PURIFICATION PROCESS FOR REMOVAL OF TYROSINE

SULFATION ANTIBODY VARIANTS; PURIFIED COMPOSITIONS

Field of the Invention

The present invention relate to compositions comprising antibodies and antigen- binding fragments thereof that lack tyrosine sulfation as well as methods of purification for preparing compositions.

Background of the Invention

Tyrosine sulfation is a post-translational modification (PTM) where a sulfate trioxide (S0 ₃ ) group is covalently bound to the hydroxyl group on the side chain of the amino acid tyrosine group. This PTM occurs in the trans-Golgi network and is catalyzed by two enzymes, tyrosyl protein sulfotransferases (TPSTs). The molecular mechanism involves the transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine, and has been found on a variety of proteins and peptides. Recent findings indicate that tyrosylprotein sulfotransferase 2 recognizes tyrosines flanked by acid residues for sulfation . This PTM is responsible for strengthening interactions between proteins and occurs on secreted and trans-membrane spanning proteins. Some chemokine receptors have been shown to be tyrosine sulfated such as at the N-terminal extracellular domain of CCR5, the principle HI V-1 and several 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 for treating various blood clotting disorders are not sulfated. Sulfation increases the mass of a biomolecule by 80 Da, which is the same mass difference as a phosphate moiety (P0 ₃ ). Unlike P0 ₃ , which forms a fairly stable P-0 bond, the S0 ₃ is very labile and readily decomposes under high temperature and low pH conditions.

The presence of different PTM variants in a therapeutic antibody preparation leads to heterogeneity which, depending on the location of the modification, can lead to variations in antibody potency, bioavailability or immunogenicity. Such issues also create issues before regulatory agencies. Though tyrosine sulfation has been described in chemokine receptors and other proteins, there is a need to identify if such modifications occur in antibody preparations and, if identified, to remove them. Summary 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) that, for example, comprises:

a light chain variable domain comprising:

CDR-L1 that comprises the amino acid sequence: ASQSLDYEGDSDMN (SEQ I D NO: 38);

CDR-L2 that comprises the amino acid sequence: GASNLES (SEQ I D NO: 39); and

CDR-L3 that comprises the amino acid sequence: QQSTEDPRT (SEQ ID NO: 40); and/or a heavy chain variable domain comprising:

CDR-H1 that comprises the amino acid sequence: DYNVD (SEQ I D NO: 33);

CDR-H2 that comprises the amino acid sequence:

DiNPNNGG iYAQKFQE (SEQ I D NO: 59);

DiNPNSGG iYAQKFQE (SEQ I D NO: 60);

DiNP DGGTIYAQKFQE (SEQ I D NO: 61 );

DiNP QGGTiYAQKFQE (SEQ I D NO: 62);

DiNPNGGGTiYAQKFQE (SEQ I D NO: 63); or

DINPNX ₁ GGTIYX ₂ QKFX ₃ X ₄ (SEQ ID NO: 64) wherein, X _I = 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 I D NO: 35); which lacks detectable levels of sulfated tyrosine on CDR-L1 . For example, in an embodiment of the invention, the antibodies or fragments in the composition further lack 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 a constant domain. In an embodiment of the invention, the antibody or fragment comprises engineered yeast or CHO N-linked glycans. In an embodiment of the invention, an anti-LAG3 antibody (e.g. , Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9) containing composition comprises one or more species of the antibody lacking tyrosine sulfation and having molecular weights of about 148590 Da, 148752 Da and/or 148914 Da (e.g. , having G0F and/or G1 F glycan species, e.g. , as set forth in Table 1 , N-terminal heavy chain glutamine 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 comprise one or more sulfated tyrosines (e.g., on CDR-L1 ) and antibodies or antigen- binding fragments lacking sulfated tyrosine comprising adjusting the pH of the mixture to about 6.5 to about 7.0 or about 6.5 to about 7.5, contacting the mixture with an anion exchange resin, and removing and retaining a non-resin bound aqueous fraction of the mixture from the resin. In an embodiment of the invention, the method comprises washing the column with an aqueous composition, e.g. , under isocratic conditions, and removing and retaining the wash composition from the resin. In an embodiment of the invention, the resin is in a column and the method comprises adding said mixture to the column and collecting the flow-through fraction from the column. In an embodiment of the invention, the method comprises equilibrating a chromatography resin, comprising a dimethylaminopropyl anion exchange ligand, in a chromatography column with 25 mM sodium phosphate pH 6.5, adjusting the pH of the mixture to about 6.5, applying the mixture to the column, collecting flow-through fraction form the column, washing the resin in the column with 25 mM sodium phosphate pH 6.5 and collecting the flow-through fraction from the wash. In an embodiment of the invention, the method comprises equilibrating a chromatography resin, comprising a quarternized polyethyleneimine anion exchange ligand, in a chromatography column with 25 mM sodium phosphate pH 7.0; optionally, 5 mM NaCI, adjusting the pH of the mixture to about 7.0, applying the mixture to the column, collecting flow-through fraction form the column, washing the resin in the column with 25 mM sodium phosphate pH 7.0; optionally, 5 mM NaCI and collecting the flow-through fraction from the wash. In an embodiment of the invention, the A ₂₈ o absorbance of the anion exchange chromatography flow-through is monitored and collected and retained when the A ₂ so first reaches at least about 2.5 absorbance units/cm; and not collected or retained when the A ₂ so falls below about 1.0 absorbance units/cm. In an embodiment of the invention, the methods of the present invention further comprise purifying the antibody or antigen-binding fragment by cation exchange chromatography, further anion exchange chromatography in bind-elute mode, hydrophobic interaction chromatography, protein-A chromatography, protein-L

chromatography, protein-G chromatography, hydroxyapatite chromatography, size exclusion chromatography, fractional precipitation, filtration, centrifugation or viral inactivation. In an embodiment of the invention, the immunoglobulin light chains and/or heavy chains of the antibody or antigen-binding fragment are expressed in a Chinese hamster ovary cell. In an embodiment of the invention, the antibody or antigen-binding fragment comprises:

a light chain variable domain comprising:

CDR-L1 that comprises the amino acid sequence: KASQS LDYEGDS DMN (SEQ ID NO: 38); CDR-L2 that comprises the amino acid sequence: GASNLES (SEQ ID NO: 39); and

CDR-L3 that comprises the amino acid sequence: QQSTEDPRT (SEQ ID NO: 40); and/or a heavy chain variable domain comprising:

CDR-H1 that comprises the amino acid sequence: DYNVD (SEQ ID NO: 33);

CDR-H2 that comprises the amino acid sequence: DINPNNGG IYAQKFQE (SEQ ID NO: 59);

DiNPNSGG iYAQKFQE (SEQ ID NO: 60);

DiNPNDGG iYAQKFQE (SEQ ID NO: 61 );

DiNPNQGGTiYAQKFQE (SEQ ID NO: 62);

DiNPNGGG iYAQKFQE (SEQ ID NO: 63); or

DINPNX ₁ GGTIYX ₂ QKFX ₃ X ₄ (SEQ ID NO: 64) wherein, 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 method are also part of the present invention. In an embodiment of the invention, an anti-LAG3 antibody (e.g., Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9) is purified by AEX chromatography wherein the antibodies lacking sulfated tyrosine, having molecular weights of about 148590 Da, 148752 Da and/or 148914 Da (e.g., having G0F and/or G1 F glycan species, e.g. , as set forth in Table 1 , N-terminal heavy chain glutamine converted to pyroglutamate and/or C-terminal heavy chain lysine removed). Brief Description of the Drawings

Figure 1. Overlay of IEX-HPLC UV profile of AEX feed (bold trace), strip (light trace) and pool fraction (dashed trace).

Figure 2. Intact mass spectrum of AEX feed, pool and strip samples.

Figure 3. Reduced light chain mass spectrum of AEX pool and strip samples.

Figure 4A-B. UV trace of reduced LysC peptide mapping of AEX pool and strip fractions.

Figure 5A-C. (A)CI D fragmentation spectrum of light chain AA25-43+80 Da in 400- 1800 m/z (B) Zoomed in m/z 300-1 100 (C) Zoomed in m/z 1200-2000.

Figure 6. ETD fragmentation of light chain peptide AA25-43+80 Da. The 80Da attached fragment ions were labeled.

Figure 7A-B. (A) Deconvoluted intact mass spectra of AEX strip fraction with and without alkaline phosphatase treatment. (B) Deconvoluted intact mass spectra of chicken ovalbumin with and without alkaline phosphatase treatment.

Figure 8A-B. (A) Normalized concentrations of mAb AEX pool and strip were subjected to reduced SDS-PAGE, probed for the human heavy (HC) and light chains (LC) by western hybridization (upper panel), then stripped and re-probed for antisulfotyrosine (lower panel). See the indications for HC and LC at the far right. (B) Normalized

concentrations of different CHO-derived mAbs in addition to AEX strip and pool are subjected to reduced SDS PAGE, probed for the human HC and LC by western

hybridization, then stripped and re-probed for anti sulfotyrosine. For both (A) and ( B) MagicMark XP was used as a protein molecular weight standard, and equal amounts of HEK293 and EGF-treated A431 cell extracts are analyzed as controls.

Figure 9A-C. SIC of (A) LC25-43+80 Da from AEX Strip Fraction, (B) Synthetic Peptide XSXSXDYEGDSDXXXXXXX (SEQ I D NO: 65)+ Phosphorylation and (C) Synthetic Peptide XSXSXDYEGDSDXXXXXXX (SEQ I D NO: 65)+Sulfation.

Figure 10. mAb tyrosine (Y31 ) site showing the CDR loops in ribbon diagram for both the heavy and light chain.

Figure 11. Predominant N-linked glycans for monoclonal antibodies produced in Chinese hamster ovary cells (CHO N-linked glycans) and in engineered yeast cells

(engineered yeast N-linked glycans): squares: N-acetylglucosamine (GlcNac); circles: mannose (Man); diamonds: galactose (Gal); triangles: fucose (Fuc).

Detailed Description of the Invention

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

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., 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, N.Y. (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, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J. ; 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).

A sulfated tyrosine includes a tyrosine having an added sulfate group, e.g., having the structure:

Chromatography

The present invention provides a method for removing contaminant variant antibodies or antigen-binding fragments (e.g., Ab1 -Ab9) thereof that comprise sulfated tyrosine from a composition, e.g., a composition that comprises a mixture of antibodies or fragments, some of which having sulfated tyrosine and some of which lacking the sulfated tyrosine to generate a composition comprising undetectable levels of tyrosine sulfated variants (e.g., tyrosine sulfated CDR-L1 , e.g., of Ab1 or Ab6). In an embodiment of the invention, the composition is treated by anion exchange (AEX) chromatography in flow- through mode to remove tyrosine sulfated variants. In an embodiment of the invention, the AEX resin has a dimethylaminopropyl ligand {i.e., a ligand that includes a

dimethylaminopropyl moiety). For example, in an embodiment of the invention, the composition that is subjected to the AEX chromatography is the product of prior protein-A chromatographic purification. In an embodiment of the invention, the composition is pH adjusted to a pH of about 6.5, e.g., with Tris (e.g., 0.5M, 0.725M or 1 M) prior to AEX treatment (e.g., having a dimethylaminopropyl ligand). In an embodiment of the invention, the AEX column (e.g., having a dimethylaminopropyl ligand) is equilibrated, e.g., with sodium phosphate, e.g. , 25 mM, e.g., sodium phosphate pH 5, 6.2 or 6.5. The column (e.g. , having a dimethylaminopropyl ligand) can, in an embodiment of the invention, be washed with buffer (e.g., with sodium phosphate, e.g. , 25 mM, e.g., sodium phosphate pH 6.5) to recover antibody or fragment within the column, but not tightly bound to the AEX resin. Flow-through, not tightly bound to the AEX resin, is collected (e.g. , in fractions) and, for example, pooled. In an embodiment of the invention, after use, the column is stripped, e.g., with 1 M NaCI.

Mass spectrometric analysis of the AEX flow-through material revealed several glycosylated species of Ab6 lacking 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 with an N-terminal glutamine on the heavy chain converted to N-terminal pyroglutamic acid (pE1) and a C-terminal lysine on the heavy chain removed (-K).

* Refer to figure 1 1 for the identity of the glycan species GOF and G1 F

The present invention includes a composition comprising anti-LAG3 antibodies (e.g., Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9; preferably Ab6) lacking detectable levels of tyrosine sulfation, e.g. , on CDR-L1 , comprising species having one or more molecular weights of about 148590, 148749, and/or 148915; and/or comprising the glycan species GOF and/or G1 F.

Flow-through mode refers to purification of a polypeptide, using a chromatography resin, by a method that does not include an elution step for the recovery of the polypeptide. In such a method, the polypeptide of interest does not bind tightly to the resin, but contaminant substances to be removed from the polypeptides of interest do bind tightly to the resin. For example, an AEX resin is used in flow-through mode in a method comprising loading a composition that comprises contaminant variant antibodies having tyrosine sulfation and antibodies lacking tyrosine sulfation onto a column containing the AEX resin and collecting and retaining the antibody or fragment in the flow-through of the column. Unbound antibody lacking sulfation can be washed out of the column (and retained) under conditions that do not lead to elution, e.g. , isocratic conditions. In such a method, the contaminant remains bound to the column and the antibody lacking the tyrosine sulfation would remain in the flow-through.

Bind/elute mode refers to purification of a polypeptide using a chromatography resin by a method that includes an elution step. In such a method, the polypeptide of interest binds tightly to the resin, but contaminant substances to be removed from the polypeptides of interest do bind tightly to the resin. With a chromatography column, the contaminants flow through the column and remain largely unbound to the resin. Bound antibodies, following an optional wash, are unbound and collected and retained when exposed to an elution buffer that causes unbinding from the resin.

A chromatography resin ligand is a substance that is fixed to a stationary phase particle (e.g., a sepharose particle), which reversibly binds a desired molecule (e.g., antibody or contaminant) present in the multi-component mobile phase.

In an embodiment of the invention, the AEX resin has the ligand quarternized polyethyleneimine (i.e., a ligand that includes a quarternized polyethyleneimine moiety). In an embodiment of the invention, the resin (e.g., having a quarternized polyethyleneimine ligand) is pre-equilibrated with 1 M NaCI. In an embodiment of the invention, the resin (e.g., having a quarternized polyethyleneimine ligand) is equilibrated with sodium phosphate, e.g., 25 mM and NaCI, e.g., 5 mM; pH about 7.0. In an embodiment of the invention, the column (e.g., having a quarternized polyethyleneimine ligand) is loaded with the feed and washed with sodium phosphate, e.g., 25 mM and NaCI, e.g., 5 mM; pH about 7.0; and the flow- through is collected, e.g. , in fractions, e.g., and pooled. In another embodiment of the invention, the method of the invention comprises equilibrating a chromatography resin, comprising an anion exchange ligand, in a chromatography column with about 10-50 mM sodium phosphate; pH about 6.5 to 7.5, adjusting the pH of the mixture to about 6.5 to 7.5, applying the mixture to the column, collecting flow-through fraction from the column, washing the resin in the column with about 10-50 mM sodium phosphate; pH about 6.5 to 7.5 and collecting flow-through fraction from the wash. In a further embodiment of the invention, the method of the invention comprises equilibrating a chromatography resin, comprising an anion exchange ligand, in a chromatography column with about 10-50 mM sodium phosphate; pH about 6.5 to 7.0, adjusting the pH of the mixture to about 6.5 to 7.0, applying the mixture to the column, collecting flow-through fraction from the column, washing the resin in the column with about 10-50 mM sodium phosphate; pH about 6.5 to 7.0 and collecting flow-through fraction from the wash.

Any suitable quantity of antibody or antigen-binding fragment can be loaded onto a chromatography resin, e.g., a chromatography column (e.g., AEX having a quarternized polyethyleneimine ligand or dimethylaminopropyl ligand). For example, in an embodiment of the invention, about 100, 1 10, 120, 130, 140, 150, 100-150, 160, 170, 180, 190, 200, 300, 150-200, 100-200, 250-350, or 280-320 grams of material, e.g. , antibody or fragment, is loaded per liter of resin (e.g., AEX having a quarternized polyethyleneimine ligand or dimethylaminopropyl ligand).

If a chromatography column is used (e.g. , containing an AEX resin having a quarternized polyethyleneimine ligand or dimethylaminopropyl ligand), any acceptable dimension can be used. For example, in an embodiment of the invention, 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.

Flow rate refers to the volume of mobile phase passing through the column (e.g., containing an AEX resin having a quarternized polyethyleneimine ligand or

dimethylaminopropyl ligand) over a period of time. In an embodiment of the invention, 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, 1 10, 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 an embodiment of the invention, the absorbance at 280 nm (A ₂₈ o) of the flow- through of the column (e.g. , containing an AEX resin having a quarternized

polyethyleneimine ligand or dimethylaminopropyl ligand) is monitored. In an embodiment of the invention, the antibody or fragment product in the major A ₂ 8o peak of the flow-through is collected and retained. In an embodiment of the invention, flow-through is collected when the A ₂ 8o reaches about 1 .0, 1.5, 2.0, 2.5 or 3.0 A ₂₈ o absorbance units per cm (path length) and collection ceases when the A ₂₈₀ drops below about 1.0, 1.5, 2.0, 2.5 or 3.0 A ₂₈₀ absorbance units per cm (path length).

In order to protect chromatography columns (e.g. , containing an AEX resin having a quarternized polyethyleneimine ligand or dimethylaminopropyl ligand) from clogging due to particulate matter in the mobile phase, a pre-column filter can be used. In an embodiment of the invention, the filter is a polyethersulfone membrane. Also, a post-column filter can be used to filter out any particulates from the flow-through. In an embodiment of the invention, the filter has a 0.2 or 0.5 μΐη pore size.

The presence of the variant having sulfated tyrosine can be confirmed, e.g. , by mass spectrograph ic analysis of flow-through fractions. Sulfated variants will have a higher mass than non-sulfated variants. For example, in an embodiment of the invention, the sulfated variant is about 80 Da heavier than variants lacking sulfation. In an embodiment of the invention, the sulfation is resistant to digestion by phosphatase and the sulfated peptide has different fragmentation pattern by electron transfer dissociation (ETD) compared to phosphorylated peptides. In an embodiment of the invention, a composition comprising antibodies (e.g., Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9; preferably Ab6) lacking tyrosine sulfation refers to a composition lacking detectable tyrosine sulfation (e.g., at CDR-L1 ). A composition comprising undetectable levels of tyrosine sulfation (e.g., at CDR-L1 ) comprises a level that cannot be observed by mass spectrometric analysis of the composition. For example, in an embodiment of the invention, mass spectrometric analysis of the composition is performed by intact and reduced mass measurement and reduced peptide mapping of the

immunoglobulin peptides of the composition. In an embodiment of the invention, the reduced peptide mapping includes denaturation and reduction of the antibody

immunoglobulin disulfide bonds and alkylation of the free cysteines, followed by enzymatic digestion (e.g., using LysC, Trypsin or GluC). The enzymatic digested peptides were analyzed by mass spectrometry. In an embodiment of the invention, an "undetectable" level refers to less than about 0.5% (less than about 0.4, 0.3, 0.2, 0.1 %) tyrosine sulfated species (e.g., on CDR-L1 ) compared to unmodified species in the composition.

Molecular weight of a polypeptide can be calculated, e.g., based on the known weights of the amino acids (modified or unmodified/sulfated or unsulfated) and known modifications (e.g. oxidation, deamidation, glycosylation, C and N terminal modification). Molecular weight can be measured by mass spectrometric analysis, e.g., when coupled with liquid chromatography. In an 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, e.g., a substance in a composition is separated from other substances in the composition by contacting the substances to a resin which acts as an adsorbent. The adsorbent which adsorbs or retains a substance more or less strongly due, e.g., to properties of the solute, such as pi, hydrophobicity, size and structure, under particular buffering conditions of the process. Chromatography can be performed by traditional methods of percolation of a composition through a bed of chormatography resin, e.g., through a column containing the resin. Batch chromatography purification includes preparing a slurry of the resin and contacting the antibody or fragment containing composition with the slurry to adsorb the substance to be separated to the resin. The solution comprising the substance not bound to the resin is separated from the slurry, e.g., by allowing the slurry to settle and removing the supernatant and the non-bound substance can be retained or discarded. The slurry is optionally subjected to one or more wash steps. If desired, the slurry can be contacted with an appropriate elution buffer to desorb resin-bound substances from the resin. The desorbed substance can be retained or discarded. In an embodiment of the invention, sulfated tyrosine variants of an antibody in a composition are bound to an anion exchange resin while non-sulfated tyrosine variants do not bind significantly to the resin. In an embodiment of the invention, an 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 IgG-type antibodies forms the basis for purification of IgG, IgG fragments containing the Fc region, and IgG subclasses. Protein-A or protein-G can be coupled to solid phase such as sepharose, which can be used for protein-A or protein-G chromatography. The present invention includes methods for making 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 the sulfated tyrosine variants by a method including AEX chromatography in flow-through mode and protein-A and/or protein-G.

In an embodiment of the invention, an antibody or antigen-binding fragment thereof is purified by multimodal chromatography (mixed-mode). Multimodal or mixed-mode protein chromatography is based on resins that have been functionalized with ligands capable of multiple modes of interaction, e.g., ion exchange, hydroxyapatite, affinity, size exclusion, and/or hydrophobic interactions. The present invention includes methods for making 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 the sulfated tyrosine variants by a method including AEX chromatography in flow-through mode and mixed mode chromatography.

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

In an embodiment of the invention, an antibody or antigen-binding fragment thereof is purified by hydrophobic interaction chromatography (HIC). HIC separates proteins with differences in hydrophobicity. Separation is based on the reversible interaction between a protein and the hydrophobic surface of a chromatography medium. The present invention includes methods for making 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 the sulfated tyrosine variants by a method including AEX chromatography in flow-through mode and HIC.

In an embodiment of the invention, an antibody or antigen-binding fragment thereof is purified by size exclusion chromatography (SEC). SEC separates proteins with differences in molecular size. The present invention includes methods for a composition comprising making 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 the sulfated tyrosine variants by a method including AEX chromatography in flow-through mode and SEC chromatography.

In an embodiment of the invention, the antibody or antigen-binding fragment is subjected to viral inactivation. For example, in an embodiment of the invention, viral inactivation is done by pH treatment of compositions including an antibody or antigen- binding fragment thereof. Specifically, direct exposure of a composition to pH extremes can be used for viral clearance. For example, pH treatment is, in an embodiment of the invention, low pH treatment (e.g., pH 3.0-3.6). In an embodiment of the invention, the antibodies or antigen-binding fragments are subject to high pH treatment. In an

embodiment of the invention, viral inactivation is performed with solvent or detergent of compositions including an antibody or antigen-binding fragment thereof. The present invention includes methods for making 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 the sulfated tyrosine variants by a method including AEX chromatography in flow-through mode and viral inactivation.

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

The phrase "ion exchange" resin refers to a solid phase that is negatively charged

{i.e., a cation exchange) or positively charged {i.e., an anion exchange).

In an embodiment of the invention, an antibody or antigen-binding fragment thereof is purified by cation exchange chromatography. A "cation exchange" resin refers to a solid phase which is negatively charged, and which has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. Any negatively charged ligand attached to the solid phase suitable to form the cation exchange resin can be used. Cation exchange materials include, but are not limited to those having the ligand: sulfopropyl (SP) - CH2-CH2-CH2-SO3- ; methyl sulfonate (S) -CH ₂ -S0 ₃ ^" ; or carboxymethyl (CM) -CH ₂ -COO ^" . The present invention includes methods for making a composition comprising 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 the sulfated tyrosine variants by a method including AEX chromatography in flow-through mode and cation exchange chromatography.

In an embodiment of the invention, an antibody or antigen-binding fragment thereof is purified by anion exchange chromatography. An "anion exchange" resin refers to a solid phase which is positively charged, thus having one or more positively charged ligands attached thereto. Any positively charged ligand attached to the solid phase suitable to form the anionic exchange resin can be used. Anion exchange materials include, but are not limited to those having the ligand: quaternary ammonium (Q) -CH ₂ -N+-(CH ₃ )3;

diethylaminoethyl (DEAE) -CH ₂ -CH2-N+-(CH2-CH ₃ )2; or diethylaminopropyl (ANX) -CH ₂ - CHOH-CH ₂ -N+-(CH ₂ -CH ₃ ) ₂ . The GoPure D 50 pm column has a dimethylaminopropyl functional group. The present invention includes methods for making 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 the sulfated tyrosine variants by a method including AEX chromatography in flow- through mode and AEX chromatography (in bind/elute mode) chromatography.

The term "solid phase" or "stationary phase" is used to mean any non-aqueous matrix to which one or more ligands (e.g., anion exchange ligands or cation exchange ligands) can adhere or alternatively, in the case of size exclusion chromatography, it can refer to the gel structure of a resin. The mobile phase is the liquid, e.g. , aqueous substance that carries the antibody or antigen-binding fragment over the solid phase is a

chromatographic purification. The mobile phase may include the loading buffer that is applied to the column. Examples of materials that can be used to form the solid phase include polysaccharides (such as agarose and cellulose) and other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles and derivatives of any of these.

An "equilibration" buffer or solution is used to adjust the pH and conductivity of the chromatography resin prior to loading with 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, e.g., such as buffers described above, and include any buffer at pH that is compatible with the selected resin used in the chromatography step for purifying the protein of interest.

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

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

Though tyrosine sulfated variant antibodies and antigen-binding fragments are contaminants, the present invention includes compositions comprising such variants e.g., bound to an AEX chromatography resin or unbound in the absence of un-tyrosine sulfated variants. The unbound variants can be obtained by eluting from the AEX column following removal from the un-tyrosine sulfated antibodies and fragments.

An "elution" buffer dissociates a molecule (e.g., an antibody or antigen-binding fragment thereof) bound to a chromatography resin.

Upstream Processing

Antibodies and antigen-binding fragments which are to be purified of contaminant tyrosine sulfated variants can be generated by host cell expression. For example, a method of the present invention includes, in an embodiment, prior to removal of the variants, the expression of the heavy and/or light immunoglobulin chains in a host cell in a culture medium under conditions favorable to such expression and isolation of the antibodies or antigen-binding fragments from the host cell and/or culture medium. The present invention includes methods for making a composition comprising an antibody or antigen-binding fragment thereof lacking detectable levels of sulfated tyrosine variants or for purifying an antibody or antigen-binding fragment thereof to remove the sulfated tyrosine variants by a method including host cell expression and AEX chromatography in flow-through mode.

The scope of the present invention includes methods for producing a composition comprising antibodies or antigen-binding fragments which are free of tyrosine sulfation (e.g. , on CDR-L1 thereof) comprising (i) introducing a polynucleotide encoding 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 to expression of the

immunoglobulin chains in the cell, e.g., wherein the antibody or antigen-binding fragment having the immunoglobulin chain(s) is secreted from the host cell into the 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 chromatography in flow-through mode as is discussed herein.

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

In an embodiment of the invention, antibodies or antigen-binding fragments are purified by methods including centrifugation. Centrifugation of a 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 an embodiment of the invention, centrifugation is performed (e.g., on a cell lysate including discarding the lysate solid fraction of the lysate) at about 40,000 to 50,000 X g for 15-30 minutes. In an embodiment of the invention, cells are removed from a liquid cell culture medium by centrifugation. For example, centrifugation using a gravitational force within a range of about 8,000 X g to about 15,000 X g (e.g., about 8000, 9000, 10000, 11000, 12000, 13000, 14000 or 15000), e.g., characterized by a Q/SIGMA ratio ranging between about 0.9 X 10 ^"8 and 2.8x 10 ⁹ . In an embodiment of the invention, the liquid centrate is depth filtered (e.g., with a pore size of 0.1 to about 0.2 μΐη). The present invention includes methods for making an antibody or antigen-binding fragment thereof lacking sulfated tyrosine variant or for purifying an antibody or antigen-binding fragment thereof to remove the sulfated tyrosine variants by a method including centrifugation and AEX chromatography in flow-through mode.

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

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

In an embodiment of the invention, antibodies or antigen-binding fragments are purified by fractional precipitation. Increased salt concentration can enhance hydrophobic interaction between proteins and result in a selective precipitation. In an embodiment of the invention, an aqueous composition comprising the 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 making an antibody or antigen- binding fragment thereof lacking sulfated tyrosine variant or for purifying an antibody or antigen-binding fragment thereof to remove the sulfated tyrosine variants by a method including AEX chromatography in flow-through mode and fractional precipitation.

In an embodiment of the invention, a host cell, in which an 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, e.g., Pichia such as Pichia pastoris or Saccharomyces cerevisiae. In an embodiment of the invention, the host cell, e.g., CHO cell, lacks glutamine synthase.

In an embodiment of the invention, the polynucleotide(s) encoding the

immunoglobulin heavy and/or light chain is/are operably linked to one or more expression control sequences such as a promoter. For example, the immunoglobulin is in an expression vector. To achieve high levels of antibody or antigen-binding fragment expression, a strong promoter/enhancer such as the cytomegalovirus (CMV) promoter and/or elongation factor alpha (EF1 a) promoter can be used to drive immunoglobulin heavy chain and/or light chain expression.

In an embodiment of the invention, an intron sequence in the 5' untranslated region is included after the promoter/enhancer to increase 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 an embodiment of the invention, a polyadenylation signal sequence is the SV40 late or early polyadenylation signal sequence or the bovine growth hormone polyadenylation sequence. In an embodiment of the invention, a consensus Kozak sequence is created by placing GCC GCC(A/G)CC (SEQ ID NO: 69) immediately in front of the first translation initiation codon to enhance translation initiation. In an embodiment of the invention, a signal peptide sequence is placed immediately in front of an immunoglobulin chain to direct antibody or fragment secretion.

The conditions of cell culture can be monitored and adjusted as needed. For example, conditions such as pH, cell count, cell viability and temperature can be monitored and adjusted. In an embodiment of the invention, the temperature of a cell culture is adjusted, e.g. , from 37°C to 30-35°C at 48 hours post-inoculation. Dissolved oxygen is, in an embodiment of the invention, monitored and/or adjusted to a set point such as 20-50%. In an embodiment of the invention, dissolved C0 ₂ is monitored and/or adjusted, e.g. , to no greater than about 120-150 mm Hg. I n an embodiment of the invention, osmolality is monitored and/or adjusted, e.g., to 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 an embodiment of the invention, the antibody or fragment comprises a sulfated tyrosine and binds to an antigen selected from: PD1 , CD27, LAG 3, CTLA4, BTLA, TI M3, ICOS, B7-H3, B7-H4, CD137, GITR, PD-L1 , PD-L2, I LT1 , I LT2 CEACAM 1 , CEACAM5, TI M3, TIGIT, VISTA, I LT3, I LT4, I LT5, ILT6, I LT7, I LT8, CD40, OX40, CD137, KI R2DL1 , KI R2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KI R2DL5B, KI R3DL1 , KI R3DL2, KI R3DL3, NKG2A, NKG2C, NKG2E, I L-10, IL-17 or TSLP.

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

Examples of the immunoglobulin chains of anti-LAG3 antibodies (e.g. , Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9 disclosed in WO2016028672) lacking tyrosine sulfation include those summarized below. For example, wherein the antibody or fragment comprises one or more of the CDRs and/or immunoglobulin chains set forth below. I n an embodiment of the invention, the contaminant antibody or antigen-binding fragment comprises a CDR-L1 having the amino acid sequence KASQS LDYEGDSDMN (SEQ I D NO: 38) wherein the Y (bold and underscored) is sulfated.

In an embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment comprises the 4A10 heavy chain immunoglobulins and/or light chain

immunoglobulins; V _H and/or V _L chains or the light chain CDRs and/or heavy chain CDRs (e.g. , 4A10 CDR-L1 , CDR-L2, CDR-L3, CDR-H1 , CDR-H2 and CDR-H3).

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

4A10- VH sequence ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTATAGGAATCAATTCAGAG GTTCAGCTGCTCCAGTC TGGGGCAGAACTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCCTCTGGCTT CAACATTGAAGACTACT ATATGCACTGGATGAAACAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATC CTGTGAATGGTGATACT GAATATGCCCCGAAGTTCCAGGGCAAGGCCACTATGACTGCAGACACATCCTCCAACACA GCCTACCTACACCTCAA CAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTAATTTCTATGATGGTTACCTCTT TGCTTTCTGGGGCCAAG GGACCCTGGTCACTGTCTCTGCA

(SEQ ID NO: 1 ; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

MKCSWIFFIMA IGINSEVQLLQSGAELVRSGASVKLSCTASGFNIEDYYMHWMKQRPEQGLEWIGWIDPVN GDT

EYAPKFQGKATMTADTSSNTAYLHLNSLTSEDTAVYYCNFYDGYLFAFWGQGTLVTV SA

(SEQ ID NO: 2; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

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

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

CDR-H3: YDGYLFAF (SEQ I D NO: 5)

4A10- Vi_ sequence

ATGAGGTGCCTAGCTGAGTTCCTGGGGCTGCTTGTGCTCTGGATCCCTGGAGCCATTGGG GATATTGTGCTGACTCA

GGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTGTCCATCTCCTGCAGGTC TAGTAAGAGTCTCCTGCATA GTGATGGCAACACTTATCTGTATTGGCTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCC TGATATATCGGATGTCC AACCTTGCCTCAGGGGTCCCAGACAGGTTCAGCGGCAGTGGGTCAGGAACTGTTTTCACA CTGAGAATCAGCAGACT GGAGGCTGAGGATGTGGGTATTTATTACTGTATGCAACATCTAGAATATCCTTTCACGTT TGGAGGGGGGACCAAGC TGGAAATAAAA

(SEQ ID NO: 6; wherein the CDRs are underscored and wherein the signal sequence is in bold font) RCI-AEFLGLLVLWIPGAIGDIVLTQAAPSVPVTPGESVSISCRSSKSLLHSDGNTYLYW LLQRPGQSPQLLIYRMS

NLASGVPDRFSGSGSGTVFTLRISRLEAEDVGIYYCMQHLEYPFTFGGGTKLEIK

(SEQ ID NO: 7; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

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

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

CDR-L3: MQHLEYPFT (SEQ I D NO: 10)

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

19E8- VH sequence

ATGGGATGGAGCTGGATCTTTCTTTTCCTCCTGTCAGGAACTGCAGGTGTCCGTTGCCAG ATCCGACTGCAGCAGTC

TGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGG GTCCTCCTTCACTGACTACT ATATAAACTGGGTGAAGCAGAAGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATC CTGGAAGCGGTAATTCT ATCTACAATGAGAACTTCAAGGCCAAGGCCACATTGACTGTAGACACATCCTCCAGCACA GCCTACATGCATCTCAG CAGCCTGACATCTGAGGACACTGCTGTCTATTTCTGTGCAAGAGAGGCTGATTACGACGA TGCTTTGGACTACTGGG GTCAAGGAACCTCGGTCACCGTCTCCTCA

(SEQ ID NO: 1 1 ; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

MG SWIFLFLLSGTAGVRCQIRLQQSGPELVKPGASVKI SCKASGSSFTDYYINWVKQKPGQGLEWIGWIYPGSGNS IYNENFKAKATLTVDTSS STAYMHLS SLTSEDTAVYFCAREADYDDALDYWGQGTSVTVS S

(SEQ ID NO: 12; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

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

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

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

19E8- Vi_ sequence

ATGGTATCCACACCTCAGTTCCTTGTATTTTTGCTTTTCTGGATTCCAGCCTCCAGAGGT CACATCTTGCTGACTCA

GTCTCCAGCCATTCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGC CAGTCAGAGCATTGGCACAA GCATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTT CTGAGTCTATCTCTGGG ATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGT GTGGAGTCAGAAGATAT TGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCAACGTACACGTTCGGAGGGGGGAC CAAGCTGGAAATAAAA

(SEQ ID NO: 16; wherein the CDRs are underscored and wherein the signal sequence is in bold font) VSTPQFLVFLLFWIPASRGHILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTN G

SPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNS WPTYTFGGGTKLEIK

(SEQ ID NO: 17; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

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

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

CDR-L3: QQSNS PTYT (SEQ I D NO: 20) In an embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment comprises the 1 1 C9 heavy chain immunoglobulins and/or light chain

immunoglobulins; V _H and/or V _L chains or the light chain CDRs and/or heavy chain CDRs (e.g. , 1 1 C9 CDR-L1 , CDR-L2, CDR-L3, CDR-H1 , CDR-H2 and CDR-H3):

1 1 C9- VH sequence

ATGAGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCAACTCCCAG GTCCAACTGCAGCAGCC

TGGGGCTGAGCTTGTGATGCCTGGGGCTTCAGCGAAGATGTCCTGCAAGGCTTCTGG CTACACACTCACTGACTACT GGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGCGATTGATA TTTCTGATAGTTATTCT AGCTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACGAATCCTCCAGCACA GCCTACATGCAGCTCAC CAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCCCCTTTCTACAATAG TAGAGGGGGGAACTACT TTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

(SEQ ID NO: 21 ; wherein the CDRs are underscored and wherein the signal sequence is in bold font) R SCIILFLVATATGVNSQVQLQQPGAELVMPGASAKMSCKASGYTLTDYW

MHWVKQRPGQGLEWIGAIDI SDSYSSYNQKFKGKATLTVDESSSTAYMQLTSLTSEDSAVYYCARSPFYNSRGGNYF DYWGQGTTLTVSS

(SEQ ID NO: 22; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

CDR-H1 : GYTLTDYWMH (SEQ I D NO: 23)

CDR-H2: AIDISDSYSSYNQKFKG (SEQ I D NO: 24)

CDR-H3: SPFYNSRGGNYFDY (SEQ I D NO: 25)

1 1 C9- Vi_ sequence

ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGT GATATCCAGATGACACA

GACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGC AAGTCAGGACATTAGCAATT ATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACAT CAAGATTACACTCAGGA GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAAC CTGGAGCAAGAAGATAT TGCCACTTACTTTTGCCAACAGGGTGATACGCTTCCTCCGTGGACGTTCGGTGGAGGCAC CAAGCTGGAAATCAAA

(SEQ ID NO: 26; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQ QKPDGTVKLLIYYTSRLHSG

VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTLPPWTFGGGTKLEIK

(SEQ ID NO: 27; wherein the CDRs are underscored and wherein the signal sequence is in bold font) CDR-L1 : RASQDisNYLN (SEQ ID NO: 28)

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

CDR-L3: QQGDTLPPWT (SEQ I D NO: 30)

In an embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment comprises the 22D2 heavy chain immunoglobulins and/or light chain

immunoglobulins; V _H and/or V _L chains or the light chain CDRs and/or heavy chain CDRs (e.g. , 22D2 CDR-L1 , CDR-L2, CDR-L3, CDR-H1 , CDR-H2 and CDR-H3):

22D2- VH sequence

ATGGGATGGACCTGGATCTTTCTCTTCTTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAG GTCCTGCTGCTACAGTC

TGGACCTGAACTGGTGAAGCCTGGGACTTCAGTGAAAATCCCCTGCAAGGCTTCTGG ATACACATTCACTGACTACA ACGTGGACTGGGTGAAGCAGCGCCATGGAAAGGGCCTTGAGTGGATTGGAGATATTAATC CAAACAATGGTGGTACT ATCTACAGTCAGAAATTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACA GCCTTCATGGAGCTCCG CAGCCTGACATCTGAGGACACTGCAGTCTATTTCTGTGCAAGGAACTATAGGTGGTTTGG TGCTATGGACCACTGGG GTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCAC TG

(SEQ ID NO: 31 ; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

MG TWIFLFFLSGTAGVLSEVLLLQSGPELVKPGTSVKI PCKASGYTFTDYNVDWVKQRHGKGLEWIGDINPN NGGTIYSQKFKGKATLTVDKSSSTAFMELRSLTSEDTAVYFCARNYRWFGAMDHWGQGTS VTVSS

(SEQ ID NO: 32; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

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

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

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

22D2- V _L sequence

ATGGAGACAGACACAATCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACTGGT GACATTGTGTTGACCCA

ATCTCCAGCTTCTTTGGCTGTGTCTCCAGGGCAGAGGGCCACCATTTCCTGCAAGGC CAGTCAAAGTCTTGATTATG AAGGTGATAGTGATATGAATTGGTACCAACAGAAACCAGGACAGCCACCCAGACTCCTCA TCTCTGGTGCATCCAAT CTAGAGTCTGGGATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTGTT AACATCCATCCTGTGGA GGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTACTGAGGATCCTCGGACGTTCGG TGGAGGCACCAAGCTGG AAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGT TAACATCTGGAGGTGCC TCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATT GATGGCAGTGAACGACA AAATGGCG

(SEQ ID NO: 36; wherein the CDRs are underscored and wherein the signal sequence is in bold font) ETDTILL VLLL VPGSTGDIVLTQSPASLAVSPGQRATISCKASQSLDYEGDSDMNWYQQKPGQPPRLLI SGASN LESGIPARFSGSGSGTDFTVNIHPVEEEDAATYYCQQSTEDPRTFGGGTKLEIK

(SEQ ID NO: 37; wherein the CDRs are underscored and wherein the signal sequence is in bold font)

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

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

CDR-L3: QQSTEDPRT (SEQ I D NO: 40).

In an embodiment of the invention, the anti-LAG3 antibody or antigen-binding fragment comprises the Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9 heavy chain

immunoglobulins and/or light chain immunoglobulins; V _H and/or V _L chains or the light chain CDRs and/or heavy chain CDRs (e.g., Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 or Ab9 CDR-L1 , CDR-L2, CDR-L3, CDR-H1 , CDR-H2 and CDR-H3): · Ab1 : humanized light chain 45AGX Humanized x [LAG3_H] mAb

(LB145.22D2.E1. D1 (VL3) ) Kappa (PX) (or the variable domain thereof) and humanized heavy chain 53AHH Humanized x [LAG3_H] mAb (LB145.22D2. E1.D1 VH6) lgG1 / Kappa (PX) (or the variable domain thereof); for example comprising: a light chain immunoglobulin comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASN LESGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 41 ); and

a heavy chain immunoglobulin comprising the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNNGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN HKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

(SEQ ID NO: 42); or

a light chain immunoglobulin variable domain comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK

(amino acids 1 -1 1 1 of SEQ ID NO: 41 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNNGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 42 (CDRs underscored)) ; or comprising the CDRs:

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

CDR-L2: GASNLES (SEQ I D 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 the variable domain thereof) and humanized heavy chain 56AHH Humanized x [LAG3_H] mAb (LB145.22D2.E1 .D1 VH6 N55S) lgG1 / Kappa (PX) (or the variable domain thereof); for example:

comprising:

a light chain immunoglobulin comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASN LESGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 43); and

a heavy chain immunoglobulin comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNSGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN HKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

(SEQ ID NO: 44); or

a light chain immunoglobulin variable domain comprising the amino acid seguence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK

(amino acids 1 -1 1 1 of SEQ ID NO: 43 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNSGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 44 (CDRs underscored))

; or comprising the CDRs:

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

CDR-L2: GASNLES (SEQ I D 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 the variable domain thereof) and humanized heavy chain 54AHH Humanized x [LAG3_H] mAb (LB145.22D2.E1 .D1 VH6 N55D) lgG1 / Kappa (PX) (or the variable domain thereof); ; for example comprising:

a light chain immunoglobulin comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 45)

a heavy chain immunoglobulin comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNDGGTIY AQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN HKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

(SEQ ID NO: 46); or

a light chain immunoglobulin variable domain comprising the amino acid seguence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK

(amino acids 1 -1 1 1 of SEQ ID NO: 45 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNDGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 46 (CDRs underscored))

; or comprising the CDRs:

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

CDR-L2: GASNLES (SEQ I D 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 the variable domain thereof) and humanized heavy chain 52AHH Humanized x [LAG3_H] mAb (LB145.22D2.E1 .D1 VH6 N55Q) lgG1 / Kappa (PX) (or the variable domain thereof); ; for example comprising:

a light chain immunoglobulin comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 47); and

a heavy chain immunoglobulin comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNQGGTIY AQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN HKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRWSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

(SEQ ID NO: 48); or

a light chain immunoglobulin variable domain comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASN LESGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK

(amino acids 1 -1 1 1 of SEQ ID NO: 47 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNQGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 48 (CDRs underscored))

; or comprising the CDRs:

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

CDR-L2: GASNLES (SEQ I D 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 the variable domain thereof) and humanized heavy chain 57AHH Humanized x [LAG3_H] mAb (LB145.22D2.E1 .D1 VH6) lgG4 S228P (PX) (or the variable domain thereof); ; for example comprising: a light chain immunoglobulin comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 49); and

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNNGGTIY AQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K

(SEQ ID NO: 50); or

a light chain immunoglobulin variable domain comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK

(amino acids 1 -1 1 1 of SEQ ID NO: 49 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNNGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 50 (CDRs underscored))

; or comprising the CDRs:

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

CDR-L2: GASNLES (SEQ I D 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 the variable domain thereof) and humanized heavy chain 73AHD Humanized x [LAG3_H] mAb (LB145.22D2.E1 .D1 VH6 N55D / VL3) lgG4 S228P / Kappa (PX) (or the variable domain thereof); for example comprising:

a light chain immunoglobulin comprising the amino acid seguence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASN LESGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWC LLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 51 ); and

a heavy chain immunoglobulin comprising the amino acid seguence: QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNDGGTIY AQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K

(SEQ ID NO: 52); or

a light chain immunoglobulin variable domain comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK

(amino acids 1 -1 1 1 of SEQ ID NO: 51 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNDGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 52 (CDRs underscored))

; or comprising the 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 the variable domain thereof) and humanized heavy chain 21AHG Humanized x [LAG3_H] mAb (LB145.22D2. E1. D1 VH6 N55S / VL3) lgG4 S228P / Kappa (PX) (or the variable domain thereof); for example comprising:

a light chain immunoglobulin comprising the amino acid seguence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASN LESGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWC LLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 53); and

a heavy chain immunoglobulin comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNSGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K

(SEQ ID NO: 54); or

a light chain immunoglobulin variable domain comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASN LESGVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK

(amino acids 1 -1 1 1 of SEQ ID NO: 53 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNSGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 54 (CDRs underscored))

; or comprising the CDRs:

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

CDR-L2: GASNLES (SEQ I D 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 the variable domain thereof) and humanized heavy chain 80AHG Humanized x [LAG3_H] mAb (LB145.22D2. E1. D1 VH6 N55Q / VL3) lgG4 S228P / Kappa (PX) (or the variable domain thereof); for example comprising:

a light chain immunoglobulin comprising the amino acid seguence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 55); and

a heavy chain immunoglobulin comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNQGGTIY AQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K

(SEQ ID NO: 56); or a light chain immunoglobulin variable domain comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK

(amino acids 1 -1 1 1 of SEQ ID NO: 55 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNQGG TIYAQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 56 (CDRs underscored))

; or comprising the CDRs:

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

CDR-L2: GASNLES (SEQ I D 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 the variable domain thereof) and humanized heavy chain 72AHD Humanized x [LAG3_H] mAb (LB145.22D2.E1 .D1

VH6 N55G / VL3) lgG4 S228P / Kappa (PX)) (or the variable domain thereof); for example comprising:

a light chain immunoglobulin comprising the amino acid seguence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

(SEQ ID NO: 57); and

a heavy chain immunoglobulin comprising the amino acid seguence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNGGGTIY AQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K

(SEQ ID NO: 58); or

a light chain immunoglobulin variable domain comprising the amino acid sequence:

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLES GVPDRFSGSGSGTDFTL KISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK (amino acids 1 -1 1 1 of SEQ ID NO: 57 (CDRs underscored)); and

a heavy chain immunoglobulin variable domain comprising the amino acid sequence:

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNGGGTIY AQKFQERVTITVDKSTS TAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS

(amino acids 1-1 19 of SEQ ID NO: 58 (CDRs underscored))

; or comprising the CDRs:

CDR-L1 : K SQSLDYEGDSDMN (SEQ ID NO: 38);

CDR-L2: GASNLES (SEQ I D 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 an embodiment of the invention, 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)

wherein,

X ₂ = A or s

X ₃ = Q or K

X ₄ = E or G

The present invention includes antibodies and antigen-binding fragments thereof (e.g. , 4A10, 19E8, 11 C9 and/or 22D2; e.g., Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 and/or Ab9) comprising N-linked glycans that 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 an embodiment of the invention, the antibody or antigen-binding fragment comprises one or more of the "engineered yeast N-linked glycans" or "CHO N-linked glycans" that are set forth in Figure 1 1 (e.g. , GO and/or G0-F and/or G1 and/or G1-F and/or G2-F and/or Man5). In an embodiment of the invention, the antibody or antigen-binding fragment comprises the engineered yeast N-linked glycans, i.e., GO and/or G1 and/or G2, optionally, further including Man5. In an embodiment of the invention, the antibody or antigen-binding fragment comprise the CHO N-linked glycans, i.e. , G0-F, G1 -F and G2-F, optionally, further including GO and/or G1 and/or G2 and/or Man5. In an embodiment of the invention, about 80% to about 95% (e.g. , about 80-90%, about 85%, about 90% or about 95%) of all N- linked glycans on the antibody or antigen-binding fragment immunoglobulin chains are engineered yeast N-linked glycans or CHO N-linked glycans. 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 an embodiment of the invention, an engineered yeast cell is GFI5.0 or YGLY8316 or strains set forth in U.S. 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 sulfation variants of anti-LAG3 antibodies (e.g., Ab1 , Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8 and/or Ab9) comprise molecular weights of about 148670 Da, 148832 Da and/or 148994 Da. Variants lacking the tyrosine sulfation comprise molecular weights of about 148590 Da, 148752 Da and/or 148914 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 produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term "isolated" is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

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

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed.

Raven Press, N.Y. (1989).

Monoclonal antibodies are substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. See Kohler et al. (1975) Nature 256: 495; U.S. Pat. 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. 1 16:731.

A chimeric antibody is an antibody having the variable domain from a first antibody and the 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, the variable domains are obtained from an antibody from an experimental animal (the "parental antibody"), such as a rodent, and the constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parental (e.g., mouse) antibody.

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

Immunoglobulins may 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 several of these may be further divided into subclasses (isotypes), e.g. lgG-1 , lgG-2, lgG-3 and lgG-4; lgA-1 and lgA-2. The invention comprises antibodies and antigen-binding fragments (e.g. , 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 γ1 , γ2, γ3, or y4 human heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen-binding fragment (e.g., anti-LAG3) comprises a light chain constant region, e.g. a human light chain constant region, such as lambda or kappa human light chain region or variant thereof. By way of example, and not limitation, the human heavy chain constant region can be γ4 and the human light chain constant region can 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 be limiting

Example 1 : Identification Of Antibody Tyrosine Sulfation Variants And

Purification Methods. In this example, the presence of tyrosine sulfation antibody variants of Ab6 were identified and a purification method for removing the variants was developed. Materials and Methods

Alkaline phosphatase was available from New England Biolabs (Ipswich, MA). Anti- tyrosine sulfation antibody was available from Millipore (Billerica, MA). Synthetic peptide was purchased from AnaSpec (Fremont, CA).

Anion exchange (AEX) chromatography

AEX chromatography was performed using POROS GoPure D Pre-packed Column, 0.5 x 5 cm, 1 mL in a flow through mode by using a GE Akta Avant system. The protein-A chromatography purified mAb was pH adjusted to pH 6.5 with 1 M Tris and was loaded on the column. Prior to protein loading, the column was equilibrated with 25 mM sodium phosphate pH 6.5, post loading the column was washed with 25 mM sodium phosphate pH 6.5 and striped with 1 M NaCI. The absorbance at 280 nm was monitored for the duration of the run. Fractions, pool and strip, and AEX load were collected and analyzed. Ion exchange HPLC

Ion exchange HPLC was performed on a MabPac SCX-10 column (4 ^χ 250 mm, 3.14 ml) at ambient temperature by using an Agilent 1600 series system. Mobile phase B was 30 mM sodium phosphate pH 8.0 and mobile 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 min. The mAb protein was then eluted from the column using a gradient of mobile phase B (14% to 80% in 18 min). The column was then cleaned with 100% mobile B for 3 min and re- equilibrated at 14% mobile phase B for the next sample analysis. The absorbance at 280 nm of the eluate was monitored throughout the LC run. Intact and reduced LC/MS

20 μg of sample was diluted to 0.5 mg/mL with 50 mM Tris buffer pH 8.0. The RP- HPLC separation was performed using Waters Acquity UPLC H-Class. The column used was Acquity UPLC BEH300 C4, 1 .7 pm, 1.0 x 100 mm (Waters, Milford, MA; -0-(Si)(CH ₃ ) ₂ - C ₄ H ₉ ligand). Mobile phases were 0.1 % formic acid (FA) in water as mobile A and 0.1 % FA in acetonitrile (ACN) as mobile 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 gradient of 4 - 15 min of 30% - 90% B. MS spectra were acquired on a Waters Xevo G2 Q-TOF system which was scanned in a range of m/z 800 - 4000.

20 pg of sample was diluted by a reducing buffer (50 mM Tris pH 8.0, containing 6 M Guanidine HCI) to a final volume of 100 pL. Two microliters of 1 M dithiothreitol (DTT)

(Sigma-Aldrich, St. Louis, MO) solution was added to each of the samples followed by incubation at 56°C for 20 minutes. The RP-UPLC separation was performed on a Waters Acquity UPLC H-Class. The column used was Acquity UPLC, BEH300 C4, 2.1 x 100mm, 1.7um (Waters). MS spectra were acquired on a Waters Xevo G2 Q-TOF system which was scanned in a range of m/z 600 - 3000. MS data was analyzed by MaxEntl of

MassLynx 4.1 .

Peptide mapping LC/MS

100 pg of a sample was buffer exchanged to 100 uL denaturing buffer containing 50 mM Tris pH 8.0, 6 M Guanidine HCI and 5 mM EDTA. The reducing reactions were conducted at 56°C for 30 minutes with 20 mM DTT in the solution. The samples were alkylated with 50 mM iodoacetamide at room temperature for 30 minutes in dark. The alkylation reaction was terminated by adding 1 μΙ_ of a 500 mM DTT solution. The reduced and alkylated samples were diluted with a digestion buffer (50 mM Tris pH 8.0) to a final volume of 300 μΙ_, before adding Lys-C enzyme (Wako, Richmond, VA) with an

enzyme: substrate ratio of 1 :20 (w:w). The solution was incubated at 37°C for 4 hour. The peptides were separated by RP-HPLC on a Waters Acquity UPLC H-Class using a HALO Peptide ES-C18, 2.1 x 150 nm, 2.7 pm column (MAC-MOD Analytical, Inc. , Chadds Ford, PA). MS spectra were acquired on a Waters Xevo G2 Q-TOF system scanned in a range of m/z 100 - 2000. MS data was analyzed by BiopharmaLynx 1.3 (Waters). Target MS/MS

LC/MS/MS of target peptide was conducted on a LTQ-Orbitrap MS system (Thermo

Fisher, Walt ham _, , MA). Resolution of 17500 in FT mode was applied for MS/MS acquisition.

The peptides were separated by Waters Acquity UPLC H-Class using a HALO Peptide ES- C18 column, 2.1X150 mm, 2.7 pm. MS/MS was scanned in m/z ranges depending on the m/z values of the precursor ions. Normalized fragmentation energy was set at 35% for CID fragmentation and 35% for ETD fragmentation. MS2 data was manually interpreted.

Alkaline phosphatase treatment

10 ug of mAb protein in AEX strip fraction were diluted in 50uL phosphatase reaction buffer. 1 uL (10 unit) alkaline phosphatase from calf intestinal (New England Biolabs, Ipswish, MA) was added for incubation at 37°C for 1 hour. Chicken ovalbumin (Sigma) was treated side by side as a positive control. 10uL solution was injected to LC/MS for mass analysis.

Western Blotting

Magic Mark XP Western Standard (Invitrogen) and specific concentrations of both monoclonal antibodies (mAb) and control cell extracts (HEK293 whole cell extract and EGF stimulated A431 Cell lysate (Millilpore)) were reduced with β-Mercaptoethanol plus heating at 95°C then resolved by Tris-glycine based SDS PAGE using a 4-20% gradient gel (Novex). Resolved proteins were subsequently electro-transferred onto nitrocellulose membrane and washed overnight in Tris-buffered saline plus .05% Tween20 (TBST) (Sigma) with rocking at 4°C. Membranes were then blocked for 1 hour in Tris-buffered saline plus 1 % BSA (TBS-BSA) (Sigma) at room temperature with continuous rocking. Primary antibodies (anti-sulfotyrosine/anti-tyrosine sulfation (Millipore) or anti-human IgG (H+L) (Jackson ImmunoResearch Labratories Inc.)) were diluted into TBS-BSA and incubated with the membrane for 2 h at room temperature. After washing with TBST, HRP- conjugated secondary antibodies (goat-anti-mouse or goat-anti-rabbit (Thermo Scientific)) were diluted into 5% Non-fat milk protein plus .05% Tween20-phosphobuffered saline (Invitrogen) and incubated at room temperature for 1 hour. After a final washing with TBST, chemilluminesence substrates (Thermo Scientific) were used for development; signals were recovered by exposure to photographic film (GE Healthcare Life Sciences) and subsequent processing. Nitrocellulose membrane stripping in between primary antibodies was done as indicated previously (Kaufmann SH, E.C., Shaper JH., The erasable Western blot. Anal Biochem., 1987. 161 (1 ): p. 89-95).

Results and Discussion

Separation of mAb molecule

Anion exchange chromatography (AEX) is typically utilized as a polishing step during monoclonal antibody purification. This step typically is operated in a flow-through mode, where the mAb flows through the column and in-process impurities (HCP, DNA) bind to the column. During the AEX development, it was noted that there was a fraction of the mAb loaded on the column bound to the resin, which affects protein recovery. The bound fraction of the protein eluted in the strip fraction of the AEX chromatography. To characterize the mAb bound to AEX column, fractions of the AEX chromatography were analyzed: "load" refer to the sample before AEX purification; "pool" refers to flow-through portion of the sample and "strip" refers to the bound fraction of the sample. Load, pool and strip fraction from AEX chromatography were initially analyzed by IEX-HPLC

chromatography. Figure 1 shows the I EX-HPLC profile of mAb in AEX load, pool and strip fraction. As shown in Figure 1 , the strip fraction had a significantly high amount of acidic variants as compared to the load and pool: -65% acid variants in strip fraction (light trace) compared to 23% in pool fraction (dashed trace) and 33% in feed fraction (bold trace). Additional difference was noted in the acidic pre-main peak. This peak was present in the AEX feed at higher levels, where in the AEX pool, this peak was minimal. The strip fraction was enriched with the acidic pre-main peak. AEX chromatography was also performed at pH 7.0 or 7.5 with the same buffer and salt conditions as described above. At pH 7.0, similar reduction of the acidic pre-main peak was also observed in the AEX pool. Analysis of intact and reduced protein by mass spectrometry

To characterize the impurities, all three fractions (AEX load, pool and strip) were analyzed by intact and reduced LC/MS using Q-TOF MS. Figure 2 shows the deconvoluted mass spectra of the intact molecule. Three main glycoforms were observed in all three fractions: G0F/G0F, G0F/G1 F and G1 F/G1 F with mass of 148591 Da, 148751 Da and 148912 Da, respectively. The calculated intact mass of this molecule with G0F/G0F is

148590 Da. The mass errors for intact mass measurement are all within 25 ppm. Additional species were only detected in AEX strip fraction. These species correspond to 80 Da mass increase (148668, 148830, 148991 Da) of the three major glycoforms (G0F/G0F, G1 F/G0F, G1 F/G1 F). To locate the modification, the light chain and heavy chain mass were measured after the disulfide bonds cleavage by reducing agent DTT. No difference was detected on heavy chain mass of strip fraction and pool fraction, suggesting the modification is not located on heavy chain (data not shown). As shown in Figure 3, light chain apo form mass (23674 Da) and glycated light chain mass (23836 Da) were detected in both fractions (strip and pool fraction). A peak with 80 Da increase of light chain was only observed at 23754 Da in the AEX strip fraction. The mass error of reduced mass measurement is within 20 ppm. These data suggest that the 80 Da modifications are located on light chain of Ab6.

Analysis of mAb antibody by peptide mapping

To further locate the modification site, AEX strip and pool fractions were reduced, alkylated and then digested by LysC enzyme. The peptide mixtures were mass mapped by Q-TOF MS. When comparing the UV trace of these two fractions, two differences were noticed. As shown in Figure 4 (a) and (b), two new peaks were detected at retention time 37.6 min and 65.2 min in AEX strip fraction. The observed m/z in the new peaks are 1165.4796 (2+) at 37.6min and 1476.7372 (4+) Da at 65.2 min. The observed masses correspond to light chain peptide AA25-43+80Da and AA25-78 +80 Da with mass error of 6.4 ppm and 9.8 ppm, respectively. Light chain peptide AA25-78 contains one mis-cleavage site. The modified and unmodified form of light chain peptide AA25-43 and AA25-78 were labeled in Figure 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 the peak area in extracted ion chromatogram (SIC).

MS/MS fragmentation of modified peptide There are two possibilities of modification with 80 Da increase in mass:

phosphorylation (+79.9663 Da) and sulfation (+79.9568 Da). The theoretical mass difference of these two modifications is only 0.0095 Da, which makes it difficult to be differentiated by mass only. Initially, the target peptide AA25-78 was fragmented by collision induced dissociation (CID) and the produced fragments were analyzed by LTQ- Orbitrap MS. As shown in Figure 5, complete loss of modification group (80Da) from precursor ion was observed. Only fragments from peptide backbone were detected, which confirms the peptide sequence of LC25-43. While no site specific information was obtained from CID fragmentation. It has been reported that sulfated tyrosine (sY) is very labile and could be easily lost under standard CID conditions (Nemeth-Cawley JF1 , K.S., Rouse JC , Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry. J Mass Spectrom., 2001 . 36(12): p. 1301-1 1 ). It was not possible to obtain site-specific information on the location of the sulfate moieties using the positive ion CID MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. In contrast, phosphorylated peptides tend to persist under CID and peptide backbone fragmentation allows for the site-specific identification of the modification (Nemeth-Cawley JF1 , K.S., Rouse JC, Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry. J Mass Spectrom., 2001. 36(12): p. 1301 -1 1 ). In Figure 5, a neutral loss of 80 Da from precursor ion was observed. It's known that the characteristic neutral loss ion for phosphorylation is -H ₃ P0 ₄ (-98 Da) and characteristic fragment ion is P0 ₃ ^" (-79 Da). While for sulfation, the characteristic neutral loss ions and fragment ions are both -S0 ₃ ion with 80 Da (Monigatti F, H.B., Steen H., Protein sulfation analysis-A primer. Biochim Biophys Acta., 2006. 1764(12): p. 1904-13). The CID MS2 data suggests the 80 Da modification is sulfation.

Another widely used fragmentation mechanism is electron transfer dissociation

(ETD). It transfers electron to a multiply protonated peptide/protein, which could lead to the cleavage of the N-Ca backbone bonds and generate c- and z-type fragment ions without loss of the information of the PTM localization (Mikesh LM, U.B., 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): p. 1811 -22). ETD can provide complementary information with CI D: ETD process allows retaining the S0 ₃ group and thus the amino acid localization, while CI D preferably fragments labile modifications. In our case, the target peptide was analyzed by LTQ-Orbitrap with ETD fragmentation and high resolution mass detection. As shown in Figure 6, partial loss of 80 Da modifications was observed on precursor ion. Fragment ions with SO3 group (80 Da) attached are labeled. Based on the detection of S0 ₃ attached fragment ions (c9, c11 , C12-16), the modification site is identified to be tyrosine 31 on light chain, which is in the CDR1 region of the mAb molecule. Alkaline phosphatase treatment

Since phosphorylation and sulfation of tyrosine are isobaric, alkaline phosphatase was used here to distinguish 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): p. 583-8). Alkaline phosphatase has been widely used for removing

phosphorylation group from proteins. Chicken albumin was used as a positive control as this protein has been widely known for its phosphorylation and glycosylation form. Chicken albumin and mAb in AEX strip fraction were treated with phosphatase and incubate at 37°C side by side. Figure 7 shows the measured intact mass of mAb and chicken ovalbumin before and after phosphatase treatment. As shown in Figure 7(a), no mass change was observed for mAb. While for chicken albumin (Figure 7 (b)), an obvious mass shift of 160 Da was observed for all the major glycoforms. Since chicken albumin contains two phosphorylation sites, the loss of 160 Da confirms the activity of alkaline phosphotase. As no mass change was detected before and after phosphatase treatment, it suggests that this mAb in AEX strip is not phosphorylated.

Western blot

Thus far, LC/MS analysis has been used to investigate the nature 80 Da adduct to tyrosine 31 on the light chain CDR. MS2 analysis and mass analysis of the mAb AEX strip fraction after phosphatase-treatment have suggested that the 80 Da adduct is sulfation on tyrosine 31. However, the ability of these mass analysis-based techniques to directly distinguish between tyrosine-sulfation and phosphorylation is problematic due to the similar molecular mass of these two groups. To begin addressing this problem, Western blotting with an anti-sulfotyrosine-specific monoclonal antibody was applied to confirm the presence of tyrosine sulfation in the mAb AEX strip 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. e561 14). In Figure 8a (upper panel), the normalized concentrations of mAb AEX pool and strip fractions were subjected to reduced SDS PAGE, probed for the human heavy and light chains by western hybridization. The increased concentrations of heavy- and light chains from pool and strip were then "stripped" of the first detecting antibodies and re-probed with an anti-sulfotyrosine-specific monoclonal antibody (lower panel). As shown in Figure 8a (lower panel), positive signals were only detected on light chain of strip fractions, suggesting that it contains tyrosine sulfation. As a control for cross reactivity with phosphorylation, lane one was loaded with a commercial source of EGF-treated A431 cell extract that is enriched with phosphorylated proteins. The lack of positive signal in lane one of the lower panel shows the anti-sulfotyrosine monoclonal antibody does not have strong cross reactivity with phosphorylation (Figure 8a, lower panel). Further, HEK 293 extract, which was suggested by the manufacturer as a positive control for the anti-sulfotyrosine monoclonal antibody, was loaded in lane 2. The positive signal below the bottom-20 KDa is consistent with manufacturer's analysis (Figure 8a, lower panel). In Figure 8b, normalized concentrations of different CHO-derived mAbs (mAbl , 2 and 3) in addition to AEX strip and pool are subjected to reduced SDS PAGE, probed for the human heavy and light chains by western hybridization (upper panel), then stripped and re-probed for antisulfotyrosine (lower panel). In agreement with Figure 8a, only the AEX mAb strip shows a positive signal at the light chain position when probed with the anti- sulfotyrosin antibody. No positive signal was observed on tyrosine sulfation for the other three CHO-derived Merck mAbs when similar amounts of protein were analyzed. This is consistent with our observation that no increased level of AEX acidic peak or tyrosine sulfation hotspot was detected on these three mAbs (data not shown).

Comparison of retention time with synthetic peptide with sulfation or phospohorylation

To further distinguish phosphorylation and sulfation, synthetic peptide with identical sequence of LC AA25-43 (XSXSXDYEGDSDXXXXXXX) (SEQ I D NO: 65) modified with either phosphorylation or sulfation on the Y31 were analyzed by LC/MS. Figure 9 shows the SIC of synthetic peptide XSXSXDYEGDSDXXXXXXX (SEQ ID NO:

65)+phosphorylation, XSXSXDYEGDSDXXXXXXX (SEQ ID NO: 65)+sulfation and AA25- 43+80Da in AEX strip. Synthetic peptide with sulfation elutes at the same retention time with AEX strip, while the synthetic peptide with phosphorylation elutes earlier than AEX strip. This further confirms our observation that Y31 on light chain is sulfated.

Structure of tyrosine sulfation site

The protein tyrosine sulfation reaction is catalyzed by the Golgi enzyme called the tyrosyl protein sulfotransferase. Previous studies indicated that TPSTs recognize accessible tyrosine residues that are usually surrounded by several acidic residues within -5 to +5 positions (Hortin G, F.R. , Gordon Jl, Strauss AW., Characterization of sites of tyrosine sulfation in proteins and criteria for predicting their occurrence. Biochem Biophys Res Commun., 1986. 141 (1 ): p. 326-33; Rosenquist GL, N.H.J. , Analysis of sequence requirements for protein tyrosine sulfation. Protein Sci. , 1993. 2(2): p. 215-22; Teramoto T1 , F.Y., 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 tyrosyl protein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction. Nat

Commun., 2013. 4: p. 1572). The acceptor tyrosine needs to have acidic residues nearby to enable the recognition of positively charged residues in TPST2 substrate binding site. The acceptor tyrosine also needs to be in an intrinsically flexible region to fit into the deep cleft of TPST2. However, no general consensus sequence for tyrosine sulfation sites has been defined. The most common features describing the sequence surroundings of sulfated tyrosine includes presence of one acidic amino acid within two residues of the tyrosine; presence of at least three acidic amino acid within 5 residues and presence of turn-inducing amino acids nearby, etc (Monigatti F, H. B., Steen H., Protein sulfation analysis-A primer. Biochim Biophys Acta., 2006. 1764(12): p. 1904-13). Figure 10 shows the structure of the mAb tyrosine site in the context of CDR loops in ribbon diagram, which was generated by MOE software (Chemical Computing Group, Montreal, Canada). The sequence near to light chain Y31 on this mAb is: XSXSXDYEGDSDXXXXXXX (SEQ ID NO: 65). In this sequence, the adjacent residues of Y31 are both 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 turn inducing residues are close to Y31 : three serine(S) and one glycine(G). The unique structure of Y31 with neighboring acidic amino acids and elements of local secondary structure play an essential role to make this modification happen.

We describe here the evidence that points to the presence of an unexpected O- linked tyrosine sulfation in a CHO produced antibody. The location of this labile

modification was found in CDR1 region of light chain, as identified by mass spectrometry with ETD fragmentation. This tyrosine sulfation was further confirmed by phosphatase treatment, Western blot experiment using anti-tyrosine sulfation antibody and retention time correlation with synthetic sulfated peptide. Structural analysis of CDR tyrosine confirms the impact of acidic residues on sulfation. The neighboring acidic amino acid residues and elements of local secondary structure might play an essential role to make Y31 a hotspot for sulfation.

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

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes. This application claims priority to U.S. provisional application No. 62/414,209 incorporated herein by reference in its entirety. All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GenelD entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GenelD entries), patent application, or patent, each of which is clearly identified in compliance with 37 C. F. R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. To the extent that the references provide a definition for a claimed term that conflicts with the definitions provided in the instant specification, the definitions provided in the instant specification shall be used to interpret the claimed invention.