RU2793400C2 - Method of purification for removing tyrosine sulphated antibodies variants, purified compositions - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for removing an antibody with sulphated tyrosine from an aqueous mixture comprising an antibody in which tyrosine is sulphated and an antibody in which tyrosine is not sulphated, is proposed. These antibodies are antibodies against human LAG3. Tyrosine sulfation takes place in the CDR-L1 region. In addition, a composition for therapy, including antibodies against human LAG3, is proposed. The composition has less than 0.5% anti-hLAG3 antibodies that contain sulphated tyrosine on CDR-L1.

EFFECT: invention provides a method for purifying an antibody composition from contaminating antibody variants comprising sulphated tyrosine.

13 cl, 18 dwg, 1 tbl, 1 ex

Description

The field of technology to which the invention belongs

The present invention relates to compositions comprising antibodies and antigen-binding fragments that lack tyrosine sulfation, as well as purification methods to obtain compositions.

Prerequisites for the creation of the invention

Tyrosine sulfation is a post-translational modification (PTM) where a sulfate trioxide (SO ₃ ) group is covalently attached to a hydroxyl group on the side chain of the tyrosine amino acid group. This PTM occurs in the trans-Golgi network and is catalyzed by two tyrosyl protein sulfotransferase (TPST) enzymes. The molecular mechanism involves the transfer of activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine, and has been found on various proteins and peptides. Recent data indicate that tyrosyl protein sulfotransferase 2 recognizes acid-flanked tyrosines for sulfation. This PTM is responsible for enhancing interactions between proteins and occurs on secreted and transmembrane proteins. Some chemokine receptors have been shown to be tyrosine sulfated, such as those in the N-terminal extracellular domain of CCR5, the major receptor for HIV-1, and some glycoprotein hormone receptors. For example, the native form of the leech-derived thrombin-inhibiting peptide hirudin is tyrosine sulfated. Interestingly, the two recombinant forms of hirudin (Revasc and Refludan) used to treat various clotting disorders are not sulfated. Sulfation increases the mass of the biomolecule by 80 Da, which is the same mass difference as the phosphate group (PO ₃ ). Unlike PO ₃ , which forms a fairly stable PO bond, SO ₃ is very labile and easily decomposes at high temperature and low pH.

The presence of different PTM variants in an antibody therapeutic preparation leads to heterogeneity, which, depending on the location of the modification, may lead to changes in the activity, bioavailability, or immunogenicity of the antibody. Such problems also create problems for government regulators. Although tyrosine sulfation has been described in chemokine receptors and other proteins, it is necessary to determine whether such modifications occur in antibody preparations and, if they are found, remove them.

The essence of the invention

The present invention provides a composition comprising an anti-LAG3 antibody or antigen-binding fragment thereof (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, or Ab9), which, for example, includes:

light chain variable domain, including:

CDR-L1 which includes the amino acid sequence: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2, which includes the amino acid sequence: GASNLES (SEQ ID NO: 39); And

CDR-L3 which includes the amino acid sequence: QQSTEDPRT (SEQ ID NO: 40); and/or

heavy chain variable domain, including:

CDR-H1, which includes the amino acid sequence: DYNVD (SEQ ID NO: 33);

CDR-H2, which includes the amino acid sequence:

DINPNNGGTIYAQKFQE (SEQ ID NO: 59);

DINPNSGGTIYAQKFQE (SEQ ID NO: 60);

DINPNDGGTIYAQKFQE (SEQ ID NO: 61);

DINPNQGGTIYAQKFQE (SEQ ID NO: 62);

DINPNGGGTIYAQKFQE (SEQ ID NO: 63); or

DINPNX ₁ GGTIYX ₂ QKFX ₃ X ₄ (SEQ ID NO: 64) where, X ₁ = D,N,S or Q, X ₂ = A or S, X ₃ = Q or K, and X ₄ = E or G; and CDR-H3: NYRWFGAMDH (SEQ ID NO: 35); in which there are no detectable levels of sulfated tyrosine on CDR-L1. For example, in one embodiment, the antibodies or fragments in the composition also do not contain detectable levels of sulfated tyrosine in one or more members selected from the group consisting of FR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, FR-L4, FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4 and constant domain. In one embodiment, the antibody or fragment comprises recombinant yeast or CHO N-linked glycans. In one embodiment, a composition comprising an anti-LAG3 antibody (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, or Ab9) comprises one or more antibodies that do not have tyrosine sulfation and have molecular weights of about 148590 Yes, 148752 Yes and/or 148914 Yes (e.g., which contain G0F and/or G1F glycan species, for example, as shown in Table 1, and in which the N-terminal glutamine in the heavy chain is converted to pyroglutamate and/or C-terminal heavy chain lysine removed).

The present invention also provides a method for removing tyrosine sulfated antibodies or antigen-binding fragments thereof (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, or Ab9) from an aqueous mixture comprising antibodies or antigen-binding fragments that include one or several sulfated tyrosines (for example, on CDR-L1), and antibodies or antigen-binding fragments that do not contain sulfated tyrosine, including bringing the pH of the mixture to about 6.5 - about 7.0 or to about 6.5 - about 7.5, contacting the mixture with the anion exchange resin; and removing and retaining the non-resin-bound aqueous fraction of the mixture from the resin. In one embodiment of the invention, the method includes washing the column with an aqueous composition, eg, under isocratic conditions, and removing and retaining the wash composition from the resin. In one embodiment of the invention, the resin is in the column and the method includes adding said mixture to the column and collecting a running fraction from the column. In one embodiment, the method includes equilibrating a chromatographic resin comprising a dimethylaminopropyl anion exchange ligand in a chromatographic column with a solution of 25 mM sodium phosphate pH 6.5, adjusting the pH of the mixture to about 6.5, applying the mixture to the column, collecting the flow fraction from the column, washing resins in a column with a solution of 25 mm sodium phosphate pH 6.5 and the collection of the flow fraction from the wash. In one embodiment, the method comprises equilibrating a chromatographic resin comprising a quaternized polyethyleneimine anion exchange ligand on a chromatographic column with a solution of 25 mM sodium phosphate pH 7.0; optionally, 5 mM NaCl, adjusting the pH of the mixture to about 7.0, applying the mixture to the column, collecting the flow fraction from the column, washing the resin in the column with a solution of 25 mM sodium phosphate pH 7.0; optionally 5 mM NaCl, and collection of the running fraction from the wash. In one embodiment, the A ₂₈₀ absorbance of the anion exchange chromatography flow through is measured and collected and stored when the A ₂₈₀ first reaches at least about 2.5 OD/cm; and are not collected and stored when A ₂₈₀ falls below about 1.0 OD/cm. In one embodiment, the methods of the present invention further comprise purifying the antibody or antigen-binding fragment by cation exchange chromatography, then by anion exchange chromatography in binding-elution mode, hydrophobic interaction chromatography, protein-A chromatography, protein-L chromatography, protein-G chromatography, hydroxyapatite chromatography, size exclusion chromatography, fractionated precipitation, filtration, centrifugation or viral inactivation. In one embodiment, the immunoglobulin light chains and/or heavy chains of the antibody or antigen-binding fragment are expressed in Chinese hamster ovary cells. In one embodiment, the antibody or antigen-binding fragment comprises:

light chain variable domain, including:

CDR-L1 which includes the amino acid sequence: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2, which includes the amino acid sequence: GASNLES (SEQ ID NO: 39); And

CDR-L3 which includes the amino acid sequence: QQSTEDPRT (SEQ ID NO: 40); and/or

heavy chain variable domain, including:

CDR-H1, which includes the amino acid sequence: DYNVD (SEQ ID NO: 33);

CDR-H2, which includes the amino acid sequence:

DINPNNGGTIYAQKFQE (SEQ ID NO: 59);

DINPNSGGTIYAQKFQE (SEQ ID NO: 60);

DINPNDGGTIYAQKFQE (SEQ ID NO: 61);

DINPNQGGTIYAQKFQE (SEQ ID NO: 62);

DINPNGGGTIYAQKFQE (SEQ ID NO: 63); or

DINPNX ₁ GGTIYX ₂ QKFX ₃ X ₄ (SEQ ID NO: 64), where X ₁ = D,N,S or Q, X ₂ = A or S, X ₃ = Q or K, and X ₄ = E or G; and CDR-H3: NYRWFGAMDH (SEQ ID NO: 35). Compositions that are the product of such a process are also part of the present invention. In one embodiment, an anti-LAG3 antibody (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, or Ab9) is purified by AEX chromatography, where the sulfated tyrosine-free antibodies have molecular weights of about 148590 Da, 148752 Yes and/or 148914 Yes (eg, contain G0F and/or G1F glycan species, for example, as shown in Table 1, N-terminal glutamine in the heavy chain converted to pyroglutamate and/or lysine removed at the C-terminus of the heavy chain).

Brief description of the drawings

Fig. 1. Overlay IEX-HPLC UV profile of the original sample (thick line), desorbed (thin line) and combined fraction (dashed line) AEX.

Fig. 2 . Spectrum of intact masses AEX of the original, pooled and desorbed samples.

Fig. 3 . Mass spectrum of the reduced light chain of the pooled and desorbed AEX samples.

Fig. 4A-B . UV spectrum obtained by LysC mapping of the reduced peptide of the pooled and desorbed AEX fractions.

Fig. 5A-C . (A) Light chain fragmentation LED spectrum AA25-43+80 Da scaled 400-1800 m/z (B) Scaled m/z 300-1100 (C) Scaled m/z 1200-2000.

Fig. 6 . DPE fragmentation of the light chain peptide AA25-43+80 Yes. 80 Da attached fragment ions are labeled.

Fig. 7A-B . (A) Intact mass spectra after AEX deconvolution of the desorbed fraction with and without alkaline phosphatase treatment. (B) Intact mass spectra after deconvolution of chicken ovalbumin with and without alkaline phosphatase treatment.

Fig. 8A-B . (A) Normalized AEX concentrations of pooled and desorbed mAb fractions were subjected to SDS-PAGE under reducing conditions, probed for human heavy (HC) and light chains (LC) by Western blot hybridization (upper panel), then desorbed and probed again for antisulfotyrosine (the bottom panel). See directions HC and LC on the right. (B) Normalized concentrations of various CHO-derived mAbs in addition to the AEX desorbed and pooled fractions were subjected to SDS-PAGE under reducing conditions, probed for human HC and LC by Western blot hybridization, then stripped and probed again for antisulfotyrosine. For both (A) and (B), MagicMark XP was used as the protein molecular weight standard and equal amounts of HEK293 and EGF-treated A431 cell extracts were analyzed as controls.

Fig. 9A-C . SIC (A) LC25-43+80 Da from AEX Desorbed Fraction, (B) XSXSXDYEGDSDXXXXXXX Synthetic Peptide (SEQ ID NO: 65) + Phosphorylation and (C) XSXSXDYEGDSDXXXXXXX Synthetic Peptide (SEQ ID NO: 65) + Sulfation.

Fig. 10 . The tyrosine (Y31) region of the mAb showing loop CDRs in a ribbon diagram for both the heavy and light chains.

Fig. 11 . Preferential N-linked glycans for monoclonal antibodies that were generated in Chinese hamster ovary cells (CHO N-linked glycans) and in engineered yeast cells (genetically engineered yeast N-linked glycans): squares: N-acetylglucosamine (GlcNac) ; circles: mannose (Man); diamonds: galactose (Gal); triangles: fucose (Fuc).

Detailed description of the invention

Some antibodies and other proteins expressed in Chinese hamster ovary (CHO) cells are contaminated with sulfated tyrosine-containing variants. Mass spectrographic analysis of such variants is characterized by an adduct of about +80 Da, which corresponds to the mass of the added sulfate group. Such adducts are also resistant to alkaline phosphatase and are able to react with antibodies against sulfated tyrosine. The present invention provides a method for purifying a composition comprising such contaminating sulfated tyrosine variants, as well as antibody compositions substantially free of such variants.

In accordance with the present invention, conventional methods of molecular biology, microbiology and recombinant DNA known to those skilled in the art can be used. Unless otherwise defined, scientific and technical terms used in connection with the present invention should have meanings that are well known to those of ordinary skill in the art. In addition, unless the context dictates otherwise, singular terms must include the plural, and plural terms must include the singular. In general, the nomenclatures used in connection with the methods of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and chemistry of proteins and nucleic acids and hybridization, which are described in this application, are well known and widely used in the art. The methods and procedures of the present invention are generally carried out in accordance with conventional methods well known in the art and as described in the various general and more specific references that are cited and discussed herein, unless otherwise indicated. See, for example, James M. Cregg (Editor), Pichia Protocols (Methods in Molecular Biology), Humana Press (2010), Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, NJ; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999), Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide to Molecular Cloning (1984).

Sulfated tyrosine includes tyrosine containing an attached sulfate group, for example having the structure:

Chromatography

The present invention provides a method for removing contaminating antibody variants or antigen-binding fragments thereof (e.g., Ab1-Ab9) that include sulfated tyrosine from a composition, e.g., a composition that includes a mixture of antibodies or fragments, some of which contain sulfated tyrosine and some of which which do not contain sulfated tyrosine, to obtain a composition that includes undetectable levels of sulfated variants of tyrosine (for example, CDR-L1 with sulfated tyrosine, for example, from Ab1 or Ab6). In one embodiment, the composition is purified by anion exchange (AEX) flow chromatography to remove sulfated tyrosine variants. In one embodiment of the invention, the AEX resin contains a dimethylaminopropyl ligand (ie, a ligand that includes a dimethylaminopropyl group). For example, in one embodiment of the invention, the composition that is subjected to AEX chromatography is a protein-A prepurification chromatography product. In one embodiment of the invention, the pH of the composition is adjusted to pH about 6.5, eg with Tris (eg 0.5M, 0.725M or 1M), prior to AEX purification (eg containing dimethylaminopropyl ligand). In one embodiment of the invention, the AEX column (eg containing dimethylaminopropyl ligand) is equilibrated with eg sodium phosphate, eg 25 mM, eg sodium phosphate pH 5, 6.2 or 6.5. The column (e.g., containing dimethylaminopropyl ligand) can, in one embodiment of the invention, be washed with a buffer (e.g., sodium phosphate, e.g. 25 mM, e.g. strongly bonded to the AEX resin. The eluate not strongly bound to the AEX resin is collected (eg as separate fractions) and eg combined. In one embodiment of the invention, after use in the column, desorption is carried out, for example, with a 1M NaCl solution.

Mass spectrometric analysis of the AEX eluate revealed some glycosylated Ab6 species with no tyrosine sulfation on CDR-L1. These species are summarized below in Table 1. These theoretical masses refer to the calculated mass of the Ab6 molecule where the N-terminal glutamine on the heavy chain is converted to N-terminal pyroglutamic acid (pE1) and the C-terminal lysine on the heavy chain is removed (-K) .

Table 1 intact mass G0F, pE1, -K,
/G0F, pE1, -K G1F, pE1, -K
/G0F, pE1, -K G1F, pE1, -K
/G1F, pE1, -K Theoretical mass (Yes) 148590 148752 148914 Observed mass in the combined fraction (Yes) 148590 148749 148915 * Refer to FIG. 11 for identification of G0F and G1F glycan species

The present invention includes a composition comprising anti-LAG3 antibodies (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, or Ab9; preferably Ab6) that do not contain detectable levels of tyrosine sulfation, e.g., on CDR-L1, comprising varieties having one or more of molecular weights of about 148590, 148749 and/or 148915; and/or including G0F and/or G1F glycan species.

Flow mode refers to the purification of a polypeptide using a chromatographic resin in a manner that does not include an elution step to isolate the polypeptide. In such a method, the polypeptide of interest does not bind strongly to the resin, but contaminants to be removed from the polypeptides of interest bind strongly to the resin. For example, AEX resin is used in a flow-through mode in a method involving loading a composition that includes impure tyrosine-sulfated and non-tyrosine-sulfated antibody variants onto a column containing AEX resin, and collecting and retaining the antibody or fragment in a flow-through fraction of the column. Unbound, non-sulfated antibody can be washed from the column (and stored) under conditions that do not result in elution, such as isocratic conditions. In such a method, the contaminant remains bound to the column and the antibody lacking tyrosine sulfation will remain in the flow fraction.

The binding/elution mode refers to the purification of a polypeptide using a chromatographic resin in a manner that includes an elution step. In such a method, the polypeptide of interest binds strongly to the resin, and contaminants to be removed from the polypeptides of interest do not bind strongly to the resin. In a chromatographic column, contaminants flow through the column and remain largely unbound to the resin. Bound antibodies, after optional washing, are released and collected and stored by exposure to an elution buffer which causes them to be released from the resin.

A chromatographic resin ligand is a substance that attaches to a stationary phase particle (eg, Sepharose particle) that reversibly binds to a desired molecule (eg, antibody or impurity) present in the multicomponent mobile phase.

In one embodiment of the invention, the AEX resin contains a quaternized polyethyleneimine ligand (ie, a ligand that includes a quaternized polyethyleneimine group). In one embodiment of the invention, the resin (eg, containing a quaternized polyethyleneimine ligand) is pre-equilibrated with a 1M NaCl solution. In one embodiment of the invention, the resin (for example, containing a quaternized polyethyleneimine ligand) is equilibrated with sodium phosphate,For example25 mM, and NaCl,For example,5 mM; pH around 7.0. In one embodiment of the invention, a column (for example, containing a quaternized polyethyleneimine ligand) is loaded with the initial mixture and washed with sodium phosphate,For example25 mM, and NaCl,For example5 mM; pH about 7.0; and the solution flowing from the column is collected,For exampleas individual factions, andFor exampleunite. In another embodiment, the method of the invention comprises equilibrating a chromatographic resin comprising an anion exchange ligand on a chromatographic column with about 10-50 mM sodium phosphate; pH about 6.5-7.5, bringing the pH of the mixture to about 6.5-7.5, applying the mixture to the column, collecting the flow fraction from the column, washing the resin in the column with a solution of sodium phosphate of about 10-50 mm; pH about 6.5-7.5 and collection of the running fraction from the wash. In another embodiment, the method of the invention comprises equilibrating a chromatographic resin comprising an anion exchange ligand on a chromatographic column with a sodium phosphate solution of about 10-50 mM; pH about 6.5-7.0, bringing the pH of the mixture to about 6.5-7.0, applying the mixture to the column, collecting the flow fraction from the column, washing the resin in the column with a sodium phosphate solution of about 10-50 mm; pH about 6.5-7.0, and collection of the running fraction from the wash.

Any suitable amount of antibody or antigen binding fragment can be loaded onto a chromatographic resin, such as a chromatographic column (eg, AEX containing quaternized polyethyleneimine ligand or dimethylaminopropyl ligand). For example, in one embodiment, about 100, 110, 120, 130, 140, 150, 100-150, 160, 170, 180, 190, 200, 300, 150-200, 100-200, 250-350, or 280 -320 grams of material, eg antibody or fragment, is loaded per liter of resin (eg AEX containing quaternized polyethyleneimine ligand or dimethylaminopropyl ligand).

If a chromatographic column is used (eg containing an AEX resin containing a quaternized polyethyleneimine ligand or dimethylaminopropyl ligand), any suitable size can be used. For example, in one embodiment, the column diameter or height is about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29 or 30 cm.

The flow rate refers to the volume of mobile phase passing through a column (eg containing AEX resin containing quaternized polyethyleneimine ligand or dimethylaminopropyl ligand) in a certain period of time. In one embodiment, the flow rate is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 liters per hour

In one embodiment of the invention, the absorbance at 280 nm (A ₂₈₀ ) of the column flow fraction (eg, containing an AEX resin containing a quaternized polyethyleneimine ligand or dimethylaminopropyl ligand) is measured. In one embodiment, the antibody or fragment product in the main A ₂₈₀ peak flow fraction is collected and stored. In one embodiment, the flow-through fraction is collected when A ₂₈₀ reaches about 1.0, 1.5, 2.0, 2.5, or 3.0 A ₂₈₀ optical units per cm (optical path length) and collection is stopped when A ₂₈₀ falls below about 1.0, 1.5, 2.0, 2.5, or 3.0 A ₂₈₀ optical units per cm (optical path length).

A pre-column filter can be used to protect chromatography columns (eg, those containing AEX resin containing quaternized polyethyleneimine ligand or dimethylaminopropyl ligand) from clogging due to particles in the mobile phase. In one embodiment of the invention, the filter is a polyethersulfone membrane. A post-column filter can also be used to filter out any particulate matter from the sheath. In one embodiment of the invention, the filter has a pore size of 0.2 or 0.5 mm.

The presence of the sulfated tyrosine variant can be confirmed, for example, by mass spectrographic analysis of the flow-through fractions. Sulfated variants will have a greater mass than non-sulfated variants. For example, in one embodiment, the sulfated version is about 80 Da heavier than the non-sulfated versions. In one embodiment, the sulfation is resistant to phosphatase cleavage and the sulfated peptide has a different pattern of electron transfer dissociation (ETD)-induced fragmentation compared to phosphorylated peptides.

In one embodiment of the invention, a composition comprising antibodies (e.g., Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, or Ab9; preferably Ab6) with no tyrosine sulfation refers to a composition that lacks detectable tyrosine sulfation (e.g., on CDR-L1). A composition comprising undetectable levels of tyrosine sulfation (eg, on CDR-L1) includes a level that cannot be observed by mass spectrometric analysis of the composition. For example, in one embodiment of the invention, mass spectrometric analysis of the composition is performed by measuring intact and reconstituted mass and mapping the reconstituted peptides of the immunoglobulin peptides of the composition. In one embodiment of the invention, mapping of reduced peptides involves denaturing and reducing disulfide bonds in the antibody molecule and alkylation of free cysteines, followed by enzymatic cleavage (eg, using LysC, Trypsin, or GluC). Enzymatically digested peptides were analyzed by mass spectrometry. In one embodiment, the "undetectable" level refers to less than about 0.5% (less than about 0.4, 0.3, 0.2, 0.1%) of species with sulfated tyrosine (e.g., on CDR-L1) by compared to unmodified molecules in the composition.

The molecular weight of a polypeptide can be calculated, for example, based on the known mass of amino acids (modified or unmodified/sulfated or unsulfated) and known modifications (eg, oxidation, deamidation, glycosylation, C- and N-terminal modification). Molecular weight can be measured by mass spectrometric analysis, for example in combination with liquid chromatography. In one embodiment of the invention, the mass spectrometry is quadrupole time-of-flight (Q-TOF) mass spectrometry or Orbitrap mass spectrometry.

The term "chromatography" refers to the process by which a solute of interest, such as a substance in a composition, is separated from other substances in the composition by contacting the substances with a resin that acts as an adsorbent. An adsorbent is a substance that adsorbs or retains the substance more or less strongly, for example, due to the properties of the solute, such as pI, hydrophobicity, size and structure, under certain process buffer conditions. Chromatography can be carried out by conventional means of percolating the composition through a layer of chromatographic resin, for example through a column containing resin. Batch chromatographic purification includes preparing a resin slurry and contacting the composition containing the antibody or fragment with the slurry to adsorb the substance to be separated from the resin. The solution including the non-resin bound material is separated from the slurry, for example by allowing the slurry to settle and removing the supernatant, and the unbound material may be retained or discarded. The slurry is optionally subjected to one or more washing steps. If desired, the slurry may be contacted with a suitable elution buffer to desorb resin-bound materials from the resin. The desorbed material can be kept or discarded. In one embodiment, the sulfated tyrosine antibody variants in the composition are bound to the anion exchange resin, while the nonsulfated tyrosine variants do not substantially bind to the resin.

In one embodiment, the antibody or antigen-binding fragment thereof is purified by protein A or protein G chromatography. Protein-G and protein-A are bacterial proteins from Group G Streptococci and Staphylococcus aureus , respectively. The affinity of protein G and protein A for the Fc region of an IgG-type antibody serves as the basis for the purification of IgG, Fc region-containing IgG fragments, and IgG subclasses. Protein-A or protein-G may be coupled to a solid phase, such as Sepharose, which may be used for protein-A or protein-G chromatography. The present invention includes methods for preparing a composition comprising an antibody or antigen-binding fragment thereof lacking detectable levels of sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method including flow-through AEX chromatography and protein-A and/or protein-G.

In one embodiment, the antibody or antigen-binding fragment thereof is purified by multimodal chromatography (mixed mode). Multimodal or mixed protein chromatography is based on resins that have been functionalized with ligands capable of multiple modes of interaction, eg based on ion exchange, hydroxyapatite, affinity, size exclusion and/or hydrophobic interactions. The present invention includes methods for preparing a composition comprising an antibody or an antigen-binding fragment thereof that lacks detectable levels of sulfated tyrosine variants, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method comprising flow-through AEX chromatography and mixed chromatography. mode.

In one embodiment, the antibody, or antigen-binding fragment thereof, is purified by protein-L chromatography. Protein L is a Peptostreptococcus magnus protein that binds immunoglobulins via the immunoglobulin light chain. Protein L binds to representatives of all classes of antibodies, including IgG, IgM, IgA, IgE and IgD. The recombinant L protein binds to the variable region of the kappa light chain of immunoglobulins and immunoglobulin fragments. Protein L binds to three of the four light chain subtypes of kappa in humans (1, 3 and 4) and kappa 1 in mice. The present invention includes methods for preparing a composition comprising an antibody or antigen-binding fragment thereof lacking detectable levels of a sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method including AEX flow chromatography and protein chromatography. L.

In one embodiment, the antibody or antigen-binding fragment thereof is purified by hydrophobic interaction chromatography (HIC). HIC separates proteins with differences in hydrophobicity. The separation is based on the reversible interaction between the protein and the hydrophobic surface of the chromatographic medium. The present invention includes methods for preparing a composition comprising an antibody or antigen-binding fragment thereof lacking detectable levels of sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method including AEX flow chromatography and HIC.

In one embodiment, the antibody, or antigen-binding fragment thereof, is purified by size exclusion chromatography (SEC). SEC separates proteins with different molecular sizes. The present invention includes methods for preparing a composition comprising an antibody or antigen-binding fragment thereof that lacks detectable levels of a sulfated tyrosine variant, or purifying the antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method involving flow-through AEX and SEC chromatography.

In one embodiment of the invention, the antibody or antigen-binding fragment is subjected to viral inactivation. For example, in one embodiment of the invention, viral inactivation is accomplished by pH treatment of compositions comprising an antibody or antigen-binding fragment thereof. In particular, for virus purification, pH limits can be used to directly affect the composition. For example, the pH treatment in one embodiment of the invention is a low pH treatment (eg, pH 3.0-3.6). In one embodiment, the antibodies or antigen-binding fragments are subjected to a high pH treatment. In one embodiment of the invention, viral inactivation of compositions comprising an antibody or antigen-binding fragment thereof is carried out using a solvent or detergent. The present invention includes methods for preparing a composition comprising an antibody or antigen-binding fragment thereof lacking detectable levels of sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method including AEX flow chromatography and viral inactivation.

"Ion exchange" separates molecules based on differences in their net surface charge. Molecules vary considerably in their charging properties and will exhibit varying degrees of interaction with charged chromatography resins according to differences in their overall charge, charge density, and surface charge distribution. In one embodiment, the antibody, or antigen-binding fragment thereof, is purified by ion exchange chromatography. "Ion exchange chromatography" includes cation exchange, anion exchange and mixed mode chromatography.

The phrase "ion exchange" resin refers to a solid phase that is negatively charged (ie cation exchange) or positively charged (ie anion exchange).

In one embodiment, the antibody, or antigen-binding fragment thereof, is purified by cation exchange chromatography. "Cation exchange" resin refers to a solid phase that is negatively charged and that has free cations to exchange with cations in aqueous solution passing over or through the solid phase. You can use any negatively charged ligand attached to the solid phase, suitable for the formation of a cation exchange resin. Cation exchange materials include, but are not limited to, materials having a ligand: sulfopropyl (SP) -CH ₂ -CH ₂ -CH ₂ -SO ₃ -, methylsulfonate (S) -CH ₂ -SO ₃ ^- or carboxymethyl (CM) -CH ₂ - ^COO- . The present invention includes methods for preparing a composition comprising an antibody or antigen-binding fragment thereof lacking detectable levels of sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method including AEX flow chromatography and cation exchange chromatography.

In one embodiment, the antibody or antigen-binding fragment thereof is purified by anion exchange chromatography. An "anion exchange" resin refers to a solid phase that is positively charged, thus having one or more positively charged ligands attached to it. You can use any positively charged ligand attached to the solid phase, suitable for the formation of an anion exchange resin. Anion exchange materials include, but are not limited to, materials having a ligand: quaternary ammonium (Q)-CH ₂ -N+-(CH ₃ ) ₃ ; diethylaminoethyl (DEAE) -CH ₂ -CH ₂ -N+-(CH ₂ -CH ₃ ) ₂ ; or diethylaminopropyl (ANX)-CH ₂ -CHOH-CH ₂ -N+-(CH ₂ -CH ₃ ) ₂ . The GoPure D 50 µm column contains a dimethylaminopropyl functional group. The present invention includes methods for preparing a composition comprising an antibody or antigen-binding fragment thereof lacking detectable levels of sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method comprising AEX flow chromatography and AEX chromatography (in binding/elution).

The term "solid phase" or "stationary phase" is used to refer to any non-aqueous matrix to which one or more ligands (e.g., anion exchange ligands or cation exchange ligands) can be attached, or alternatively, in the case of size exclusion chromatography, this can refer to the gel structure of the resin. . The mobile phase is a liquid, such as an aqueous substance, which carries the antibody or antigen-binding fragment over a solid phase for chromatographic purification. The mobile phase may include a loading buffer that is injected into the column. Examples of materials that can be used to form a solid phase include polysaccharides (such as agarose and cellulose) and other mechanically stable matrices such as silica (such as controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles, and derivatives of any of these substances.

A "balancing" buffer or solution is used to adjust the pH and conductivity of the chromatographic resin prior to loading the mixture containing the antibody or antigen binding fragment for purification. Suitable buffers or solutions that can be used for this purpose are well known in the art, such as those described above, and include any buffer at pH that is compatible with the selected resin used in the chromatography step to purify the protein of interest.

A "loading" buffer or solution is used to load a mixture containing an antibody or antigen binding fragment onto a purification resin (eg, an anion exchange resin or a cation exchange resin). Any suitable solution can be used as loading buffer. In one embodiment of the invention, the loading buffer is obtained from a buffered mixture from a previous purification step, such as an elution buffer.

The term "wash" buffer or solution means a composition used to elute one or more impurities from a purification resin (eg, an anion exchange resin or cation exchange resin) prior to eluting the antibody or antigen-binding fragment. The term "washing" describes passing the appropriate composition through or over a chromatographic resin. In one embodiment of the invention, the wash is isocratic. Under isocratic washing conditions, the mobile phase of the chromatography remains essentially the same.

While antibodies and antigen-binding fragments with sulfated tyrosine variant are contaminants, the present invention includes compositions containing such variants, for example, bound to AEX chromatography resin or unbound in the absence of variants without sulfated tyrosine. Unbound variants can be obtained by elution from the AEX column after removal from antibodies and fragments not containing sulfated tyrosine.

The "elution" buffer dissociates the molecule (eg, an antibody or antigen-binding fragment thereof) bound to the chromatographic resin.

Previous processing steps

Antibodies and antigen-binding fragments to be purified from contaminating sulfated tyrosine variants can be generated by expression in a host cell. For example, the method of the present invention comprises, in one embodiment, before removing such variants, expressing immunoglobulin heavy and/or light chains in a host cell in a culture medium under conditions favorable for such expression, and isolating antibodies or antigen-binding fragments from the host cell. and/or culture medium. The present invention includes methods for preparing a composition comprising an antibody or an antigen-binding fragment thereof lacking detectable levels of sulfated tyrosine variants, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants, by a method comprising expression in a host cell and AEX chromatography in flow mode.

Within the scope of the present invention are methods for preparing a composition comprising antibodies or antigen-binding fragments that lack tyrosine sulfation (e.g., on their CDR-L1), comprising (i) administering a polynucleotide encoding the immunoglobulin light and/or heavy chains of said antibodies or fragments, into a host cell (e.g., a CHO cell); and (ii) culturing the host cell under conditions favorable for the expression of immunoglobulin chains in the cell, for example, wherein an antibody or antigen-binding fragment having an immunoglobulin chain(s) is secreted from the host cell into culture medium, and (iii) isolating the immunoglobulin chain polypeptide(s) from the host cell and/or culture medium by a method that includes anion exchange flow chromatography as discussed above.

For example, antibodies or fragments can be released from the host cell by lysis, for example, by methods such as grinding/rubbing (eg with glass beads), cell lysis using a French press, enzymatic digestion, or sonication. Lysed cells, including soluble and insoluble materials from them, form a cell lysate. The present invention includes methods for producing an antibody or antigen-binding fragment lacking a sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method involving cell lysis and AEX flow chromatography.

In one embodiment, the antibodies or antigen-binding fragments are purified by methods including centrifugation. Centrifugation of the cell lysate or other suspension removes most particulate matter, such as cell debris, from the aqueous fraction containing the antibody or fragment. For example, in one embodiment of the invention, centrifugation is performed (eg, on the cell lysate, including removing the solid fraction of the lysate) at about 40,000-50,000 g for 15-30 minutes. In one embodiment of the invention, cells are removed from the liquid culture medium by centrifugation. For example, centrifugation using a gravity force in the range of about 8000xg to about 15000xg (eg, about 8000, 9000, 10000, 11000, 12000, 13000, 14000, or 15000), for example, has a Q/SIGMA ratio ranging from about 0.9×10 ^-8 to 2.8×10 ⁹ . In one embodiment of the invention, the liquid from the centrifuge is subjected to depth filtration (eg, with a pore size of 0.1 to about 0.2 microns). The present invention includes methods for producing an antibody or antigen-binding fragment lacking a sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method including centrifugation and AEX flow chromatography.

In one embodiment, immunoglobulin heavy and light chains are expressed in a host cell fused to a secretion signal sequence and secreted from the host cells into the culture medium of the host cells.

In one embodiment, the antibodies or antigen-binding fragments are purified by filtration (eg, before or after AEX chromatographic purification). For example, in one embodiment, an aqueous composition comprising an antibody or antigen binding fragment is filtered to remove particulate material, such as through a filter having a pore size of about 1 µm, 0.45 µm, or 0.22 µm. In one embodiment of the invention, the filter is made from cellulose acetate or polyvinylidene fluoride (PVDF). The present invention includes methods for preparing an antibody or antigen-binding fragment thereof lacking a sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method including AEX flow chromatography and filtration.

In one embodiment, the antibodies or antigen-binding fragments are purified by fractional precipitation. An increased salt concentration can improve the hydrophobic interaction between proteins and lead to selective precipitation. In one embodiment, an aqueous composition comprising an antibody or fragment is precipitated in the presence of ammonium sulfate, dextran sulfate, polyvinylpyrrolidine, polyethylene glycol (PEG; e.g., PEG4000), acetone, polyethyleneimine, protamine sulfate, streptomycin sulfate, or caprylic acid. The present invention includes methods for preparing an antibody or antigen-binding fragment thereof lacking a sulfated tyrosine variant, or for purifying an antibody or antigen-binding fragment thereof to remove sulfated tyrosine variants by a method including AEX flow chromatography and fractional precipitation.

In one embodiment, the host cell in which the immunoglobulin chain is expressed is a mammalian cell such as a Chinese Hamster Ovary (CHO) cell, a mouse myeloma cell, a PER cell, a hybridoma cell, or a fungal or yeast cell such as Pichia . like Pichia pastoris , or Saccharomyces cerevisiae . In one embodiment of the invention, the host cell, for example a CHO cell, does not contain glutamine synthase.

In one embodiment of the invention, a polynucleotide(s) encoding an immunoglobulin heavy and/or light chain is operably linked to one or more expression control sequences, such as a promoter. For example, an immunoglobulin is in an expression vector. To achieve high levels of expression of antibodies or antigen-binding fragments, a strong promoter/enhancer, such as the cytomegalovirus (CMV) promoter and/or elongation factor alpha (EF1α) promoter, can be used to stimulate immunoglobulin heavy chain and/or light chain expression.

In one embodiment of the invention, an intron sequence in the 5'-untranslated region is included after a promoter/enhancer to increase the export of transcribed mRNA to the cytoplasm from the nucleus, and one or more 3'-polyadenylation signal sequences are included to maximize mRNA levels. In one embodiment of the invention, the polyadenylation signal sequence is an SV40 late or early polyadenylation signal sequence or a bovine growth hormone polyadenylation sequence. In one embodiment, a Kozak consensus sequence is created by placing GCC GCC(A/G)CC (SEQ ID NO: 69) immediately before the first translation initiation codon to enhance translation initiation. In one embodiment of the invention, a signal peptide sequence is placed immediately upstream of the immunoglobulin chain to direct secretion of the antibody or fragment.

Cell culture conditions can be controlled and adjusted as needed. For example, conditions such as pH, cell count, cell viability, and temperature can be controlled and adjusted. In one embodiment of the invention, the temperature of the cell culture is adjusted, for example, from 37 to 30-35°C 48 hours after inoculation. Dissolved oxygen, in one embodiment of the invention, is controlled and/or adjusted to a predetermined value, such as 20-50%. In one embodiment, the dissolved CO ₂ is controlled and/or adjusted to, for example, no more than about 120-150 mm Hg. In one embodiment, the osmolality is controlled and/or adjusted to, for example, about 270-330 mOsm/kg.

Antibodies

The present invention provides compositions comprising antibodies and antigen-binding fragments thereof that lack detectable levels of sulfated tyrosine, as well as methods for isolating compositions comprising such antibodies and fragments. For example, in one embodiment, the antibody or fragment comprises sulfated tyrosine and binds to an antigen selected from: PD1, CD27, LAG3, CTLA4, BTLA, TIM3, ICOS, B7-H3, B7-H4, CD137, GITR, PD-L1 ,PD-L2, ILT1, ILT2 KIR3DL2, KIR3DL3, NKG2A, NKG2C, NKG2E, IL-10, IL-17 or TSLP.

The term "LAG3", in relation to the polypeptide to which the antibodies and antigen-binding fragments of the present invention bind, refers to human and cynomolgous monkey LAG3, e.g., Macaca fascicularis or Macaca mulatta , as well as fragments thereof, such as their mature fragment lacking the signal peptide.

Examples of anti-LAG3 antibody immunoglobulin chains (eg, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9 as disclosed in WO2016028672) lacking tyrosine sulfation include the examples below. For example, where the antibody or fragment includes one or more of the CDRs and/or immunoglobulin chains below. In one embodiment, the contaminating antibody or antigen-binding fragment comprises a CDR-L1 having the amino acid sequence KASQSLD Y of EGDSDMN (SEQ ID NO: 38), where Y (in bold and underlined) is sulfated.

In one embodiment, the anti-LAG3 antibody or antigen-binding fragment comprises immunoglobulin heavy chains and/or immunoglobulin light chains 4A10; V _H and/or V _L chain or light chain CDRs and/or heavy chain CDRs (eg CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 4A10).

In one embodiment, for any of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, or Ab9, any N-terminal glutamine in the heavy chain is converted to pyroglutamate and/or any C-terminal lysine in the heavy chain is removed. .

4A10-V _H subsequence

ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTATAGGAATCAATTCA GAGGTTCAGCTGCTCCAGTCTGGGGCAGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCCTCT GGCTTCAACATTGAAGACTACTATATGCAC TGGATGAAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGA TGGATTGATCCTGTGAATGGTGATACTGAATATGCCCCGAAGTTCCAGGGC AAGGCCACTATGACTGCAGACACATCCTCCAACACAGCCTACCTACACCTCAACAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATTTC TATGATGGTTACCTCTTTGCTTTC TGGGGCCAAGGGACCCTGGTCACTGTCTCTGCA

(SEQ ID NO: 1; where CDRs are underlined and where the signal sequence is in bold)

MKCSWVIFFLMAVVIGINS EVQLLQSGAELVRSGASVKLSCTAS GFNIEDYYMH W M KQRPEQGLEWIG WIDPVNGDTEYAPKFQG KAT M TADTSSNTAYLHL NS LTSEDTAVYYCNF YDGYLFAF WGQGTLVTVSA

(SEQ ID NO: 2; where CDRs are underlined and where the signal sequence is in bold)

CDR-H1: GFNIEDYYMH (SEQ ID NO: 3)

CDR-H2: WIDPVNGDTEYAPKFQG (SEQ ID NO: 4)

CDR-H3: YDGYLFAF (SEQ ID NO: 5)

4A10-V _L subsequence

ATGAGGTGCCTAGCTGAGTTCCTGGGGCTGCTTGTGCTCTGGATCCCTGGAGCCATTGGG GATATTGTGCTGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTGTCCATCTCCTGC AGGTCTAGTAAGAGTCTCCTGCATAGTGATGGCAACACTTATCTGTAT TGGCTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATAT CGGATGTCCAACCTTGCCTCA GGGGTCCCAGACAGGTTCAGCGGCAGTGGGTCAGGAACTGTTTTCACACTGAGAATCAGCAGACTGGAGGCTGAGGATGTGGGTATTTATTACTGT ATGCAACATCTAGAATATCCTTTCACG TTTGGAGGGGGGACCAAGCTGGAAATAAAA

(SEQ ID NO: 6; where CDRs are underlined and where the signal sequence is in bold)

MRCLAEFLGLLVLWIPGAIG DIVLTQAAPSVPVTPGESVSISC RSSKSLLHSDGNTYLY WLLQRPGQSPQLLI YRMSNLAS GVPDRFSGSGSGTVFTLRISRLEAEDVGIYYC MQHLEYPFT FGGGTKLEIK

(SEQ ID NO: 7; where CDRs are underlined and where the signal sequence is in bold)

CDR-L1: RSSKSLLHSDGNTYLY (SEQ ID NO: 8)

CDR-L2: YRMSNLAS (SEQ ID NO: 9)

CDR-L3: MQHLEYPFT (SEQ ID NO: 10)

In one embodiment, the anti-LAG3 antibody or antigen-binding fragment comprises immunoglobulin heavy chains and/or 19E8 immunoglobulin light chains; V _H and/or V _L chain or light chain CDRs and/or heavy chain CDRs (e.g. CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 19E8):

19E8-V _H subsequence

ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTGTCCGTTGC CAGATCCGACTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCT GGGTCCTCCTTCACTGACTACTATATAAAC TGGGTGAAGCAGAAGCCTGGACAGGGACTTGAGTGGATTGGA TGGATTTATCCTGGAAGCGGTAATTCTATCTACAATGAGAACTTCAAGGCC AAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCATCTCAGCAGCCTGACATCTGAGGACACTGCTGTCTATTTCTGTGCAAGA GAGGCTGATTACGACGATGCTTTGGACTAC TGGGGTCAAGGAACCTCGGTCACCGTCTCCTCA

(SEQ ID NO: 11; where CDRs are underlined and where the signal sequence is in bold)

MGWSWIFLFLLSGTAGVRC QIRLQQSGPELVKPGASVKISCKAS GSSFTDYYIN WVKQKPGQGLEWIG WIYPGSGNSIYNENFKA KATLTVDTSSSTAY M HLSSLTSEDTAVYFCAR EADYDDALDY WGQGTSVTVSS

(SEQ ID NO: 12; where CDRs are underlined and where the signal sequence is in bold)

CDR-H1: GSSFTDYYIN (SEQ ID NO: 13)

CDR-H2: WIYPGSGNSIYNENFKA (SEQ ID NO: 14)

CDR-H3: EADYDDALDY (SEQ ID NO: 15)

19E8-V _L subsequence

ATGGTATCCACACCTCAGTTCCTTGTATTTTTGCTTTTCTGGATTCCAGCCTCCAGAGGT CACATCTTGCTGACTCAGTCTCCAGCCATTCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGC AGGGCCAGTCAGAGCATTGGCACAAGCATACAC TGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAG TATGCTTCTGAGTCTATCTCT GGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCAGAAGATATTGCAGATTATTACTGT CAACAAAGTAATAGCTGGCCAACGTACACG TTCGGAGGGGGGACCAAGCTGGAAATAAAA

(SEQ ID NO: 16; where CDRs are underlined and where the signal sequence is in bold)

MVSTPQFLVFLLFWIPASRG HILLTQSPAILSVSPGERVSFSC RASQSIGTSIH WYQQRT NG S PRRLLIK YASESIS GIPSRFSGSGSGTDFTLSI NS VESEDIADYYC QQSNS WPTYT FGGGTKLEIK

(SEQ ID NO: 17; where CDRs are underlined and where the signal sequence is in bold)

CDR-L1: RASQSIGTSIH (SEQ ID NO: 18)

CDR-L2: YASESIS (SEQ ID NO: 19)

CDR-L3: QQSNSWPTYT (SEQ ID NO: 20)

In one embodiment, the anti-LAG3 antibody or antigen-binding fragment comprises immunoglobulin heavy chains and/or 11C9 immunoglobulin light chains; V _H and/or V _L chain or light chain CDRs and/or heavy chain CDRs (e.g. CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 11C9):

11C9-V _H subsequence

ATGAGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCAACTCC CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGATGCCTGGGGCTTCAGCGAAGATGTCCTGCAAGGCTTCT GGCTACACACTCACTGACTACTGGATGCAC TGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGA GCGATTGATATTTCTGATAGTTATTCTAGCTACAATCAAAAGTTCAAGGGC AAGGCCACATTGACTGTAGACGAATCCTCCAGCACAGCCTACATGCAGCTCACCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGA TCCCCTTTCTACAATAGTAGAGGGGGGAACTACTTTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

(SEQ ID NO: 21; where CDRs are underlined and where the signal sequence is in bold)

MRWSCIILFLVATATGVNS QVQLQQPGAELV M PGASAK M SCKAS GYTLTDYW MH WVKQRPGQGLEWIG AIDISDSYSSYNQKFKG KATLTVDESSSTAY M QLTSLTSEDSAVYYCAR SPFYNSRGGNYFDY WGQGTTLTVSS

(SEQ ID NO: 22; where CDRs are underlined and where the signal sequence is in bold)

CDR-H1: GYTLTDYWMH (SEQ ID NO: 23)

CDR-H2: AIDISDSYSSYNQKFKG (SEQ ID NO: 24)

CDR-H3: SPFYNSRGGNYFDY (SEQ ID NO: 25)

11C9-V _L subsequence

ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGT GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGC AGGGCAAGTCAGGACATTAGCAATTATTTAAAC TGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTAC TACACATCAAGATTACACTCA GGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGC CAACAGGGTGATACGCTTCCTCCGTGGACG TTCGGTGGAGGCACCAAGCTGGAAATCAAA

(SEQ ID NO: 26; where CDRs are underlined and where the signal sequence is in bold)

MMSSAQFLGLLLLCFQGTRC DIQMTQTTSSLSASLGDRVTISC RASQDISNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGDTLPPWT FGGGTKLEIK

(SEQ ID NO: 27; where CDRs are underlined and where the signal sequence is in bold)

CDR-L1: RASQDISNYLN (SEQ ID NO: 28)

CDR-L2: YTSRLHS (SEQ ID NO: 29)

CDR-L3: QQGDTLPPWT (SEQ ID NO: 30)

In one embodiment, the anti-LAG3 antibody or antigen-binding fragment comprises immunoglobulin heavy chains and/or 22D2 immunoglobulin light chains; V _H and/or V _L chain or light chain CDRs and/or heavy chain CDRs (e.g. CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 22D2):

22D2-V _H subsequence

ATGGGATGGACCTGGATCTTTCTCTTCTTCCTGTCAGGAACTGCAGGTGTCCTCTCT GAGGTCCTGCTGCTACAGTCTGGACCTGAACTGGTGAAGCCTGGGACTTCAGTGAAAATCCCCTGCAAGGCTTCT GGATACACATTCACTGACTACAACGTGGAC TGGGTGAAGCAGCGCCATGGAAAGGGCCTTGAGTGGATTGGA GATATTAATCCAAACAATGGTGGTACTATCTACAGTCAGAAATTCAAGGGC AAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTTCATGGAGCTCCGCAGCCTGACATCTGAGGACACTGCAGTCTATTTCTGTGCAAGG AACTATAGGTGGTTTGGTGCTATGGACCAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTG

(SEQ ID NO: 31; where CDRs are underlined and where the signal sequence is in bold)

MGWTWIFLFFLSGTAGVLS EVLLLQSGPELVKPGTSVKIPCKASGYTFT DYNVD WVKQRHGKGLEWIG DINPN NGGTIYSQKFKG KATLTVDKSSSTAFMELRSLTSEDTAVYFCAR NYRWFGAMDH WGQGTSVTVSS

(SEQ ID NO: 32; where CDRs are underlined and where the signal sequence is in bold)

CDR-H1: DYNVD (SEQ ID NO: 33)

CDR-H2: DINPNNGGTIYSQKFKG (SEQ ID NO: 34)

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

22D2-V _L subsequence

ATGGAGACAGACACAATCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACATTGT GTTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCCAGGGCAGAGGGCCACCATTTCCTGC AAGGCCAGTCAAAGTCTTGATTATGAAGGTGATAGTGATATGAAT TGGTACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTCT GGTGCATCCAATCTAGAGTCT GGGATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTGTTAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGT CAGCAAAGTACTGAGGATCCTCGGACG TTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCG

(SEQ ID NO: 36; where CDRs are underlined and where the signal sequence is in bold)

METDTILLWVLLLWVPGSTG DIVLTQSPASLAVSPGQRATISC KASQSLDYEGDSDMN WYQQKPGQPPRLLIS GASNLES GIPARFSGSGSGTDFTVNIHPVEEEDAATYYC QQSTEDPRT FGGGTKLEIK

(SEQ ID NO: 37; where CDRs are underlined and where the signal sequence is in bold)

CDR-L1: KASQSLD Y EGDSDMN (SEQ ID NO: 38)

CDR-L2: GASNLES (SEQ ID NO: 39)

CDR-L3: QQSTEDPRT (SEQ ID NO: 40).

In one embodiment, the anti-LAG3 antibody or antigen-binding fragment comprises immunoglobulin heavy chains and/or immunoglobulin light chains Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9; V _H and/or V _L chains or light chain CDRs and/or heavy chain CDRs (e.g. CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9):

Ab1 : humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 53AHH Humanized x[LAG3_H] mAb (LB145.22D2. E1.D1 VH6) IgG1/Kappa (PX) (or its variable domain); for example, including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 41); And

an immunoglobulin heavy chain containing the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNNGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 42); or

an immunoglobulin light chain variable domain that includes the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 41 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNNGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 of SEQ ID NO: 42 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNNGGTIYAQKFQE (SEQ ID NO: 59); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

Ab2 : humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 56AHH Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6 N55S) IgG1/Kappa (PX) (or its variable domain); for example: including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 43); And

an immunoglobulin heavy chain containing the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNSGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 44); or

variable domain of an immunoglobulin light chain, including the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 43 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNSGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 of SEQ ID NO: 44 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNSGGTIYAQKFQE (SEQ ID NO: 60); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

Ab3 : humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 54AHH Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6 N55D) IgG1/Kappa (PX) (or its variable domain); for example, including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 45)

an immunoglobulin heavy chain containing the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNDGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 46); or

an immunoglobulin light chain variable domain that includes the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 45 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNDGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 of SEQ ID NO: 46 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNDGGTIYAQKFQE (SEQ ID NO: 61); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

Ab4 : humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 52AHH Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6 N55Q) IgG1/Kappa (PX) (or its variable domain); for example, including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 47); And

an immunoglobulin heavy chain containing the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNQGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 48); or

an immunoglobulin light chain variable domain that includes the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 47 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNQGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 of SEQ ID NO: 48 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNQGGTIYAQKFQE (SEQ ID NO: 62); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

Ab5 : Humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 57AHH Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6) IgG4 S228P (PX) (or its variable domain); for example, including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 49); And

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNNGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO: 50); or

an immunoglobulin light chain variable domain that includes the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 49 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNNGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 SEQ ID NO: 50 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNNGGTIYAQKFQE (SEQ ID NO: 59); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

Ab6 : humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 73AHD Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6 N55D/VL3) IgG4 S228P/Kappa (PX) (or its variable domain); for example, including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 51); And

an immunoglobulin heavy chain containing the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNDGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO: 52); or

an immunoglobulin light chain variable domain that includes the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 51 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNDGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 of SEQ ID NO: 52 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNDGGTIYAQKFQE (SEQ ID NO: 61); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

Ab7 : humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 21AHG Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6 N55S/VL3) IgG4 S228P/Kappa (PX) (or its variable domain); for example, including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 53); And

an immunoglobulin heavy chain containing the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNSGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO: 54); or

an immunoglobulin light chain variable domain that includes the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 53 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNSGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 of SEQ ID NO: 54 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNSGGTIYAQKFQE (SEQ ID NO: 60); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

Ab8 : humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 80AHG Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6 N55Q/VL3) IgG4 S228P/Kappa (PX) (or its variable domain); for example, including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 55); And

an immunoglobulin heavy chain containing the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNQGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO: 56); or

an immunoglobulin light chain variable domain that includes the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 55 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNQGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 of SEQ ID NO: 56 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNQGGTIYAQKFQE (SEQ ID NO: 62); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

or

Ab9 : humanized light chain 45AGX Humanized x [LAG3_H] mAb (LB145.22D2.E1.D1 (VL3)) Kappa (PX) (or its variable domain) and humanized heavy chain 72AHD Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6 N55G/VL3) IgG4 S228P/Kappa (PX)) (or its variable domain); for example, including:

an immunoglobulin light chain containing the amino acid sequence:

DIVMTQTPLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQRGDSKDSTYSLSSTLECSKADYSFEKHKVY

(SEQ ID NO: 57); And

an immunoglobulin heavy chain containing the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNGGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO: 58); or

an immunoglobulin light chain variable domain that includes the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(amino acids 1-111 SEQ ID NO: 57 (CDRs underlined)); And

variable domain of an immunoglobulin heavy chain, including the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNGGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(amino acids 1-119 of SEQ ID NO: 58 (CDRs underlined));

or including CDRs:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ ID NO: 39);

CDR-L3: QQSTEDPRT (SEQ ID NO: 40);

CDR-H1: DYNVD (SEQ ID NO: 33);

CDR-H2: DINPNGGGTIYAQKFQE (SEQ ID NO: 63); And

CDR-H3: NYRWFGAMDH (SEQ ID NO: 35)

In one embodiment, the CDR-H2 of any anti-LAG3 antibody or antigen-binding fragment thereof of the present invention comprises the amino acid sequence:

DINPNX ₁ GGTIYX ₂ QKFX ₃ X ₄ (SEQ ID NO: 64)

Where,

X ₁ = D,N,S or Q

X ₂ = A or S

X ₃ \u003d Q or K

X ₄ = E or G

The present invention includes antibodies and their antigen-binding fragments (for example, 4A10, 19E8, 11C9 and/or 22D2; for example, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 and/or Ab9), including N-linked glycans, which are typically added to immunoglobulins produced in Chinese hamster ovary cells (CHO N-linked glycans) or to engineered yeast cells (engineered yeast N-linked glycans), such as, for example, Pichia pastoris . For example, in one embodiment, the antibody or antigen-binding fragment comprises one or more "engineered yeast" N-linked glycans or "CHO N-linked glycans" as shown in FIG. 11 (eg G0 and/or G0-F and/or G1 and/or G1-F and/or G2-F and/or Man5). In one embodiment, the antibody or antigen-binding fragment comprises N-linked glycans from engineered yeast, i.e. G0 and/or G1 and/or G2, optionally further including Man5. In one embodiment, the antibody or antigen-binding fragment comprises N-linked CHO glycans, ie. G0-F, G1-F and G2-F optionally further including G0 and/or G1 and/or G2 and/or Man5. In one embodiment, about 80% to about 95% (e.g., about 80-90%, about 85%, about 90%, or about 95%) of all N-linked glycans on the immunoglobulin chain of an antibody or antigen-binding fragment are N -linked glycans of genetically engineered yeast or N-linked glycans CHO. See Nett et al. Yeast. 28(3): 237-252 (2011); Hamilton et al. Science. 313(5792): 1441-1443 (2006); Hamilton et al. Curr Opin Biotechnol. 18(5): 387-392 (2007). For example, in one embodiment, the engineered yeast cell is GFI5.0 or YGLY8316 or the strains described in US Patent No. 7,795,002 or Zha et al. Methods Mol Biol. 988:31-43 (2013). See also International Patent Application Publication No. WO2013/066765.

Tyrosine sulfated anti-LAG3 antibody variants (eg, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, and/or Ab9) include molecular weights of about 148,670 Da, 148,832 Da, and/or 148,994 Da. Options with no tyrosine sulfation include molecular weights of about 148,590 Da, 148,752 Da, and/or 148,914 Da.

"Isolated" antibodies, or antigen-binding fragments thereof, are at least partially free of other biological molecules from the cells or cell culture from which they are derived. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth media. The isolated antibody, or antigen-binding fragment thereof, may also be at least partially free of components of the expression system, such as biological molecules from the host cell or its growth medium. In general, the term "isolated" does not imply the complete absence of such biological molecules or the absence of water, buffers or salts or components of a pharmaceutical composition that includes antibodies or fragments.

An antigen-binding fragment of an antibody is the portion of an antibody that retains the ability to specifically bind to the antigen to which the full-length antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ and Fv fragments; diabody; single chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

Typically, the basic structural unit of an antibody comprises a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. See Fundamental Immunology Ch. for a general description. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989).

Monoclonal antibodies are essentially homogeneous antibodies, i. the antibody molecules that form the population are identical in amino acid sequence, except for possible natural mutations, which may be present in small quantities. See Kohler et al. (1975) Nature 256: 495; US Patent No. 4,816,567; Clackson et al. (1991) Nature 352: 624-628; Marks et al. (1991) J. Mol. Biol. 222:581-597; and Presta (2005) J. Allergy Clin. Immunol. 116:731.

A chimeric antibody is an antibody containing a variable domain from a first antibody and a constant domain from a second antibody, where the first and second antibodies are from different species. (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). Typically, variable domains are derived from an antibody from an experimental animal ("parent antibody"), such as a rodent species, and constant domain sequences are derived from human antibodies, such that the resulting chimeric antibody is less likely to elicit an adverse immune response in humans compared to parental (eg, mouse) antibody.

A humanized antibody contains sequences from both a human and a non-human antibody (eg, mouse or rat). Typically, a humanized antibody will include substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to hypervariable loops from a non-human immunoglobulin, and all or substantially all of the framework regions (FR ) are derived from a human immunoglobulin sequence. The humanized antibody may optionally include at least a portion of a human immunoglobulin (Fc) constant region.

Immunoglobulins can be assigned to different classes depending on the amino acid sequences of the constant domain of their heavy chains. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The invention includes antibodies and antigen-binding fragments (eg, anti-LAG3) of any of these classes or subclasses of antibodies.

In one embodiment, the antibody or antigen binding fragment (e.g., anti-LAG3) comprises a heavy chain constant region, e.g., a human constant region, such as a γ1, γ2, γ3, or γ4 human heavy chain constant region, or a variant thereof. In another embodiment, the antibody or antigen binding fragment (eg, anti-LAG3) comprises a light chain constant region, eg, a human light chain constant region, such as a human lambda or kappa light chain region, or a variant thereof. By way of example, and not limitation, the human heavy chain constant region may be γ4 and the human light chain constant region may be kappa. In an alternative embodiment, the Fc region of the antibody is γ4 with a Ser228Pro mutation (Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).

Examples

These examples illustrate the present invention and are not intended to limit it.

Example 1 : Identification of tyrosine sulfation antibody variants and purification methods.In this example, the presence of tyrosine-sulfated Ab6 antibody variants was detected and a purification method was developed to remove such variants.

Materials and methods

Alkaline phosphatase was purchased from New England Biolabs (Ipswich, MA). Tyrosine sulfation antibody was purchased from Millipore (Billerica, MA). The synthetic peptide was purchased from AnaSpec (Fremont, CA).

Anion exchange (AEX) chromatography

AEX chromatography was performed using a pre-packed POROS GoPure D, 0.5 x 5 cm, 1 ml column in flow mode using a GE Akta Avant system. The protein-A purified mAb was adjusted to pH 6.5 with 1M Tris and loaded onto the column. Before loading the protein, the column was equilibrated with a solution of 25 mM sodium phosphate pH 6.5; after loading, the column was washed with a solution of 25 mM sodium phosphate pH 6.5 and desorbed with a solution of 1 M NaCl. Absorbance at 280 nm was monitored throughout the experiment. Fractions, pooled and desorbed, and AEX load were collected and analyzed.

Ion exchange HPLC

Ion exchange HPLC was performed on a MabPac SCX-10 column (4 x 250 mm, 3.14 ml) at ambient temperature using an Agilent 1600 series system. Mobile phase B was 30 mM sodium phosphate pH 8.0 and mobile phase A was 25 mM MES pH 5.8. The column was first equilibrated at 14% mobile phase B at a flow rate of 1.0 ml/min for 10 minutes. The mAb protein was then eluted from the column using a mobile phase B gradient (14% to 80% over 18 min). The column was then purified using 100% mobile phase B for 3 min and re-equilibrated at 14% mobile phase B to analyze the next sample. The absorbance at 280 nm of the eluate was monitored throughout the LC analysis.

LC/MS analysis of intact and reconstituted sample

A 20 μg sample was diluted to 0.5 mg/ml with 50 mM Tris buffer pH 8.0. RP-HPLC separation was performed using a Waters Acquity UPLC H-Class. An Acquity UPLC BEH300 C4 column, 1.7 μm, 1.0×100 mm (Waters, Milford, MA; -O-(Si)(CH ₃ ) ₂ -C ₄ H ₉ ligand) was used. The mobile phases were 0.1% formic acid (FA) in water as mobile phase A and 0.1% FA in acetonitrile (ACN) as mobile phase B. The LC flow rate was 0.08 ml/min, and the column temperature was maintained at 80°C. The antibody was eluted using a 4-15 min gradient of 30% - 90% B. MS spectra were obtained on a Waters Xevo G2 Q-TOF system, scanning in the range m/z 800-4000.

20 μg of sample was diluted with reconstitution buffer (50 mM Tris pH 8.0 containing 6 M Guanidine HCl) to a final volume of 100 μl. Two microliters of a 1M dithiothreitol (DTT) solution (Sigma-Aldrich, St. Louis, MO) was added to each of the samples, followed by incubation at 56° C. for 20 minutes. RP-UPLC separation was performed on a Waters Acquity UPLC H-Class. An Acquity UPLC column, BEH300 C4, 2.1×100 mm, 1.7 µm (Waters) was used. MS spectra were obtained on a Waters Xevo G2 Q-TOF system, scanning in the range m/z 600-3000. MS data were analyzed using the MaxEnt1 program MassLynx 4.1.

Peptide mapping using LC/MS

100 μg of sample was buffer exchanged with 100 μl of denaturing buffer containing 50 mM Tris pH 8.0, 6 M Guanidine HCl and 5 mM EDTA. Reduction reactions were carried out at 56° C. for 30 minutes with 20 mM DTT in solution. Samples were alkylated using 50 mM iodoacetamide at room temperature for 30 minutes in the dark. The alkylation reaction was stopped by adding 1 μl of 500 mM DTT solution. Reduced and alkylated samples were diluted with hydrolysis buffer (50 mM Tris pH 8.0) to a final volume of 300 μl before addition of Lys-C enzyme (Wako, Richmond, VA) at an enzyme:substrate ratio of 1:20 (wt:wt). The solution was incubated at 37°C for 4 hours. Peptides were separated by RP-HPLC on a Waters Acquity UPLC H-Class using a HALO Peptide ES-C18, 2.1×150 nm, 2.7 µm column (MAC-MOD Analytical, Inc., Chadds Ford, PA). MS spectra were obtained on a Waters Xevo G2 Q-TOF system, scanning in the range m/z 100-2000. MS data were analyzed using BiopharmaLynx 1.3 (Waters).

MS/MS target peptide

LC/MS/MS analysis of the target peptide was performed on an LTQ-Orbitrap MS system (Thermo Fisher, Waltham, MA). To obtain MS/MS data, a resolution of 17500 with Fourier transform was used. Peptides were separated on a Waters Acquity UPLC H-Class using a HALO Peptide ES-C18, 2.1×150 mm, 2.7 µm column. MS/MS scanned in m/z ranges depending on the m/z values of the parent ions. The normalized fragmentation energy was set at 35% for CID (collision induced dissociation) fragmentation and 35% for DTE (electron transfer dissociation) fragmentation. MS2 data was processed manually.

Treatment with alkaline phosphatase

10 μg of protein mAb in the AEX desorbed fraction was diluted in 50 μl of phosphatase reaction buffer. 1 μl (10 units) of calf intestine alkaline phosphatase (New England Biolabs, Ipswish, MA) was added to incubate at 37° C. for 1 hour. Chicken ovalbumin (Sigma) was treated simultaneously as a positive control. 10 μl of the solution was injected into the LC/MS system for mass analysis.

Western blotting

Magic Mark XP Western Standard (Invitrogen) and specific concentrations of both monoclonal antibodies (mAb) and control cell extracts (HEK293 total cell extract and EGF stimulated A431 cell lysate (Millilpore)) were reconstituted with β-mercaptoethanol plus heating at 95°C , then separated by Tris-glycine SDS-PAGE using a 4-20% gradient gel (Novex). The separated proteins were then electrotransferred to a nitrocellulose membrane and washed overnight in Tris buffered saline plus 0.05% Tween20 (TBST) (Sigma) with shaking at 4°C. The membranes were then blocked for 1 hour in Tris buffered saline plus 1% BSA (TBS-BSA) (Sigma) at room temperature with constant agitation. Primary antibodies (anti-sulfotyrosine/anti-tyrosine sulfation (Millipore) or anti-human IgG (H+L) (Jackson ImmunoResearch Labratories Inc.)) were diluted in TBS-BSA and incubated with the membrane for 2 hours at room temperature. After washing with TBST, HRP-conjugated secondary antibodies (goat-anti-mouse or goat-anti-rabbit (Thermo Scientific)) were diluted in 5% milk protein from skimmed milk plus 0.05% Tween20-phosphobuffer saline (Invitrogen) and incubated at room temperature within 1 hour. After a final wash with TBST, chemiluminescent substrates (Thermo Scientific) were used for development; signals were recovered by exposure to photographic film (GE Healthcare Life Sciences) and post-processing. Purification of the nitrocellulose membrane between primary antibodies was performed as previously described (Kaufmann SH, E.C., Shaper JH., The erasable Western blot. Anal Biochem., 1987. 161(1): p. 89-95).

Results and discussion

Separation of mAb molecules

Anion exchange chromatography (AEX) is commonly used as a post-purification step in the purification of monoclonal antibodies. This step typically operates in a flow-through mode where the mAb flows through the column and impurities released during the process (host cell proteins, DNA) are bound to the column. During the AEX, it was observed that there is a resin-bound fraction of mAbs loaded onto the column that affects protein recovery. The bound protein fraction was eluted in the stripped fraction of AEX chromatography. To characterize the mAbs bound to the AEX column, the following fractions of AEX chromatography were analyzed: "loaded" refers to a sample prior to AEX purification; "combined" refers to the flow path of the sample, and "desorbed" refers to the bound fraction of the sample. Loaded, combined and desorbed fractions from AEX chromatography were first analyzed by IEX-HPLC chromatography. 1 shows the IEX-HPLC profile of the mAb in the loaded, pooled and desorbed AEX fraction. As shown in FIG. 1, the stripped fraction had significantly more acid variants compared to the loaded and pooled fraction: ~65% acid variants in the stripped fraction (thin line) compared to 23% in the pooled fraction (dashed line) and 33% in the loaded fraction (thick line). ). An additional difference was noted in the acid peak before the main component peak. This peak was present in the original AEX sample at higher levels, while this peak was minimal in the pooled AEX fraction. The desorbed fraction was enriched in the acidic peak before the peak of the main component. AEX chromatography was also carried out at pH 7.0 or 7.5 using the same conditions for buffers and salts as described above. At pH 7.0, a similar recovery of the acidic peak before the main component peak was also observed in the pooled AEX fraction.

Analysis of intact and reduced protein by mass spectrometry

To characterize impurities, all three AEX fractions (original, pooled and desorbed) were analyzed by LC/MS to determine intact and reduced protein using Q-TOF MS (time of flight mass spectrometry). Fig. 2 shows the mass spectra of the intact molecule after deconvolution. Three main glycoforms were observed in all three fractions: G0F/G0F, G0F/G1F and G1F/G1F with a mass of 148591 Da, 148751 Da and 148912 Da, respectively. The calculated intact mass of this molecule with G0F/G0F is 148590 Da. Errors in measurements of intact mass were all within 25 ppm. Additional species were found only in the AEX desorbed fraction. These species correspond to an increase in mass of 80 Da (148668, 148830, 148991 Da) of the three main glycoforms (G0F/G0F, G1F/G0F, G1F/G1F). To determine the position of the modification, the weight of the light chain and heavy chain was measured after cleavage of disulfide bonds with the reducing agent DTT. No difference was found in the weight of the heavy chain in the desorbed fraction and the combined fraction, suggesting that the modification is not on the heavy chain (data not shown). As shown in FIG. 3, the mass of the apo light chain (23674 Da) and the mass of the glycated light chain (23836 Da) were found in both fractions (desorbed and pooled fractions). A peak with an 80 Da increase in the light chain was observed only at 23754 Da in the desorbed fraction of AEX. The errors in the measurements of the recovered mass were within 20 ppm. These data suggest that 80 Da modifications are present on the Ab6 light chain.

Analysis of mAb antibodies by peptide mapping

To further clarify the site of modification, the desorbed and combined AEX fractions were reduced, alkylated, and then cleaved with LysC enzyme. Peptide mixtures were analyzed by mass spectrometric mapping using a Q-TOF MS mass spectrometer. When comparing the UV spectra of these two fractions, two differences were found. As shown in FIG. 4(a) and (b), two new peaks were found at retention times of 37.6 minutes and 65.2 minutes in the stripped AEX fraction. The m/z observed in the new peaks were 1165.4796 (2+) at 37.6 min and 1476.7372 (4+) Da at 65.2 min. The observed masses correspond to the light chain peptide AA25-43+80Da and AA25-78+80 Da with mass errors of 6.4 ppm and 9.8 ppm, respectively. The AA25-78 light chain peptide contains one miscleavage site. The modified and unmodified form of the light chain peptide AA25-43 and AA25-78 are labeled in FIG. 4. The level of this modified peptide was estimated to be 20.9% and 21.6% for AA25-43 and AA25-78 compared to their apo forms based on extraction ion chromatogram (SIC) peak area.

MS/MS fragmentation of the modified peptide

There are two possibilities for modification with an increase in mass by 80 Da: phosphorylation (+79.9663 Da) and sulfation (+79.9568 Da). Theoretically, the difference in mass between these two modifications is only 0.0095 Da, which makes it difficult to establish a difference only in mass. First, the target AA25-78 peptide was fragmented by collision-induced dissociation (CID) and the resulting fragments were analyzed by LTQ-Orbitrap MS. As shown in FIG. 5, complete loss of the modification group (80Da) from the parent ion was observed. Only fragments from the peptide backbone were found, confirming the peptide sequence of LC25-43. However, no site-specific information was obtained from the fragmentation LED. It has been reported that sulfated tyrosine (sY) is very labile and can easily be lost under standard LED conditions (Nemeth-Cawley JF1, KS, Rouse JC., Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry. J Mass Spectrom., 2001 36(12): pp. 1301-11). It was not possible to obtain site-specific information on the localization of sulfate groups using SID/MS/MS in positive ion mode, since none of the original parent ions were present at the time of fragmentation of the peptide backbone. In contrast, phosphorylated peptides are usually preserved under SID conditions, and fragmentation of the peptide backbone allows site-specific identification of the modification (Nemeth-Cawley JF1, KS, Rouse JC., Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry. J Mass Spectrom., 2001. 36(12): pp. 1301-11). On FIG. 5 there is a neutral loss of 80 Da from the parent ion. It is known that the characteristic ion with a neutral loss for phosphorylation is -H ₃ PO ₄ (-98 Da), and the characteristic fragmentary ion is PO ₃ ^- (-79 Da). Whereas for sulfation the characteristic ions with neutral loss and fragment ions are both -SO ₃ ions with 80 Da (Monigatti F, HB, Steen H., Protein sulfation analysis--A primer. Biochim Biophys Acta., 2006. 1764(12) : pp. 1904-13). The MS2 LED data indicates that the 80 Da modification is sulfation.

Another widely used fragmentation mechanism is electron transfer dissociation (EDT). It transfers an electron to a multiply protonated peptide/protein, which can lead to cleavage of N-Cα bonds in the main chain and the formation of c- and z-type fragment ions without loss of PTM localization information (Mikesh LM, UB, Chi A, Coon JJ, Syka JE, Shabanowitz J, Hunt DF., The utility of ETD mass spectrometry in proteomic analysis Biochim Biophys Acta., 2006. 1764(12): pp. 1811-22. DPE can provide complementary information to SID: The DPE process allows the retention of the SO ₃ group and thus amino acid localization, while SID preferentially fragments labile modifications. In this case, the target peptide was analyzed using LTQ-Orbitrap with DPE fragmentation and high resolution mass detection. As shown in FIG. 6, partial loss of 80 Da modifications was observed on the parent ion. Fragment ions with an attached SO ₃ group (80 Da) are labeled. Based on the detection of fragment ions with an attached SO ₃ group (c9, c11, c12-16), the modification site was identified as tyrosine 31 on the light chain, which is located in the CDR1 region of the mAb molecule.

Treatment with alkaline phosphatase

Since tyrosine phosphorylation and sulfation are isobaric, alkaline phosphatase was used to distinguish between these two modifications (Yu Y, H.A., Moore KL, Leary JA., Determination of the sites of tyrosine O-sulfation in peptides and proteins. Nat Methods, 2007. 4( 7): pp. 583-8). Alkaline phosphatase is widely used to remove a phosphorylation group from proteins. Chicken albumin was used as a positive control as this protein is well known for its form of phosphorylation and glycosylation. Chicken albumin and mAb in the desorbed AEX fraction were treated with phosphatase and incubated at 37° C. simultaneously. Fig. 7 shows the measured intact mass of mAb and chicken ovalbumin before and after phosphatase treatment. As shown in FIG. 7(a), no weight change was observed for the mAb. With respect to chicken albumin (FIG. 7(b)), a clear weight change of 160 Da was observed for all major glycoforms. Since hen albumin contains two phosphorylation sites, the loss of 160 Da confirms the activity of alkaline phosphatase. Since no weight change was detected before and after phosphatase treatment, this indicates that this mAb in the desorbed AEX fraction is not phosphorylated.

Western blot

To date, LC/MS analysis has been used to investigate the nature of the 80 Da adduct to tyrosine 31 in the light chain CDR. MS2 and AEX mass analysis of the desorbed mAb fraction following phosphatase treatment suggested that the 80 Da adduct is sulfation on tyrosine 31. However, the ability of these mass analysis-based methods to directly distinguish between tyrosine sulfation and phosphorylation is problematic due to the similar molecular weight these two groups. To begin addressing this issue, Western blotting with an anti-sulfotyrosine-specific monoclonal antibody was used to confirm the presence of tyrosine sulfation in the AEX desorbed mAb fraction (Xu J, D.X., Tang M, Li L, Xiao L, Yang L, Zhong J, Bode AM, Dong Z, Tao Y, Cao Y., Tyrosylprotein sulfotransferase-1 and tyrosine sulfation of chemokine receptor 4 are induced by Epstein-Barr virus encoded latent membrane protein 1 and associated with the metastatic potential of human nasopharyngeal carcinoma.PLoS One. , 2013. 8(3): p.e56114). On FIG. 8a (upper panel), normalized concentrations of mAb AEX pooled and desorbed fractions were subjected to SDS-PAGE under reducing conditions, analyzed for human heavy and light chains by Western blot hybridization. Elevated concentrations of heavy and light chains from the pooled and desorbed fractions were then "purified" of the first detectable antibodies and re-analyzed with an anti-sulfotyrosine-specific monoclonal antibody (lower panel). As shown in FIG. 8a (bottom panel), positive signals were found only on the light chain of the desorbed fractions, indicating that it contains tyrosine sulfation. As a control for phosphorylation cross-reactivity, lane 1 was loaded with a commercial source of EGF-treated A431 cell extract that is enriched in phosphorylated proteins. The absence of a positive signal in lane 1 of the lower panel indicates that the anti-sulfotyrosine monoclonal antibody does not have strong cross-reactivity with phosphorylation (Fig. 8a, lower panel). In addition, an extract of HEK 293, which was proposed by the manufacturer as a positive control for an anti-sulfotyrosine monoclonal antibody, was loaded into lane 2. A positive signal below the lower 20 kDa value was consistent with the manufacturer's analysis (Fig. 8a, bottom panel). On FIG. 8b, normalized concentrations of various CHO-derived monoclonal antibodies (mAb1, 2 and 3), in addition to the AEX desorbed and pooled fractions, were subjected to SDS-PAGE under reducing conditions, examined for human HC and LC by Western blot hybridization (upper panel). ), then desorbed and again examined for antisulfotyrosine (lower panel). In accordance with FIG. 8a, only the AEX desorbed mAb fraction shows a positive signal at the light chain position when probed with an anti-sulfotyrosine antibody. No positive tyrosine sulfation signal was observed for three other CHO Merck-derived mAbs when assaying similar amounts of protein. This is consistent with the observation that no elevated AEX acid peak or tyrosine sulfation "hot spot" was detected on these three mAbs (data not shown).

Retention Time Comparison Using a Synthetic Peptide with Sulfation or Phosphorylation

To further distinguish between phosphorylation and sulfation, a synthetic peptide with sequence identical LC AA25-43 (XSXSXD Y EGDSDXXXXXXX) (SEQ ID NO: 65) modified with either Y31 phosphorylation or sulfation was analyzed by LC/MS. Fig. 9 shows the SIC of the synthetic peptide XSXSXD Y EGDSDXXXXXXX (SEQ ID NO: 65)+phosphorylation, XSXSXD Y EGDSDXXXXXXX (SEQ ID NO: 65)+sulfation and AA25-43+80Da in the desorbed AEX fraction. The sulfated synthetic peptide eluted at the same retention time as the desorbed AEX fraction, while the phosphorylated synthetic peptide eluted earlier than the desorbed AEX fraction. This is further support for the observation made by the present inventors that Y31 on the light chain is sulfated.

Tyrosine sulfation site structure

The protein tyrosine sulfation reaction is catalyzed by a Golgi enzyme called tyrosyl protein sulfotransferase (TPST). Previous studies have shown that TPST recognizes available tyrosine residues that are usually surrounded by several acidic residues ranging from -5 to +5 positions (Hortin G, FR, Gordon JI, Strauss AW., Characterization of sites of tyrosine sulfation in proteins and criteria for predicting their occurrence Biochem Biophys Res Commun., 1986. 141(1): pp. 326-33 Rosenquist GL, NHJ, Analysis of sequence requirements for protein tyrosine sulfation, Protein Sci., 1993. 2(2): p. 215-22; Teramoto T1, FY, Kawaguchi Y, Kurogi K, Soejima M, Adachi R, Nakanishi Y, Mishiro-Sato E, Liu MC, Sakakibara Y, Suiko M, Kimura M, Kakuta Y, Crystal structure of human tyrosylprotein sulfotransferase -2 reveals the mechanism of protein tyrosine sulfation reaction, Nat Commun., 2013. 4: p. 1572). The acceptor tyrosine must have acidic residues nearby to allow recognition of positively charged residues at the TPST2 substrate binding site. The acceptor tyrosine must also be in a substantially flexible region to fit into the deep cleft of TPST2. However, a common consensus sequence for tyrosine sulfation sites has not been determined. The most typical features describing the environment of a sulfated tyrosine in a sequence include the presence of one acidic amino acid within two residues of a tyrosine; the presence of at least three acidic amino acids within 5 residues and the presence of bend-inducing amino acids nearby, and so on. (Monigatti F, HB, Steen H., Protein sulfation analysis--A primer. Biochim Biophys Acta., 2006. 1764(12): p. 1904-13). Fig. 10 shows the structure of the mAb tyrosine region in the context of CDR loops in a strip diagram generated by MOE (Chemical Computing Group, Montreal, Canada). The sequence next to the Y31 light chain on this mAb is: XSXSXD Y EGDSDXXXXXXX (SEQ ID NO: 65). In this sequence, the residues adjacent to Y31 are acidic: aspartic acid (D) and glutamic acid (E). A total of four acidic residues are within five residues of Y31: three D and one E. Four bend-inducing residues are close to Y31: three serines (S) and one glycine (G). The unique structure of Y31 with neighboring acidic amino acids and local secondary structure elements is essential for this modification to occur.

The inventors describe in this application evidence indicating the presence of unexpected O-linked tyrosine sulfation in CHO-produced antibodies. The position of this labile modification was found in the CDR1 region of the light chain, which was identified by mass spectrometry with DPE fragmentation. This tyrosine sulfation was further confirmed by phosphatase treatment, Western blotting using an anti-tyrosine sulfation antibody, and retention time correlation with a synthetic sulfated peptide. Structural analysis of tyrosine CDR confirms the effect of acidic residues on sulfation. Neighboring acidic amino acid residues and local secondary structure elements may play a significant role in making Y31 a hotspot for sulfation.

The scope of the present invention should not be limited to the specific embodiments described in this application. Indeed, the scope of the present invention includes the embodiments specifically set forth in this application and other embodiments not specifically set forth in this application; the embodiments specifically set forth in this application need not be exhaustive. Various modifications of the invention in addition to those described in this application will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be covered by the scope of the claims.

Throughout this application, references are made to patents, patent applications, publications, product descriptions, and protocols, the disclosures of which are incorporated herein by reference in their entirety for all purposes. The present application claims priority to U.S. Provisional Application No. 62/414209, incorporated herein by reference in its entirety. All referenced documents cited in this application are incorporated by reference to the extent that each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application or patent were specifically and individually designated for inclusion as links. Statement of incorporation by reference, pursuant to 37 C.F.R. 1.57(b)(1), as Applicants intend, refers to each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is clearly identified under 37 C.F.R. 1.57(b)(2), even if there is no specific indication of incorporation by reference when such reference is made immediately adjacent. The inclusion of specific indications of inclusion by reference, if any, in the description of the invention in no way weakens this general statement of inclusion by reference. The citation of referenced documents in this application is not intended to be an admission that the referenced document is prior art and does not constitute any admission as to the content or date of those publications or documents. In the event that the referenced documents provide a definition of a claimed term that conflicts with the definitions provided in this specification, the definitions provided in this specification should be used to interpret the claimed invention.

--->

SEQUENCE LIST

<110> Merck Sharp & Dohme Corp.

Zhao, Jia

Rios, Sandra

Dukleska Schussler, Svetlana

<120> Purification method for removing sulfated antibody variants

tyrosine; purified compositions

<130> 24380

<150> 62/414.209

<151> 2016-10-28

<160> 65

<170>PatentIn version 3.5

<210> 1

<211> 408

<212> DNA

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 1

atgaaatgca gctgggtcat cttcttcctg atggcagtgg ttataggaat caattcagag

60

gttcagctgc tccagtctgg ggcagaactt gtgaggtcag gggcctcagt caagttgtcc

120

tgcacagcct ctggcttcaa cattgaagac tactatatgc actggatgaa acagaggcct

180

gaacagggcc tggagtggat tggatggatt gatcctgtga atggtgatac tgaatatgcc

240

ccgaagttcc agggcaaggc cactatgact gcagacacat cctccaacac agcctaccta

300

cacctcaaca gcctgacatc tgaggacact gccgtctatt actgtaattt ctatgatggt

360

tacctctttg ctttctgggg ccaagggacc ctggtcactg tctctgca

408

<210> 2

<211> 136

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 2

Met Lys Cys Ser Trp Val Ile Phe Phe Leu Met Ala Val Val Ile Gly

1 5 10 15

Ile Asn Ser Glu Val Gln Leu Leu Gln Ser Gly Ala Glu Leu Val Arg

20 25 30

Ser Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile

35 40 45

Glu Asp Tyr Tyr Met His Trp Met Lys Gln Arg Pro Glu Gln Gly Leu

50 55 60

Glu Trp Ile Gly Trp Ile Asp Pro Val Asn Gly Asp Thr Glu Tyr Ala

65 70 75 80

Pro Lys Phe Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn

85 90 95

Thr Ala Tyr Leu His Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Asn Phe Tyr Asp Gly Tyr Leu Phe Ala Phe Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ala

130 135

<210> 3

<211> 10

<212> Protein

<213> Mus

<400> 3

Gly Phe Asn Ile Glu Asp Tyr Tyr Met His

1 5 10

<210> 4

<211> 17

<212> Protein

<213> Mus

<400> 4

Trp Ile Asp Pro Val Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln

1 5 10 15

gly

<210> 5

<211> 8

<212> Protein

<213> Mus

<400> 5

Tyr Asp Gly Tyr Leu Phe Ala Phe

15

<210> 6

<211> 396

<212> DNA

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 6

atgaggtgcc tagctgagtt cctggggctg cttgtgctct ggatccctgg agccattgggg

60

gatattgtgc tgactcaggc tgcaccctct gtacctgtca ctcctggaga gtcagtgtcc

120

atctcctgca ggtctagtaa gagtctcctg catagtgatg gcaacactta tctgtattgg

180

ctcctgcaga ggccaggcca gtctcctcag ctcctgatat atcggatgtc caaccttgcc

240

tcaggggtcc cagacaggtt cagcggcagt gggtcaggaa ctgttttcac actgagaatc

300

agcagactgg aggctgagga tgtgggtatt tattactgta tgcaacatct agaatatcct

360

ttcacgtttg gaggggggac caagctggaa ataaaa

396

<210> 7

<211> 132

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 7

Met Arg Cys Leu Ala Glu Phe Leu Gly Leu Leu Val Leu Trp Ile Pro

1 5 10 15

Gly Ala Ile Gly Asp Ile Val Leu Thr Gln Ala Ala Pro Ser Val Pro

20 25 30

Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser

35 40 45

Leu Leu His Ser Asp Gly Asn Thr Tyr Leu Tyr Trp Leu Leu Gln Arg

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Val Phe

85 90 95

Thr Leu Arg Ile Ser Arg Leu Glu Ala Glu Asp Val Gly Ile Tyr Tyr

100 105 110

Cys Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys

115 120 125

Leu Glu Ile Lys

130

<210> 8

<211> 16

<212> Protein

<213> Mus

<400> 8

Arg Ser Ser Lys Ser Leu Leu His Ser Asp Gly Asn Thr Tyr Leu Tyr

1 5 10 15

<210> 9

<211> 8

<212> Protein

<213> Mus

<400> 9

Tyr Arg Met Ser Asn Leu Ala Ser

15

<210> 10

<211> 9

<212> Protein

<213> Mus

<400> 10

Met Gln His Leu Glu Tyr Pro Phe Thr

15

<210> 11

<211> 414

<212> DNA

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 11

atgggatgga gctggatctt tcttttcctc ctgtcaggaa ctgcaggtgt ccgttgccag

60

atccgactgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaagatatcc

120

tgcaaggctt ctgggtcctc cttcactgac tactatataa actgggtgaa gcagaagcct

180

ggacagggac ttgagtggat tggatggatt tatcctggaa gcggtaattc tatctacaat

240

gagaacttca aggccaaggc cacattgact gtagacacat cctccagcac agcctacatg

300

catctcagca gcctgacatc tgaggacact gctgtctatt tctgtgcaag agaggctgat

360

tacgacgatg ctttggacta ctggggtcaa ggaacctcgg tcaccgtctc ctca

414

<210> 12

<211> 138

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 12

Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly

1 5 10 15

Val Arg Cys Gln Ile Arg Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe

35 40 45

Thr Asp Tyr Tyr Ile Asn Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Trp Ile Tyr Pro Gly Ser Gly Asn Ser Ile Tyr Asn

65 70 75 80

Glu Asn Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser

85 90 95

Thr Ala Tyr Met His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Phe Cys Ala Arg Glu Ala Asp Tyr Asp Asp Ala Leu Asp Tyr Trp

115 120 125

Gly Glyn Gly Thr Ser Val Thr Val Ser Ser

130 135

<210> 13

<211> 10

<212> Protein

<213> Mus

<400> 13

Gly Ser Ser Phe Thr Asp Tyr Tyr Ile Asn

1 5 10

<210> 14

<211> 17

<212> Protein

<213> Mus

<400> 14

Trp Ile Tyr Pro Gly Ser Gly Asn Ser Ile Tyr Asn Glu Asn Phe Lys

1 5 10 15

Ala

<210> 15

<211> 10

<212> Protein

<213> Mus

<400> 15

Glu Ala Asp Tyr Asp Asp Ala Leu Asp Tyr

1 5 10

<210> 16

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 16

atggtatcca cacctcagtt ccttgtattt ttgcttttct ggattccagc ctccagaggt

60

cacatcttgc tgactcagtc tccagccatt ctgtctgtga gtccaggaga aagagtcagt

120

ttctcctgca gggccagtca gagcattggc acaagcatac actggtatca gcaaagaaca

180

aatggttctc caaggcttct cataaagtat gcttctgagt ctatctctgg gatcccttcc

240

aggtttagtg gcagtggatc agggacagat tttactctta gcatcaacag tgtggagtca

300

gaagatattg cagattatta ctgtcaacaa agtaatagct ggccaacgta cacgttcgga

360

ggggggacca agctggaaat aaaa

384

<210> 17

<211> 128

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 17

Met Val Ser Thr Pro Gln Phe Leu Val Phe Leu Leu Phe Trp Ile Pro

1 5 10 15

Ala Ser Arg Gly His Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser

20 25 30

Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser

35 40 45

Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro

50 55 60

Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn

85 90 95

Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn

100 105 110

Ser Trp Pro Thr Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 18

<211> 11

<212> Protein

<213> Mus

<400> 18

Arg Ala Ser Gln Ser Ile Gly Thr Ser Ile His

1 5 10

<210> 19

<211> 7

<212> Protein

<213> Mus

<400> 19

Tyr Ala Ser Glu Ser Ile Ser

15

<210> 20

<211> 10

<212> Protein

<213> Mus

<400> 20

Gln Gln Ser Asn Ser Trp Pro Thr Tyr Thr

1 5 10

<210> 21

<211> 426

<212> DNA

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 21

atgagatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt caactcccag

60

gtccaactgc agcagcctgg ggctgagctt gtgatgcctg gggcttcagc gaagatgtcc

120

tgcaaggctt ctggctacac actcactgac tactggatgc actgggtgaa gcagaggcct

180

ggacaaggcc ttgagtggat cggagcgatt gatatttctg atagttattc tagctacaat

240

caaaagttca agggcaaggc cacattgact gtagacgaat cctccagcac agcctacatg

300

cagctcacca gcctgacatc tgaggactct gcggtctatt actgtgcaag atcccctttc

360

tacaatagta gaggggggaa ctactttgac tactggggcc aaggcaccac tctcacagtc

420

tcctca

426

<210> 22

<211> 142

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 22

Met Arg Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val Asn Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Met

20 25 30

Pro Gly Ala Ser Ala Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Leu

35 40 45

Thr Asp Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Ile Ser Asp Ser Tyr Ser Ser Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Pro Phe Tyr Asn Ser Arg Gly Gly Asn Tyr

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135 140

<210> 23

<211> 10

<212> Protein

<213> Mus

<400> 23

Gly Tyr Thr Leu Thr Asp Tyr Trp Met His

1 5 10

<210> 24

<211> 17

<212> Protein

<213> Mus

<400> 24

Ala Ile Asp Ile Ser Asp Ser Tyr Ser Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

gly

<210> 25

<211> 14

<212> Protein

<213> Mus

<400> 25

Ser Pro Phe Tyr Asn Ser Arg Gly Gly Asn Tyr Phe Asp Tyr

1 5 10

<210> 26

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 26

atgatgtcct ctgctcagtt ccttggtctc ctgttgctct gttttcaagg taccagatgt

60

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc

120

atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca

180

gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca

240

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa

300

gaagatattg ccacttactt ttgccaacag ggtgatacgc ttcctccgtg gacgttcggt

360

ggaggcacca agctggaaat caaa

384

<210> 27

<211> 128

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 27

Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln

1 5 10 15

Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp

35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val

50 55 60

Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser

85 90 95

Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asp

100 105 110

Thr Leu Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 28

<211> 11

<212> Protein

<213> Mus

<400> 28

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 29

<211> 7

<212> Protein

<213> Mus

<400> 29

Tyr Thr Ser Arg Leu His Ser

15

<210> 30

<211> 10

<212> Protein

<213> Mus

<400> 30

Gln Gln Gly Asp Thr Leu Pro Pro Trp Thr

1 5 10

<210> 31

<211> 447

<212> DNA

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 31

atgggatgga cctggatctt tctcttcttc ctgtcaggaa ctgcaggtgt cctctctgag

60

gtcctgctgc tacagtctgg acctgaactg gtgaagcctg ggacttcagt gaaaatcccc

120

tgcaaggctt ctggatacac attcactgac tacaacgtgg actgggtgaa gcagcgccat

180

ggaaagggcc ttgagtggat tggagatatt aatccaaaca atggtggtac tatctacagt

240

cagaaattca agggcaaggc cacattgact gttgacaagt cctccagcac agccttcatg

300

gagctccgca gcctgacatc tgaggacact gcagtctatt tctgtgcaag gaactatagg

360

tggtttggtg ctatggacca ctggggtcaa ggaacctcag tcaccgtctc ctcagccaaa

420

acaacagccc catcggtcta tccactg

447

<210> 32

<211> 138

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 32

Met Gly Trp Thr Trp Ile Phe Leu Phe Phe Leu Ser Gly Thr Ala Gly

1 5 10 15

Val Leu Ser Glu Val Leu Leu Leu Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Thr Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asp Tyr Asn Val Asp Trp Val Lys Gln Arg His Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Ile Tyr Ser

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Phe Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val

100 105 110

Tyr Phe Cys Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp

115 120 125

Gly Glyn Gly Thr Ser Val Thr Val Ser Ser

130 135

<210> 33

<211> 5

<212> Protein

<213> Mus

<400> 33

Asp Tyr Asn Val Asp

15

<210> 34

<211> 17

<212> Protein

<213> Mus

<400> 34

Asp Ile Asn Pro Asn Asn Gly Gly Thr Ile Tyr Ser Gln Lys Phe Lys

1 5 10 15

gly

<210> 35

<211> 10

<212> Protein

<213> Mus

<400> 35

Asn Tyr Arg Trp Phe Gly Ala Met Asp His

1 5 10

<210> 36

<211> 547

<212> DNA

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 36

atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ttccactggt

60

gacattgtgt tgacccaatc tccagcttct ttggctgtgt ctccagggca gagggccacc

120

atttcctgca aggccagtca aagtcttgat tatgaaggtg atagtgatat gaattggtac

180

caacagaaac caggacagcc acccagactc ctcatctctg gtgcatccaa tctagagtct

240

gggatcccag ccaggttcag tggcagtggg tctgggacag acttcactgt taacatccat

300

cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtactga ggatcctcgg

360

acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc

420

atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg

480

aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa

540

aatggcg

547

<210> 37

<211> 131

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 37

Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Pro Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser

35 40 45

Leu Asp Tyr Glu Gly Asp Ser Asp Met Asn Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Arg Leu Leu Ile Ser Gly Ala Ser Asn Leu Glu Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Val Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Ser Thr Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210> 38

<211> 15

<212> Protein

<213> Mus

<400> 38

Lys Ala Ser Gln Ser Leu Asp Tyr Glu Gly Asp Ser Asp Met Asn

1 5 10 15

<210> 39

<211> 7

<212> Protein

<213> Mus

<400> 39

Gly Ala Ser Asn Leu Glu Ser

15

<210> 40

<211> 9

<212> Protein

<213> Mus

<400> 40

Gln Gln Ser Thr Glu Asp Pro Arg Thr

15

<210> 41

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 41

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 42

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 42

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 43

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 43

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 44

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 44

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Ser Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 45

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 45

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 46

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 46

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asp Gly Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 47

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 47

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 48

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 48

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Gln Gly Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 49

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 49

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 50

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 50

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 51

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 51

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 52

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 52

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asp Gly Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 53

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 53

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 54

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 54

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Ser Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 55

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 55

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 56

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 56

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Gln Gly Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 57

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 57

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Leu Asp Tyr Glu

20 25 30

Gly Asp Ser Asp Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro

35 40 45

Gln Leu Leu Ile Tyr Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Thr

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 58

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized mouse

<400> 58

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Val Asp Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Gly Gly Gly Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Arg Trp Phe Gly Ala Met Asp His Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 59

<211> 17

<212> Protein

<213> Mus

<400> 59

Asp Ile Asn Pro Asn Asn Gly Gly Thr Ile Tyr Ala Gln Lys Phe Gln

1 5 10 15

Glu

<210> 60

<211> 17

<212> Protein

<213> Mus

<400> 60

Asp Ile Asn Pro Asn Ser Gly Gly Thr Ile Tyr Ala Gln Lys Phe Gln

1 5 10 15

Glu

<210> 61

<211> 17

<212> Protein

<213> Mus

<400> 61

Asp Ile Asn Pro Asn Asp Gly Gly Thr Ile Tyr Ala Gln Lys Phe Gln

1 5 10 15

Glu

<210> 62

<211> 17

<212> Protein

<213> Mus

<400> 62

Asp Ile Asn Pro Asn Gln Gly Gly Thr Ile Tyr Ala Gln Lys Phe Gln

1 5 10 15

Glu

<210> 63

<211> 17

<212> Protein

<213> Mus

<400> 63

Asp Ile Asn Pro Asn Gly Gly Gly Thr Ile Tyr Ala Gln Lys Phe Gln

1 5 10 15

Glu

<210> 64

<211> 17

<212> Protein

<213> Mus

<220>

<221> OTHER_INDICATOR

<222>(6)..(6)

<223> X is D,N,S or Q

<220>

<221> OTHER_INDICATOR

<222> (12)..(12)

<223> X is A or S

<220>

<221> OTHER_INDICATOR

<222> (16)..(16)

<223> X is Q or K

<220>

<221> OTHER_INDICATOR

<222> (17)..(17)

<223> X is E or G

<400> 64

Asp Ile Asn Pro Asn Xaa Gly Gly Thr Ile Tyr Xaa Gln Lys Phe Xaa

1 5 10 15

xaa

<210> 65

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide

<220>

<221> other_characteristic

<222> (1)..(1)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_characteristic

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_characteristic

<222> (5)..(5)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> other_characteristic

<222> (13)..(19)

<223> Xaa can be any naturally occurring amino acid

<400> 65

Xaa Ser Xaa Ser Xaa Asp Tyr Glu Gly Asp Ser Asp Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa

<---

Claims (14)

1. A method for removing an antibody with sulfated tyrosine from an aqueous mixture, including an antibody in which tyrosine is sulfated, and an antibody in which tyrosine is not sulfated, including adjusting the pH of the mixture to 6.5 - 7.5, contacting the aqueous mixture with an anion exchange resin and retaining the resin-free aqueous fraction of the resin mixture, wherein the antibody in which tyrosine is sulfated or the antibody in which tyrosine is not sulfated is an anti-human LAG3 antibody that contains the heavy chain amino acid sequence of SEQ ID NO: 52 and the amino acid sequence of the light chain SEQ ID NO: 51; or the heavy chain amino acid sequence of SEQ ID NO: 52 and the light chain amino acid sequence of SEQ ID NO: 51 with the heavy chain N-terminal glutamine converted to pyroglutamate and/or the heavy chain C-terminal lysine removed; and where tyrosine sulfation occurs in the CDR-L1 region. 2. The method of claim 1 wherein the step of adjusting the pH of the mixture comprises adjusting the pH of the mixture to 6.5-7.0. 3. The method according to p. 1, including balancing the anion-exchange chromatographic resin, which includes an anion-exchange ligand, in a chromatographic column with a solution of sodium phosphate 10-50 mm pH 6.5-7.5, bringing the pH of the aqueous mixture to 6.5-7.5 , applying the mixture to the column, collecting the flow fraction from the column, washing the resin in the column with a solution of sodium phosphate 10-50 mm pH 6.5-7.5 and collecting the flow fraction from the wash. 4. The method of claim 1 or 2, further comprising washing the resin with the aqueous composition under isocratic conditions and removing and retaining the rinse composition from the resin. 5. The method according to p. 2, including balancing the anion-exchange chromatographic resin, which includes dimethylaminopropyl anion-exchange ligand, in a chromatographic column with a solution of 25 mm sodium phosphate pH 6.5, bringing the pH of the aqueous mixture to 6.5, applying the mixture to the column, collecting the flow fraction from the column, washing the resin in the column with a solution of 25 mm sodium phosphate pH 6.5 and collecting the flow fraction from the wash. 6. The method according to p. 2, including balancing an anion-exchange chromatographic resin, which includes a quaternized polyethyleneimine anion-exchange ligand, in a chromatographic column with a solution of 25 mm sodium phosphate and 5 mm NaCl pH 7.0, bringing the pH of the aqueous mixture to 7.0, applying the mixture to column, collecting the running fraction from the column, washing the resin in the column with a solution of 25 mM sodium phosphate and 5 mM NaCl pH 7.0, and collecting the running fraction from the wash. 7. The method according to any one of paragraphs. 1-6 where the anion exchange chromatography flow-through is collected at A ₂₈₀ uptake when A ₂₈₀ first reaches at least 2.5 AU/cm and continues until A ₂₈₀ falls below 1.0 AU density/cm. 8. The method according to any one of paragraphs. 1-7, further comprising purifying the antibody or antigen-binding fragment by cation exchange chromatography, anion exchange chromatography in binding-elution mode, hydrophobic interaction chromatography, protein-A chromatography, protein-L chromatography, protein-G chromatography, hydroxyapatite chromatography, size exclusion chromatography , fractionated precipitation, filtration, centrifugation or viral inactivation. 9. The method according to any one of paragraphs. 1-8, where the light chains and heavy chains of immunoglobulin antibody fragments are expressed in a Chinese hamster ovary cell. 10. The method according to any one of paragraphs. 1-8, wherein the antibody in which the tyrosine is not sulfated is an antibody having a molecular weight of 148590 Da, 148752 Da and/or 148914 Da. 11. Composition for therapy, comprising an antibody that specifically binds to human lymphocyte activation gene 3 (hLAG3) (anti-hLAG3), comprising: the amino acid sequence of the heavy chain of SEQ ID NO: 52 and the amino acid sequence of the light chain of SEQ ID NO: 51, where anti-hLAG3 antibody expressed in Chinese hamster ovary cells; and the composition has less than 0.5% anti-hLAG3 antibodies that contain sulfated tyrosine on CDR-L1 compared to anti-hLAG3 antibodies in which the tyrosine is not sulfated. 12. The composition of claim 11 wherein the anti-hLAG3 antibody that does not contain sulfated tyrosine is a glycosylated antibody species having molecular weights of about 148590 Da, 148752 Da or 148914 Da. 13. The composition of claim 11 wherein the anti-hLAG3 antibody has an N-terminal heavy chain glutamine converted to pyroglutamate and/or a C-terminal heavy chain lysine removed. 14. The composition according to any one of paragraphs. 11-13, where anti-hLAG3 antibodies that contain sulfated tyrosine on CDR-L1 or anti-hLAG3 antibodies that do not contain sulfated tyrosine are detected by mass spectroscopy.

Images