavy (VH) and light chain (VL) of HA anti-pSer413 tau mAb are shown below in Table 1 (both LALA and YTE mutations are indicated in bold). The nucleic acid sequences of the heavy and light chain (with signal peptide) also are shown in Table 1.

[0057] HA anti-pSer413 tau antibody may be produced using any method known in the art. For example, the antibody may be produced recombinantly. Nucleic acids encoding the antibody molecules of the invention (e.g., heavy and light chain) may be inserted into a vector and expressed in a recombinant host cell. Several methods for producing recombinant antibodies are known in the art.

[0058] Mammalian cell lines that can be used as hosts for expression of the antibodies or antibody fragments (e.g., VHand VL) are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and several other cell lines. Among these, cell lines that have high expression levels may be more desirable. Other cell lines that may be used are insect cell lines (e.g., Sf9 cells), amphibian cells, bacterial cells, plant cells, and fungal cells. When recombinant expression vectors encoding the heavy chain or antigen-binding fragment thereof and the light chain or antigen-binding fragment thereof are introduced into host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.

[0059] Antibodies can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions.

[0060] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. In some cases, antibodies with a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because these antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem.278: 3466-3473 (2003); U.S. Patent Nos.6,946,292 and 7,214,775).25845 Sequences Table 1 SEQ Sequence Sequence (SEQ ID NOs: 6 and 11 are nucleotide sequences. ID Description All other are amino acid sequences)25845 SEQ Sequence Sequence (SEQ ID NOs: 6 and 11 are nucleotide sequences. ID Description All other are amino acid sequences) NO: P A V L P K25845 SEQ Sequence Sequence (SEQ ID NOs: 6 and 11 are nucleotide sequences. ID Description All other are amino acid sequences) NO:Antibody crystallization

[0061] The invention is based, in part, on the identification of methods for crystallization of HA anti-pSer413 tau mAb. The crystallization methods comprise a unique combination of: (1) a precipitant solution which comprises a high salt concentration or PEG and has an acidic pH (4.2 - 5.0; and (2) a temperature between about 18 °C to about 35 °C. The pH may be maintained within the required range by including a buffering agent in the precipitant solution. Suitable buffering agents include, e.g., Tris-HCl, ammonium hydrogen phosphate, histidine, and ammonium hydroxide. The pH of the precipitant solution is preferably determined for the temperature at which the crystallization is to be performed. Antibody solution is mixed with the preciptatnt solution to obtain a crystallization mixture, to which a crystallization process is applied. The methods of the invention are compatible with several crystallization processes and can produce crystalline forms of the HA anti-pSer413 tau antibody in a variety of lengths, (e.g., 1 to 20 microns and 5 to 100 microns) and diffraction resolution (low resolution, e.g., 3.5 Å or high resolution, e.g., 2.3 Å), depending on the intended use of the crystals. Crystallization processes25845 contemplated include hanging drop vapor diffusion, sitting drop vapor diffusion, dialysis, and batch (microbatch).

[0062] Various methods of protein crystallization are known. See Giege et al. (1994) Acta Crystallogr. D50:339; and McPherson (1990) Eur. J. Biochem.189:1. Such methods include hanging drop vapor diffusion (McPherson (1976) J. Biol. Chem.251:6300), sitting drop vapor diffusion, microbatch, and dialysis.

[0063] Both hanging drop and sitting drop vapor diffusion entail a droplet containing purified protein, buffer, and precipitant, being allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. In some instances, the reservoir may contain the same solution as the precipitant solution, which, when mixed with the protein solution in the droplet becomes diluted, and the droplet comes to contain the precipitant solution at a relatively lower concentration. In these instances, the terms “reservoir solution” and “precipitant solution” may be used interchangeably. Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water vaporizes from the drop and transfers to the reservoir, the precipitant concentration increases to a level optimal for crystallization. Since the system is in equilibrium, these optimum conditions are maintained until the crystallization is complete. The hanging drop method differs from the sitting drop method in the vertical orientation of the protein solution drop within the system.

[0064] In the micro batch method, polypeptide is mixed with precipitants to achieve supersaturation, and the vessel is sealed and set aside until crystals appear.

[0065] In the dialysis method, polypeptide is retained on one side of a dialysis membrane which is placed into contact with a solution containing precipitant. Equilibration across the membrane increases the precipitant concentration thereby causing the polypeptide to reach supersaturation levels.

[0066] Some of these techniques were used to prepare HA anti-pSer413 tau mAb crystals of the invention, as described in greater detail in Examples 1-6. Of note, the high-throughput screening conditions described in Example 1 are better suited for identifying the precipitant solution for successful crystallization, than for large scale crystal production.

[0067] Typically, about 10 to 100 mg / ml of HA anti-pSer413 mAb in about 10 mM histidine buffer, pH of about 5.4, was used for crystallization experiments. However, it is contemplated that the mAb concentration could be higher, for example, 150 to 200 mg / ml.

[0068] For large scale production of HA anti-pSer413 mAb crystals, e.g., for therapeutic use, batch crystallization may be used. This method involves preparing a crystallization mixture comprising the antibody and a precipitant. An additive may also be added. Examples of additives25845 include 3% 1,5 di-amino pentane di-hydrochloride, 3% isopropanol, and 4% propylene glycol. Crystals may be harvested from a batch crystallization mixture using methods known in the art, such as centrifugation, dialysis, and various filtration methods, including hollow fiber tangential flow filtration.

[0069] The HA anti-pSer413 mAb crystals may be analyzed by various methods to examine or characterize their physical properties, such as crystal size, shape, surface morphology, total surface area, and porosity. Such analytical techniques include, e.g., electron diffraction, solid state nuclear magnetic resonance (ssNMR), light microscopy, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and various light scattering techniques.

[0070] The biological activity and / or biophysical properties of the HA anti-pSer413 mAb in crystals of the invention may be analyzed by “re-dissolving” or solubilizing the antibody crystal in a buffer suitable for the desired analytical technique. For example, the solubilized HA anti- pSer413 mAb may be analyzed by one or more of ELISA, size exclusion chromatography, SDS PAGE, and dynamic light scattering. It is contemplated that the methods described herein will be useful in batch crystallization techniques to prepare crystalline suspensions of HA anti-pSer413 mAb.

[0071] HA anti-pSer413 mAb that has been solubilized from antibody crystals prepared in accordance with the invention should retain the properties of the pre-crystallization starting material within acceptable tolerances. Acceptable tolerances for the various functional parameters may vary based on the intended use. Regarding binding affinity or biological activity, it is contemplated that the HA anti-pSer413 mAb, solubilized from crystals, would retain at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of the original (non-crystallized) affinity or activity. For example, the ability of the HA anti-pSer413 mAb to bind tau peptides phosphorylated at serine 413 may be measured by surface plasmon resonance as described in WO 2021 / 262791. Also, as described in WO 2021 / 262791, the ability of the HA anti-pSer413 mAb to bind FcRn with a higher affinity due to the presence of the YTE mutation may be measured using surface plasmon resonance. Pharmaceutical Compositions

[0072] To prepare pharmaceutical compositions, the HA anti-pSer413 crystals of the invention, or HA anti-pSer413 mAb solubilized from such crystals, are mixed with at least one pharmaceutically acceptable excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia:National Formulary, Mack Publishing Company, Easton, Pa. (1984). It is not required that the HA anti-pSer413 crystals used in a pharmaceutical composition of the invention25845 have any particular diffraction quality, as long as the biological activity and stability of the antibody are maintained within the desired range.

[0073] The excipient(s) may be added directly to the crystallization liquor during or after crystallization. Alternatively, the crystals may first be harvested from the liquor, washed by suspension in a stabilizing solution, harvested from the stabilizing solution and then suspended in a liquid solution comprising the excipient(s). The composition of the liquid may be any pharmaceutically acceptable medium, and may include, e.g., aqueous solutions and water in oil mixtures.

[0074] Pharmaceutical compositions of crystals in a solid form may be prepared by drying a liquid suspension comprising the crystals and the desired excipient(s), e.g., by passing a stream of nitrogen, air or inert gas over the crystals, by air drying, vacuum drying, or lyophilization. The moisture content in the final product will typically be less than 10%, less than 7%, less than 5%, or less than 3% by weight.

[0075] A pharmaceutical composition comprising HA anti-pSer413 mAb that has been solubilized from HA anti-pSer413 mAb crystals in a liquid suspension or in a dried solid may be prepared by adding a desired quantity of the crystals to a pharmaceutically acceptable dissolution buffer and incubating at 4° C until the crystals have dissolved. Particulates in the resulting composition may be removed prior to administration, e.g., by centrifugation or filtration. Treatment Methods

[0076] Determination of the appropriate dose of a pharmaceutical composition of the invention for treating a tauopathy in a particular patient may be made by the clinician, e.g., by using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. For example, the physician may choose to initiate treatment with a dose that is somewhat less than the optimum dose or the approved dose and then increase the dose by small increments until the desired or optimum effect is achieved relative to any negative side effects.

[0077] The dose and administration route of the composition will provide a median exposure to HA anti-pSer413 mAb that is substantially similar to that provided by un-crystallized HA anti- pSer413 mAb. Embodiments

[0078] The invention is set forth more specifically in the following.25845

[0079] In one aspect, the invention provides a crystal of an anti-pSer413 tau monoclonal antibody (HA anti-pSer413 tau mAb) having a light chain amino acid sequence of SEQ ID NO: 3 and a heavy chain amino acid sequence of SEQ ID NO: 8.

[0080] In one embodiment, the crystal has unit cell dimensions of a =140.36 Å, b =186.66 Å, c =85.19 Å, α =90°, β =90°, γ = 90° and space group P21212. The crystal may have a length in the range of 0.5 to 300 microns.

[0081] In one embodiment, the crystal diffracts X-rays to a maximum resolution of about 2.5 Å -2.76 Å. In one embodiment, the crystal diffracts X-rays to a maximum resolution of about 2.76 Å.

[0082] In some embodiments, the HA anti-pSer413 tau mAb used for obtaining the crystal is produced by recombinant DNA technology.

[0083] Further provided herein is a pharmaceutical composition comprising the crystal of any one of the preceding embodiments and at least one pharmaceutically acceptable excipient.

[0084] In one embodiment, in the pharmaceutical composition, the HA anti-pSer413 tau mAb crystals are suspended in a liquid and the concentration of the antibody in the composition is at least about 100 mg / ml. In alternative embodiments, the concentration of the antibody in the composition is in the range of about 50-150 mg / ml, about 75-175 mg / ml, about 100-200 mg / ml, about 125-225 mg / ml, about 150-250 mg / ml, about 175-275 mg / ml, about 200-300 mg / ml, about 225-325 mg / ml, 250-350 mg / ml, 275-375 mg / ml, 300-400 mg / ml, 325-425 mg / ml.350-450 mg / ml, 375-475 mg / ml, 400-500 mg / ml, 425-525 mg / ml, 50-100 mg / ml, 75-125 mg / ml, 100-150 mg / ml.125-175 mg / ml, 150-200 mg / ml, 175-225 mg / ml, 200-250 mg / ml, 225-275 mg / ml, 250- 300 mg / ml, 275-325 mg / ml, 300-350 mg / ml, 325-375 mg / ml, 350-400 mg / ml, 375-425 mg / ml, 400-450 mg / ml, 425-475 mg / ml, 450-500 mg / ml, 475-525 mg / ml, 50-525 mg / ml, 100-500 mg / ml, 150-450 mg / ml, 200-400 mg / ml or 250-350 mg / ml.

[0085] Also provided herein is method for producing crystalline HA anti-pSer413 tau mAb of any of the preceding embodiments. This method comprises: (i) mixing a solution comprising the antibody with a precipitant solution, thereby obtaining a crystallization mixture, (ii) applying a crystallization process to the crystallization mixture for a duration sufficient for crystal formation at a temperature of about 18 °C to about 35 °C, and (iii) optionally harvesting the crystalline antibody from the mixture, such that the precipitant solution comprises (a) about 1.4 mM ammonium sulphate and (b) about 100 mM sodium citrate having a pH of about 4.8, and the crystallization process is vapor diffusion.25845

[0086] In one embodiment, in the above method for producing crystalline HA anti-pSer413 tau mAb, the crystallization process occurs at about 22 °C. In one embodiment, the concentration of the mAb in the solution comprising the mAb is about 60 mg / ml. In one embodiment, the solution comprising the antibody is a buffered solution having a pH of about 4.0 to about 8.0. In one embodiment, HA anti-pSer413 tau mAb is provided in 10 mM Histidine, having a pH of about 5.4.

[0087] In one embodiment, provided herein is a method of treating tauopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the crystal of any one of the preceding embodiments and at least one pharmaceutically acceptable excipient.

[0088] The tauopathy may be one of Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, argyrophilic grain dementia (argyrophilic grain disease), multiple system tauopathy with presenile dementia (MSTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), dementia with neurofibrillary tangles, diffuse neurofibrillary tangle with calcification (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), frontotemporal lobar degeneration with tau pathology (FTLD-tau), Economo’s encephalitis sequela, subacute sclerosing panencephalitis, and boxer's encephalopathy.

[0089] In one embodiment, the tauopathy is Alzheimer’s disease.

[0090] In another aspect, the invention provides a method for producing crystalline anti- pSer413 tau monoclonal antibody (HA anti-pSer413 tau mAb), the antibody having a light chain amino acid sequence of SEQ ID NO: 3 and a heavy chain amino acid sequence of SEQ ID NO: 8 comprising: (i) mixing a solution comprising the antibody with a precipitant solution, thereby obtaining a crystallization mixture, (ii) applying a crystallization process to the crystallization mixture for a duration sufficient for crystal formation at a temperature between about 18 °C to about 35 °C, and (iii) optionally harvesting the crystalline HA anti-pSer413 tau mAb from the mixture, wherein the precipitant solution has a pH of about 4.0 to about 5.0 and comprises (a) 50 mM to about 2500 mM ammonium sulphate or ammonium citrate dibasic, or a combination thereof, and optionally, and one or more components selected from the group consisting of (b) about 50 mM to about 200 mM sodium acetate, (c) about 100 mM to about 250 mM sodium chloride,25845 (d) about 1% to about 40% weight per volume (w / v) of polyethylene glycol (PEG) ranging from PEG 1000 to PEG 5000, and (e) about 50 mM to about 150 mM sodium citrate or potassium citrate having a pH of about 4.0 to about 5.0.

[0091] In one embodiment, step (i) of paragraph

[0088] further comprises adding a seed crystal of HA anti-pSer413 tau mAb prior to mixing.

[0092] In one embodiment, step (ii) of paragraphs

[0088] or

[0089] comprises a process selected from the group consisting of (i) hanging drop vapor diffusion, (ii) sitting drop vapor diffusion, (iii) microbatch crystallization, and (iv) batch crystallization.

[0093] In some embodiments the PEG used is PEG 3350 / 4000.

[0094] In one embodiment, the ratio of the volume of the solution comprising the antibody and the precipitant solution is 1:3, 1:2, 1:1, 2:1, or 3:1.

[0095] In some embodiments, the concentration of HA anti-pSer413 tau mAb in the solution comprising the antibody is about 5 to about 220 mg / ml. In some embodiments, the concentration of the HA anti-pSer413 tau mAb in the solution comprising the antibody is about 40 to about 100 mg / ml. In some embodiments, the solution comprising HA anti-pSer413 tau mAb is a buffered solution having a pH of 4.0 to 8.0. In some embodiments, the solution comprising HA anti- pSer413 tau mAb is a histidine or citrate buffered solution having a pH of about 4.0 to about 8.0.

[0096] In some embodiments, step (ii) is carried out at about 22 °C. In other embodiments, step (ii) is carried out at about 30 °C.

[0097] In one embodiment, the precipitant solution comprises (a) about 1.2 M ammonium sulphate, (b) about 200 mM NaCl, and (c) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about pH 4.5, the temperature is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise PEG.

[0098] In one embodiment, the precipitant solution comprises (a) about 1.2 M ammonium sulphate, and (b) about 100 mM sodium citrate, and wherein the precipitant solution has a pH of about pH 4.8, the temperature is about 22 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise PEG or NaCl.

[0099] In one embodiment, the precipitant solution comprises (a) about 1.3 M ammonium sulphate, and (b) about 100 mM sodium citrate, and wherein the precipitant solution has a pH of about pH 4.9, the temperature is about 22 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise PEG or NaCl.25845

[0100] In one embodiment, the precipitant solution comprises (a) about 2.0 M ammonium sulphate, (b) about 200 mM NaCl, and (c) about 100 mM acetate, and wherein the precipitant solution has a pH of about pH 4.6, the temperature is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise PEG.

[0101] In one embodiment, the precipitant solution comprises (a) about 1.5 M ammonium citrate dibasic, and (b) about 2% w / v of PEG 3350, and wherein the precipitant solution has a pH of about pH 4.8, the temperature is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise NaCl.

[0102] In one embodiment, the precipitant solution comprises (a) about 1.5 M ammonium citrate dibasic, and (b) about 2% w / v of PEG 4000, and wherein the precipitant solution has a pH of about pH 4.0, the temperature is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise NaCl.

[0103] In one embodiment, the precipitant solution comprises (a) about 1.8 M ammonium citrate dibasic, pH 4.8, and (b) about 2% w / v of PEG 4000, wherein the temperature is about 22 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise NaCl.

[0104] In one embodiment, the precipitant solution comprises (a) about 1.4 M ammonium sulphate, and (b) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about pH 4.5, the temperature is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise PEG. In some embodiments, the precipitant solution does not comprise NaCl. In some embodiments, the precipitant solution does not comprise PEG or NaCl.

[0105] In one embodiment, the precipitant solution comprises (a) about 1.0 M ammonium sulphate and (b) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about pH 4.6, the temperature is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise PEG. In some embodiments, the precipitant solution does not comprise NaCl. In some embodiments, the precipitant solution does not comprise PEG or NaCl.

[0106] In one embodiment, the precipitant solution comprises (a) about 2.0 M ammonium sulphate, and (b) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about pH 4.6, the temperature is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion. In some embodiments, the precipitant solution does not comprise PEG. In some25845 embodiments, the precipitant solution does not comprise NaCl. In some embodiments, the precipitant solution does not comprise PEG or NaCl.

[0107] In one embodiment, the preceding embodiments of this aspect of the invention further comprises, in step (i), adding a suspension comprising seed crystals of HA anti-pSer413 tau mAb prior to mixing, wherein the precipitant solution comprises about 50 mM ammonium sulphate, about 25% to about 40% w / v of PEG 4000, and about 100 mM sodium acetate having pH between about 4.5 and about 5.0, and the crystallization process is vapor diffusion.

[0108] In one embodiment, PEG 4000 is at about 34 % w / v.

[0109] In one embodiment, the suspension comprising seed crystals of the preceding two embodiments is produced following the method described above in paragraph

[0088] , wherein the precipitant solution for producing the suspension comprises about 0.5 to about 1.5 M ammonium sulphate, and about 100 mM sodium citrate, and has a pH of about 4.5 to about 5.5, the crystallization process is vapor diffusion, and step (iii) comprises combining drops having crystals of the antibody, and optionally agitating the combined drops. In one embodiment, the combined drops are agitated to reduce crystal size. In some embodiments, the precipitant solution does not comprise PEG. In some embodiments, the precipitant solution does not comprise NaCl. In some embodiments, the precipitant solution does not comprise sodium acetate. In some embodiments, the precipitant solution does not comprise PEG, sodium acetate, or NaCl.

[0110] In one embodiment, the invention provides crystalline HA anti-pSer413 tau mAb produced by the any of methods of this aspect of the invention. The antibody may be produced by recombinant DNA technology.

[0111] In one embodiment, the invention provides a pharmaceutical composition comprising the crystal of any of the two preceding embodiments and at least one pharmaceutically acceptable excipient. In one embodiment, the HA anti-pSer413 tau mAb crystal is suspended in a liquid and the concentration of the antibody in the composition is at least 100 mg / ml. In alternative embodiments, the concentration of the antibody in the composition is in the range of about 50- 150 mg / ml, about 75-175 mg / ml, about 100-200 mg / ml, about 125-225 mg / ml, about 150-250 mg / ml, about 175-275 mg / ml, about 200-300 mg / ml, about 225-325 mg / ml, 250-350 mg / ml, 275-375 mg / ml, 300-400 mg / ml, 325-425 mg / ml.350-450 mg / ml, 375-475 mg / ml, 400-500 mg / ml, 425-525 mg / ml, 50-100 mg / ml, 75-125 mg / ml, 100-150 mg / ml.125-175 mg / ml, 150-200 mg / ml, 175-225 mg / ml, 200-250 mg / ml, 225-275 mg / ml, 250-300 mg / ml, 275-325 mg / ml, 300- 350 mg / ml, 325-375 mg / ml, 350-400 mg / ml, 375-425 mg / ml, 400-450 mg / ml, 425-475 mg / ml, 450-500 mg / ml, 475-525 mg / ml, 50-525 mg / ml, 100-500 mg / ml, 150-450 mg / ml, 200-400 mg / ml or 250-350 mg / ml.

[0112] In another aspect of the invention, a method of treating tauopathy in a subject in need thereof is provided, the method comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition described above in paragraph

[0109] . The tauopathy may be one of Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, argyrophilic grain dementia (argyrophilic grain disease), multiple system tauopathy with presenile dementia (MSTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), dementia with neurofibrillary tangles, diffuse neurofibrillary tangle with calcification (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), frontotemporal lobar degeneration with tau pathology (FTLD-tau), Economo’s encephalitis sequela, subacute sclerosing panencephalitis, and boxer's encephalopathy. In one embodiment, the tauopathy is Alzheimer’s disease. Exemplification

[0113] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments of the invention and are not intended to limit the invention. Example 1: High throughput screening for identifying conditions of crystallization of HA anti- pSer413 tau mAb:

[0114] High throughput screening of crystallization conditions was carried out to increase the chances of successful crystallization. Six commercially available screens, designed specifically for crystallizing monoclonal antibodies, were used (see list below). Screening was performed in 96-well plates at 22 °C and 30 °C using vapor diffusion in sitting drop method and involved testing different reagents and different drop ratios of mAb concentration to precipitants. Specifically, Intelliplate 3 drop LVR 96 well crystallization plates (Hampton Research) were used and three protein wells: complimentary reservoir (precipitant) solution drop ratios (0.1 µl: 0.3 µl, 0.2 µl: 0.2 µl, and 0.3 µl: 0.1 µl) were employed over 80 µl of complimentary reservoir solutions. Mosquito®crystallization robot (SPT Labtech, Inc.) was used for liquid handling. The plates were observed under a microscope to monitor crystal formation over different time periods, including 1 day, 7 days, 2 weeks, 3 weeks, and 1 month. HA anti-pSer413 tau mAb was used at 60 mg / ml (in 10 mM Histidine, pH 5.4). The screens and hit conditions used in the study were as follows: 1. JBS Wizard 1 & 2 2. JBS Wizard 3 & 43. Hampton Research GRAS Screens 1-8 4. JBS Classic 1 & 2 5. JBS Classic 3 & 4 6. Additive Screen

[0115] Conditions that yielded crystals (i.e., hits) are listed in Table 1 below. Table 1 Hit Screen type and Temperature Protein: Reservoir / Precipitant solution No. conditionoC reservoir d ti

[0116] For Hit No.1, crystals were observed after 1 week under the crystallization condition, 1.2 M ammonium sulphate, 100 mM sodium acetate, pH 4.6, and 200 mM NaCl (JBS Wizard screen 1+2, condition C06). Crystals were visualized using a microscopic inspection system. Photomicrographs of the observed crystals are shown in Fig.1. Example 2: Further development of crystallization condition of Hit No.1 (high salt)

[0117] Crystallization based on the condition of Hit No.1 (JBS Wizard screen 1+2, condition C06 - 1.26 M Ammonium sulphate, 100 mM sodium acetate, pH 4.5, 200 mM sodium chloride at 22oC) was further developed as described in the following.

[0118] Ammonium sulphate concentration was varied from 0.5-1.5 M. Sodium citrate was used instead of sodium acetate and the pH was varied from 4.0 to 6.5. Crystals grew under ammonium25845 sulphate concentration range of 1.0-1.3 M and within the pH range 4.2 to 5.5 at all drop ratios (1:3, 1:1, and 3:1) within one week. As noted in Example 2, the concentration of HA anti- pSer413 tau mAb was 60 mg / ml (in 10 mM Histidine, pH 5.4).

[0119] Examples of crystals obtained are shown in the photomicrographs depicted in Figs.2A and 2B. The photomicrograph in Fig.2A was taken at 100× magnification after one week of setting up of crystallization. The crystals depicted were obtained by vapor diffusion using a precipitant solution of 1.2 M ammonium sulphate and 100 mM sodium citrate pH 4.8 at 22 °C. The photomicrograph in Fig.2B was also taken at 100× magnification after one week of setting up of crystallization. The crystals depicted were obtained by vapor diffusion using a precipitant solution of 1.3 M ammonium sulphate and 100 mM sodium citrate pH 4.9 at 22 °C. Example 3: Further development of crystallization condition of Hit No.3 (PEG)

[0120] Crystallization based on the condition of Hit No.3 (Hampton GRAS4, condition - 1.5 M ammonium citrate dibasic, pH 4.8, 2% PEG 3350 at 22oC) was further developed as described in the following.

[0121] Ammonium citrate dibasic concentration (1.0 – 2.0 M) was varied against the concentration of PEG4000 / PEG3350 (0 – 4 % w / v) and the concentration of ammonium citrate tribasic (0 – 0.1 M). As noted in Example 2, the concentration of HA anti-pSer413 tau mAb was 60 mg / ml (in 10 mM Histidine, pH 5.4). Antibody: precipitant drop ratios used were 300 µl:100 µl, 200 µl: 200 µl or 100 µl:300 µl.

[0122] One example of crystals obtained following the above procedure is shown in the photomicrograph of Fig.3. The photomicrograph was taken after one week at 100× magnification. Specifically, the crystals were obtained by vapor diffusion using a precipitant solution of 2% PEG 4000 and 1.8 M ammonium citrate dibasic pH 4.8 at 22 °C. Example 4: Development of crystallization condition using seed crystal

[0123] In this Example, crystal seeds obtained using a high salt condition were used to nucleate crystal formation under high PEG-low salt conditions. The process involved three steps: (1) production of antibody crystals using high ammonium sulphate concentration (high salt) for use as crystals for seeding, (2) production of seed crystals using the crystals from step (1), and (3) seeding crystallization mixtures comprising varying amounts of PEG 4000 and having varying pH with the seed crystals from step (2) and allowing crystals to form by vapor diffusion.25845

[0124] Step 1 - HA anti-pSer413 tau mAb antibody at 90 mg / ml in 5 mM histidine buffer was used. Ammonium sulphate concentration was varied between 0.5 and 1.5 M and pH was varied between 4.5 and 5.5 using 100 mM sodium citrate. Drop ratio of antibody solution to precipitant / reservoir solution was 0.2 µl: 0.2 µl. Incubation was done at 22 °C in a Swiss-SCI 96 well plate and 35 µl of a solution of 1.5 M ammonium sulphate and 100 mM sodium citrate pH 5.0 was used in the wells. Crystals producing drops were combined.

[0125] Step 2 - The combined crystal producing drops from step (1) were added to 300 µl of a solution of 1.5 M ammonium sulphate and 100 mM sodium citrate pH 5.0 (precipitant / reservoir solution). Seed stocks suitable for step 3 were generated by vortexing (four cycles of 30 seconds vortex, 30 seconds on ice) in the presence of a Molecular Dimensions MicroSeed Bead.

[0126] Step 3 - HA anti-pSer413 tau mAb antibody at 61 mg / ml, 10 mM Histidine, pH 5.4 was used in this step. Seed stock was added to the mAb solution. Reservoir / precipitant solutions used were 50 mM ammonium sulphate, 100 mM sodium acetate with the pH varying between 4.5 and 5.0, and PEG 4000 varying between 25 – 40% w / v. Crystallization was set up in 96 well SWISS- SCI plates using 0.1 µl of mAb solution + 0.26 µl of reservoir solution + 0.04 µl of seed stock, at 22 °C with each well containing 35 µl of the reservoir solution. Crystals grew in several drops, i.e., under several conditions. An example of crystals obtained following the above screening procedure is shown in the photomicrograph of Fig.4. The photomicrograph was taken at 100× magnification after one week. Specifically, the crystals shown in this photomicrograph were obtained under the condition in which the reservoir solution (0.26 µl) used was 50 mM ammonium sulphate,100 mM sodium acetate pH 4.5 and 34% w / v PEG 4000. Example 5: X-ray diffraction analysis

[0127] Crystals were grown using HA anti-pSer413 tau mAb (60 mg / ml, 10 mM Histidine, pH 5.4) and 1.4 M ammonium sulphate and 100 mM sodium citrate pH, 4.8 as precipitant. Crystallization was setup at 22 °C using a hanging drop technique. Crystals were grown, harvested, and cryo-protected using a 20% ethylene glycol solution in 2.0 M ammonium sulphate. X-ray diffraction data were collected using synchrotron radiation at ID-17 (Argonne), processed, and scaled using autoPROC (XDS). The final dataset was merged from two crystals grown in the same drop (same condition as above) with a maximum resolution of 2.76 Å. Crystals of HA anti-pSer413 tau mAb belong to the P21212 system with a =140.36 Å, b =186.66 Å, c =85.19 Å, α =90, β =90, γ = 90 °.25845 Example 6: X-ray characterization

[0128] The structure was solved using molecular replacement procedures. As search models, the individual variable and constant domains from the in-house structure 5DK3, with sugars and waters removed, were used. Refinement was carried out initially with REFMAC and subsequently with autoBUSTER. Model building was done in COOT. The final model contains the full antibody (two light chains and two heavy chains; residues 223-225, 238-241, 247-277, 425-426, 450-451 of one heavy chain were not visible in the density), one 7-residue and one 8- residue sugar chains, and 10 sulphate ions. Incorporation by reference

[0129] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. Equivalents

[0130] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the invention. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

25845 WHAT IS CLAIMED IS:

1. A crystal of an anti-pSer413 tau monoclonal antibody (HA anti-pSer413 tau mAb) having a light chain amino acid sequence of SEQ ID NO: 3 and a heavy chain amino acid sequence of SEQ ID NO:

8.

2. The crystal of claim 1 having unit cell dimensions of a =140.36 Å, b =186.66 Å, c =85.19 Å, α =90°, β =90°, γ = 90° and space group P21212.

3. The crystal of claim 2, characterized by having a length in the range of 0.5 to 300 microns.

4. The crystal of claim 2 or claim 3, capable of diffracting X-rays to a maximum resolution of about 2.76 Å.

5. The crystal of any one of claims 1-4, wherein HA anti-pSer413 tau mAb is produced by recombinant DNA technology.

6. A pharmaceutical composition comprising the crystal of any one of claims 1-5 and at least one pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, wherein the HA anti-pSer413 tau mAb crystal is suspended in a liquid and the concentration of the antibody in the liquid is at least about 100 mg / ml.

8. A method for producing the crystalline HA anti-pSer413 tau mAb of any one of claims 1- 5, the method comprising, (i) mixing a solution comprising the antibody with a precipitant solution, thereby obtaining a crystallization mixture, (ii) applying a crystallization process to the crystallization mixture for a duration sufficient for crystal formation at a temperature of about 18 °C to about 35 °C, and (iii) optionally harvesting the crystalline antibody from the mixture,25845 wherein the precipitant solution comprises (a) about 1.4 mM ammonium sulphate and (b) about 100 mM sodium citrate having a pH of about 4.8, and the crystallization process is vapor diffusion.

9. The method of claim 8, wherein the crystallization process occurs at about 22 °C.

10. The method of claim 8 or claim 9, wherein the concentration of HA anti-pSer413 tau mAb in the solution comprising the antibody is about 60 mg / ml.

11. The method of any one of claims 8-10, wherein the solution comprising HA anti-pSer413 tau mAb is a buffered solution having a pH of about 4.0 to about 8.

0.

12. The method of claim 11, wherein HA anti-pSer413 tau mAb is provided in 10 mM Histidine, having a pH of about 5.

4.

13. A method of treating tauopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 6 or claim 7.

14. The method of claim 13, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, argyrophilic grain dementia (argyrophilic grain disease), multiple system tauopathy with presenile dementia (MSTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), dementia with neurofibrillary tangles, diffuse neurofibrillary tangle with calcification (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), frontotemporal lobar degeneration with tau pathology (FTLD-tau), Economo’s encephalitis sequela, subacute sclerosing panencephalitis, and boxer's encephalopathy.

15. The method of claim 14, wherein the tauopathy is Alzheimer’s disease.

16. A method for producing crystalline anti-pSer413 tau monoclonal antibody (HA anti- pSer413 tau mAb), the antibody having a light chain amino acid sequence of SEQ ID NO: 3 and a heavy chain amino acid sequence of SEQ ID NO: 8, the method comprising25845 (i) mixing a solution comprising the antibody with a precipitant solution, thereby obtaining a crystallization mixture; (ii) applying a crystallization process to the crystallization mixture for a duration sufficient for crystal formation at a temperature between about 18 °C to about 35 °C; and (iii) optionally harvesting the crystalline HA anti-pSer413 tau mAb from the mixture, wherein the precipitant solution has a pH of about 4.0 to about 5.0; and comprises (a) 50 mM to about 2500 mM ammonium sulphate or ammonium citrate dibasic, or a combination thereof, and optionally one or more components selected from the group consisting of (b) about 50 mM to about 200 mM sodium acetate, (c) about 100 mM to about 250 mM sodium chloride, (d) about 1% to about 40% weight per volume (w / v) of polyethylene glycol (PEG) ranging from PEG 1000 to PEG 5000, and (e) about 50 mM to about 150 mM sodium citrate or potassium citrate having a pH of about 4.0 to about 5.

0.

17. The method of claim 16, wherein the PEG used is PEG 3350 / 4000.

18. The method of claim 16 or claim 17 wherein step (i) further comprises adding a seed crystal of HA anti-pSer413 tau mAb prior to mixing.

19. The method of any of claims 16-18, wherein the crystallization process is selected from the group consisting of (i) hanging drop vapor diffusion, (ii) sitting drop vapor diffusion, (iii) microbatch crystallization, and (iv) batch crystallization.

20. The method of any one of claims 16-19, wherein the ratio of the volume of the solution comprising HA anti-pSer413 tau mAb and the precipitant solution is 1:3, 1:2, 1:1, 2:1, or 3:

1.

21. The method of any one of claims 16-20, wherein the concentration of the HA anti- pSer413 tau mAb in the solution comprising the antibody is about 5 to about 220 mg / ml.

22. The method of claim 21, wherein the concentration of HA anti-pSer413 tau mAb in the solution comprising the antibody is about 40 to about 100 mg / ml.25845 23. The method of any of claims 16-22, wherein the solution comprising HA anti-pSer413 tau mAb is a buffered solution having a pH of about 4.0 to about 8.

0.

24. The method of claim 23, wherein the solution comprising HA anti-pSer413 tau mAb is a histidine or citrate buffered solution having a pH of about 4.0 to about 8.

0.

25. The method of any one of claims 16-24, wherein the temperature of step (ii) is about 22 °C.

26. The method of any one of claims 16-24, wherein the temperature of step (ii) is about 30 °C.

27. The method of any one of claims 16-26, wherein the precipitant solution comprises (a) about 1.2 M ammonium sulphate, (b) about 200 mM NaCl, and (c) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about 4.6, the temperature of step (ii) is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion.

28. The method of any of claims 16-26, wherein the precipitant solution comprises (a) about 1.2 M ammonium sulphate and (b) about 100 mM sodium citrate, and wherein the precipitant solution has a pH of about pH 4.8, the temperature of step (ii) is about 22 °C, and the crystallization process is vapor diffusion.

29. The method of any of claims 16-26, wherein the precipitant solution comprises (a) about 1.3 M ammonium sulphate and (b) about 100 mM sodium citrate, and wherein the precipitant solution has a pH of about pH 4.9, the temperature of step (ii) is about 22 °C, and the crystallization process is vapor diffusion.

30. The method of any of claims 16-26, wherein the precipitant solution comprises (a) about 2.0 M ammonium sulphate, (b) about 200 mM NaCl, and (c) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about 4.6, the temperature of step (ii) is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion.

31. The method of any of claims 16-26, wherein the precipitant solution comprises (a) about 1.5 M ammonium citrate dibasic and (b) about 2% w / v of PEG 3350 / 4000, and wherein the25845 precipitant solution has a pH of about 4.0 to about 4.8, the temperature of step (ii) is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion.

32. The method of any of claims 16-26, wherein the precipitant solution comprises (a) about 1.8 M ammonium citrate dibasic and (b) about 2% w / v of PEG 4000, and wherein the precipitant solution has a pH of about 4.8, the temperature of step (ii) is about 22 °C, and the crystallization process is vapor diffusion.

33. The method of any of claims 16-26, wherein the precipitant solution comprises (a) about 1.4 M ammonium sulphate and (b) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about 4.5, the temperature of step (ii) is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion.

34. The method of any of claims 16-26, wherein the precipitant solution comprises (a) about 1.0 M ammonium sulphate and (b) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about 4.6, the temperature of step (ii) is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion.

35. The method of any of claims 16-26, wherein the precipitant solution comprises (a) about 2.0 M ammonium sulphate and (b) about 100 mM sodium acetate, and wherein the precipitant solution has a pH of about 4.6, the temperature of step (ii) is about 22 °C or about 30 °C, and the crystallization process is vapor diffusion.

36. The method of any of claims 16-26, further comprising, in step (i), adding a suspension comprising seed crystals of HA anti-pSer413 tau mAb prior to mixing, wherein the precipitant solution comprises about 50 mM ammonium sulphate, (b) about 25% to about 40% w / v of PEG 4000, and (c) about 100 mM sodium acetate having pH between about 4.5 and about 5.0, and the crystallization process is vapor diffusion.

37. The method of claim 36, wherein PEG 4000 is at about 34% w / v.

38. The method of claim 36 or claim 37, wherein the suspension comprising seed crystals is produced according to the method of claim 16, wherein the precipitant solution for producing the suspension comprises about 0.5 to about 1.5 M ammonium sulphate and about 100 mM sodium25845 citrate, having a pH of about 4.5 to about 5.5, and wherein the crystallization process is vapor diffusion, and step (iii) comprises combining drops producing crystals of the antibody, and optionally agitating the combined drops.

39. Crystalline HA anti-pSer413 tau mAb produced by the method of any one of claims 16- 38.

40. The crystalline HA anti-pSer413 tau mAb of claim 39, wherein HA anti-pSer413 tau mAb is produced by recombinant DNA technology.

41. A pharmaceutical composition comprising the crystalline HA anti-pSer413 tau mAb of claim 39 or claim 40 and at least one pharmaceutically acceptable excipient.

42. The pharmaceutical composition of claim 41, wherein the crystalline HA anti-pSer413 tau mAb is suspended in a liquid and the concentration of the antibody in the composition is at least about 100 mg / ml.

43. A method of treating tauopathy in a subject in need thereof, the method comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition of claim 41.

44. The method of claim 43, wherein the tauopathy is selected from the group consisting of Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, Pick's disease, argyrophilic grain dementia (argyrophilic grain disease), multiple system tauopathy with presenile dementia (MSTD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), dementia with neurofibrillary tangles, diffuse neurofibrillary tangle with calcification (DNTC), white matter tauopathy with globular glial inclusions (WMT-GGI), frontotemporal lobar degeneration with tau pathology (FTLD-tau), Economo’s encephalitis sequela, subacute sclerosing panencephalitis, and boxer's encephalopathy.

45. The method of claim 44, wherein the tauopathy is Alzheimer’s disease.

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