RU2822192C2 - Combined formulations of anti-lag3 antibody and anti-pd-1 antibody - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to versions of a stable pharmaceutical composition containing an anti-LAG3 antibody, an anti-PD-1 antibody, sucrose, polysorbate 80, histidine buffer, L-arginine or its pharmaceutically acceptable salt and L-methionine.

EFFECT: reduced self-association, improved colloidal stability of anti-LAG3 antibody, as well as improved stability of joint formulations of anti-PD-1 and anti-LAG3 antibodies.

14 cl, 51 dwg, 20 tbl, 11 ex

Description

FIELD OF THE INVENTION TO WHICH THE INVENTION RELATES

The present invention relates generally to therapeutic antibody compositions, and their use in the treatment of various disorders.

BACKGROUND OF THE ART

Antibodies may differ somewhat in the amino acid sequence of their constant domains or in their framework sequences in the variable domains, but they usually differ most widely in their CDR sequences. Even antibodies that bind to the same protein, the same polypeptide, or even potentially the same epitope can contain completely different CDR sequences. Therapeutic antibodies for use in humans can also be derived from human germline antibody sequences or from non-human (eg, rodent) germline antibody sequences, such as humanized antibodies, resulting in even greater diversity of potential sequences. These sequence differences may result in potentially different stability in solution and different responses to solution parameters. In addition, small changes in amino acid arrangement or changes in one or more amino acid residues can result in dramatically different antibody stability and antibody susceptibility to sequence-specific degradation pathways. As a consequence, it is currently impossible to predict the solution conditions required to optimize antibody stability. Each antibody must be studied individually to determine the optimal composition of the solution. Bhambhani et al. (2012) J. Pharm. Sci. 101:1120.

Antibodies are also fairly large proteins (~150,000 Da), for example, compared to other therapeutic proteins such as hormones and cytokines. Antibody drug products must be stable during storage to ensure potency and consistent dosing, so it is critical that any formulation chosen maintains the required properties such as high concentration, clarity and acceptable viscosity, and also maintains these properties and drug efficacy for an acceptably long shelf life under standard storage conditions.

LAG3 (CD223) is a cell surface molecule expressed on activated T cells (Huard et al. Immunogenetics 39: 213-217, 1994), NK cells (Triebel et al. J Exp Med 171: 1393-1405, 1990) , B cells (Kisielow et al. Eur J Immunol 35: 2081–2088, 2005), and plasmacytoid dendritic cells (Workman et al. J Immunol 182: 1885–1891, 2009), which play an important role in the function of this lymphocyte subset . In addition, the interaction between LAG3 and its major ligand, MHC class II, is believed to play a role in modulating dendritic cell function (Andreae et al. J Immunol 168: 3874-3880, 2002). Recent preclinical studies have documented a role for LAG-3 in CD8 T cell depletion (Blackburn et al. Nat Immunol 10: 29-37, 2009).

As with chronic viral infection, tumor antigen-specific CD4 ⁺ and CD8 ⁺ T cells exhibit impaired effector function and an exhausted phenotype characterized by reduced production of proinflammatory cytokines and hyporesponsiveness to antigenic re-stimulation. This is mediated by extrinsic cellular mechanisms such as regulatory T cells (Tregs) and intrinsic cellular mechanisms such as inhibitory molecules that are activated on exhausted tumor-infiltrating lymphocytes (TILs). These inhibitory mechanisms represent a serious barrier to effective antitumor immunity.

LAG is expressed on tolerized TILs, suggesting that they contribute to tumor-mediated immune suppression. Inhibition of LAG3 may lead to increased activation of antigen-specific T cells, from which therapeutic benefits may be obtained.

PD-1 is recognized as an important molecule in immune regulation and maintenance of peripheral tolerance. PD-1 is moderately expressed on naïve T, B, and NKT cells and is activated by T/B receptor signaling on lymphocytes, monocytes, and myeloid cells. Two known ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human cancers arising in various tissues. In large samples, such as ovarian, kidney, colorectal, pancreatic, liver, and melanoma cancers, PD-L1 expression has been shown to correlate with poor prognosis and reduce overall survival regardless of subsequent treatment. Similarly, PD-1 expression on tumor-infiltrating lymphocytes has been found to mark dysfunctional T cells in breast cancer and melanoma and correlates with poor prognosis in kidney cancer. Thus, it has been proposed that PD-L1-expressing tumor cells interact with PD-1-expressing T cells to attenuate T cell activation and evade immune surveillance, thereby contributing to an impaired immune response against the tumor.

Several monoclonal antibodies that inhibit the interaction between PD-1 and one or both of its ligands, PD-L1 and PD-L2, are approved by the FDA for the treatment of cancer. It has been suggested that the effectiveness of these antibodies can be increased if they are administered in combination with other approved or experimental cancer treatments, such as radiation, surgery, chemotherapeutic agents, targeted therapies, agents that inhibit other signaling pathways that are regulated in tumors with disorders, and other agents that enhance immunity.

As a consequence, there is a need for stable co-formulations of anti-LAG3 antibodies and anti-PD-1 antibodies. These stable formulations will preferably exhibit stability for months to years under conditions typical for storing self-administered drugs, i.e. at refrigerator temperature in the syringe, which ensures a long shelf life of the corresponding medicinal product.

SUMMARY OF THE INVENTION

The present invention relates to co-formulations of anti-LAG3 antibodies or antigen binding fragments thereof and anti-PD-1 antibodies or antigen binding fragments thereof. In one embodiment, the formulation contains: approximately 16-22 mg/ml of an anti-LAG3 antibody or antigen-binding fragment thereof; approximately 3-7 mg/ml anti-PD-1 antibody or antigen-binding fragment thereof; approximately 30-120 mg/ml non-reducing disaccharide; approximately 0.02-2.0 mg/ml polysorbate 80 or polysorbate 20; buffer at pH approximately 4.5-6.5; and about 40-150 mM L-arginine or a pharmaceutically acceptable salt thereof, wherein the anti-LAG3 antibody or antigen binding fragment thereof comprises a light chain variable region comprising CDRL1 SEQ ID NO:39, CDRL2 SEQ ID NO:40 and CDRL3 SEQ ID NO: 41, and a heavy chain variable region comprising CDRH1 SEQ ID NO:42, CDRH2 SEQ ID NO:59 and CDRH3 SEQ ID NO:44, and an anti-PD-1 antibody or antigen binding fragment thereof comprises a light chain variable region comprising CDRL1 SEQ ID NO:1, CDRL2 SEQ ID NO:2 and CDRL3 SEQ ID NO:3, and a heavy chain variable region comprising CDRH1 SEQ ID NO:6, CDRH2 SEQ ID NO:7 and CDRH3 SEQ ID NO:8. In one embodiment, the composition further contains about 5-15 or about 5-10 mM L-methionine. Surprisingly, anti-LAG3/anti-PD-1 co-formulations have better stability than single antibody formulations. The formulations may be lyophilized to dissolve, or they may be in liquid form.

In another aspect of the present invention, the composition contains: about 3-300 mg/ml anti-LAG3 antibody or antigen binding fragment thereof and about 3-300 mg/ml anti-PD-1 antibody or antigen binding fragment thereof at a molar ratio of 4:1-5 :1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen-binding fragments thereof), one or more excipients selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine, or a pharmaceutically acceptable compound thereof salts, NaCl, KCl, LiCl, CaCl ₂ , MgCl ₂ , ZnCl ₂ and FeCl ₂ , with a total concentration of excipients of approximately 10-1000 mM, and a buffer at a pH of approximately 5-8.

The present invention also relates to a method of treating cancer or infection, comprising administering a dissolved or liquid composition (formulation in solution) to a subject in need. In additional embodiments, the composition is used in the treatment of a chronic infection. It is also contemplated that the solution or lyophilized formulation will be used in the manufacture of a medicinal product for the treatment of cancer or infection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Acidic variants of Ab6 as determined by HP-IEX stability data at storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 2: Major Ab6 types as determined by HP-IEX stability data under storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 3: Major Ab6 variants according to HP-IEX stability data under storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 4: Acid variants of MK-3475 as determined by HP-IEX stability data at storage conditions of 5°C, 25°C and 40°C for Ab6A drug product

Figure 5: Major types of MK-3475 according to HP-IEX stability data at storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 6: Major variants of MK-3475 according to HP-IEX stability data at storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 7: High molecular weight compounds according to UP-SEC stability data at storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 8: Monomer according to UP-SEC stability data at storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 9: Intact IgG as determined by non-reducing CE-SDS stability data at storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 10: Total small molecule compounds according to non-reducing CE-SDS stability data under storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 11: Homology model of the anti-LAG3 antibody Ab6 showing the effect on surface tryptophan. The surface of Trp102 is exposed as measured by the available surface area (85.25 Å ² ) calculated using a homology model.

Figure 12: Reduction of self-interaction (KD) and improvement of colloidal stability (OD350) and relative solubility (% PEGmid-point) of Anti-LAG3 antibody Ab6 in the presence of salt (50 mM NaCl) and in the presence of L-arginine hydrochloride (40 mM) in 10 mM histidine buffer pH 5.6.

Figure 13: Effect of L-arginine hydrochloride on the diffusion parameter (KD) and turbidity (OD350 at 50 mg/ml) of anti-LAG3 antibody Ab6 in 10 mM histidine buffer at pH 5.8 and pH 6.0.

Figure 14: pH range studies of anti-LAG3 antibody Ab6 (5.3-6.4).

Figure 15: Diffusion parameter (kD) of anti-LAG3 antibody Ab6 (25 mg/ml in 10 mM L-histidine pH 5.8) in the presence of L-arginine, L-histidine or sodium chloride.

Figure 16: Relative solubility of anti-LAG3 antibody Ab6 (25 mg/ml in 10 mM L-histidine pH 5.8) in the presence of L-arginine, L-histidine or sodium chloride.

Figure 17: Change in percentage of charged particles of anti-LAG3 antibody Ab6 (25 mg/ml in 10 mM L-histidine pH 5.8) in the presence of L-arginine, L-histidine or sodium chloride.

Figure 18: Optimization of a 25 mg/ml formulation of anti-LAG3 antibody Ab6 in 10 mM L-histidine pH 5.8 buffer with L-arginine, sodium chloride, or a mixture thereof, using the second virial coefficient measurement (B ₂₂ ).

Figure 19: Colloidal stability (OD350) of 25 mg/ml anti-LAG3 antibody Ab6 formulation in 10 mM L-histidine pH 5.8 buffer in the presence of L-arginine, sodium chloride or a mixture thereof.

Figure 20: Viscosity of anti-LAG3 antibody Ab6 (60 mg/ml) in 10 mM L-histidine pH 5.8 buffer in the presence of one of L-arginine and sodium chloride or a mixture thereof.

Figure 21: Osmolarity of anti-LAG3 antibody Ab6 (25 mg/ml) in 10 mM L-histidine pH 5.8 buffer in the presence of one of L-arginine and sodium chloride or a mixture thereof.

Figure 22: Analysis of turbidity of anti-LAG3 antibody Ab6 formulations (F1-F6) over time under storage conditions at 40°C and 25°C.

Figure 23: Mixed mode chromatographic analysis of anti-LAG3 antibody Ab6 (F1-F6) formulations over time under storage conditions at 40°C. The percentage change in monomer amount for each mAb (anti-LAG3 and anti-PD-1) was plotted over time for formulations F1-F6.

Figure 24: Percentage change in high molecular weight (HMW) species, monomer and low molecular weight (LMW) compounds of a 25 mg/ml formulation of anti-LAG3 antibody Ab6 (10 mM L-histidine pH 5.8 buffer) in the presence of 70 mM L-arginine hydrochloride with 2.5%-9% stabilizers or 70 mM sodium chloride with 2.5%-9.0% stabilizers.

Figure 25: Change in percentage of charged particles of 25 mg/ml anti-LAG3 antibody Ab6 formulation (10 mM L-histidine pH 5.8 buffer) in the presence of 70 mM L-arginine hydrochloride with 2.5%-9% stabilizers or 70 mM sodium chloride with 2.5%-9.0% stabilizers.

Figure 26: Tm1, Tm2 and Tonset 25 mg/ml anti-LAG3 antibody formulation Ab6 (10 mM L-histidine pH 5.8) in the presence of 70 mM L-arginine with 2.5%-9% stabilizers or 70 mM sodium chloride with 2.5%-9% stabilizers.

Figure 27: Colloidal stability (OD350) of 25 mg/ml anti-LAG3 antibody Ab6 formulation (10 mM L-histidine, 70 mM L-arginine, 5% w/v sucrose, pH 5.8) in the presence of various concentrations of polysorbate 80 at stress caused by mixing.

Figure 28: Percentage change in high molecular weight (HMW) species, monomer and low molecular weight (LMW) compounds of 25 mg/ml anti-LAG3 antibody Ab6 formulation (10 mM L-histidine pH 5.8 buffer, 70 mM L-arginine hydrochloride, 5% w/v sucrose) in the presence of different concentrations of polysorbate 80 under agitation stress.

Figure 29: Change in percentage of charged particles of 25 mg/ml anti-LAG3 antibody Ab6 formulation (10 mM L-histidine, 70 mM L-arginine, 5% w/v sucrose, pH 5.8) in the presence of various concentrations of polysorbate 80 at stress caused by mixing.

Figure 30: Colloidal stability (OD350) of 25 mg/ml anti-LAG3 antibody formulation Ab6 (10 mM L-histidine, 70 mM L-arginine, 5% w/v sucrose, pH 5.8) alone and in the presence of increasing concentrations of L -methionine.

Figure 31: Percentage change in high molecular weight (HMW) compounds and monomer of 25 mg/ml anti-LAG3 antibody Ab6 formulation (10 mM L-histidine, 70 mM L-arginine, 5% w/v sucrose, pH 5.8) separately and in the presence of increasing concentrations of L-methionine.

Figure 32: Change in percentage of charged particles of 25 mg/ml anti-LAG3 antibody Ab6 formulation (10 mM L-histidine, 70 mM L-arginine, 5% w/v sucrose, pH 5.8) alone and in the presence of increasing concentrations of L -methionine.

Figure 33: Percentage change in oxidation of 25 mg/ml anti-LAG3 antibody Ab6 formulation (10 mM L-histidine, 70 mM L-arginine, 5% w/v sucrose, pH 5.8) alone and in the presence of increasing concentrations of L- methionine.

Figure 34: Percentage change in turbidity (OD350) of 200 mg/ml anti-LAG3 antibody Ab6 in the presence of 10 mM buffer with 70 mM L-arginine hydrochloride, pH 5.8 alone and in the presence of various stabilizers.

Figure 35: Percentage change in high molecular weight (HMW) compounds, monomer and low molecular weight (LMW) compounds of 200 mg/ml anti-LAG3 antibody Ab6 in the presence of 10 mM buffer with 70 mM L-arginine hydrochloride, pH 5.8 alone and in the presence of various stabilizers.

Figure 36: Change in percentage of charged particles of 200 mg/ml anti-LAG3 antibody Ab6 in the presence of 10 mM buffer with 70 mM L-arginine hydrochloride, pH 5.8 alone and in the presence of various stabilizers.

Figure 37: Percentage change in turbidity (OD ₃₅₀ ) of 25 mg/ml anti-LAG3 antibody Ab6 in the presence of 40-70 mM salt (sodium chloride) and 20-70 mM amino acids alone and in some combinations.

Figure 38: Percentage change in high molecular weight (HMW) compounds, monomer and low molecular weight (LMW) compounds of 25 mg/ml anti-LAG3 antibody Ab6 in the presence of 40-70 mM salt (sodium chloride) and 20-70 mM amino acids alone and in some combinations .

Figure 39: Change in percentage of charged particles of 25 mg/ml anti-LAG3 antibody Ab6 in the presence of 40-70 mM salt (sodium chloride) and 20-70 mM amino acids alone and in some combinations.

Figure 40: Percentage change in hydrodynamic diameter of 25 mg/ml anti-LAG3 antibody Ab6 in the presence of 40-70 mM salt (sodium chloride) and 20-70 mM amino acids alone and in some combinations.

Figure 41: 10-day (10D) aggregation screening study of the co-formulated anti-LAG3 antibody Ab6 and anti-PD-1 antibody MK-3475 on % high molecular weight (HMW) at 5°C, 40°C or 50°C.

Figure 42: 10-day (10D) aggregation screening study of the co-formulated anti-LAG3 antibody Ab6 and anti-PD-1 antibody MK-3475 on % major peak (monomer) at 5°C, 40°C or 50°C.

Figure 43: Heavy chain+light chain stability data by reduced CE-SDS under storage conditions at 5°C, 25°C and 40°C for Ab6A drug product.

Figure 44: Cumulative amounts of impurities based on stability data by reconstituted CE-SDS under storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 45: MK-3475 overall preliminary peaks from HIC stability data at 5°C, 25°C and 40°C storage conditions for Ab6A drug product.

Figure 46: A350 turbidity stability data under storage conditions of 5°C, 25°C and 40°C for Ab6A drug product.

Figure 47: Representative charge profile chromatogram obtained for Ab6A drug product, full scale (top) and enlarged scale (bottom).

Figure 48: Fluorescence signals obtained after oxidation and fluorogenic labeling of DOPA products. Solutions of individual components and combined compositions are incubated at room temperature for 72 hours before subjecting them to AAPH oxidation and ABS labeling reactions.

Figure 49: Example of FRET measurement for solutions of MK-3475 and Ab6 co-formulation in the presence and absence of arginine and PS80. Experimental data (circles). Nonlinear approximation of fittings (lines).

Figure 50: Comparison of M105 oxidation levels by HIC-SEC analysis in Ab6A and MK-3475 composition samples thermally stressed at 25°C in the same composition matrix.

Figure 51: Comparison of M105 oxidation levels by HIC-SEC analysis in Ab6A and MK-3475 composition samples thermally stressed at 40°C in the same composition matrix.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms include the corresponding plural references unless the context clearly requires otherwise. Unless otherwise indicated, the proteins and entities mentioned in the present invention are human proteins and humans and not other species.

Definitions

As used in the present invention, unless otherwise specified, "antigen binding fragment" refers to antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to specifically bind to an antigen that binds to a full-length antibody, for example fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments.

A "Fab fragment" consists of one light chain and CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fab fragment" may be a papain cleavage product of the antibody.

The "Fc" region contains two heavy chain fragments containing the CH3 and CH2 domains of the antibody. The two heavy chain moieties are held together by two or more disulfide bonds and hydrophobic interactions of CH3 domains.

A "Fab' fragment" contains one light chain and a portion or fragment of one heavy chain that contains a VH domain and a CH1 domain and also extends between the CH1 and CH2 domains so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments. the formation of the F(ab')2 molecule.

The "F(ab')2 fragment" contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. The "F(ab')2 fragment" may be a pepsin cleavage product of the antibody.

The "Fv region" contains variable regions from both the heavy and light chains, but does not contain constant regions.

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region contains amino acid residues from the “complementarity determining region” or “CDR” ( for example, residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain and residues 31-35 ( CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.) and/or residues from the “hypervariable loop” ( i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987) J. Mol. Biol . 196: 901-917). or "FR" residues refers to variable domain residues other than hypervariable region residues, defined herein as CDR residues. The above residue numbering refers to the Kabat numbering system and does not necessarily correspond in detail to the sequential numbering in the accompanying sequence listing.

“Proliferative activity” includes activity that is necessary for, or that is specifically associated with, for example, normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis and angiogenesis.

The terms “cancer,” “tumor,” “cancerous,” and “malignant” refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, including adenocarcinoma, lymphoma, blastoma, melanoma, sarcoma and leukemia. More specific examples of these cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as liver carcinoma and hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, myeloma (eg, multiple myeloma), mucosal gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms tumors, basal cell carcinoma, melanoma , prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer and various types of head and neck cancer.

As cancer cells grow and multiply, they form a mass of cancerous tissue, which is a tumor that invades and destroys normal nearby tissue. Malignant tumors are cancer. Malignant tumors can usually be removed, but they can grow back. Malignant tumor cells can invade and damage nearby tissues and organs. In addition, cancer cells can break away from the cancer and enter the bloodstream or lymphatic system, which is a way for cancer cells to spread from the primary tumor (ie, the original cancer) to form new tumors in other organs. The spread of cancer in the body is called metastasis (What You Need to Know About Cancer-an Overview, NIH Publication No. 00-1566; posted Sept. 26, 2000, updated Sept. 16, 2002 (2002)).

As used herein, the term “solid tumor” refers to an abnormal growth or mass of tissue that does not typically contain cysts or fluid areas. Solid tumors can be benign (noncancerous) or malignant (cancerous). The different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Leukemias (blood cancers) do not usually form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

As used herein, the term “carcinomas” refers to epithelial cell cancers, which are the cells that cover the surface of the body, produce hormones, and make up glands. Examples of carcinomas include skin, lung, colon, stomach, breast, prostate and thyroid cancers.

Definitions of Pharmaceutical Compositions

As used in the present invention, an “aqueous” pharmaceutical composition is a composition suitable for pharmaceutical use, wherein the aqueous carrier is sterile water for injection. A composition suitable for pharmaceutical use may be sterile, homogeneous and/or isotonic. In some embodiments, the aqueous pharmaceutical compositions of the present invention are suitable for parenteral administration to humans. In a specific embodiment, the aqueous pharmaceutical compositions of the present invention are suitable for intravenous and/or subcutaneous administration.

The term "about" when changing the amount (e.g., mM, or M) of a substance or composition, the percentage (v/v or w/v) of a component of a composition, the pH of a solution/formulation, or the value of a parameter characterizing a step in a method, or the like, refers to a change in numerical quantity, which may occur, for example, through standard measurement, processing and sampling procedures involved in the preparation, characterization and/or use of the substance or composition; due to instrumental error in these procedures; due to differences in manufacture, source or purity of ingredients used to make or use the compositions or perform the procedures; and the like. In some embodiments, “about” may mean a change of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

As used in the present invention, “x% (w/v)” is equivalent to x g/100 ml (eg, 5% w/v equals 50 mg/ml).

The term "buffer" includes agents that maintain a solution pH within an acceptable range in a liquid formulation prior to lyophilization and/or after dissolution, and may include, but is not limited to, succinate (sodium or potassium), histidine, acetate, phosphate (sodium or potassium), tris (tris(hydroxymethyl)aminomethane), diethanolamine, citrate (sodium) and the like.

“Co-formulated” or “co-formulation” as used herein refers to at least two different antibodies or antigen binding fragments thereof that are formulated together and stored as a combination product in a single vial or vessel (e.g., injection device ), rather than formulated and stored individually and then mixed before administration or administered separately. In one embodiment, the co-formulation contains two different antibodies or antigen-binding fragments thereof.

“Glycol” refers to an alkyl with two hydroxyl groups.

“Sugar alcohol” refers to polyols derived from sugar and have the general formula HOCH ₂ (CHOH) _n CH ₂ OH, n = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Examples include , but not limited to, mannitol, sorbitol, erythritol, xylitol and glycerin.

As used in the present invention, “polyol” includes glycerin and sugar alcohol.

The terms "lyophilization" and "lyophilized" and "freeze-dried" refer to a process in which the material to be dried is first frozen and then the ice or frozen solvent is removed by vacuum sublimation. An excipient may be included in pre-lyophilized formulations to improve the storage stability of the lyophilized product.

A "non-reducing sugar" is a sugar that is unable to act as a reducing agent because it does not contain or cannot be converted to a free aldehyde group or a free ketone group. Examples of non-reducing sugars include, but are not limited to, disaccharides such as sucrose and trehalose.

The term "pharmaceutical formulation" refers to preparations that are in a form that allows the active ingredients to be effective and that do not contain additional components that are toxic to the subjects to whom the formulation will be administered.

"Pharmaceutically acceptable" excipients (vehicles, additives) are excipients that can be reasonably administered to a mammal to provide an effective dose of the active ingredient used.

"Dissolution time" is the time required for the lyophilized formulation to be rehydrated by the solution to a clear, particulate-free solution.

A "stable" formulation is one in which the protein therein substantially retains its physical stability and/or chemical stability and/or biological activity upon storage. Various analytical methods for measuring protein stability are available in the art and are discussed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For example, in one embodiment, a stable composition is a composition without significant changes observed at refrigeration temperature (2-8°C) for at least 12 months. In another embodiment, a stable composition is a composition without significant changes observed at refrigeration temperature (2-8°C) for at least 18 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 3 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 6 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 12 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 18 months. The stability criteria for the antibody composition are as follows. Typically, no more than 10%, preferably 5%, of the antibody monomer is degraded, as measured by SEC-HPLC. Typically the composition is colorless or clear or slightly opalescent when visually analyzed. As a rule, the change in concentration, pH and osmolarity of the composition is no more than +/- 10%. Efficiency is usually in the range of 60-140%, preferably 80-120% of control or reference. Typically, no more than 10%, preferably 5%, antibody cutting is observed, i.e., % low molecular weight compounds as determined by, for example, HP-SEC. Typically no more than 10%, preferably 5%, of antibody aggregates are formed, i.e. % high molecular weight compounds as defined by e.g. HP-SEC.

A “surfactant” is a surfactant that is amphipathic in properties.

An antibody "maintains its physical stability" in a pharmaceutical formulation if it does not show a significant increase in aggregation, sedimentation and/or denaturation when examined visually for color and/or clarity, or when measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering. Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines the tertiary structure of the protein, and by FTIR spectroscopy, which determines the secondary structure of the protein.

An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or cutting (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping coupled with mass spectroscopy or MALDI/TOF/MS) , deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement), and isomerization (assessed by isoaspartic acid measurement, peptide mapping, etc.).

An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody over a given period of time is within the predetermined range of biological activity exhibited at the time the formulation is prepared. The biological activity of an antibody can be determined, for example, using an antigen binding assay. In one embodiment, the biological activity of the stable antibody formulation over 12 months is within 60-140% of the reference.

The term "isotonic" means that the composition of interest has essentially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of approximately 270-328 mOsm. Slightly hypotonic pressure is 250-269, and slightly hypertonic pressure is 328-350 mOsm. Osmotic pressure can be measured, for example, using a vapor pressure osmometer or a freezing point osmometer.

A "dissolved" formulation is a formulation that has been prepared by dissolving a lyophilized protein in a diluent such that the protein is dispersed within the dissolved formulation. The dissolved composition is suitable for administration (eg, parenteral administration) and may optionally be suitable for subcutaneous administration.

“Total concentration of excipients” refers to the sum of the molar concentrations of specified ionizable excipients (e.g., histidine, aspartate, glutamine, glycine, proline, methionine, arginine or pharmaceutically acceptable salts thereof, NaCl, KCl, LiCl, CaCl ₂ , MgCl ₂ , ZnCl ₂ and FeCl ₂ ), without taking into account the concentrations of excipients indicated as buffers.

When a range of pH values is specified, such as “pH between 5.0 and 6.0,” the range is intended to include the stated values. PH is usually measured at 25°C using a standard pH meter with a glass bulb. As used in the present invention, a solution containing "histidine buffer at pH X" refers to a solution at pH X and containing a histidine buffer, i.e. pH is intended to indicate the pH of a solution.

Analytical methods

Analytical methods suitable for assessing product stability include size exclusion chromatography (SEC), dynamic light scattering (DLS), differential scanning calorimetry (DSC), iso-asp quantification, activity, UV at 350 nm, UV spectroscopy and FTIR. SEC (J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); J. Pharm Bio. Anal., 14:1133-1140 (1986)) measures the percentage of monomer in the product and provides information on the amount of soluble aggregates. DSC (Pharm. Res., 15:200 (1998); Pharm. Res., 9:109 (1982)) provides information on the protein denaturation temperature and glass transition temperature. DLS (American Lab., November (1991)) measures the average diffusion coefficient and provides information on the number of soluble and insoluble aggregates. UV at 340 nm measures the intensity of scattered light at 340 nm and provides information on the amount of soluble and insoluble aggregates. UV spectroscopy measures absorbance at 278 nm and provides information about protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45: 231 (1998); Pharm. Res., 12: 1250 (1995); J. Pharm. Scien., 85: 1290 (1996); J. Pharm. Scien. , 87: 1069 (1998)) measures the IR spectrum in the amide region and provides information about the secondary structure of the protein.

The iso-asp content of the samples was measured using an Isoquant isoaspartate detection system (Promega). The kit uses the protein isoaspartyl methyltransferase (PIMT) enzyme to specifically detect the presence of isoaspartic acid residues in a target protein. PIMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to isoaspartic acid at the α-carboxyl position, forming S-adenosyl-L-homocysteine (SAH) in the process. It is a relatively small molecule and can usually be isolated and quantified by reverse phase HPLC using the SAH HPLC standards supplied in the kit.

The effectiveness or bioidentity of an antibody can be measured by its ability to bind to its antigen. The specific binding of an antibody to its antigen can be quantified by any method known to those skilled in the art, for example, an immunoassay such as an ELISA (enzyme-linked immunosorbent assay).

Anti-LAG3 antibodies

CDR amino acid residues are highly variable between different antibodies and can be derived from human germline sequences (in the case of fully human antibodies) or from non-human germline sequences (eg, rodents). The framework regions can also differ significantly from antibody to antibody. Constant regions will vary depending on whether the antibody selected has a lambda (λ) or kappa (κ) light chain, and depending on the class (or isotype) of the antibody (IgA, IgD, IgE, IgG, or IgM) and subclass (eg , IgG1, IgG2, IgG3, IgG4).

The following examples of LAG3 antibodies have CDR sequences derived from non-human germline (in this case mouse) or human germline sequences. Germline sequences include the repertoire of sequences from which antibody CDR sequences are derived, in addition to changes resulting from somatic hypermutation, and as a consequence, CDRs derived starting from the mouse germline can be expected to be systematically different from CDRs derived from the germline. human lines. The use of human germline sequences is often justified on the grounds that CDR sequences from human germlines will be less immunogenic in humans than sequences derived from other species, reflecting the underlying belief that CDRs will vary systematically depending on their species origin. Although increasing CDR diversity increases the likelihood of discovering an antibody with desirable properties, such as high affinity, it further increases the difficulty in developing a stable formulation in the resulting antibody solution.

Even antibodies that bind to the same antigen can differ dramatically in sequence, and are not necessarily more closely related in sequence than antibodies to completely separate antigens. Based on low sequence similarity, it cannot be assumed that the chemical properties of the antibodies, and therefore their propensity to degrade, are similar despite their common target.

As discussed above, antibodies are large, highly complex polypeptide complexes that are subject to various forms of degradation and instability in solution. The diversity of the sequence and, therefore, the structure of the antibody determines a wide range of chemical properties. In addition to the obvious sequence-specific differences in antigen binding specificity, antibodies exhibit varying sensitivities to different degradation, aggregation, and precipitation pathways. Amino acid side chains vary in the presence or absence of reactive groups, such as carboxy- (D, E), amino- (K), amide- (N, Q), hydroxy- (S, T, Y), sulfhydryl- (C) , thioether (M) groups, as well as potentially chemically reactive sites on histidine, phenylalanine and proline residues. Amino acid side chains directly involved in antigen binding interactions are obvious candidates for inactivation by side chain modification, but degradation at other positions may also influence factors such as CDR steric orientation (e.g., changes in scaffold residues), effector function (e.g. , changes in the Fc region - see, for example, Liu et al . (2008) Biochemistry 47:5088) or self-association/aggregation.

Antibodies are subject to any number of potential degradation pathways. Oxidation of methionine residues in antibodies, especially in CDRs, can be a problem if it interferes with antigen binding. Presta (2005) J. Allergy Clin. Immunol. 116: 731; Lam et al. (1997) J. Pharm. Sci. 86:1250. Other potential degradation pathways include asparagine deamidation (Harris et al. (2001) Chromatogr., B 752:233; Vlasak et al . (2009) Anal. Biochem. 392:145) tryptophan oxidation (Wei et al. (2007) Anal. Chem. 79:2797), cysteinylation (Banks et al. (2008) J. Pharm. Sci. 97:775), glycation (Brady et al. (2007) Anal. Chem. 79:9403), pyroglutamate formation (Yu et al. al. (2006) J. Pharm. Biomed. Anal. 42:455), disulfide rearrangement (Liu et al. (2008) J. Biol. Chem. 283:29266) and hydrolysis (Davagnino et al. (1995) J. Immunol Methods 185:177). Discussed in Ionescu & Vlasak (2010) Anal. Chem. 82:3198. See also Liu et al. (2008) J. Pharm. Sci. 97:2426. Some potential degradation pathways depend not only on the presence of a particular amino acid residue, but also on the surrounding sequence. Deamidation and isoaspartate formation can arise from spontaneous intramolecular rearrangement of the peptide bond after the (C-terminal to) N or D residues, with the NG and DG sequences being particularly sensitive Reissner & Aswad (2003) CMLS Cell. Mol. Life Sci. 60:1281.

Antibodies are also subject to sequence-dependent non-enzymatic fragmentation during storage. lasak & Ionescu (2011) mAbs 3:253. The presence of reactive side chains such as D, G, S, T, C or N can lead to intramolecular cleavage reactions that break the polypeptide backbone. These sequence-specific hydrolysis reactions are typically pH dependent, ibid . Antibodies can also undergo sequence-dependent aggregation, for example when CDRs contain a large number of hydrophobic residues. Perchiacca et al. (2012) Prot. Eng. Des. Selection 25:591. Aggregation is particularly problematic for antibodies that need to be formulated at high concentrations for subcutaneous administration, and even leads some to modify the antibody sequence by adding charged residues to increase solubility, ibid .

Reflecting the diversity of potential problems with sequence-specific stability of antibodies, potential antibody compositions are also diverse. Antibody sequence variability results in chemical heterogeneity of the resulting antibody, resulting in a wide range of potential degradation pathways. The formulations may vary, for example, in antibody concentration, buffer, pH, presence or absence of a surfactant, presence or absence of tonic agents (ionic or nonionic), presence or absence of a phase exclusion agent. Commercially available therapeutic antibodies are sold in a wide range of formulations in solution, phosphate buffer (eg, adalimumab), phosphate/glycine buffer (eg, basilixumab), Tris buffer (eg, ipilimumab), histidine buffer (eg, ustekinumab), sodium citrate (eg rituximab); and from pH 4.7 (eg, certolizumab) and pH 5.2 (eg, adalimumab) to pH 7.0-7.4 (eg, cetuximab). They are also available in formulations optionally containing disodium edetate (eg, alemtuzumab), mannitol (eg, ipilimumab), sorbitol (eg, golimumab), sucrose (eg, ustekinumab), sodium chloride (eg, rituximab), potassium chloride (eg, alemtuzumab), trehalose (eg ranibizumab); all with and without polysorbate-80, ranging from 0.001% (eg, abcixmab) to 0.1% (eg, adalimumab).

Biological activity of humanized anti-LAG3 and anti-PD-1 antibodies

The compositions of the present invention contain anti-LAG3 antibodies and fragments thereof and anti-PD-1 antibodies and fragments thereof, which are biologically active when dissolved or in a liquid formulation.

Examples of anti-LAG3 antibodies are provided below (described in WO 2016/028672, incorporated by reference in its entirety):

Ab1 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35); And

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNNGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO:36); or

immunoglobulin light chain variable domain containing the amino acid sequence : DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

immunoglobulin heavy chain variable domain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNNGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:38 (CDR underlined));

Or containing a CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

CDR-H2: DINPNNGGTIYAQKFQE (SEQ ID NO:43); And

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

Ab2 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35); And

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNSGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO:45); or

immunoglobulin light chain variable domain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

immunoglobulin heavy chain variable domain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNSGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:46 (CDR underlined));

or containing CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

CDR-H2: DINPNSGGTIYAQKFQE (SEQ ID NO:47); And

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

Ab3 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35)

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNDGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO:48); or

immunoglobulin light chain variable domain containing the amino acid sequence : DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

immunoglobulin heavy chain variable domain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNDGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:49 (CDRs underlined))

; or containing CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

CDR-H2: DINPNDGGTIYAQKFQE (SEQ ID NO:50); And

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

Ab4 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35); And

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNQGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO:51); or

immunoglobulin light chain variable domain containing the amino acid sequence : DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

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

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNQGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:52 (CDRs underlined));

or containing CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

CDR-H2: DINPNQGGTIYAQKFQE (SEQ ID NO:53); And

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

Ab5 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35); And

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNNGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO:54); or

immunoglobulin light chain variable domain containing the amino acid sequence: DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

immunoglobulin heavy chain variable domain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNNGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:55 (CDR underlined));

or containing CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

CDR-H2: DINPNNGGTIYAQKFQE (SEQ ID NO:56); And

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

Ab6 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35); And

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNDGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO:57); or

immunoglobulin light chain variable domain containing the amino acid sequence : DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

immunoglobulin heavy chain variable domain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNDGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:58 (CDR underlined));

or containing CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

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

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

Ab7 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35); And

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNSGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO:60); or

immunoglobulin light chain variable domain containing the amino acid sequence : DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

immunoglobulin heavy chain variable domain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNSGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:61 (CDR underlined));

or containing CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

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

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

Ab8 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35); And

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNQGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO:63); or

immunoglobulin light chain variable domain containing the amino acid sequence : DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

immunoglobulin heavy chain variable domain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNQGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:64 (CDR underlined));

or containing CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

CDR-H2: DINPNQGGTIYAQKFQE (SEQ ID NO:65); And

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

Ab9 : immunoglobulin light chain containing the amino acid sequence :

DIVMTQTPLSLSVTPGQPASISCKASQSLDYEGDSDMNWYLQKPGQPPQLLIYGASNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO:35); And

immunoglobulin heavy chain containing the amino acid sequence :

QMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNVDWVRQARGQRLEWIGDINPNGGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDHWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

(SEQ ID NO:66); or

immunoglobulin light chain variable domain containing the amino acid sequence: DIVMTQTPLSLSVTPGQPASISC KASQSLDYEGDSDMN WYLQKPGQPPQLLIY GASNLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQSTEDPRT FGGGTKVEIK

(SEQ ID NO:37 (CDR underlined)); And

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

QMQLVQSGPEVKKPGTSVKVSCKASGYTFT DYNVD WVRQARGQRLEWIG DINPNGGGTIYAQKFQE RVTITVDKSTSTAYMELSSLRSEDTAVYYCAR NYRWFGAMDH WGQGTTVTVSS

(SEQ ID NO:67 (CDR underlined));

or containing CDR:

CDR-L1: KASQSLDYEGDSDMN (SEQ ID NO:39);

CDR-L2: GASNLES (SEQ ID NO:40);

CDR-L3: QQSTEDPRT (SEQ ID NO:41);

CDR-H1: DYNVD (SEQ ID NO:42);

CDR-H2: DINPNGGGTIYAQKFQE (SEQ ID NO:68); And

CDR-H3: NYRWFGAMDH (SEQ ID NO:44)

As used herein, “Ab6 variant” means a monoclonal antibody that contains heavy chain and light chain sequences that are substantially identical to those in Ab6 (as described below and in WO2016028672, incorporated by reference in its entirety), for except for the presence of three, two, or one conservative amino acid substitutions at positions that are located outside the light chain CDR and six, five, four, three, two, or one conservative amino acid substitutions that are located outside the heavy chain CDR, for example, the variant positions are located in the regions FR or constant region, and optionally has a deletion of the C-terminal heavy chain lysine residue. In other words, Ab6 and the Ab6 variant contain identical CDR sequences but differ from each other due to the presence of a conserved amino acid substitution at no more than three or six other positions in their full-length light and heavy chain sequences, respectively. The Ab6 variant is substantially similar to Ab6 with respect to the following properties: binding affinity for human LAG3 and the ability to block binding of human LAG3 to human MHC class II.

The present invention provides anti-LAG3 antibody compositions that contain two identical light chains of SEQ ID NO:35 and two identical heavy chains of SEQ ID NO:36, 45, 48, 51, 54, 57, 60, 63 or 66. The present invention also provides anti-LAG3 antibody compositions that contain two identical light chains of SEQ ID NO:35 and two identical heavy chains of SEQ ID NO:57.

The present invention relates to anti-LAG3 antibody or antigen binding fragment compositions that comprise a light chain variable region sequence SEQ ID NO:37 and a heavy chain variable region sequence SEQ ID NO:38, 46, 49, 52, 55, 58, 61, 64 or 67. The present invention also provides anti-LAG3 antibody or antigen binding fragment compositions that comprise the light chain variable region sequence of SEQ ID NO:37 and the heavy chain variable region sequence of SEQ ID NO:58. The present invention also provides anti-LAG3 antibody or antigen binding fragment compositions comprising the light chain variable region sequence CDRL1 of SEQ ID NO:39, the CDRL2 sequence of SEQ ID NO:40 and the CDRL3 sequence of SEQ ID NO:41, and the heavy chain variable region sequence of CDRH1 SEQ ID NO:42, CDRH2 sequence SEQ ID NO:43, 47, 50, 53, 56, 59, 62, 65 or 68, and CDRH3 sequence SEQ ID NO:44. The present invention also provides anti-LAG3 antibody or antigen binding fragment compositions comprising the light chain variable region sequence CDRL1 of SEQ ID NO:39, the CDRL2 sequence of SEQ ID NO:40 and the CDRL3 sequence of SEQ ID NO:41, and the heavy chain variable region sequence of CDRH1 SEQ ID NO:42, CDRH2 sequence SEQ ID NO:59 and CDRH3 sequence SEQ ID NO:44.

Other anti-LAG3 antibodies that may be included include BMS-986016, described in WO2014008218; IMP731, and IMP701. Therefore, the present invention provides anti-LAG3 antibody or antigen binding fragment compositions that comprise the light chain variable region sequence of SEQ ID NO:69 and the heavy chain variable region sequence of SEQ ID NO:70. The present invention also provides anti-LAG3 antibody or antigen binding fragment compositions comprising the light chain variable region sequence CDRL1 of SEQ ID NO:71, the CDRL2 sequence of SEQ ID NO:72 and the CDRL3 sequence of SEQ ID NO:73, and the heavy chain variable region sequence of SEQ ID NO:74, CDRH2 sequence SEQ ID NO:75 and CDRH3 sequence SEQ ID NO:76.

The composition further contains an anti-PD-1 antibody or antigen binding fragment, as follows by way of example.

Table 1. Approximate sequences of PD-1 antibodies

Antibody sign Amino acid sequence SEQ ID NO. Pembrolizumab light chain CDR1 RASKGVSTSGYSYLH 1 CDR2 LASYLES 2 CDR3 QHSRDLPLT 3 Variable region EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK 4 Light chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 5 Pembrolizumab heavy chain CDR1 NYYMY 6 CDR2 GINPSNGGTNFNEKFKN 7 CDR3 RDYRFDMGFDY 8 Variable region QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS 9 Heavy chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 10 Nivolumab light chain CDR1 RASQSVSSYLA eleven CDR2 DASNRAT 12 CDR3 QQSSNWPRT 13 Variable region EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK 14 Light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 15 Nivolumab heavy chain CDR1 NSGMH 16 CDR2 VIWYDGSKRYYADSVKG 17 CDR3 NDDY 18 Variable region QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS 19 Heavy chain QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 20

Table 2. Additional anti-PD-1 antibodies and antigen binding fragments useful in the compositions, methods and applications of the present invention.

A. Antibodies and antigen binding fragments containing hPD-1.08A light and heavy chain CDRs in WO2008/156712 (incorporated by reference in its entirety) CDRL1 SEQ ID NO:21 CDRL2 SEQ ID NO:22 CDRL3 SEQ ID NO:23 CDRH1 SEQ ID NO:24 CDRH2 SEQ ID NO:25 CDRH3 SEQ ID NO:26 C. Antibodies and antigen binding fragment containing the mature h109A heavy chain variable region and one of the K09A mature light chain variable regions in WO 2008/156712 Heavy chain VR SEQ ID NO:27 Light chain VR SEQ ID NO:28 or SEQ ID NO:29 or SEQ ID NO:30 D. Antibodies and antigen binding fragments containing the 409 mature heavy chain and one of the K09A mature light chains in WO 2008/156712 Heavy chain SEQ ID NO:31 Light chain SEQ ID NO:32 or SEQ ID NO:33 or SEQ ID NO:34

As used herein, “pembrolizumab variant” means a monoclonal antibody that contains heavy chain and light chain sequences that are substantially identical to those in pembrolizumab except for the presence of three, two, or one conservative amino acid substitutions at positions that are located outside the light CDR. chain, and six, five, four, three, two or one conservative amino acid substitutions that are located outside the heavy chain CDR, for example, the variant positions are located in FR or constant region regions, and optionally has a deletion of the C-terminal lysine residue of the heavy chain. In other words, pembrolizumab and the pembrolizumab variant contain identical CDR sequences but differ from each other due to the presence of a conserved amino acid substitution at no more than three or six other positions in their full-length light and heavy chain sequences, respectively. The pembrolizumab variant is substantially similar to pembrolizumab with respect to the following properties: binding affinity for PD-1 and the ability to block the binding of each of PD-L1 and PD-L2 to PD-1.

In another aspect of the present invention, the composition comprises an anti-LAG3 antibody or antigen binding fragment comprising a light chain variable region sequence of SEQ ID NO:37 and a heavy chain variable region sequence of SEQ ID NO:58; and an anti-PD-1 antibody or antigen binding fragment comprising the light chain variable region sequence of SEQ ID NO:4 and the heavy chain variable region sequence of SEQ ID NO:9. In another embodiment, the composition comprises an anti-LAG3 antibody comprising the light chain sequence of SEQ ID NO:35 and the heavy chain sequence of SEQ ID NO:57; and an anti-PD-1 antibody comprising the light chain sequence of SEQ ID NO:5 and the heavy chain sequence of SEQ ID NO:10. The present invention also provides compositions of anti-LAG3 antibodies or antigen binding fragments thereof containing the light chain sequence CDRL1 SEQ ID NO:39, CDRL2 sequence SEQ ID NO:40 and CDRL3 sequence SEQ ID NO:41, and the heavy chain sequence CDRH1 SEQ ID NO :42, CDRH2 sequence SEQ ID NO:59, and CDRH3 sequence SEQ ID NO:44; and an anti-PD-1 antibody comprising the light chain sequence CDRL1 SEQ ID NO:1, CDRL2 sequence SEQ ID NO:2, and CDRL3 sequence SEQ ID NO:3, and the heavy chain sequence CDRH1 SEQ ID NO:6, CDRH2 sequence SEQ ID NO:7, and CDRH3 sequence SEQ ID NO:8. In some formulations of the present invention, the anti-LAG3 antibody is an Ab6 or Ab6 variant.

In a further aspect of the present invention, the compositions comprise an anti-LAG3 antibody or antigen binding fragment that comprises a light chain variable region sequence of SEQ ID NO:69 and a heavy chain variable region sequence of SEQ ID NO:70, and an anti-PD-1 antibody or antigen binding fragment , which contains the light chain variable region sequence of SEQ ID NO:14 and the heavy chain variable region sequence of SEQ ID NO:19. The present invention also provides anti-LAG3 antibody or antigen binding fragment compositions comprising the light chain variable region sequence CDRL1 of SEQ ID NO:71, the CDRL2 sequence of SEQ ID NO:72, and the CDRL3 sequence of SEQ ID NO:73, and the heavy chain variable region sequence CDRH1 sequence SEQ ID NO:74, CDRH2 sequence SEQ ID NO:75 and CDRH3 sequence SEQ ID NO:76, and an anti-PD-1 antibody or antigen binding fragment containing the light chain variable region sequence CDRL1 SEQ ID NO:11, CDRL2 sequence SEQ ID NO:12, and CDRL3 sequence of SEQ ID NO:13, and CDRH1 heavy chain variable region sequence SEQ ID NO:16, CDRH2 sequence of SEQ ID NO:17, and CDRH3 sequence of SEQ ID NO:18.

The antibody or antigen binding fragments of the composition may comprise a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region comprises SEQ ID NO:4 or a variant of SEQ ID NO:4, and the heavy chain variable region comprises SEQ ID NO:9 or a variant of SEQ ID NO:9. In further embodiments, the light chain variable region comprises SEQ ID NO:14 or a variant of SEQ ID NO:14, and the heavy chain variable region comprises SEQ ID NO:19 or a variant of SEQ ID NO:19. In further embodiments, the heavy chain variable region comprises SEQ ID NO:27 or a variant of SEQ ID NO:27 and the light chain variable region comprises SEQ ID NO:28 or a variant of SEQ ID NO:28, SEQ ID NO:29 or a variant of SEQ ID NO:29, or SEQ ID NO:30, or a variant of SEQ ID NO:30. In these embodiments, the sequence of the light chain or heavy chain variable region of the variant is identical to the control sequence except for one, two, three, four, or five amino acid substitutions. In some embodiments, the replacements are in the framework region (ie, outside the CDR). In some embodiments, one, two, three, four, or five amino acid substitutions are conservative.

In another embodiment, the compositions of the present invention contain an antibody or antigen binding fragment that contains a V _L domain and/or a V _H domain with at least 95%, 90%, 85%, 80%, 75% or 50% homology to sequence with one of the VL domains or VH domains described above and exhibits specific binding to PD-1 or LAG3. In another embodiment, the antibody or antigen binding fragment of the compositions of the present invention contains V _L - and V _H domains containing up to 1, 2, 3, 4 or 5 or more amino acid substitutions, and exhibits specific binding to PD-1 or LAG3.

In embodiments of the present invention, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:5 and a heavy chain containing or consisting of an amino acid sequence as set forth in SEQ ID NO:10. In alternative embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:15 and a heavy chain containing or consisting of an amino acid sequence as set forth in SEQ ID NO:20. In further embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:32 and a heavy chain containing or consisting of an amino acid sequence as set forth in SEQ ID NO:31. In further embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:33 and a heavy chain containing or consisting of an amino acid sequence as set forth in SEQ ID NO:31. In still further embodiments, the antibody is an anti-PD-1 antibody comprising a light chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:34 and a heavy chain containing or consisting of an amino acid sequence as set forth in SEQ ID NO:31. In some formulations of the present invention, the anti-PD-1 antibody is pembrolizumab or a variant of pembrolizumab.

Typically, the amino acid sequence variants of the anti-PD-1 or anti-LAG3 antibody and antigen binding fragments of the present invention will contain an amino acid sequence having at least 75% amino acid sequence identity with the amino acid sequence of the reference antibody or antigen binding fragment (e.g., heavy chain , light chain, _VH , _VL , framework or humanized sequence), more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably, at least 95, 98 or 99%. Sequence identity or homology is defined in the present invention as the percentage of amino acid residues in a candidate sequence that are identical to anti-PD-1 or anti-LAG3 residues, after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering conservative substitutions as part of sequence identity. None of the N-terminal, C-terminal or internal extensions, deletions or insertions in the antibody sequence should be construed as affecting sequence identity or homology.

In one embodiment, the ratio of anti-LAG3 antibody to anti-PD-1 antibody in the composition is 1:1, 1:2, or 1:3. In another embodiment, the molar ratio of anti-LAG3 antibody to anti-PD-1 antibody in the composition is 1:1, 2:1, 3:1, 3.5:1, 4:1, 5:1, or 6:1 . In one embodiment, the molar ratio of anti-LAG3 antibody to anti-PD-1 antibody in the formulation is 4:1. In another embodiment, the molar ratio of anti-LAG3 antibody to anti-PD-1 antibody in the composition is 5:1.

Compositions

In some aspects of the present invention, the compositions of the present invention minimize the formation of antibody aggregates (high molecular weight compounds) and particles, increase colloidal stability, minimize fragmentation (low molecular weight compounds), or ensure that the antibody maintains its biological activity over time. In one aspect, the formulation contains: about 3-300 mg/ml anti-LAG3 antibody or antigen binding fragment thereof and about 3-300 mg/ml anti-PD-1 antibody or antigen binding fragment thereof at a molar ratio of 4:1-5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof), one or more excipients selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof , NaCl, KCl, LiCl, CaCl ₂ , MgCl ₂ , ZnCl ₂ and FeCl ₂ , with a total excipient concentration of approximately 10-1000 mM, and a buffer at a pH of approximately 5-8. In one embodiment, the anti-LAG3 antibody or antigen binding fragment thereof and the anti-PD-1 antibody or antigen binding fragment thereof have a molar ratio of 4:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof). In another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof and the anti-PD-1 antibody or antigen binding fragment thereof have a molar ratio of 5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof). In another aspect, the formulation contains: about 4-200 mg/ml of an anti-LAG3 antibody or an antigen-binding fragment thereof and about 4-200 mg/ml of an anti-PD-1 antibody or an antigen-binding fragment thereof. In one embodiment, one or more excipients selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl ₂ , MgCl ₂ , ZnCl ₂ and FeCl ₂ are present at a total concentration of excipients of approximately 25-250 mM. In another embodiment, one or more excipients selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl ₂ , MgCl ₂ , ZnCl ₂ and FeCl ₂ are present at a total concentration of excipients of approximately 40-250 mM.

In one aspect, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 15-250 mM. In one aspect, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 25-250 mM. In another embodiment, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 40-150 mM. In another embodiment, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 40-100 mM. In another embodiment, the excipient is L-arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 70 mM. In another embodiment, the excipient is arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 70-150 mM. Examples of pharmaceutically acceptable arginine salt (L or D form) include, but are not limited to, L-arginine hydrochloride and L-arginine succinate. In other aspects of the embodiments above, the composition further comprises a non-ionic surfactant, a sugar or polyol, or glutamine, glycine, proline or methionine.

In another aspect, the excipients are NaCl and arginine or a pharmaceutically acceptable salt thereof with a total excipient concentration of approximately 25-250 mM. In a further embodiment, the excipients are NaCl and arginine or a pharmaceutically acceptable salt thereof with a total excipient concentration of approximately 70-100 mM. In one embodiment, the ratio of NaCl to arginine concentrations is 1:1. In another embodiment, the NaCl concentration is approximately 35 mM, and the arginine concentration is approximately 35 mM. In another embodiment, the NaCl concentration is approximately 50 mM, and the arginine concentration is approximately 50 mM.

In a further aspect, the excipient is NaCl, KCl or LiCl at approximately 40-150 mM. In a further embodiment, the excipient is NaCl, KCl or LiCl at approximately 40-100 mM. In a further embodiment, the excipient is NaCl, KCl or LiCl at approximately 70-130 mM. In a further embodiment, the excipient is NaCl, KCl or LiCl at approximately 70-100 mM. In a further embodiment, the excipient is NaCl at approximately 70 mM. In other aspects of the embodiments above, the composition further comprises a nonionic surfactant.

In a further aspect, the excipient is L-histidine at approximately 25-200 mM. In a further embodiment, L-histidine is present at approximately 50-200 mM. In yet another embodiment, L-histidine is present at approximately 40-100 mM.

In a further aspect, the excipient is L-glutamine, L-glycine, L-proline or L-methionine, or a combination thereof at approximately 25-200 mM. In a further embodiment, the excipient is present at approximately 50-200 mM. In yet another embodiment, the excipient is present at approximately 40-100 mM. In yet another embodiment, the excipient is present at approximately 70 mM.

In one embodiment, the excipient is L-glutamine, L-glycine, L-aspartate, or a combination thereof at approximately 25-200 mM. In another embodiment, the excipient is present at approximately 20-50 mM. In a further embodiment, the excipient is present at approximately 20 mM. In yet another embodiment, the excipient is present at approximately 40-100 mM. In yet another embodiment, the excipient is present at approximately 70 mM. In another embodiment, the excipients are approximately 20 mM L-aspartate and approximately 50 mM L-glycine. In another embodiment, the excipients are approximately 20 mM L-glutamine and approximately 50 mM L-glycine.

In one embodiment, the co-formulated composition contains a buffer having a neutral or slightly acidic pH (pH 4.5-8) and arginine or a pharmaceutically acceptable salt thereof. In one embodiment, a buffer pH of approximately 5.5-6.5 is used in the composition. In one embodiment, a buffer pH of approximately 4.5-6.5 is used in the composition. In another embodiment, a buffer pH of approximately 5.5-6.0 is used in the composition. In a further embodiment, a pH buffer of approximately 5.0-6.0 is used in the composition. The buffer may have a concentration of approximately 5-1000 mM. In another embodiment, the buffer may have a concentration of approximately 5-150 mM. In a further embodiment, the buffer may have a concentration of approximately 5-300 mM. In a further embodiment, the buffer has a concentration of approximately 1-300 mM. In another embodiment, the buffer may have a concentration of approximately 1-30 mM. In yet another embodiment, the buffer may have a concentration of 5-30 mM. In yet another embodiment, the buffer may have a concentration of approximately 5-20 mM. In yet another embodiment, the buffer may have a concentration of approximately 8-12 mM. In one embodiment, the buffer is a histidine, acetate or citrate buffer. The preferred buffer contains approximately 10 mM histidine, acetate or citrate.

In one embodiment, the formulation contains approximately 3-300 mg/ml anti-LAG3 antibody or antigen binding fragment thereof and 3-300 mg/ml anti-PD-1 antibody or antigen binding fragment thereof in a molar ratio of 4:1-5:1 ( anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof), sugar or polyol; nonionic surfactant, histidine buffer or acetate buffer at a pH of about 4.5-8, about 10-1000 mM arginine or a pharmaceutically acceptable salt thereof and optionally methionine (L or D form), EDTA, DTPA, tryptophan (L or D form) or pyridoxine. In another embodiment, the formulation contains about 4-250 mg/ml anti-LAG3 antibody or antigen binding fragment thereof and about 4-250 mg/ml anti-PD-1 antibody or antigen binding fragment thereof at a molar ratio of 4:1-5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof), sugar or polyol; nonionic surfactant, about 50-500 mM histidine buffer at pH about 5-8, about 10-1000 mM salt of monovalent cations selected from NaCl, KCl and LiCl or salt of polyvalent cations selected from CaCl ₂ , MgCl ₂ , ZnCl ₂ , FeCl ₂ and FeCl ₃ , optionally about 10-1000 mM arginine or a pharmaceutically acceptable salt thereof, and optionally methionine (D or L form), EDTA, DTPA, tryptophan and pyridoxine. In another aspect, the formulation contains: about 4-200 mg/ml of an anti-LAG3 antibody or an antigen-binding fragment thereof and about 4-200 mg/ml of an anti-PD-1 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-LAG3 antibody or antigen binding fragment thereof and the anti-PD-1 antibody or antigen binding fragment thereof have a molar ratio of 4:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof). In another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof and the anti-PD-1 antibody or antigen binding fragment thereof have a molar ratio of 5:1 (anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof).

The composition may contain about 1-100 μM, about 1-30 μM, about 1-20 μM, about 10 μM-30 μM DTPA or EDTA. The composition may also contain approximately 1-30 mM L-methionine. In one embodiment, the composition may also contain approximately 1-20 mM L-methionine. The composition may also contain approximately 5-15 mM L-methionine. The composition may also contain approximately 5-15 mM L-methionine. The composition may also contain approximately 5-20 mM L-methionine. The composition may also contain about 10 mM, or at least about 10 mM L-methionine. Sometimes a nitrogen topcoat (covering, for example, only 5% or 10% residual O ₂ when adding a nitrogen topcoat) is applied during the preparation steps and/or before closing the vial to stabilize the antibody against oxidation.

In another aspect of the present invention, the composition further contains a sugar, a polyol or a nonionic surfactant, or a combination thereof. In one embodiment, the sugar is selected from the group consisting of glucose, sucrose, trehalose and lactose, or a combination thereof. In one embodiment, the sugar is a disaccharide such as sucrose, trehalose and maltose. In one embodiment, the sugar is a non-reducing sugar. In another embodiment, the sugar is a non-reducing disaccharide such as sucrose or trehalose, or a combination thereof. In one embodiment, the sugar is present at a concentration of approximately 10-200 mg/ml. In another embodiment, the sugar is present at a concentration of approximately 30-120 mg/ml. In another embodiment, the sugar is present at a concentration of approximately 30-80 mg/ml. In a further embodiment, the sugar is present at a concentration of approximately 50-90 mg/ml.

In one embodiment, the polyol is selected from the group consisting of mannitol, sorbitol, glycerol and polyethylene glycol. In another embodiment, the polyol is a sugar alcohol. In one embodiment, the sugar and polyol are selected from the group consisting of sucrose, trehalose, sorbitol, glycerol and polyethylene glycol. In a further embodiment, the polyol is a glycol. In one embodiment, the glycol is selected from the group consisting of ethylene glycol, propylene glycol and polyethylene glycol. In one embodiment, the polyol is present at a concentration of approximately 10-200 mg/ml. In another embodiment, the polyol is present at a concentration of approximately 10-50 mg/ml. In a further embodiment, the polyol is present at a concentration of approximately 5-30 mg/ml.

In one embodiment, the composition contains approximately 10-250 mg/ml sucrose or trehalose. In another embodiment, the composition contains approximately 20-200 mg/ml sucrose or trehalose. In a further embodiment, the composition contains approximately 50-80 mg/ml sucrose or trehalose. In a further embodiment, the composition contains approximately 30-80 mg/ml sucrose or trehalose. In another embodiment, the composition contains approximately 50-90 mg/ml sucrose or trehalose. In yet another embodiment, the composition contains approximately 70-80 mg/ml sucrose or trehalose. In yet another embodiment, the composition contains at least about 50 mg/ml sucrose or trehalose. In another embodiment, the composition contains approximately 20-200 mg/ml sorbitol, PEG400 or glycerol. In a further embodiment, the formulation contains approximately 20-50 mg/ml sorbitol, PEG400 or glycerol.

In one embodiment, the nonionic surfactant is selected from the group consisting of polysorbate and poloxamer. In yet another embodiment, the surfactant is selected from the group consisting of Tween80® (polysorbate 80), Tween20® (polysorbate 20), PluronicF88®, Pluoronic F-127®, PluronicF68®, Triton X-100®. In a preferred embodiment, the surfactant is polysorbate 20 or polysorbate 80, and the sugar is sucrose or trehalose. The surfactant polysorbate 80 or polysorbate 20 may be present in the composition in an amount of from about 0.005 to about 1 mg/ml. The surfactant polysorbate 80 or polysorbate 20 may be present in the composition in an amount of from about 0.02 to about 2 mg/ml. The surfactant polysorbate 80 or polysorbate 20 may be present in the composition in an amount of from about 0.05 to about 1 mg/ml. The surfactant polysorbate 80 or polysorbate 20 may be present in the composition in an amount of from about 0.1 to about 0.5 mg/ml. In another embodiment, the surfactant polysorbate 80 or polysorbate 20 may be present in the formulation in an amount of at least about 0.005 mg/ml. The surfactant polysorbate 80 or polysorbate 20 may also be present in the composition in an amount of at least about 0.1 mg/ml. The surfactant polysorbate 80 may be present in the formulation in an amount of about 0.2 mg/ml.

In other aspects of the formulations above, at 50°C, the % high molecular weight (HMW) content is less than 5% in the co-formulated anti-LAG3 antibody and anti-PD-1 antibody after 10 days, as measured by size exclusion chromatography.

The present invention relates to the following embodiments:

1. Composition containing:

approximately 16-22 mg/ml anti-LAG3 antibody or antigen-binding fragment thereof; approximately 3-7 mg/ml anti-PD-1 antibody or antigen-binding fragment thereof; approximately 30-120 mg/ml non-reducing disaccharide; approximately 0.02-2.0 mg/ml polysorbate 80 or polysorbate 20; buffer at pH approximately 4.5-6.5; and about 40-150 mM L-arginine or a pharmaceutically acceptable salt thereof, wherein the anti-LAG3 antibody or antigen binding fragment thereof comprises a light chain variable region comprising CDRL1 SEQ ID NO:39, CDRL2 SEQ ID NO:40 and CDRL3 SEQ ID NO: 41, and a heavy chain variable region comprising CDRH1 SEQ ID NO:42, CDRH2 SEQ ID NO:59 and CDRH3 SEQ ID NO:44, and an anti-PD-1 antibody or antigen binding fragment thereof comprises a light chain variable region comprising CDRL1 SEQ ID NO:1, CDRL2 SEQ ID NO:2, and CDRL3 SEQ ID NO:3, and a heavy chain variable region comprising CDRH1 SEQ ID NO:6, CDRH2 SEQ ID NO:7, and CDRH3 SEQ ID NO:8.

2. The composition of embodiment 1 containing approximately 18-22 mg/ml anti-LAG3 antibody; approximately 4-7 mg/ml anti-PD-1 antibody; approximately 50-90 mg/ml sucrose or trehalose; approximately 0.05-1.0 mg/ml polysorbate 80 or polysorbate 20; approximately 3-30 mM histidine buffer at pH approximately 5.0-6.5; and about 40-100 mM L-arginine or a pharmaceutically acceptable salt thereof.

3. The composition of embodiment 1 containing approximately 18-20 mg/ml anti-LAG3 antibody; approximately 4-7 mg/ml anti-PD-1 antibody; approximately 50-60 mg/ml sucrose or trehalose; approximately 0.05-1.0 mg/ml polysorbate 80 or polysorbate 20; approximately 8-12 mM histidine buffer at pH approximately 5.0-6.5; and about 40-70 mM L-arginine or a pharmaceutically acceptable salt thereof.

4. The composition of any of embodiments 1-3, further comprising approximately 5-15 mM L-methionine.

5. The composition of any of embodiments 1-3, further comprising approximately 5-10 mM L-methionine.

6. The composition of embodiment 1 containing approximately 18.75 mg/ml anti-LAG3 antibody; approximately 6.25 mg/ml anti-PD-1 antibody; approximately 55 mg/ml sucrose; approximately 0.2 mg/ml polysorbate 80; approximately 10 mM histidine buffer at pH approximately 5.8; and about 52.5 mM L-arginine or a pharmaceutically acceptable salt thereof; and additionally containing approximately 7.5 mM L-methionine.

7. The composition of embodiment 1 containing approximately 20 mg/ml anti-LAG3 antibody; approximately 5 mg/ml anti-PD-1 antibody; approximately 54 mg/ml sucrose; approximately 0.2 mg/ml polysorbate 80; approximately 10 mM histidine buffer at pH approximately 5.8; and about 56 mM L-arginine or a pharmaceutically acceptable salt thereof; and additionally containing approximately 8 mM L-methionine.

8. The composition of embodiment 1 containing approximately 20.83 mg/ml anti-LAG3 antibody; approximately 4.17 mg/ml anti-PD-1 antibody; approximately 53 mg/ml sucrose; approximately 0.2 mg/ml polysorbate 80; approximately 10 mM histidine buffer at pH approximately 5.8; and approximately 58.3 mM L-arginine or a pharmaceutically acceptable salt thereof; and additionally containing approximately 8.3 mM L-methionine.

9. The composition of embodiment 1 containing approximately 18.02 mg/ml anti-LAG3 antibody; approximately 4.505 mg/ml anti-PD-1 antibody; approximately 50 mg/ml sucrose; approximately 0.2 mg/ml polysorbate 80; approximately 10 mM histidine buffer at pH approximately 5.8; and about 70 mM L-arginine or a pharmaceutically acceptable salt thereof; and additionally containing approximately 10 mM L-methionine.

The following embodiments also represent aspects of the present invention:

1. A composition containing: approximately 3-300 mg/ml anti-LAG3 antibody or antigen-binding fragment thereof and approximately 3-300 mg/ml anti-PD-1 antibody or antigen-binding fragment thereof at a molar ratio of 4:1-5:1 ( anti-LAG3 antibody to anti-PD-1 antibody, or antigen binding fragments thereof), one or more excipients selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl ₂ , MgCl ₂ , ZnCl ₂ and FeCl _{2 ,} with a total excipient concentration of approximately 10-1000 mM, and a buffer at a pH of approximately 4.5-8.

2. The composition of embodiment 1, comprising: approximately 10-200 mg/ml of an anti-LAG3 antibody or antigen binding fragment thereof containing a light chain variable region containing CDRL1 SEQ ID NO:39, CDRL2 SEQ ID NO:40, and CDRL3 SEQ ID NO:41, and a heavy chain variable region comprising CDRH1 SEQ ID NO:42, CDRH2 SEQ ID NO:59, and CDRH3 SEQ ID NO:44, and approximately 4-200 mg/ml anti-PD-1 antibody or antigen binding fragment thereof , containing a light chain variable region containing CDRL1 SEQ ID NO:1, CDRL2 SEQ ID NO:2 and CDRL3 SEQ ID NO:3, and a heavy chain variable region containing CDRH1 SEQ ID NO:6, CDRH2 SEQ ID NO:7, and CDRH3 SEQ ID NO:8, one or more of the excipients selected from the group consisting of histidine, aspartate, glutamine, glycine, proline, methionine, arginine or a pharmaceutically acceptable salt thereof, NaCl, KCl, LiCl, CaCl ₂ , MgCl ₂ , ZnCl ₂ and FeCl _2, at a concentration of approximately 25-250 mM, and a buffer at a pH of approximately 5-8.

3. The composition of embodiment 1 or 2, wherein the excipient is L-arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 25-250 mM.

4. The composition of embodiment 1 or 2, wherein the excipient is L-arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 40-100 mM.

5. The composition of embodiment 1 or 2, wherein the excipients are a combination of NaCl and L-arginine or a pharmaceutically acceptable salt thereof at a concentration of approximately 25-250 mM.

6. The composition of embodiment 5, wherein the concentration ratio of NaCl to L-arginine is 1:1.

7. The composition of embodiment 6, wherein the concentration of NaCl is approximately 35 mM, and the concentration of L-arginine is approximately 35 mM.

8. The composition of embodiment 6, wherein the concentration of NaCl is approximately 50 mM, and the concentration of L-arginine is approximately 50 mM.

9. The composition of embodiment 1 or 2, where e excipients are NaCl, KCl and/or LiCl at approximately 40-150 mM.

10. Formulation of embodiment 1 or 2, wherein the excipient is L-histidine at approximately 25-200 mM.

11. The composition of embodiment 10, wherein L-histidine is present at approximately 40-100 mM.

12. The composition of any one of embodiments 1-11, wherein the buffer is an L-histidine, acetate or citrate buffer.

13. The composition of embodiment 12, wherein the buffer has a concentration of approximately 1-300 mM.

14. The composition of any of embodiments 1-13, further comprising a sugar, a polyol or a nonionic surfactant, or a combination thereof.

15. The composition of embodiment 14, wherein the sugar is a non-reducing disaccharide.

16. The composition of embodiment 15, wherein the sugar is trehalose or sucrose, or a combination thereof.

17. The composition of embodiments 15 or 16, wherein the sugar is present at a concentration of approximately 10-200 mg/ml.

18. The composition of embodiment 14, wherein the polyol is a sugar alcohol.

19. The composition of embodiment 14, wherein the polyol is selected from the group consisting of mannitol, sorbitol, glycerol and polyethylene glycol.

20. The composition of embodiments 18 or 19, wherein the polyol is present at a concentration of approximately 10-200 mg/ml.

21. The composition of embodiment 14, wherein the nonionic surfactant is selected from the group consisting of polysorbate and poloxamer.

22. The composition of embodiment 14, wherein the nonionic surfactant is selected from the group consisting of Tween80®, Tween20®, PluronicF88®, Pluoronic F-127®, PluronicF68® and Triton X-100®.

23. The composition of embodiment 14, wherein the nonionic surfactant is polysorbate 80 or polysorbate 20.

24. The composition of any of embodiments 1-13, further comprising the surfactant compound polysorbate 20 or polysorbate 80, and the sugar sucrose or trehalose, or a combination thereof.

25. The composition of any of embodiments 1-11, further comprising approximately 10-250 mg/ml of sucrose, trehalose, mannitol, sorbitol, polyethylene glycol or glycerol; approximately 0.005-2.0 mg/ml polysorbate 80 or polysorbate 20; and approximately 3-300 mM L-histidine, acetate or citrate buffer at a pH of approximately 5.0-6.5.

26. The composition of any of embodiments 1-11, further containing approximately 30-120 mg/ml sucrose or trehalose; approximately 0.05-1.5 mg/ml polysorbate 80 or polysorbate 20; approximately 3-150 mM L-histidine, acetate or citrate buffer at pH approximately 5.0-6.5.

27. The composition of any of embodiments 1-11, further containing approximately 50-90 mg/ml sucrose or trehalose; approximately 0.05-1.0 mg/ml polysorbate 80; approximately 5-30 mM L-histidine, acetate or citrate buffer at pH approximately 5.0-6.5.

28. The composition of embodiment 1 or 2 containing approximately 20-220 mg/ml of anti-LAG3 antibody or antigen binding fragment thereof; approximately 50-90 mg/ml sucrose or trehalose; approximately 0.05-1.0 mg/ml polysorbate 80 or polysorbate 20; approximately 5-20 mM L-histidine, acetate or citrate buffer at pH approximately 5.0-6.5; and about 40-150 mM L-arginine or a pharmaceutically acceptable salt thereof.

29. The composition of embodiment 1 or 2 containing approximately 20-220 mg/ml of anti-LAG3 antibody or antigen binding fragment thereof; approximately 20-200 mg/ml glycerol, sorbitol or PEG400; approximately 0.05-1.0 mg/ml polysorbate 80 or polysorbate 20; approximately 3-150 mM L-histidine, acetate or citrate buffer at pH approximately 5.0-6.5; and about 40-150 mM L-arginine or a pharmaceutically acceptable salt thereof.

30. The composition of embodiment 1 or 2 containing approximately 20-220 mg/ml of anti-LAG3 antibody or antigen binding fragment thereof; approximately 40-150 mM L-glutamine, L-glycine, L-proline or L-methionine; approximately 0.05-1.0 mg/ml polysorbate 80 or polysorbate 20; approximately 3-150 mM L-histidine, acetate or citrate buffer at pH approximately 5.0-6.5; and about 40-150 mM L-arginine or a pharmaceutically acceptable salt thereof.

31. The composition of embodiment 1 or 2 containing approximately 20-220 mg/ml of anti-LAG3 antibody or antigen binding fragment thereof; approximately 0.05-1.0 mg/ml polysorbate 80 or polysorbate 20; approximately 3-150 mM L-histidine, acetate or citrate buffer at pH approximately 5.0-6.5; and about 40-150 mM NaCl or a pharmaceutically acceptable salt thereof.

32. The composition of any of embodiments 1-31, further comprising approximately 3-150 mM L-methionine.

33. The composition of any of embodiments 1-31, further comprising approximately 5-70 mM L-methionine.

34. The composition of embodiment 1 or 2 containing approximately 25 mg/ml of anti-LAG3 antibody or antigen binding fragment thereof; approximately 50 mg/ml sucrose; approximately 0.2 mg/ml polysorbate 80; approximately 10 mM L-histidine buffer at pH approximately 5.8; approximately 70 mM L-arginine or a pharmaceutically acceptable salt thereof; and approximately 10 mM L-methionine.

35. The composition of embodiment 1 or 2, containing: about 3-250 mg/ml anti-LAG3 antibody or antigen binding fragment thereof, sugar, polyol, nonionic surfactant, histidine or acetate buffer at a pH of about 5-8, and about 10-1000 mM L-arginine or a pharmaceutically acceptable salt thereof.

36. The composition of embodiment 1 or 2 containing approximately 20 mg/ml of anti-LAG3 antibody or antigen binding fragment thereof; approximately 50 mg/ml sucrose; approximately 0.2 mg/ml polysorbate 80; approximately 10 mM L-histidine buffer at pH approximately 5.8-6.0; and approximately 70 mM L-arginine or L-arginine-HCl.

37. A composition containing: approximately 10 mg/ml anti-LAG3 antibody or antigen binding fragment; approximately 10 mg/ml anti-PD-1 antibody or antigen binding fragment; approximately 50 mg/ml sucrose; approximately 0.2 mg/ml polysorbate 80; approximately 10 mM histidine buffer at pH approximately 5.8; approximately 70 mM L-arginine or a pharmaceutically acceptable salt thereof; and approximately 10 mM L-methionine; wherein the anti-LAG3 antibody or antigen binding fragment thereof comprises a light chain variable region comprising CDRL1 SEQ ID NO:39, CDRL2 SEQ ID NO:40 and CDRL3 SEQ ID NO:41, and a heavy chain variable region comprising CDRH1 SEQ ID NO:42 , CDRH2 SEQ ID NO:59, and CDRH3 SEQ ID NO:44, and the anti-PD-1 antibody or antigen binding fragment thereof contains a light chain variable region containing CDRL1 SEQ ID NO:1, CDRL2 SEQ ID NO:2 and CDRL3 SEQ ID NO:3, and a heavy chain variable region comprising CDRH1 SEQ ID NO:6, CDRH2 SEQ ID NO:7 and CDRH3 SEQ ID NO:8.

38. The composition of any one of embodiments 1-37, which is a liquid composition.

39. The composition of any of embodiments 1-37, which is frozen at least below -70°C.

40. The formulation of any one of embodiments 1-37, which is a reconstituted solution from a lyophilized formulation.

41. The composition of any one of embodiments 1-40, wherein at 50°C, the % high molecular weight (HMW) compound is less than 5% after 10 days, as measured by size exclusion chromatography.

42. The composition of any one of embodiments 1-41, wherein at 5°C, the % monomer of the anti-LAG3 antibody or antigen binding fragment thereof and the anti-PD-1 antibody or antigen binding fragment thereof is ≥99.5% after 3 months as measured size exclusion chromatography.

43. The composition of any one of embodiments 1-41, wherein at 40°C, the % monomer of the anti-LAG3 antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof is ≥98% after 3 months, as measured by size exclusion chromatography .

44. The composition of any one of embodiments 1-43, wherein at 5°C, the % acidic variants of the anti-LAG3 antibody or antigen binding fragment thereof is less than 15% after 3 months, as measured by ion exchange chromatography, the % acidic variants of the anti-PD-1 antibody or antigen-binding fragment thereof is less than 20% after 3 months, as measured by ion exchange chromatography.

45. The composition of any one of embodiments 1-43, wherein at 25°C/60% relative humidity (RH) inverse, the % acidic variant anti-LAG3 antibody or antigen binding fragment thereof is less than 20% after 3 months, as measured by ion exchange chromatography, the % acidic variant of the anti-PD-1 antibody or antigen-binding fragment thereof is less than 25% after 3 months, as measured by ion exchange chromatography.

46. The composition of any one of embodiments 1-43, wherein at 40°C/75% RH reverse, the % acidic variant of the anti-LAG3 antibody or antigen binding fragment thereof is less than 50% after 3 months, as measured by ion exchange chromatography, % acidic variant anti-PD-1 antibody or antigen-binding fragment thereof is less than 53% at 3 months, as measured by ion exchange chromatography.

47. The composition of any one of embodiments 1-46, wherein the anti-LAG3 antibody or antigen binding fragment comprises: a light chain variable region sequence of SEQ ID NO:37 and a heavy chain variable region sequence of SEQ ID NO:58.

48. The composition of any one of embodiments 1-46, wherein the anti-LAG3 antibody or antigen binding fragment thereof comprises the light chain sequence of SEQ ID NO:35 and the heavy chain sequence of SEQ ID NO:57.

49. The composition of any one of embodiments 1-46, wherein the anti-LAG3 Antibody is an Ab6 or an Ab6 variant.

50. The composition of embodiment 47, wherein the anti-PD-1 antibody or antigen binding fragment thereof comprises a heavy chain variable region of SEQ ID NO:9 and a light chain variable region of SEQ ID NO:4.

51. The composition of embodiment 48, wherein the anti-PD-1 antibody comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:5.

52. The composition of embodiment 49, wherein the anti-PD-1 antibody is pembrolizumab or a variant of pembrolizumab.

Lyophilized formulations

Lyophilized formulations of therapeutic proteins provide several advantages. Lyophilized formulations generally have better chemical stability than solution formulations and thus increase shelf life. The lyophilized formulation can also be reconstituted at different concentrations depending on clinical factors such as route of administration or dosage. For example, the lyophilized formulation can be reconstituted at a high concentration (ie, in a small volume), if necessary, for subcutaneous administration, or at a low concentration, if administered intravenously. High concentrations may also be necessary if a high dosage is required for a particular subject, especially when administered subcutaneously where the volume of injection must be minimized. Subcutaneous administration of antibody-based drugs offers the possibility of self-administration. Self-administration avoids the time and expense associated with visiting a medical facility for administration, such as intravenous administration. Subcutaneous administration is limited by the volume of solution that can be practically delivered to the injection site in one injection, which is usually approximately 1-1.5 ml. This limitation often requires a relatively high concentration of solution to deliver the required amount of drug. Subcutaneous self-administration is usually accomplished using a prefilled syringe or auto-injector filled with a liquid solution of the drug formulation rather than a lyophilized form so that the patient does not have to resuspend the drug before injection.

Typically, the lyophilized formulation is prepared prior to reconstitution at a high drug product (DP) concentration, ie, prior to reconstitution in a liquid volume. Subsequent dilution with water or isotonic buffer can easily be used to dilute the DP to a lower concentration. Typically, the excipients are included in the lyophilized formulation of the present invention in amounts that will result in an approximately isotonic composition when reconstituted at a high DP concentration, for example, for subcutaneous administration. Dilution in a larger volume of water to obtain a lower concentration of DP will necessarily reduce the tonicity of the reconstituted solution, but this reduction may have little effect when administered subcutaneously as a mixture with an isotonic solution (0.9% sodium chloride, USP or 5% dextrose solution, USP). If isotonicity is desired at a lower DP concentration, the lyophilized powder can be reconstituted in a standard small volume of water and then further diluted with an isotonic diluent such as 0.9% sodium chloride.

The lyophilized compositions of the present invention are prepared by lyophilization (freeze drying) of the lyophilization solution. Freeze drying is carried out by freezing the composition and subsequent sublimation of water at a temperature suitable for primary drying. Under these conditions, the temperature of the product is below the eutectic point or temperature of destruction of the composition. Typically, the primary drying shelf temperature will be in the range of about -30 to -25 °C (assuming the product remains frozen during primary drying) at a suitable pressure, typically in the range of about 50 to 250 mTorr. The composition, size and type of container in which the sample is contained (e.g., glass vial), and the volume of the composition that can be lyophilized will determine the time required for drying, which can vary from several hours to several days (40-60 hours). Secondary drying can be carried out at approximately 0-40 °C, depending primarily on the type and size of the container and the type of protein used. The duration of secondary drying is dictated by the desired level of residual moisture in the product and typically takes at least approximately 5 hours. Typically, the moisture content of the lyophilized formulation is less than about 5%, and preferably less than about 3%. The pressure can be the same as during the primary drying stage. Freeze-drying conditions can be varied depending on the composition and size of the bottle.

In some cases, it may be desirable to lyophilize the protein formulation in a container in which the protein must be dissolved to avoid a transfer step. The container in this case can be, for example, a bottle with a volume of 3, 5, 10, 20, 50 or 100 ml.

The lyophilized formulations of the present invention are reconstituted before administration. Protein can be reduced at concentrations of about 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or about 100 mg/ml or higher concentrations such such as about 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml and up to about 500 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof after reconstitution is approximately 4-7 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof after reconstitution is approximately 3-7 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof after reconstitution is approximately 4 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof after reconstitution is approximately 5 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof after reconstitution is approximately 6 mg/ml. In one embodiment, the concentration of the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 18-22 mg/ml. In one embodiment, the concentration of the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 20 mg/ml. In one embodiment, the concentration of the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 16-22 mg/ml. In one embodiment, the anti-PD-1 or anti-LAG3 antibody or antigen binding fragment concentration after reconstitution is approximately 3-300 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof or the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 4-250 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof or the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 150-250 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof or the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 180-220 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof or the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 50-150 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof or the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 50 mg/ml. In one embodiment, the concentration of the anti-PD-1 antibody or antigen binding fragment thereof or the anti-LAG3 antibody or antigen binding fragment thereof after reconstitution is approximately 25 mg/ml. High protein concentrations are particularly useful where subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration, such as intravenous administration, lower protein concentrations (eg, approximately 5-25 mg/ml) may be desirable.

Reduction usually occurs at approximately 25°C to ensure complete hydration, although other temperatures can be used if desired. The time required for reconstitution will depend, for example, on the type of diluent, the amount of excipient(s) and protein. Examples of diluents include sterile water, bacteriostatic water for injection (BWFI), pH-buffered solution (eg, phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

In one embodiment of the present invention, an anti-LAG3 antibody (or an antigen binding fragment thereof) and an anti-PD-1 antibody (or an antigen binding fragment thereof) are formulated together as a lyophilized powder for intravenous administration. In another embodiment of the present invention, the co-formulated product is a lyophilized powder for subcutaneous administration. In some embodiments, the antibody (or antigen binding fragment thereof) is provided at approximately 100-1200 mg/vial, and reconstituted with sterile water for injection prior to use. In other embodiments, the antibody (or antigen binding fragment thereof) is provided at approximately 200 mg/vial, and reconstituted with sterile water for injection prior to use. In one embodiment, the target pH of the reconstituted formulation is approximately 6.0. In various embodiments, the lyophilized formulation of the present invention provides reconstitution of the anti-LAG3 antibody or anti-PD-1 antibody, or antigen binding fragments thereof, to concentrations such as about 5, 10, 20, 25, 30, 40, 50, 60, 75 , 100, 150, 200, 250 or more mg/ml. In other embodiments, the concentration of anti-LAG3 antibody or anti-PD-1 antibody or antigen binding fragments thereof after reconstitution is approximately 3-300, 10-300, 20-250, 150-250, 180-220, 20-200, 40 -100 or 50-150 mg/ml. In other embodiments, the concentration of anti-LAG3 antibody or anti-PD-1 antibody or antigen binding fragments thereof prior to lyophilization is approximately 3-300, 10-300, 150-250, 180-220, 10-100, 10-50, or 25 -50 mg/ml.

In other embodiments, the lyophilized composition of the anti-LAG3 antibody or antigen binding fragment and the anti-PD-1 antibody or antigen binding fragment are determined in terms of the reconstituted solution obtained from the lyophilized composition. Reconstituted solutions may contain the antibody or antigen binding fragment thereof at concentrations of approximately 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg/ml or higher concentrations such as 150 mg /ml, 200 mg/ml, 250 mg/ml, or up to about 300 mg/ml. In one embodiment, the reconstituted formulation may contain approximately 3-300 mg/ml of antibody or antigen-binding fragment thereof. In another embodiment, the reconstituted formulation may contain approximately 10-200 mg/ml of antibody or antigen binding fragment thereof. In another embodiment, the reconstituted formulation may contain approximately 10-100 mg/ml of antibody or antigen binding fragment thereof. In another embodiment, the reconstituted formulation may contain about 10-60 or about 15-50 mg/ml of antibody or antigen binding fragment thereof. In another embodiment, the reconstituted formulation may contain approximately 10-25 mg/ml of antibody or antigen binding fragment thereof. In a preferred embodiment, the reconstituted formulation may contain approximately 20-30 or 25 mg/ml of antibody or antigen-binding fragment thereof.

Liquid composition

A liquid antibody formulation can be prepared by using the drug, which is found in, for example, an aqueous pharmaceutical formulation, and a buffer, replacing it with the desired buffer at the last stage of the purification process. In this embodiment, there is no lyophilization step. The drug substance in the final buffer is concentrated to the required concentration. Excipients such as stabilizers and surfactants are added to the drug substance and diluted using an appropriate buffer to the final protein concentration. The final formulated drug substance is filtered using a 0.22 µm filter and filled into a final container (eg glass vials). The formulation may be stored in a vial and delivered through an injection device or vessel.

In another aspect of the present invention, for a liquid co-formulated composition containing an anti-PD-1 antibody and an anti-LAG3 antibody (or antigen binding fragments thereof), the anti-LAG3 antibody or antigen binding fragment thereof has a concentration of approximately 3-300 mg/ml. In another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has a concentration of approximately 4-250 mg/ml. In another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has a concentration of approximately 40-100 mg/ml. In a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has a concentration of approximately 10-60 mg/ml. In a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has a concentration of approximately 20-30 mg/ml. In a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has a concentration of approximately 10-30 mg/ml. In a further embodiment, the anti-LAG3 antibody or antigen binding fragment thereof has a concentration of approximately 15-50 mg/ml. In another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is present at a concentration of approximately 10-100 mg/ml. In yet another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is present at a concentration of approximately 20-30 or 25 mg/ml. In yet another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is present at a concentration of approximately 16-22 mg/ml. In yet another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is present at a concentration of approximately 18-22 mg/ml. In yet another embodiment, the anti-LAG3 antibody or antigen binding fragment thereof is present at a concentration of approximately 20 mg/ml.

In another aspect of the present invention, the anti-PD-1 antibody or antigen binding fragment thereof in the liquid formulation has a concentration of approximately 3-300 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 4-250 mg/ml. In another embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 40-100 mg/ml. In a further embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 10-60 mg/ml. In a further embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 20-30 mg/ml. In a further embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 10-30 mg/ml. In a further embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 15-50 mg/ml. In another embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 10-100 mg/ml. In another embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 20-30 or 25 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 3-7 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof is present at a concentration of approximately 4-7 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof has a concentration of approximately 4 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof has a concentration of approximately 5 mg/ml. In one embodiment, the anti-PD-1 antibody or antigen binding fragment thereof has a concentration of approximately 6 mg/ml.

In one embodiment, the liquid composition contains a buffer at a pH of approximately 4.5-8, 5.0-6.5, 5.5-6.5, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 or 6.2 and arginine or a pharmaceutically acceptable salt thereof. In one embodiment, the liquid composition contains a buffer at a pH of approximately 5-8. In one embodiment, the liquid composition contains a buffer at a pH of approximately 5.0-6.5. In one embodiment, the liquid composition contains a buffer at a pH of approximately 5.0-6.0. In other embodiments, the buffer is histidine. In another embodiment, the buffer is citrate or acetate. In a further embodiment, the liquid formulation contains an acetate buffer at a pH of approximately 5-8, 5.0-6.5, 5.5-6.5, 5.5, 5.6, 5.7, 5.8, 5 .9, 6.0, 6.1 or 6.2 and arginine or a pharmaceutically acceptable salt thereof.

The liquid antibody composition of the present invention is suitable for parenteral administration, such as intravenous, intramuscular, intraperitoneal or subcutaneous administration; Particularly suitable for subcutaneous administration.

Dosing and Administration

Toxicity should be considered when selecting the correct dosage of a therapeutic agent such as a humanized anti-LAG3 or anti-PD-1 antibody (or antigen-binding fragments thereof). The toxicity and therapeutic efficacy of antibody compositions, administered alone or in combination with an immunosuppressive agent, can be determined by standard pharmaceutical methods in cell cultures or experimental animals, for example, to determine the LD50 (dose lethal to 50% of the population) and ED50 (dose therapeutically effective in 50% of the population). The dose relationship between toxic and therapeutic effects represents the therapeutic index and can be expressed as the ratio of LD50 to ED50. Antibodies with high therapeutic indices are preferred. Data obtained from these cell culture assays and animal studies can be used to determine dose ranges for use in humans. The dosage of these compounds is preferably within a circulating concentration range that includes the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used.

Suitable routes of administration may, for example, include parenteral administration, including intramuscular, intradermal, subcutaneous, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal. Drugs can be administered by various conventional routes, such as intraperitoneal, parenteral, intra-arterial or intravenous administration. Administration modes where solution volume must be limited (eg, subcutaneous administration) require that the lyophilized formulation provide reconstitution at a high concentration.

Alternatively, the antibody can be administered locally rather than systemically, for example by injecting the antibody directly into a pathogen-induced lesion characterized by immunopathology, often in the form of a depot or sustained release formulation. In addition, the antibody can be administered in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, a pathogen-induced lesion characterized by immunopathology. The liposomes will be targeted and selectively captured by the affected tissue.

The choice of therapeutic regimen depends on several factors, including the rate of serum or tissue metabolism of the molecule, the level of symptoms, the immunogenicity of the molecule, and the availability of target cells in the biological matrix. Preferably, the administration regimen maximizes the amount of therapeutic agent delivered to the patient, consistent with an acceptable level of side effects. Accordingly, the amount of biological agent delivered depends in part on the specific molecule and the severity of the condition being treated. Guidelines for selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002).

Determination of the appropriate dose is made by the clinician, for example, using parameters or factors known or suspected in the art to influence or be expected to influence treatment. The appropriate dosage ("therapeutically effective amount") of protein will depend, for example, on the condition being treated, the severity and course of the condition, whether the protein is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to protein, the type of protein used and at the discretion of the attending physician. Typically, the dose starts at an amount slightly less than the optimal dose and is then increased in small increments until the desired or optimal effect relative to any negative side effects is achieved. Important diagnostic indicators include the degree of symptoms, such as inflammation, or the levels of inflammatory cytokines produced. The protein is suitably administered to the patient once or repeatedly. The protein may be administered alone or in combination with other drugs or therapies.

Antibodies or antibody fragments can be provided by continuous infusion or dosing at intervals, for example, one day, 1-7 times a week, one week, two weeks, three weeks, monthly, bimonthly, etc. The preferred dosing protocol is one that includes the maximum dose or frequency of dosing that will avoid significant unwanted side effects.

In some embodiments, the pharmaceutical compositions of the present invention will be administered by intravenous (IV) infusion or injection.

In other embodiments, the pharmaceutical compositions of the present invention will be administered by subcutaneous administration. Subcutaneous administration can be accomplished by injection using a syringe or using other injection devices (eg, Inject-easy® devices); syringe pens; or needleless devices (eg MediJector and BioJector®).

Subcutaneous administration can be accomplished by injection using a syringe, autoinjector, pen, or needleless injection device. Intravenous administration can be done after diluting the formulation with a suitable commercial diluent such as saline or 5% dextrose in water.

Although the high concentration solution formulations of the present invention are particularly advantageous for applications requiring high concentrations of antibody, there is no reason why the formulations cannot be used at lower concentrations of antibody in circumstances where high concentrations of antibody are not required or desired. Lower concentrations of antibody may be suitable when administered subcutaneously at low doses or in other modes of administration (eg, intravenous administration) where the volume that can be delivered is substantially greater than 1 ml. These lower concentrations may include about 15, 10, 5, 2, 1 mg/ml or less.

Applications

The present invention relates to lyophilized or liquid formulations of an anti-LAG3 antibody or antigen binding fragment thereof and an anti-PD-1 antibody or antigen binding fragment for use in the treatment of cancer and infection.

Those skilled in the art will understand that the term "cancer" is the name of diseases in which the cells of the body become abnormal and divide uncontrollably. Cancers that can be treated with the compounds, compositions and methods of the present invention include, but are not limited to: heart: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; lungs: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal tract: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, VIPoma), small intestine (adenocarcinoma, lymphoma a , carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma; large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma) colorectal; genitourinary tract (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma); , leukemia ), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testes (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; bone: osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell chordoma, osteochondroma (osteocartilaginous exostoses), benign chondroma, lastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), neurofibroma of the spinal cord, meningioma, glioma, sarcoma); gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, precancerous cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma , malignant teratoma, vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma), fallopian tubes (carcinoma), breast; hematological: blood (myeloid leukemia [acute and Chronic], acute lymphoblastic leukemia, chronic lymphocyte, myelopoliferative diseases, multiple myeloma, myelodislastic syndrome), Hodgkin's disease, non -podzhkinsky lymphoma [malignant lymphoma]; Nevovs, lipoma, angioma, dermatofibroma, keloids, psoriasis; and adrenal glands: neuroblastoma. In one embodiment, the cancer is selected from colorectal cancer, gastric cancer, and head and neck cancer.

EXAMPLES

Example 1: Studies of Conditions to Reduce Self-Association of Anti-LAG3 Antibody Ab6

Measurement of the diffusion interaction parameter (k _D )

B ₂₂ in 10 mM histidine pH 5.6 was negative, indicating the inherent ability of the molecule to self-associate. The presence of 50 mM sodium chloride in 10 mM histidine pH 5.6 was found to increase the diffusion interaction parameter (KD) or reduce self-interaction, improve the relative solubility and reduce the turbidity (OD350) of the anti-LAG3 antibody Ab6 (SEQ ID NO: 35 and 57 , light and heavy chains) as seen in Figure 12. The stability of anti-LAG3 antibody in 10 mM histidine pH 5.6 was examined in the presence of 40 mM L-arginine hydrochloride using the diffusion interaction parameter (KD), turbidity (OD350) and test by relative solubility (% PEGmid-point).

As seen in Figure 13, self-interaction and turbidity were found to be dramatically reduced, whereas the relative solubility of anti-LAG3 antibody was found to increase significantly with the addition of 40 mM L-arginine hydrochloride in 10 mM histidine buffer at pH 5.6. A study of a range of L-arginine hydrochloride (40 mM-100 mM) with 50 mg/ml anti-LAG3 antibody was carried out at pH 5.8 and at pH 6.0, and the concentration of L-arginine hydrochloride was found to be 70 mM ( 14.7 mg/ml) is effective in reducing self-interaction and improving colloidal stability (Figure 13).

Anti-LAG3 antibody was found to phase separate in low ionic strength (10 mM) buffers. To evaluate the self-association properties, as well as the colloidal, physical, chemical, as well as thermal stability of the anti-LAG3 antibody in the presence of three different charged species [L-arginine, L-histidine and sodium chloride (NaCl)] at different concentrations (mM), nine various compositions were prepared as listed in Table 3 below. Unformulated anti-LAG3 (~37 mg/ml) in 10 mM L-histidine, 70 mM L-arginine hydrochloride pH 5.8 was dialyzed against three 10 mM histidine pH 5.8 buffers; each buffer solution containing 100 mM L-arginine, 100 mM sodium chloride or 100 mM L-histidine. Anti-LAG3 antibody was formulated at 25 mg/ml using appropriately formulated dialysates. Formulations containing 40 mM to 130 mM L-arginine or sodium chloride were prepared by diluting the appropriate anti-LAG3 antibody stock solution with L-histidine buffer at pH 5.8 and concentrating the anti-LAG3 antibody to 25 mg/ml.

Table 3 High-throughput screening of anti-LAG3 antibody preformulations with charged compounds (L-arginine, L-histidine and sodium chloride) Compound# Excipient concentration Description of composition 1 40 mM L-Arginine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 40 mM L-arginine, pH 5.8 2 70 mM L-Arginine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine, pH 5.8 3 100 mM L-Arginine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 100 mM L-arginine, pH 5.8 4 40 mM L-Histidine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 40 mM L-histidine, pH 5.8 5 70 mM L-Histidine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-histidine, pH 5.8 6 100 mM L-Histidine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 100 mM L-histidine, pH 5.8 7 40 mM Sodium Chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 40 mM sodium chloride, pH 5.8 8 70 mM Sodium Chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM sodium chloride, pH 5.8 9 100 mM Sodium Chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 100 mM sodium chloride, pH 5.8

The diffusion interaction parameter (k _D ) of the nine formulations was estimated using dynamic light scattering (DLS) at 20°C for five samples. The interaction parameter (kD) was calculated from the slope and y-intercept of the plot of recorded diffusion coefficient values (cm ² /s) against a series of diluted concentrations (mg/ml) of the respective formulations. A positive diffusion interaction parameter (kD) indicates a repulsive interaction. With increasing concentration (>40 mM) of L-arginine, L-histidine, or sodium chloride, the anti-LAG3 antibody shows an increase in kD, indicating decreased molecular self-association (less molecular aggregation). The effect is comparatively pronounced for L-arginine, followed by sodium chloride and L-histidine in relative order (see Figure 15).

Relative solubility studies

Automated relative solubility screening of nine formulations was assessed using polyethylene glycol (PEG)-induced precipitation requiring a 10 mg/mL protein concentration. 40% (w/v) PEG 6000 was prepared in each buffer solution, followed by 2%-36% (w/v) PEG-6000 solutions in various increments using a JANUS G3 automated liquid handling system. The 10 mg/mL protein solution was added to the PEG solutions in a Costar 96-well clear plate to obtain a final concentration of 1 mg/mL. The plate was equilibrated at temperature overnight, transferred to an Abgene PCR plate, and centrifuged at 4600 rpm for 30 min to cause a protein pellet to settle at the bottom of each well. The supernatant was transferred from each well to a fresh 96-well clear plate. The plate was read on a SpectraMax M5 reader at 280 and 320 nm to determine protein loss due to precipitation during overnight incubation. Absorbance data (280-320) relative to PEG concentration was analyzed to determine %PEG _midpt .

The anti-LAG3 antibody showed improved relative solubility in the presence of increasing concentrations of charged molecules such as L-arginine, L-histidine or sodium chloride (40 mM up to 100 mM), suggesting reduced molecular crowding of the anti-LAG3 antibody at these concentrations. See Figure 16. The magnitude of the increase in relative solubility was similar for all three charged compounds.

Charged Particle Measurement Research

The change in charge heterogeneity and isoelectric point (pI) of anti-LAG3 antibody in the presence of L-arginine, L-histidine or sodium chloride was assessed using a ProteinSimple capillary isoelectric focusing (cIEF) system. Samples were mixed with ampholyte vehicle before injection into the capillary. When an electric field was applied to the capillary, the ampholyte carrier in the capillary created a pH gradient and the protein molecules migrated to a location in the capillary where the local pH value equaled the isoelectric pH (pI) values. Detection of separated proteins was performed by full scanning of the entire capillary using the iCE system (iCE3 from ProteinSimple). The last image obtained by the instrument was used to quantify the data. The percent area of resolved peaks was estimated by dividing the area of individual shapes by the total area of the protein. The pI value of a protein is estimated by linearly calibrating the distance between two pI markers delimiting the protein. Operating parameters included autosampler temperature 10°C; fluorocarbon (FC) coated cartridge, detection wavelength 280 nm, focusing period 1 minute at 1500 V. Nine formulations were transferred to a 96-well plate and assessed for charge change (% acidic variants, % major peak and % basic variants) at the initial time point using cIEF. The remaining samples of the nine formulations were transferred to another 96-well plate, tightly sealed, and placed under thermal stress for 10 days at 50°C. Under stress, the change in charged particles was assessed again. The data in Figure 17 shows the % change (difference) in acidic variants, % change in the main peak, and % change in the main variants under thermal stress compared to the original.

Sodium chloride showed the least change in % acidic variants and % basic peak for the anti-LAG3 antibody formulation, followed by L-arginine and L-histidine. Sodium chloride showed improved chemical stability in the concentration range from 40 to 100 mM, especially at ≥70 mM. L-arginine showed better chemical stability at a concentration of 70 mM, while L-histidine showed better chemical stability up to a concentration of 100 mM.

To evaluate the self-associating properties as well as the colloidal stability of the anti-LAG3 antibody in the presence of L-arginine or sodium chloride (NaCl), twelve different formulations were prepared, listed in Table 4. Unformulated anti-LAG3 antibody (~37 mg/ml) at 10 mM L-histidine 70 mM L-arginine hydrochloride pH 5.8 was dialyzed against four buffer solutions of 10 mM histidine pH 5.8; each buffer solution containing either 150 mM L-arginine, 150 mM sodium chloride, or a mixture of 35 mM L-arginine and 35 mM sodium chloride, or a mixture of 50 mM L-arginine and 50 mM sodium chloride. Anti-LAG3 antibody was formulated at 25 mg/ml using appropriately formulated dialysates. Formulations containing 40-130 mM L-arginine or sodium chloride were prepared by diluting the appropriate anti-LAG3 antibody stock solution with L-histidine buffer at pH 5.8 and concentrating the anti-LAG3 antibody to 25 mg/ml.

Table 4 Optimization of composition with L-arginine, sodium chloride and their mixture Compound# Excipient concentration Description of composition 1 40 mM L-Arginine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 40 mM L-arginine, pH 5.8 2 70 mM L-Arginine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine, pH 5.8 3 100 mM L-Arginine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 100 mM L-arginine, pH 5.8 4 130 mM L-Arginine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 130 mM L-arginine, pH 5.8 5 150 mM L-Arginine 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 150 mM L-arginine, pH 5.8 6 40 mM Sodium Chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 40 mM sodium chloride, pH 5.8 7 70 mM Sodium Chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM sodium chloride, pH 5.8 8 100 mM Sodium Chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 100 mM sodium chloride, pH 5.8 9 130 mM Sodium Chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 130 mM sodium chloride, pH 5.8 10 150 mM Sodium Chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 150 mM sodium chloride, pH 5.8 eleven 35 mM: 35 mM L-Arginine: sodium chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 35 mM L-arginine, 35 mM sodium chloride, pH 5.8 12 50 mM:50 mM L-Arginine:sodium chloride 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 50 mM L-arginine, 50 mM sodium chloride, pH 5.8

Measuring the second virial coefficient (B22)

Measurement of the second virial coefficient (B22) for each of the twelve formulations was carried out at 5 mg/ml using dynamic light scattering (DLS). Automated measurements were performed at 20°C using 173° backscatter.

A positive second virial coefficient (B22) suggests repulsive interactions between protein molecules (less crowding) in the composition matrix. Both L-arginine and sodium chloride at concentrations greater than 40 mM were beneficial in reducing molecular crowding. See Figure 18.

Turbidity measurement (OD ₃₅₀ )

To evaluate the colloidal stability of the anti-LAG3 antibody in the formulation matrix, the turbidity (OD ₃₅₀ ) of twelve formulations was assessed using an ultraviolet (UV) absorbance spectrophotometer. The UV absorbance of the samples was measured in a 96-well transparent co-star plate at a wavelength of 350 nm, corrected for the absorbance of the plate.

Anti-LAG3 antibody shows increased colloidal stability (OD ₃₅₀ ) with increasing concentrations of either L-arginine or sodium chloride with comparable values for both. See Figure 19. The equivalent ratio of L-arginine to sodium chloride (35:35 or 50:50) in the anti-LAG3 antibody formulation matrix also shows comparable colloidal stability.

Viscosity measurement

To evaluate the concentrateability of anti-LAG3 in different formulation matrices, twelve anti-LAG3 antibody formulations listed in Table 4 were concentrated to 60 mg/ml using an Eppendorf centrifuge at 3000 rpm at 15°C. The viscosities of twelve formulations were measured at 20°C using a RheoSense VROC® Initium viscometer on a 96-well plate.

The viscosities of anti-LAG3 antibody at 60 mg/ml in the presence of a mixture of L-arginine or sodium chloride were comparable in the concentration range from 40 to 150 mM. Viscosities at 60 mg/ml in the presence of an equivalent ratio of L-arginine to sodium chloride (35:35 or 50:50) showed similar viscosities. See Figure 20.

Osmolarity measurement

Anti-LAG3 antibody osmolarity was measured using a Vapro Vapor Pressure 5520 Osmometer. The cell was calibrated with 100 mmol/kg, 290 mmol/kg and 1000 mmol/kg calibration standards before measurement.

The osmolarities of the twelve anti-LAG3 antibody formulations listed in Table 4 were found to be comparable in the presence of either L-arginine or sodium chloride. Osmolarities in the presence of an equivalent ratio of L-arginine to sodium chloride (50:50) showed similar viscosity values, whereas an equivalent ratio of 35:35 showed lower osmolarity values. See Figure 21.

Example 2: Preliminary Screening of Charged Compound Formulation (Salt and Amino Acid) of Anti-LAG3 Antibody Ab6 Formulation

To evaluate the stability of the anti-LAG3 antibody in the presence of charged compounds (salt and amino acids), ten formulations listed in Table 5 were prepared and screened for changes in the physicochemical properties of the anti-LAG3 antibody by a high-throughput assay. The formulations were suitably sealed in a 96-well plate and stressed at 50°C for 10 days in a dry heat oven. Samples subjected to thermal stress were also analyzed for changes in the physicochemical properties of the anti-LAG3 antibody. The 20 mM concentration of L-aspartic acid or L-glutamic acid was chosen based on solubility limitations.

Table 5 Compound# Sample nomenclature Description of composition 1 L-Asp 20 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 20 mM L-aspartic acid, pH 5.8 2 L-Glu 20 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 20 mM L-glutamic acid, pH 5.8 3 L-Arg 40 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 40 mM L-arginine, pH 5.8 4 NaCl 40 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 40 mM sodium chloride, pH 5.8 5 L-His 40 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 40 mM L-histidine, pH 5.8 6 L-Arg 70 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine, pH 5.8 7 NaCl 70 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM sodium chloride, pH 5.8 8 L-His 70 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-histidine, pH 5.8 9 L-Asp/Gly 20 mM/50 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 20 mM L-aspartic acid, 50 mM glycine, pH 5.8 10 L-Glu/Gly 20 mM/50 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 20 mM L-glutamic acid, 50 mM glycine, pH 5.8

Turbidity Protocol (OD350)

Turbidity (OD350) of nine formulations was assessed using an ultraviolet (UV) absorption spectrophotometer. The UV absorbance of the samples was measured in a 96-well transparent co-star plate at a wavelength of 350 nm, corrected for the absorbance of the plate.

As seen in Figure 37, under heat stress, the change in colloidal stability (OD ₃₅₀ ) of anti-LAG3 antibody was comparable between 40 mM sodium chloride or L-arginine and 20 mM L-aspartic acid or L-glutamic acid. Likewise, the change in colloidal stability (OD ₃₅₀ ) of anti-LAG3 between 70 mM sodium chloride or L-arginine was comparable to the combination of 20 mM L-aspartic acid or L-glutamic acid with 50 mM glycine (70 mM total strength). The change in colloidal stability (OD ₃₅₀ ) of anti-LAG3 antibody in the presence of either 40 mM or 70 mM L-histidine was relatively high.

UP-SEC

Sample purity was assessed by UP-SEC, which determined the percentage of monomer, as well as the percentage of high molecular weight compounds (HMW) and late elution peaks (LMW compounds). UP-SEC was performed on Acquity H class (DS) by diluting samples to 1.0 mg/mL in the mobile phase (100 mM phosphate, 100 mM sodium chloride, pH 7.0). The column temperature was maintained at 25±3°C and the flow rate was maintained at 0.5 ml/min using isocratic elution. Diluted samples were injected (1 μL) into a UPLC equipped with a Waters BEH200 column and UV detector. Proteins in the sample were separated by size and detected by UV absorbance at 214 nm.

As can be seen in Figure 38, under heat stress, the change in the amounts of soluble aggregates (% high molecular weight compounds, HMW) and the change in % monomer for the anti-LAG3 antibody were comparable between 40-70 mM sodium chloride and 20-70 mM amino acids alone and in some combinations. The change in low molecular weight compounds was relatively low for 20 mM L-aspartic acid or L-glutamic acid, 40 mM L-histidine, 70 mM sodium chloride, and the combination of 20 mM L-aspartic acid and 50 mM glycine.

cIEF

The change in charge heterogeneity and isoelectric point (pI) of anti-LAG3 in the presence of L-arginine, L-histidine or sodium chloride was assessed using the ProteinSimple capillary isoelectric focusing (cIEF) system. Samples were mixed with ampholyte vehicle before injection into the capillary. When an electric field was applied to the capillary, the ampholyte carrier in the capillary created a pH gradient and the protein molecules migrated to a location in the capillary where the local pH value equaled the isoelectric pH (pI) values. Detection of separated proteins was performed by full scanning of the entire capillary using the iCE system (iCE3 from ProteinSimple). The last image obtained by the instrument was used to quantify the data. The percent area of resolved peaks was estimated by dividing the area of individual shapes by the total area of the protein. The pI value of a protein is estimated by linearly calibrating the distance between two pI markers delimiting the protein. Operating parameters included autosampler temperature 10°C; fluorocarbon (FC) coated cartridge, detection wavelength 280 nm, focusing period 1 minute at 1500 V.

The data in Figure 39 shows the change (difference) in % acidic variants, % change in the main peak, and % change in the main variants under heat stress, compared to the initial ones.

As seen in Figure 39, the change in % acidic variants and major peak of anti-LAG3 antibody was comparable between 20 mM L-aspartic acid or L-glutamic acid, 40 mM L-histidine, and 40 mM L-arginine. The change was smallest for 40 mM sodium chloride. Likewise, the change in % acidic variants and main peak at 70 mM was comparable between L-histidine and the combination of either 20 mM L-aspartic acid or L-glutamic acid with 50 mM glycine. The change was smallest for 70 mM sodium chloride and 70 mM L-arginine. The % change in major variants was minimal for all ten formulations listed in Table 5.

DLS

Hydrodynamic diameter measurements were performed using a Wyatt dynamic light scattering (DLS) instrument on a 96-well glass bottom plate. The sample was diluted to a protein concentration of 5 mg/mL and analyzed in automated mode using 158° scatter detection at 20°C, analysis duration 5 seconds for five measurements.

As can be seen in Figure 40, the percentage change in anti-LAG3 antibody diameter was minimal for 20 mM L-aspartic acid or L-glutamic acid and their combination with 50 mM glycine. The change in diameter was between 6 and 11% for sodium chloride (40-70 mM), L-arginine (40-70 mM) and L-histidine (40-70 mM) and within the assay variability.

Example 3: Anti-LAG3 Antibody Ab6 Anti-LAG3 Composition Stabilizer Screening

To evaluate the stability of anti-LAG3 antibody Ab6 (25 mg/ml in 10 mM L-histidine, 70 mM L-arginine hydrochloride or 70 mM sodium chloride at pH 5.8) in the presence of various stabilizers such as sugars and polyols, eleven compositions listed in Table 6.

Table 6 Optimization of anti-LAG3 antibody composition with stabilizers Compound# Sample nomenclature Description of composition 1 L-Arg70 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 2 NaCl70 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM sodium chloride (NaCl), pH 5.8 3 L-Arg70+Suc5% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8, 5% (w/v) sucrose 4 L-Arg70+Suc9% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8, 9% (w/v) sucrose 5 L-Arg70+Treh5% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8, 5% (w/v) trehalose 6 L-Arg70+Treh9% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8, 9% (w/v) trehalose 7 L-Arg70+Sorb2.5% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8, 2.5% (w/v) sorbitol 8 L-Arg70+PEG400 2% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8, 2.0% (w/v) PEG400 9 L-Arg70+Glycer 2.5% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8, 2.5% (w/v) glycerol 10 NaCl70+Suc5% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM sodium chloride, pH 5.8, 5.0% (w/v) sucrose eleven NaCl70+Suc9% 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM sodium chloride, pH 5.8, 9.0% (w/v) sucrose

Ultra performance exclusion chromatography (UP-SEC)

Sample purity was assessed by UP-SEC, which determined the percentage of monomer, as well as the percentage of high molecular weight compounds (HMW) and late elution peaks (LMW compounds). UP-SEC was performed on a Waters Acquity UPLC H-Class Bio system by diluting samples to 1.0 mg/mL in the mobile phase (100 mM phosphate, 100 mM sodium chloride, pH 7.0). The column temperature was maintained at 25±3°C and the flow rate was maintained at 0.5 ml/min using isocratic elution. Diluted samples were injected (5 μL) into a UPLC equipped with a Waters BEH200 column and UV detector. Proteins in the sample were separated by size and detected by UV absorbance at 214 nm.

As can be seen in Figure 24, it was found that the change in % HMW and % monomer of anti-LAG3 antibody (25 mg/ml in 10 mM L-histidine pH 5.8, 70 mM L-arginine) was less in the presence of stabilizers, such as sucrose, trehalose, PEG 400 and glycerol. The effect was expressed at higher concentrations of sucrose and trehalose (9% w/v) compared with anti-LAG3 antibody (25 mg/ml in 10 mM L-histidine pH 5.8, 70 mM L-arginine hydrochloride) alone. Similarly, the change in % HMW and % monomer of anti-LAG3 antibody (25 mg/ml in 10 mM L-histidine pH 5.8, 70 mM sodium chloride) was found to be less in the presence of sucrose with a greater effect at higher concentrations. sucrose and trehalose (9% w/v), compared with anti-LAG3 antibody (25 mg/ml in 10 mM L-histidine pH 5.8, 70 mM sodium chloride) alone.

Charged particle change (cIEF)

The change in charge heterogeneity and isoelectric point (pI) of anti-LAG3 antibody in the presence of L-arginine, L-histidine or sodium chloride was assessed using a ProteinSimple capillary isoelectric focusing (cIEF) system. Samples were mixed with ampholyte vehicle before injection into the capillary. When an electric field was applied to the capillary, the ampholyte carrier in the capillary created a pH gradient and the protein molecules migrated to a location in the capillary where the local pH value equaled the isoelectric pH (pI) values. Detection of separated proteins was performed by full scanning of the entire capillary using the iCE system (iCE3 from ProteinSimple). The last image obtained by the instrument was used to quantify the data. The percent area of resolved peaks was estimated by dividing the area of individual shapes by the total area of the protein. The pI value of a protein is estimated by linearly calibrating the distance between two pI markers delimiting the protein. Operating parameters included autosampler temperature 10°C; fluorocarbon (FC) coated cartridge, detection wavelength 280 nm, focusing period 1 minute at 1500 V.

Eleven formulations were filled into sterile 2 ml vials (2.0 ml fill), sealed and visually inspected. At the initial time point, eleven formulations were stored at 2 to 8°C (protected from light), and heat stress samples were placed inverted in a container, protected from light, for 10 days at 50°C in an oven. The data in Figure 25 reports the % change (difference) in acidic variants, % change in base peak, and % change in base variants under heat stress compared to baseline.

As shown in Figure 25, anti-LAG3 antibody (25 mg/ml in 10 mM L-histidine, 70 mM L-arginine pH 5.8) shows less chemical lability in the presence of 5% sucrose. The stabilizing effects of trehalose (5% w/v and 10% w/v), sorbitol, PEG 400 and glycerol were comparable. Anti-LAG3 antibody (25 mg/ml in 10 mM L-histidine, 70 mM sodium chloride pH 5.8) showed better chemical stability in the absence of sucrose.

DSK

The heat capacities (cp) in kcal/°C of the eleven anti-LAG3 antibody formulations listed in Table 6 were measured using a differential scanning microcalorimeter (DSC) at 1 mg/ml. T _m1 , T _m2 and T _onset for eleven compositions were determined from a plot of cp (cal/mol/°C) versus temperature (°C).

As seen in Figure 26, based on T _m1 , T _m2 and T _onset values, sucrose and trehalose (each at 5% w/v-9% w/v) had a stabilizing effect on anti-LAG3 (25 mg/mL in 10 mM L-histidine, 70 mM L-arginine hydrochloride pH 5.8), as well as anti-LAG3 antibody (25 mg/ml in 10 mM L-histidine, 70 mM sodium chloride pH 5.8). The stabilizing effects of sorbitol, PEG 400 and glycerol were comparable.

Example 4: Polysorbate Concentration Range Study of Anti-LAG3 Antibody Ab6 Composition

To determine the optimal concentration of polysorbate 80 in the formulation matrix (25 mg/ml anti-LAG3 antibody in 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% w/v sucrose, pH 5.8), eight different formulations were prepared. each containing polysorbate ranging from 0 mg/ml up to 1.0 mg/ml, as shown in Table 7. The formulations were shaken at 300 rpm for up to 7 days. Two formulations consisted of placebo (0.1 mg/ml or 1.0 mg/ml polysorbate 80 in the same formulation matrix without anti-LAG3 antibody i.e., formulation #1 in Table 7).

Table 7 Screening study of surfactants Compound# Polysorbate 80 (PS80) amount in mg/ml Polysorbate 80 (PS80) amount in % (w/v) Description of composition 1 0 0 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, pH 5.8 2 0.05 0.005 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, 0.05 mg/ml polysorbate 80, pH 5.8 3 0.1 0.01 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, 0.1 mg/ml polysorbate 80, pH 5.8 4 0.2 0.02 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, 0.2 mg/ml polysorbate 80, pH 5.8 5 0.5 0.05 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, 0.5 mg/ml polysorbate 80, pH 5.8 6 1.0 0.1 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, 1.0 mg/ml polysorbate 80, pH 5.8 7 0.1 0.01 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, 0.1 mg/ml polysorbate 80, pH 5.8 (Placebo) 8 1.0 0.1 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, 1.0 mg/ml polysorbate 80, pH 5.8 (Placebo)

Turbidity

To evaluate the colloidal stability of the anti-LAG3 antibody in a formulation matrix containing various concentrations of polysorbate 80, the turbidity (OD350) of the eight formulations was assessed using an ultraviolet (UV) absorbance spectrophotometer. The UV absorbance of the samples was measured in a 96-well transparent co-star plate at a wavelength of 350 nm, corrected for the absorbance of the plate.

As can be seen in Figure 27, in the absence of polysorbate 80, the anti-LAG3 antibody in the matrix formulation (25 mg/ml anti-LAG3 in 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% w/v sucrose, pH 5, 8) showed an increase in turbidity with shaking. In the presence of concentrations of 0.1 mg/ml-1.0 mg/ml polysorbate 80 in a matrix formulation (25 mg/ml anti-LAG3 antibody in 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% w/v sucrose , pH 5.8), the anti-LAG3 antibody was found to be stable. There was no effect on colloidal stability of placebo from 0.1 mg/mL to 1.0 mg/mL.

UP-SEC

Sample purity was assessed by UP-SEC, which determined the percentage of monomer, as well as the percentage of high molecular weight compounds (HMW) and late elution peaks (LMW compounds). UP-SEC was performed on Waters Acquity liquid chromatography systems by diluting samples to 1.0 mg/mL in the mobile phase (0.1 M sodium dihydrogen phosphate monohydrate, 0.1 M sodium hydrogen phosphate dihydrate, 0.1 M L-arginine, pH 7 ,0). Diluted samples were injected (5 μl) into a liquid chromatograph equipped with a Protein BEH SEC column and a UV detector. Proteins in the sample were separated by size and detected by UV absorbance at 214 nm.

As can be seen in Figure 28, there was no change in the level of soluble aggregates (% HMW) or fragmentation (% LMW) or change in % monomer in the presence of polysorbate 80 (0.1-1.0 mg/mL concentration). It was found that the anti-LAG3 antibody was colloidally stable in the presence of polysorbate 80 in the formulation matrix.

HP-IEX

High performance ion exchange chromatography (HP-IEX) was used to determine changes in anti-LAG3 antibody composition variants. The analysis was performed using a Dionex MabPac® SCX-10.10 μm 4x250 mM column and a mobile phase gradient from 25 mM MES, 14 mM Tris, pH 6.25 to 25 mM MES, 22 mM Tris, 100 mM LiCl pH 6, 85. UV detection was performed at 280 nm. This method also includes an optional stripping buffer (15 mM EDTA, 40 mM Tris, 10 mM CHES, 500 mM NaCl, pH 8.1) to improve assay reliability and stability. The sample was obtained at a rate of 5 mg/ml with an injection volume of 10 μl.

As can be seen in Figure 29, there was no change in the level of charged species (% acidic variants, % basic peak, % basic variants) in the presence of polysorbate 80 (concentration 0.1-1.0 mg/ml). It was found that the anti-LAG3 antibody was colloidally stable in the presence of polysorbate 80 in the formulation matrix.

Example 5: pH Range Study of Anti-LAG3 Antibody Ab6

Composition A anti-LAG3 antibody Ab6: 25 mg/ml anti-LAG3 antibody; 50 mg/ml sucrose; 0.2 mg/ml polysorbate 80; 10 mM histidine buffer; 70 mM L-Arginine-HCl was screened in the pH range of 5.3-6.4, taking into account the target formulation pH of 5.8. As can be seen in Figure 14, there was no significant change in concentration, turbidity, HMW %, LMW %, or monomer % between pH 5.3 and pH 6.4. Decrease in % of acidic variants (for example, 17.28% at pH 5.5-14.10% at pH 6.0) against the background of an increase in % of the main peak (for example, 64.0% at pH 5.5-66.9% at pH 6.0) were observed from pH 5.3 up to pH 6.4. The change in hydrodynamic diameter along the Z axis was minimal (less than 1 nm) from pH 5.3 to pH 6.4. A small increase in the number of invisible particles per milliliter (size range ≥5 µm, ≥10 µm and ≥25 µm) was noted at pH closer to the isoelectric point of ~6.3 (i.e., pH 6.0-pH 6.4) . Overall, the anti-LAG3 antibody was found to be stable at pH 5.3 to 6.4, supporting the selection of pH 5.8 as the target pH of the formulation.

Example 6: Antioxidant Screening of Anti-LAG3 Antibody Ab6 Composition

To determine the effect of antioxidant on anti-LAG3 antibody formulation (25 mg/ml anti-LAG3 antibody in 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% w/v sucrose, pH 5.8), three different concentrations L-methionine was evaluated in the composition. The four different formulations listed in Table 8 were filled (2.2 ml) into a 2 ml type 1 glass vial and properly sealed. The four formulations were exposed to 0.2 ICH, 0.5 ICH and 1ICH light (ultraviolet and cool white light or visible light). The dark control (foil covered) for each of the four formulations (control) was also exposed to light stress up to 1 ICH.

Table 8 Antioxidant Screening for Anti-LAG3 Antibody Composition L-methionine concentration (mM) Description of composition control 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% (w/v) sucrose, 0.2 mg/ml polysorbate 80, pH 5.8 (control) 5 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5 mM L-methionine, 5% (w/v) sucrose, 0.2 mg/ml polysorbate 80, pH 5.8 7 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 7 mM L-methionine, 5% (w/v) sucrose, 0.2 mg/ml polysorbate 80, pH 5.8 10 mM 25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine hydrochloride, 10 mM L-methionine, 5% (w/v) sucrose, 0.2 mg/ml polysorbate 80, pH 5.8

Turbidity

The turbidity (OD350) of the four formulations was assessed using an ultraviolet (UV) absorption spectrophotometer. The UV absorbance of the samples was measured in a 96-well transparent co-star plate at a wavelength of 350 nm, corrected for the absorbance of the plate.

As seen in Figure 30, L-methionine (5 mM-10 mM) was found to colloidally stabilize anti-LAG3 antibody (25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine, 5% w/v sucrose, pH 5.8) compared to control as listed in Table 8; with 10 mM L-methionine being the optimal amount. There was no effect on colloidal instability for the dark control sample when exposed to 1 ICH light.

UP-SEC

Sample purity was assessed by UP-SEC, which determined the percentage of monomer, as well as the percentage of high molecular weight compounds (HMW) and late eluting peaks (LMW compounds). UP-SEC was performed on an acquity H class UPLC system by diluting samples to 1.0 mg/mL in the mobile phase (100 mM phosphate and 100 mM sodium chloride, pH 7.0). Diluted samples were injected (5 μL) into a liquid chromatograph equipped with a Protein BEH SEC column and UV detector at a flow rate of 0.5 mL/min. Proteins in the sample were separated by size and detected by UV absorbance at 214 nm and 280 nm.

As seen in Figure 31, L-methionine (5 mM-10 mM) was found to reduce the formation of soluble aggregates (%HMW) in anti-LAG3 antibody (25 mg/ml anti-LAG3 antibody, 10 mM L-histidine, 70 mM L-arginine, 5% w/v sucrose, pH 5.8) compared to control as listed in Table 8; with 10 mM L-methionine being the optimal amount. No formation of soluble aggregates was observed for the dark control sample after exposure to 1 ICH light.

HP-IEX

High performance ion exchange chromatography (HP-IEX) was used to determine variant variation in anti-LAG3 antibody formulations. The analysis was performed using a Dionex MabPac® SCX-10.10 μm 4x250 mm column and a mobile phase gradient from 25 mM MES, 14 mM Tris, pH 6.25 to 25 mM MES, 22 mM Tris, 100 mM LiCl pH 6, 85. UV detection was performed at 280 nm. This method also includes an optional stripping buffer (15 mM EDTA, 40 mM Tris, 10 mM CHES, 500 mM NaCl, pH 8.1) to improve assay reliability and stability. The sample was obtained at 5 mg/ml with an injection volume of 10 μl and a flow rate of 0.5-1.0 ml/min.

As seen in Figure 32, it was found that 5 mM-10 mM L-methionine was effective in reducing the increase in charged species (% acidic variants and % basic variants) up to exposure to 1 ICH light for anti-LAG3 antibody (25 mg/ ml anti-LAG3 in 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% w/v sucrose, 0.2 mg/ml polysorbate 80) compared to control. For the dark control sample 1 ICH, no effect on charged compounds was observed.

Mapping of reduced peptides

Changes in the level of oxidative post-translational modifications of the anti-LAG3 antibody were assessed using reduced peptide mapping. Reduced peptide mapping was performed on a Waters Acquity H Bio system with mobile phase A (0.1% trifluoroacetic acid in water LC/MS grade), with mobile phase B (0.1% trifluoroacetic acid in acetonitrile LC/MS grade). The injection volume was 50 μL, equipped with a HALO Peptide ES-C18 column with a flow rate of 0.2 mL/min and a detection absorbance of 214 nm. Mass spectrometry consisted of 3.0 capillary, 30 sample cone, 120 °C source temperature, 30 cone gas, desolvation gas, m/z range 100–200, MS collected from 2 to 110 min. Before running through the column, samples were reduced and alkylated with appropriate reagents. Blank (non-sample) digestion was performed to identify non-sample peaks eluting in the region of interest.

As seen in Figure 33, it was found that 5 mM-10 mM L-methionine was effective in reducing the oxidation of post-translational modifications of anti-LAG3 antibody (25 mg/ml anti-LAG3 antibody in 10 mM L-histidine, 70 mM L-arginine hydrochloride, 5% w/v sucrose, 0.2 mg/ml polysorbate 80) under 1 ICH light exposure, compared to control.

Example 7: High Concentration Anti-LAG3 Antibody Ab6 Studies

To evaluate the suitability of high concentration (200 mg/ml) anti-LAG3 antibody in three different buffers at pH 5.8 (histidine, acetate and citrate; each containing 70 mM L-arginine hydrochloride) and a formulation containing 70 mM L-histidine L-arginine hydrochloride, pH 5.8 in the presence of various stabilizers, nine formulations were prepared as listed in Table 9. Each of the nine formulations was filled into 96-well plates and sealed in a suitable manner. The compositions were stressed at 50°C for 10 days in a dry-heat oven. The analysis was carried out for the original samples and samples subjected to stress.

Table 9 Suitability of high concentration anti-LAG3 antibody formulation Compound# Anti-LAG3 Antibody Concentration (mg/ml) Description of composition Stabilizer 1 200 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 No stabilizer 2 200 10 mM acetate, 70 mM L-arginine hydrochloride, pH 5.8 No stabilizer 3 200 10 mM citrate, 70 mM L-arginine hydrochloride, pH 5.8 No stabilizer 4 200 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 5% (w/v) sucrose 5 200 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 5% (w/v) glycerin 6 200 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 70 mM L-glutamine 7 200 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 70 mM L-glycine 8 200 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 70 mM proline 9 200 10 mM L-histidine, 70 mM L-arginine hydrochloride, pH 5.8 70 mM L-methionine

Turbidity

Turbidity (OD350) of nine formulations was assessed using an ultraviolet (UV) absorption spectrophotometer. The UV absorbance of the samples was measured in a 96-well transparent co-star plate at a wavelength of 350 nm, corrected for the absorbance of the plate.

As can be seen in Figure 34, the colloidal stability (OD350) of anti-LAG3 antibody (200 mg/ml anti-LAG3 antibody in 10 mM L-histidine 70 mM L-arginine, pH 5.8) was lower in the presence of 70 mM L-methionine as a stabilizer compared to the control (200 mg/ml anti-LAG3 antibody in 10 mM L-histidine 70 mM L-arginine, pH 5.8). The stabilizing effect (colloidal) of 70 mM L-glutamine and 70 mM proline in 200 mg/ml anti-LAG3 antibody was comparable. Likewise, the stabilizing effect (colloidal) of 5% w/v glycerol and 70 mM L-glycine in 200 mg/mL anti-LAG3 antibody was comparable. The colloidal stability of 200 mg/ml anti-LAG3 antibody was comparatively high in the presence of 5% w/v sucrose.

UP-SEC

Sample purity was assessed by UP-SEC, which determined the percentage of monomer, as well as the percentage of high molecular weight compounds (HMW) and late eluting peaks (LMW compounds). UP-SEC was performed on an Acquity H grade (DS) by diluting samples to 1.0 mg/mL in the mobile phase (100 mM phosphate, 100 mM sodium chloride, pH 7.0). The column temperature was maintained at 25±3°C and the flow rate was maintained at 0.5 ml/min using isocratic elution. Diluted samples were injected (5 μL) into a UPLC equipped with a Waters BEH200 column and UV detector. Proteins in the sample were separated by size and detected by UV absorbance at 214 nm.

As seen in Figure 35, 10 mM L-histidine buffer in the presence of 70 mM L-arginine hydrochloride is effective in reducing the amounts of soluble aggregates compared to 10 mM L-acetate or 10 mM citrate buffer for a high concentration of anti-LAG3 antibody formulation (200 mg/ml). 5% (w/v) sucrose is effective as a stabilizer in further reducing the amounts of soluble aggregates, followed by 5% (w/v) glycerol. The stabilizing effect of amino acids, i.e., 70 mM L-glutamine, 70 mM L-glycine, 70 mM proline and 70 mM L-methionine, was comparable.

Charged particle change (cIEF)

The change in charge heterogeneity and isoelectric point (pI) of anti-LAG3 antibody in the presence of L-arginine, L-histidine or sodium chloride was assessed using a ProteinSimple capillary isoelectric focusing (cIEF) system. Samples were mixed with ampholyte vehicle before injection into the capillary. When an electric field was applied to the capillary, the ampholyte carrier in the capillary created a pH gradient and the protein molecules migrated to a location in the capillary where the local pH value equaled the isoelectric pH (pI) values. Detection of separated proteins was performed by full scanning of the entire capillary using the iCE system (iCE3 from ProteinSimple). The last image obtained by the instrument was used to quantify the data. The percent area of resolved peaks was estimated by dividing the area of individual shapes by the total area of the protein. The pI value of a protein is estimated by linearly calibrating the distance between two pI markers delimiting the protein. Operating parameters included autosampler temperature 10°C; fluorocarbon (FC) coated cartridge, detection wavelength 280 nm, focusing period 1 minute at 1500 V.

The chemical stability of 200 mg/ml anti-LAG3 antibody was comparable in 10 mM L-histidine as well as 10 mM citrate buffer compared to 10 mM acetate buffer in the presence of 70 mM L-arginine hydrochloride at pH 5.8. 5% (w/v) glycerol was effective in reducing charged species variation (% acidic and basic variants), followed by 5% (w/v) sucrose (% basic variants). The stabilizing effect of amino acids, i.e., 70 mM L-glutamine, 70 mM L-glycine, 70 mM proline and 70 mM L-methionine, was comparable.

Example 8: stability study of the combined composition of anti-PD-1 and anti-LAG3 antibodies

Co-formulations of anti-PD-1 antibody (MK-3475, pembrolizumab, heavy chain SEQ ID NO:10 and light chain SEQ ID NO:5) and anti-LAG3 antibody Ab6 (heavy chain SEQ ID NO:57, and light chain SEQ ID NO:57 ID NO:35) was obtained as in Table 10.

Table 10

form no. Anti-LAG3 mg/ml) Anti-PD-1 (mg/ml) Histidine (mM) Arginine (mM) Sucrose (mg/ml) Methionine (mM) PS-80 (mg/ml) pH F1 20 0 10 70 50 0 0.2 5.8 F2 0 20 10 70 50 0 0.2 5.8 F3 20 20 10 70 50 0 0.2 5.8 F4 0 20 10 0 70 0 0.2 5.8 F5 20 20 10 70 50 10 0.2 5.8 F6 20 20 10 40 50 10 0.2 5.8

Thermal stability study

Thermal stability studies were performed using 1.0 ml liquid formulations F1-F6 in 2 ml tubes with a 13 mm penicillin stopper for up to 12 weeks under storage conditions of 5°C (room humidity), 25°C (60% humidity), and 40 °C (75% relative humidity). The stability of the samples was assessed by turbidity and mixed mode chromatography (MMC).

Mixed mode chromatography

Mixed-mode chromatography allowed the separation of individual antibodies (anti-LAG3 antibody and anti-PD-1 antibody) in co-formulations, and also provided monitoring of anti-LAG3 antibody aggregates and anti-PD-1 antibody aggregates and oxidation of co-formulations. In MMC, the percentage of monomer for each mAb was determined from the main peak area of each mAb. For anti-LAG3 antibodies, the percentage of high molecular weight (aggregates) and low molecular weight compounds (fragments) was calculated. For anti-PD-1 antibody, the percentages of high molecular weight (aggregates) and low molecular weight compounds (fragments), as well as oxidized compounds (Ox1 and Ox2), were calculated based on the area of the individual peaks corresponding to each compound. Mixed mode chromatography was performed by diluting samples to 1.0 mg/mL in mobile phase (PBS, pH 7.4). The column temperature was maintained at 25°C and the flow rate was maintained at 0.5 mL/min using isocratic elution. The diluted samples were injected (15 μL) into an HPLC equipped with a Sepax Zenix SEC-300 custom column. The various components in the sample were separated by both size and hydrophobicity and detected by UV absorbance at 280 nm.

Turbidity measurement

Turbidity analysis was performed on stability samples using UV-visible spectroscopy on a SpectrMax M5 Plate reader. Absorbance was measured at 350 nm and 500 nm. Turbidity was calculated by subtracting the absorbance at 500 nm from the absorbance at 350 nm.

Conclusion

The combined formulations (F3, F5 and F6) showed similar or better stability than the single compound formulations (Figures 22 and 23). Formulation F2 showed comparatively lower turbidity and monomer loss than formulation F4 over time when stored at 40°C, indicating that the anti-PD-1 antibody shows better stability when formulated with arginine (Figures 22 and 23). F5 and F6 co-formulations showed minimal change in monomer amount over time after 12 weeks of storage at 40°C compared to single compound formulations. L-Methionine helped to minimize monomer loss in the co-formulations (F5, F6) (Figure 23).

Drug substances (DS) anti-LAG3 antibody Ab6 and pembrolizumab (MK-3475) were mixed in the following ratios: 1:1 (12D), 3:1 (12E), 4:1 (12F) and 5:1 (12G). Formulations 12A and 12C are Ab6-only (SE) and MK-3475y (SE) controls, respectively, in their original composition. Formulation 12B is the control MK-3475 SE in excipient composition 12A. 12F-12H are co-formulations with a 4:1 ratio of Ab6 to MK-3475, with (12H) and without diluent (12F). A diluent was added to the composition of the composition to reconstitute the Ab6 composition (with a concentration of 70 mm arginine).

Table 11 Compositions

Composition ID # Ab6 ratio: MK-3475 Final composition 12A 1:0 Ab6 25 mg/ml, 70 mM L-arginine HCl, 5% sucrose, 10 mM methionine, 0.02% PS-80, 10 mM histidine buffer 12B 0:1 MK-3475 25 mg/ml, 70 mM L-arginine HCl, 5% sucrose, 10 mM methionine, 0.02% PS-80, 10 mM histidine buffer 12C 0:1 MK-3475 25 mg/ml, 7% sucrose, 0.02% PS-80, 10 mM histidine buffer 12D 1:1 Ab6 10 mg/ml with MK-3475 10 mg/ml, 70 mM L-arginine HCl, 5% sucrose, 10 mM methionine, 0.02% PS-80, 10 mM histidine buffer 12E 3:1 Ab6 18.75 mg/ml with MK-3475 6.25 mg/ml, 52.5 mM L-arginine HCl, 5.5% sucrose, 7.5 mM methionine, 0.02% PS-80, 10 mM histidine buffer 12F 4:1 Ab6 20 mg/ml with MK-3475 5 mg/ml, 56 mM L-arginine HCl, 5.4% sucrose, 8.0 mM methionine, 0.02% PS-80, 10 mM histidine buffer 12G 5:1 Ab6 20.83 mg/ml with MK-3475 4.17 mg/ml, 58.3 mM L-arginine HCl, 5.3% sucrose, 8.3 mM methionine, 0.02% PS-80, 10 mM histidine buffer 12H 4:1 Ab6 18.02 mg/ml with MK-3475 4.505 mg/ml, 70 mM L-arginine HCl, 5% sucrose, 10 mM methionine, 0.02% PS-80, 10 mM histidine buffer

The formulations were filtered through a 0.22 μm cellulose acetate membrane filter. The filtrate for each formulation was filled into 2R tubes with 2.2 ml fill/tube. The stability of the formulations was assessed after 10 days at 5°C, 25°C and 40°C. The stability of the formulations was analyzed by testing quality attributes including turbidity, total protein concentration, aggregation, oxidation for MK-3475, purity and charge profile. Formulations 12A, 12B, 12F and 12H were mainly evaluated for stability.

Turbidity and total protein concentration

Turbidity was measured using a Spectramax UV absorption spectrophotometer (MRK0406661). 200 μL samples were placed in a 96-well clear Co-star plate, absorbance was measured at 350 nm and 500 nm, and the path was adjusted to plate absorbance of 0.025 and 0.020, respectively.

Concentration was measured using SoloVPE Variable Pathlength Extension (MRK0416083) using Quick Slope. A weighted average ratio-dependent extinction coefficient (1.42 for MK-3475 and 1.45 for Ab6) and a wavelength of 280 nm were used to measure the total protein concentration.

Table 12: Turbidity and total protein concentration

Sample name Turbidity, OD 350 Turbidity, OD 350-500 10 days - 5°C 12A 0.073 0.052 12B 0.081 0.059 12C 0.062 0.044 12D 0.067 0.049 12E 0.079 0.056 12F 0.074 0.055 12G 0.074 0.054 12H 0.067 0.049 10 days - 40°C 12A 0.076 0.055 12B 0.083 0.062 12C 0.066 0.048 12D 0.073 0.054 12E 0.083 0.062 12F 0.080 0.060 12G 0.078 0.059 12H 0.071 0.052 10 days - 50°C 12A 0.083 0.063 12B 0.118 0.091 12C 0.076 0.059 12D 0.092 0.070 12E 0.096 0.073 12F 0.093 0.072 12G 0.09 0.069 12H 0.081 0.061

All samples were clear, colorless and free of visible particles. No effect of the Ab6:MK-3475 ratio on turbidity was observed. All prototypes with the Ab6 dose range of 200-1000 mg in the co-formulation showed the same turbidity under all stability conditions. Matrix composition had no effect on turbidity.

UP-SEC Aggregation Analysis

High-performance size exclusion chromatography (UP-SEC) was used to evaluate net protein aggregation and determine high molecular weight (HMW), low molecular weight (LMW) and total monomer content. Both samples and control material were diluted to 5 mg/ml and 200 μl of the diluted sample was used. The mobile phase consisted of 50 mM Na phosphate, 450 mM arginine HCl, pH 7.0. An isocratic flow rate of 0.5 mL/min, a detection wavelength of 280 nm, an injection volume of 6 μL, and a column temperature of 30 °C were used. For each test sample, the peak area percentage of monomer (Mon), HMW compounds, and LMW compounds (formerly known as late eluting compounds) was calculated.

% monomer=PMon x 100 ÷ Σ P (all peaks)

% HMW compounds=PHMW x 100 ÷ Σ P (all peaks)

% LMW compounds=PLMW x 100 ÷ Σ P (all peaks)

PMon, PHMW, PLMW are the individual peak areas, and ΣP is the sum of all peak areas (excluding artifact peaks, peaks appearing in empty volume, and peaks associated with buffer).

Formulation #12B with only MK-3475 showed the highest amount of High Molecular Weight (HMW) compounds (see Figures 41 and 42). The presence of 70 mM arginine resulted in increased amounts of HMW compounds MK-3475. As the ratio of Ab6:MK-3475 in the co-formulation increases from (1:1) to (5:1), the number of HMW compounds decreases. Increased concentration of Ab6 appears to stabilize MK-3475 against aggregation (HMW formation). MK-3475 in the 12C formulation has a higher tendency to aggregate compared to the 12A Ab6 formulation. No measurable differences were observed for the 800:200 co-formulation prototypes with (12H) or without diluent (12F).

Particle size analysis by microflow imaging (MFI)

Invisible particles in the range >1 µm, >2 µm, >5 µm, >10 µm, >25 µm, >50 µm were analyzed using MFI by pooling enough samples for analysis of a minimum of 3 replicates using a Brightwell Micro microflow imaging system.

Table 13: Invisible particles

# Particles/ml, ≥10 µm 12A 12B 12C 12D 12E 12F 12G 12H 5ºC-10day 10 10 7 8 14 5 1 4 40ºC-10day 5 4 12 8 23 3 10 5 50ºC-10day 581 6 9 6 8 41 33 10 # Particles/ml, ≥25 µm 12A 12B 12C 12D 12E 12F 12G 12H 5ºC-10day 1 4 0 1 1 2 0 1 40ºC-10day 1 1 2 3 0 2 2 1 50ºC-10day 206 2 1 0 2 2 3 0

Particle counts were higher for high concentration formulations (single component and co-formulation) compared to low concentration formulations (12D and 12E). Significant increases in abundance for all particle sizes were observed for the individual component Ab6 (12A) at 50ºC for 10 days. Likewise, co-formulations with higher protein concentrations (12F, 12G and 12H) showed higher counts of all particle sizes under all storage conditions compared to formulations 12D and 12E. However, the numbers of particles ≥10µm and ≥25ºM were significantly lower than those observed for the individual component Ab6 (12A) at 50ºC.

Hydrophobic interaction chromatography (HIC) to quantify the oxidation of MK-3475 in a co-formulation

HIC was used to assess the identity and quantities of all MK-3475 oxidation products in the co-formulation. The column used was Tosoh Phenyl-5PW 10 μm 7.5×75 mm and a column temperature of 30°C was used. Samples were diluted to obtain a total protein concentration of 5 mg/mL with a total sample volume of at least 200 μL. A gradient method was used with a combination of mobile phase A: 2% ACN in 5 mM Na ₃ (PO ₄ ), pH 7.0, and mobile phase B: 2% ACN in 400 mM ammonium sulfate, 5 mM Na ₃ (PO ₄ ), The pH is 6.9 and the injection volume is 10 µl. Detection wavelengths of 280 and 214 nm were used, and results were reported at 280 nm only. For results reporting, the relative % area for Pre-Peak 3 is equal to % of Pre-Peak 3. Pre-Peaks 1 and 2 are summed to equal % of Pre-Peak 1+2.

Regarding the results, the % relative area for pre-peak 3 is equal to % of pre-peak 3. Pre-peaks 1 and 2 are summed to equal % of pre-peak 1+2.

Table 14: Oxidation of MK-3475 in co-formulation

Compound# Total area % prep 1+2 (1 Fab Met Ox) % prepeak 3 % basic % post-peak 1 % post-peak 2 12A 0 0 0 12B-4°C 7889980 5.40 0.62 86.0 6.17 1.83 12B-40°C 7946807 8.25 0.71 83.0 6.03 1.96 12B-50°C 7617017 7.70 0.52 83.8 5.95 2.04 12F-4°C 1683729 5.48 0.28 82.2 7.81 4.2 12F-40°C 1688135 8.06 0.54 78.6 8.73 4.06 12F-50°C 1574991 7.72 0.70 79.6 8.02 3.94 12H-4°C 1692349 5.78 0.33 81.8 8.02 4.04 12H-40°C 1675188 9.05 0.59 78.2 8.17 4.02 12H-50°C 1543496 9.26 0.98 78.9 7.93 2.97

Ab6/MK-3475 co-formulation (12H) showed the highest degree of Fab oxidation (Met 105) in MK-3475. (12F) composition showed the lowest degree of oxidation.

Ion exchange chromatography (IEX) to assess changes in the charge distribution of individual mAbs in a co-formulation

Ion exchange chromatography (IEX) was used to assess the identity and abundance of all charged and basic variants in each of the coformulated mAbs. Each of the samples was diluted to obtain a total amount of total protein of 50 μg with a maximum injection volume of 125 μl. A Dionnex MabPac SCX-10 10 µm, 4x250 mm column was used at a column temperature of 35°C. The mobile phase used was the combination of (A): 5 mM MES, 14 mM Tris, pH 6.25 (B): 20 mM Na ₃ (PO ₄ ), 95 mM NaCl, pH 8.0, 4% acetonitrile (ACN) and (C): 15 mM EDTA, 40 mM Tris, 10 mM CHES, 500 mM NaCl, pH 8.1. A gradient method with a total run time of 70 minutes was used with a detection wavelength of 280 nm. Using this method, acidic, basic and base variants of each of the mAbs in the co-formulation were identified and quantified. In general, the charge profile changes with increasing temperature, with the largest change observed at 50°C (see Table 15). No noticeable difference was observed in the base peak or acidic and basic variants after 10 days at 5°C, 40°C and 50°C for the 800:200 co-formulation prototypes with (12H) and without diluent (12F).

Table 15: IEX Charge Profile

F# Acid group Ab6
% Main peak Ab6
% Ab6 main group
% Acid group
MK-3475
% Main peak
MK-3475
% Main group
MK-3475
% MK-3475 total % Ab6 total % 12A-4°C 13.2 77.1 9.7 12A-40°C 18.6 71.9 9.5 12A-50°C 33.1 56.2 10.7 12B-4°C 18.7 61.6 19.71 12B-40°C 24.4 57.2 18.43 12B-50°C 37.2 47.0 15.75 12F-4°C 10.7 60.5 8.1 4.1 11.8 4.83 20.8 79.2 12F-40°C 15.3 55.3 8.6 5.2 11.0 4.57 20.8 79.2 12F-50°C 26.5 43.0 9.7 7.9 8.9 4.06 20.9 79.1 12G-4°C 10.7 60.5 8.1 4.0 11.8 4.79 20.7 79.3 12G-40°C 15.2 55.3 8.5 5.3 11.0 4.65 20.9 79.1 12G-50°C 26.0 43.6 9.7 7.7 8.9 4.10 20.7 79.3 12H-4°C 10.7 60.5 8.1 4.0 11.8 4.8 12H-40°C 15.2 55.3 8.5 5.3 11.0 4.7 12H-50°C 26.0 43.6 9.7 7.7 8.9 4.1

Purity analysis by capillary electrophoresis-SDS (CE-SDS)

Analysis of mAb purity was performed using non-reducing CE-SDS. Samples were prepared according to the method's in-house protocols and incubated at 70°C for 10 minutes on a heat block. Samples were then equilibrated to ambient temperature, centrifuged at 10,000 g, and 95 μL of sample was used for analysis.

Table 16: CE-SDS purity

NR-CE-SDS Stability Analysis 40°C Composition ID IgG purity (%) LMW (%) HMW (%) 12A 98.5 1.5 - 12B 98.2 1.7 0.1 12C 98.3 1.7 - 12D 98.1 1.9 - 12E 98.1 1.9 0.05 12F 98.4 1.6 - 12G 98.1 1.9 0.04 12H 98.1 1.9 - NR-CE-SDS Stability Analysis 50°C Composition ID IgG Purity (%) LMW (%) HMW (%) 12A 97.1 2.9 0.1 12B 97.1 2.7 0.2 12C 97.0 2.8 0.1 12D 97.1 2.7 0.1 12E 97.1 2.8 0.2 12F 96.8 3.1 0.2 12G 97.0 2.9 0.1 12H 97.1 2.8 0.1

From the above table, it can be seen that the Ab6:MK-3475 ratio does not affect the purity.

Example 9: Long-term stability studies of Ab6A drug product co-formulation (4:1 ratio of anti-LAG3 antibody to anti-PD-1 antibody)

Ab6A injection is a sterile, preservative-free solution that requires dilution for intravenous infusion. Ab6A is a fixed dose combination of the anti-LAG3 antibody Ab6 and the anti-PD-1 antibody MK-3475 (pembrolizumab, heavy chain SEQ ID NO:10 and light chain SEQ ID NO:5), each single-dose vial contains 40 mg of Ab6 and 10 mg MK-3475 in 2.0 ml filling. The composition of the medicinal product is 20.0 mg/ml Ab6, 5.0 mg/ml MK-3475 in 0.56 mg/ml L-histidine, 1.35 mg/ml L-histidine monohydrochloride monohydrate, 11.80 mg/ ml L-arginine hydrochloride, 1.19 mg/ml L-methionine, 54.0 mg/ml sucrose, 0.20 mg/ml polysorbate 80 at pH 5.8 (composition 12F in example 8). Recommended storage conditions are 5°C±3°C (2-8°C) protected from light.

Three-month stability data for Ab6A in glass vials remained virtually unchanged when stored at the recommended storage conditions of 5°C (5°C ±3°C). Minor changes were observed in some characteristics for up to three months when stored under forced conditions of 25°C (25°C, 60% relative humidity). Changes under stress conditions of 40°C (40°C, 75% RH) were more significant than changes at 25°C. The following assays remain virtually unchanged over three months under all temperature conditions (including stress conditions of 40°C, 75% RH): appearance and visible particles, clarity and degree of opalescence, color, potency, protein concentration, pH and particulate matter Available data stability is confirmed by the use of this product when stored at 5 °C ±3°C with a shelf life of 12 months.

The following summarizes the three-month stability data for Ab6A drug product under storage conditions of 5 °C ± 3 °C (reversed), under forced conditions of 25 °C (25 °C ± 2 °C, 60% relative humidity, reversed), and under stress conditions 40 °C (40 °C±2 °C, 75% relative humidity, reversed) in accordance with ICH directives.

Appearance and visible particles

Appearance and visible particles were determined using a light box equipped with a white light source for samples in a transparent vial. Ab6A drug product appearance and visible particle test results under all conditions evaluated to date: “The liquid is substantially free of visible particles.” Observations do not change with storage conditions or time.

Transparency and degree of opalescence

Transparency and degree of opalescence were measured using a HACH™ 2100AN turbidimeter. HACH™ StablCal standard solutions (<0.1, 1.0, 3.0, 6.0, 10.0, 18.0 and 30.0 NTU) and purified water (as a blank) were used to test suitability system (SST) before sample analysis. There was no change in the clarity and degree of opalescence of the Ab6A drug product from the initial time point to the three-month time point in all conditions.

Options with HP-IEX charge

High performance ion exchange chromatography (HP-IEX) was performed on a Waters e2695 separation module with 2489 UV-vis detection using a Dionex MabPac® SCX-10.10 µm 4x250 mm, part # 074625. Mobile phase A contains 20 mM MES , 14 mM Tris (pH 6.25). Mobile phase B contains 20 mM NaPO ₄ , 96 mM NaCl, 4% acetonitrile (pH 8.0), and mobile phase C contains 15 mM EDTA, 40 mM Tris, 10 mM CHES, 500 mM NaCl, pH 8.1. The column and sample temperatures were set to 35°C and 4°C, respectively. A total run of 70 minutes with a gradient setting at a flow rate of 0.5 ml/min with UV detection at 280 nm. For each chromatogram of the molecule (Ab6 and MK-3475), the relative percentage area for the 5 peaks (acidic 2, acidic 1, basic, basic 1 and basic) was calculated and combined to have only three categories (acidic variants, basic and basic variants ) for each antibody. In addition, the % of each group for each antibody is normalized so that the % of all peaks equals 100% for each antibody as calculated. The acidic and basic peaks of MK-3475 and Ab6 were separated by an ion exchange column and eluted either earlier (acidic variants) or later (basic variants) relative to the basic peak. Figure 47 shows a typical chromatogram.

The HP-IEX method is a combination method that separates charged variants for both Ab6 and MK-3475. For the acidic variants of both Ab6 (Figure 1) and MK-3475 (Figure 4), there was no change under 5°C conditions for the 3 month stability data. There was a slight increase for the acidic variants of both Ab6 and MK-3475 at 25°C conditions and a sharp increase for both at 40°C conditions.

Core compounds for both Ab6 and MK-3475 at 5°C showed no change over 3 months (Figure 2 and Figure 5). There was a slight decrease in basic compounds at 25°C for both, which correlated with an increase in acidic variants, and a sharp decrease in basic compounds at 40°C for both Ab6 and MK-3475.

The major variants for Ab6 showed no change in both 5°C and 25°C conditions up to 3 months (Figure 3). At 40°C for Ab6 core variants, there was a slight increase in core variants up to 3 months. The main variants for MK-3475 also showed no change under 5°C conditions up to 3 months (Figure 6). The main variants for MK-3475 at 25°C showed a small decrease and 40°C showed a larger decrease over 3 months of stability (Table 17, Table 18 and Table 19).

Table 17. Summary of charged variants for Ab6A at 5°C reversed

Point in time (months) analysis initial 1 2 3 charged options %
by HP-IEX Ab6 sour variants 12.5 12.7 12.9 12.3 Ab6 basic in total 77.0 77.0 76.8 76.9 Ab6 main options 10.5 10.4 10.3 10.8 MK-3475 sour variants 17.8 17.4 17.4 18.4 MK-3475 basic in total 58.4 59.9 60.1 58.2 MK-3475 main variants 23.8 22.6 22.5 23.4

Table 18. Summary of charged options for Ab6A at 25°C/60%RH reversed

Point in time (months) analysis initial 1 2 3 charged options % according to HP-IEX Ab6 sour variants 12.5 14.0 16.6 17.7 Ab6 basic in total 77.0 75.4 72.9 71.2 Ab6 Basic options 10.5 10.5 10.5 11.0 MK-3475 Sour variants 17.8 19.0 21.3 23.7 MK-3475 basic in total 58.4 59.0 57.3 54.5 MK-3475 Main variants 23.8 22.1 21.3 21.8

Table 19. Summary of charged options for Ab6A at 40°C/75%RH reversed

Point in time (months) analysis initial 1 2 3 charged options % according to HP-IEX Ab6 sour variants 12.5 27.2 41.8 49.0 Ab6 basic in total 77.0 61.2 46.1 38.4 Ab6 Basic options 10.5 11.6 12.1 12.6 MK-3475 sour variants 17.8 31.1 44.5 51.7 MK-3475 basic in total 58.4 49.0 38.1 31.0 MK-3475 main variants 23.8 19.9 17.3 17.3

Purity according to UP-SEC

Size exclusion chromatography is a size separation technique. Ab6 and MK-3475 monomers are similar in size, so the monomers elute together. Sufficient separation of HMW particles from co-eluting monomers is achieved and therefore this method is suitable for determining the purity of Ab6A drug product (DP). Ab6A DP contains low concentrations of high molecular weight (HMW) compounds permitted by UP-SEC. Low molecular weight (LMW) compounds are usually detected, but at low concentrations. Reverse phase high performance liquid chromatography (RP-HPLC) was performed using Waters H-Class Acquity UPLC with HALO C4 UHPLC COLUMN, 2.1 x 75 mm. The mobile phase was water with 0.1% (v/v) trifluoroacetic acid (mobile phase A) and acetonitrile with 0.1% (v/v) trifluoroacetic acid (mobile phase B). Reference standards MK-3475 and Ab6 were prepared at 4 mg/mL with water and used to generate a standard curve, and all samples were diluted to 4 mg/mL and injected at 5 μL for measurement. The column and autosampler were maintained at 75°C and 4°C, respectively. UV detection at 280 nm and Water Empower software were used for data analysis. The concentrations of MK-3475 and Ab6 in general can be based on the standard curve obtained from the measurement.

UP-SEC for Ab6A showed no change at 5°C for high molecular weight compounds and only a slight increase at 25°C with a corresponding small decrease in monomer over 3 months of stability (Figure 7 and Figure 8). Under 40°C conditions there was an increase in high molecular weight compounds with a corresponding decrease in % monomer up to 3 months.

Non-reducing (NR) CE-SDS and reducing (R) CE-SDS

Non-reducing (NR) CE-SDS and reducing (R) CE-SDS analyzes use a Beckman PA800 Plus CE instrument and uncoated fused silicon capillaries (total length 30.2 cm and internal diameter 50 µm) with a 100 × 200 µm orifice. Samples were prepared using the IgG Purity/Heterogeneity Assay Kit and processed prior to on-instrument analysis according to forward-looking protocols, where under reducing conditions mAb samples were denatured in the presence of 1.0% SDS and reduced using 5% β-mercaptoethanol, and under non-reducing conditions conditions, mAb samples were denatured in the presence of 1.0% SDS and treated with N-ethylmaleimide (NEM), followed by heating for 10 min at 70°C. Separation was performed at 15 kV for 40 min and UV detection was performed at 220 nm and Water Empower software was used for data analysis. For NR-CE-SDS, intact IgG (purity) and total low molecular weight impurities were reported as percentages. For R-CE-SDS, the total purity (heavy chain+light chain) and total impurities were reported as a percentage.

Non-reducing CE-SDS was performed on the Ab6A drug product for up to 3 months for stability. At 5°C no changes were observed (Figure 9 and Figure 10). At 25°C, a slight increase in the total amount of low molecular weight compounds was observed with a corresponding decrease in intact IgG. A sharper increase in the total amount of low molecular weight compounds was observed at 40°C with a sharp decrease in intact IgG up to 3 months for stability.

Reducing CE-SDS was performed on the Ab6A drug product for up to 3 months for stability. At 5°C no changes were observed (Figure 43 and Figure 44). At 25°C, a slight increase in the total amount of impurities was observed with a corresponding decrease in the heavy chain+light chain. There was a sharper increase in total impurities at 40°C with a sharp decrease in heavy chain+light chain up to 3 months stability.

Particulate matter

Characterization of invisible particles was performed using a Brightwell Micro-Flow imaging system. Each sample was pooled from multiple containers (a minimum of three) into 50 mL polypropylene tubes by carefully pouring the tubes and leaving the tubes undisturbed under ambient conditions for 30 minutes before testing. Invisible particles in the range >1 µm, >2 µm, >5 µm, >10 µm, >25 µm, >50 µm were analyzed using the standard filter in the MVSS instrument software (version 2-R5.0.0.43.5864).

Particulate matter HIAC data were calculated for the Ab6A drug product. At the 5°C, 25°C and 40°C conditions, results were well below the acceptance criteria: ≤6000 particles per container for ≥10 µm and ≤600 particles per container for ≥25 µm from the initial period to 3 months). For particle sizes ≥2 µm and ≥5 µm, which are the reported results, no significant trends were observed in the data for all conditions up to 3 months.

Oxidation of MK-3475 by HP-HIC

HPLC hydrophobic interaction chromatography (HP-HIC) was performed on an Agilent 1260 system using a Tosoh Phenyl-5PW 10 µm, 7.5 x 75 mm column (PN 05753). Mobile phases were 2.0% acetonitrile in 5 mM sodium phosphate at pH 7.0 (mobile phase A) and 2.0% acetonitrile in 400 mM ammonium sulfate and 5 mM sodium phosphate at pH 6.9 (mobile phase B). The flow rate was 0.5 ml/min. The column and autosampler were maintained at 30°C and 4°C, respectively. Samples were diluted to 5 mg/mL with Milli-Q water, and 10 μL were loaded onto the column (total injection 50 μg). For analysis, the detection wavelength was set to 280 nm. Report the relative area in % for pre-peak 3. Pre-peaks 1 and 2 are summed to equal the % of pre-peaks 1+2. In addition, the relative % area for post-peaks 1 and 2 are summed to be equal to the % hydrophobic variants.

HP-HIC assay was performed to evaluate the oxidation of MK-3475 in the Ab6A drug product. There was no change in the total number of pre-peaks at 5°C (Figure 45). At 25°C there was a small increase in the total number of prepeaks, and at 40°C there was a larger increase in the total number of prepeaks up to 3 months of stability.

Turbidity A350

Turbidity was measured using a Spectramax UV absorption spectrophotometer. 200 μL samples were placed in a 96-well clear Co-star plate, absorbance was measured at 350 nm and 500 nm, and the path was checked by adjusting for plate absorbance of 0.025 and 0.020, respectively.

A350 turbidity was assessed for Ab6A drug product. No significant changes were observed under conditions at 5°C up to 3 months on stability (Figure 46). However, there was a slight increase in ulsoviya at 25°C and an even more marked increase at 40°C up to 3 months.

Example 10: Tyrosine Assay and FRET Assay for Protein-Protein Interaction Detection

Tyrosine analysis

Ab6 and MK-3475 were mixed in the presence of 10 mM histidine, 56 mM arginine, 54.0 mg/ml sucrose, 0.20 mg/ml polysorbate 80, at pH 5.8. Three solutions were prepared with MK-3475:Ab6 protein ratios of 0:1, 1:0, 1:1, where the concentrations of Ab6, MK-3475 and Ab6/MK-3475 concentrations were 20 mg/ml, 20 mg/ml and 10 mg /ml, respectively. The fluorescence results obtained for the single protein and co-formulations were comparable only if the total protein concentration was the same for all samples. The solutions are incubated at room temperature for 72 hours, aliquots are removed at regular intervals and subjected to oxidation followed by a fluorogenic labeling reaction.

Samples collected at predetermined time points are mixed with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and incubated at 37°C for 3 hours in the dark to allow protein oxidation. This reaction is stopped by adding methionine. The next fluorogenic reaction consists of mixing oxidized protein samples with (2-aminomethyl)benzenesulfonic acid (ABS) in the presence of a mild oxidizing agent (K ₃ Fe(CN) ₆ ).

The oxidation reaction of AAPH results in the formation of 3,4-dihydroxyphenylalanine (DOPA) at tyrosine residues. The fluorogenic labeling reaction makes it possible to specifically label DOPA products. The final fluorophore consists of a benzoxazole group that is resonant with the benzene moiety of the ABS molecule. This fluorophore makes it possible to monitor the formation of DOPA by fluorescence at λ _em =490 nm after excitation of the final samples at λ _ex =360 nm.

If protein aggregation occurs when the sample is stored for up to 72 hours, a decrease in the fluorescence signal is expected (Figure 48, bars) depending on the time when aliquots are taken from different protein compositions. The decrease in fluorescence can be interpreted as a change in protein conformation, limiting the availability of certain tyrosine residues for oxidation reactions and/or fluorogenic labeling. The fluorescence signal obtained from the Ab6+MK-3475 co-formulation is stable over 72 hours of incubation (Figure 48, dots). The latter leads to the conclusion that in the 1:1 co-formulation there is no interaction between MK-3475 and Ab6, which could affect the stability of the protein in solution.

FRET analysis

A widely used fluorescent technique for studying bimolecular interactions is FRET (Förster resonance energy transfer), which employs non-radiative (dipole-dipole) energy transfer from a fluorescent donor to an acceptor, which can only occur when the two fluorophores are separated by <10 nm. In the case of two proteins labeled with donor and acceptor tags, this means that FRET occurs only if and when the two proteins interact with each other.

Purified Ab6 and MK-3475 proteins were modified with fluorescent dyes Dylight-488® and Dylight-596®, respectively. Dylight-488® and Dylight-596® are amine-reactive dyes that react preferentially with the primary amine of lysine residues. Fluorescently labeled Ab6 and MK-3475 are named Ab6-488 and MK-3475-596, where labels 488 and 596 refer to the fluorescent dyes Dylight-488® and Dylight-596®, respectively.

Ab6-488 and MK-3475-596 were mixed in the presence of 10 mM histidine, 54.0 mg/ml sucrose at pH 5.8, in the absence or presence of i) arginine (56 mM) and/or ii) polysorbate 80 (0 ,20 mg/ml). The ratios of MK-3475-596/Ab6-488 are 1:4, 1:1 and 4:1. Due to the sensitivity of the assay, the molar concentration of fluorogenic tagged proteins is 3.2 μM or 800 nM depending on the protein ratio. FRET fluorescence signals are recorded for 72 hours at λ _em =620 nm (λ _ex =488 nm) (Figure 49).

If protein interaction occurs, we would expect to see an increase in the fluorescence signal as a function of time (Figure 49). The interaction is assessed by the rate of increase in fluorescence and the maximum of the fluorescence signal. No interaction (or reduced interaction) is characterized by: i) a low rate of increase in the fluorescence signal and ii) a low maximum plateau. The fluorescence signal of a composition with a ratio of MK-3475/Ab6 1/4 in the presence of 10 mM histidine, 56 mM arginine, 54.0 mg/ml sucrose, 0.20 mg/ml polysorbate 80, at pH 5.8 is within the baseline range fluorescence lines (Figure 49). Baseline fluorescence is measured in the absence of labeled protein. The combination of arginine and PS80 eliminates the protein interaction between Ab6 and MK-3475 at a 4:1 mixing ratio.

Based on data from the tyrosine assay and FRET assay, when a co-formulation of MK-3475 and Ab6 is prepared in the presence of 10 mM histidine, 54.0 mg/ml sucrose, 56 mM arginine and 0.20 mg/ml polysorbate 80 at pH 5.8, the stability of the co-formulation is improved compared to the antibody-only formulation for the same excipient matrix. For the 1:1 and 1:4 co-formulations, based on tyrosine analysis and FRET analysis, respectively, the co-formulation is unlikely to cause protein-protein interactions, hence no increase in protein aggregation.

Example 11: Oxidation Study of Joint and Single Component Formulations

Table 20

Ab6A Ab6 (20 mg/ml)+MK-3475 (5 mg/ml),
0.56 mg/ml L-histidine, 1.35 mg/ml L-histidine hydrochloride, 11.8 mg/ml L-arginine hydrochloride, 1.19 mg/ml L-methionine, 54.0 mg/ml sucrose, 0.20 mg/ml polysorbate 80, pH 5.8 MK-3475 MK-3475 (5 mg/ml), 0.56 mg/ml L-histidine, 1.35 mg/ml L-histidine hydrochloride, 11.8 mg/ml L-arginine hydrochloride, 1.19 mg/ml L- methionine, 54.0 mg/ml sucrose, 0.20 mg/ml polysorbate 80, pH 5.8

Comparison of Ab6A and MK-3475 formulation samples subjected to thermal stress at 25°C and 40°C in the same formulation matrix shows that the level of M105 oxidation of MK-3475 in Ab6A formulation was lower compared to MK-3475 alone ( see figures 50 and 51). Therefore, the presence of Ab6 in the Ab6A composition results in reduced oxidation of M105 by MK-3475. The 4:1 Ab6:MK-3475 co-formulation has improved oxidation resistance compared to MK-3475 alone in the same formulation matrix. The oxidation level of M105 was measured by HIC and SEC assays described in Example 9.

--->

LIST OF SEQUENCES

<110> MERK SHARP AND DOME CORP.

ANTOSHCHUK, Valentin

DESAI, Preeti, J.

KRISHNAMAHARI, Yogita

SANGANI, Sahil, S.

<120> COMBINED FORMULATIONS OF ANTI-LAG3 ANTIBODIES AND ANTI-PD-1 ANTIBODIES

<130> 24667-WO-PCT

<150> 62/756,678

<151> 2018-11-07

<160> 76

<170> Patent In version 3.5

<210> 1

<211> 15

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-CDR1 light chain

<400> 1

Arg Ala Ser Lys Gly Val Ser Thr Ser Gly Tyr Ser Tyr Leu His

1 5 10 15

<210> 2

<211> 7

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Light chain CDR2

<400> 2

Leu Ala Ser Tyr Leu Glu Ser

15

<210> 3

<211> 9

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Light chain CDR3

<400> 3

Gln His Ser Arg Asp Leu Pro Leu Thr

15

<210> 4

<211> 111

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Light chain variable region

<400> 4

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

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

50 55 60

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

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

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

100 105 110

<210> 5

<211> 218

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Light chain

<400> 5

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

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

50 55 60

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

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 6

<211> 5

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Heavy chain CDR1

<400> 6

Asn Tyr Tyr Met Tyr

15

<210> 7

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Heavy chain CDR2

<400> 7

Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 8

<211> 11

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Heavy chain CDR3

<400> 8

Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr

1 5 10

<210> 9

<211> 120

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Heavy chain variable region

<400> 9

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

1 5 10 15

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

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 10

<211> 447

<212>PRT

<213> Synthesized sequence

<220>

<223> Pembrolizumab-Heavy chain

<400> 10

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

1 5 10 15

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

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 11

<211> 11

<212>PRT

<213> Homo sapiens

<400> 11

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 12

<211> 7

<212>PRT

<213> Homo sapiens

<400> 12

Asp Ala Ser Asn Arg Ala Thr

15

<210> 13

<211> 9

<212>PRT

<213> Homo sapiens

<400> 13

Gln Gln Ser Ser Asn Trp Pro Arg Thr

15

<210> 14

<211> 107

<212>PRT

<213> Homo sapiens

<400> 14

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 214

<212>PRT

<213> Homo sapiens

<400> 15

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 16

<211> 5

<212>PRT

<213> Homo sapiens

<400> 16

Asn Ser Gly Met His

15

<210> 17

<211> 17

<212>PRT

<213> Homo sapiens

<400> 17

Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 18

<211> 4

<212>PRT

<213> Homo sapiens

<400> 18

Asn Asp Asp Tyr

1

<210> 19

<211> 113

<212>PRT

<213> Homo sapiens

<400> 19

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 20

<211> 440

<212>PRT

<213> Homo sapiens

<400> 20

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

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

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

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

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

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

370 375 380

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

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 21

<211> 15

<212>PRT

<213> Synthesized sequence

<220>

<223> hPD-1.08A Light chain CDR1

<400> 21

Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Phe Ser Tyr Leu His

1 5 10 15

<210> 22

<211> 7

<212>PRT

<213> Synthesized sequence

<220>

<223> hPD-1.08A Light chain CDR2

<400> 22

Leu Ala Ser Asn Leu Glu Ser

15

<210> 23

<211> 9

<212>PRT

<213> Synthesized sequence

<220>

<223> hPD-1.08A Light chain CDR3

<400> 23

Gln His Ser Trp Glu Leu Pro Leu Thr

15

<210> 24

<211> 5

<212>PRT

<213> Synthesized sequence

<220>

<223> hPD-1.08A Heavy chain CDR1

<400> 24

Ser Tyr Tyr Leu Tyr

15

<210> 25

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> hPD-1.08A Heavy chain CDR2

<400> 25

Gly Val Asn Pro Ser Asn Gly Gly Thr Asn Phe Ser Glu Lys Phe Lys

1 5 10 15

Ser

<210> 26

<211> 11

<212>PRT

<213> Synthesized sequence

<220>

<223> hPD-1.08A Heavy chain CDR3

<400> 26

Arg Asp Ser Asn Tyr Asp Gly Gly Phe Asp Tyr

1 5 10

<210> 27

<211> 120

<212>PRT

<213> Synthesized sequence

<220>

<223> h109A heavy chain variable region

<400> 27

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

1 5 10 15

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

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 28

<211> 111

<212>PRT

<213> Synthesized sequence

<220>

<223> K09A light chain variable region

<400> 28

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

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

50 55 60

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

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

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

100 105 110

<210> 29

<211> 111

<212>PRT

<213> Synthesized sequence

<220>

<223> K09A light chain variable region

<400> 29

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

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

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 30

<211> 111

<212>PRT

<213> Synthesized sequence

<220>

<223> K09A light chain variable region

<400> 30

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

1 5 10 15

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

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

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

50 55 60

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

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 31

<211> 447

<212>PRT

<213> Synthesized sequence

<220>

<223> heavy chain mature 409

<400> 31

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

1 5 10 15

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

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 32

<211> 218

<212>PRT

<213> Synthesized sequence

<220>

<223> mature K09A light chain

<400> 32

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

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

50 55 60

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

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 33

<211> 218

<212>PRT

<213> Synthesized sequence

<220>

<223> mature K09A light chain

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

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

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 34

<211> 218

<212>PRT

<213> Synthesized sequence

<220>

<223> mature K09A light chain

<400> 34

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

1 5 10 15

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

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

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

50 55 60

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

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His Ser Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 35

<211> 218

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin light chain Ab1

<400> 35

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 36

<211> 449

<212>PRT

<213> Synthesized sequence

<220>

<223> Immunoglobulin heavy chain Ab 1

<400> 36

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 37

<211> 111

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin light chain variable domain Ab1

<400> 37

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 38

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab1

<400> 38

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 39

<211> 15

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab1 light chain CDR1

<400> 39

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

1 5 10 15

<210> 40

<211> 7

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab1 light chain CDR2

<400> 40

Gly Ala Ser Asn Leu Glu Ser

15

<210> 41

<211> 9

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab1 light chain CDR3

<400> 41

Gln Gln Ser Thr Glu Asp Pro Arg Thr

15

<210> 42

<211> 5

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab1 heavy chain CDR1

<400> 42

Asp Tyr Asn Val Asp

15

<210> 43

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab1 heavy chain CDR2

<400> 43

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

1 5 10 15

Glu

<210> 44

<211> 10

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab1 heavy chain CDR3

<400> 44

Asn Tyr Arg Trp Phe Gly Ala Met Asp His

1 5 10

<210> 45

<211> 449

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain Ab2

<400> 45

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 46

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab2

<400> 46

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 47

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab2 heavy chain CDR2

<400> 47

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

1 5 10 15

Glu

<210> 48

<211> 449

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain Ab3

<400> 48

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 49

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab3

<400> 49

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 50

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab3 heavy chain CDR2

<400> 50

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

1 5 10 15

Glu

<210> 51

<211> 449

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain Ab4

<400> 51

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 52

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab4

<400> 52

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 53

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab4 heavy chain CDR2

<400> 53

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

1 5 10 15

Glu

<210> 54

<211> 446

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain Ab5

<400> 54

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 55

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab5

<400> 55

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 56

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab5 heavy chain CDR2

<400> 56

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

1 5 10 15

Glu

<210> 57

<211> 446

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain Ab6

<400> 57

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 58

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab6

<400> 58

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 59

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab6 heavy chain CDR2

<400> 59

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

1 5 10 15

Glu

<210> 60

<211> 446

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain Ab7

<400> 60

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 61

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab7

<400> 61

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 62

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab7 heavy chain CDR2

<400> 62

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

1 5 10 15

Glu

<210> 63

<211> 446

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain Ab8

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 64

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab8

<400> 64

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 65

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab8 heavy chain CDR2

<400> 65

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

1 5 10 15

Glu

<210> 66

<211> 446

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain Ab9

<400> 66

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 67

<211> 119

<212>PRT

<213> Synthesized sequence

<220>

<223> immunoglobulin heavy chain variable domain Ab9

<400> 67

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 68

<211> 17

<212>PRT

<213> Synthesized sequence

<220>

<223> Ab9 heavy chain CDR2

<400> 68

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

1 5 10 15

Glu

<210> 69

<211> 214

<212>PRT

<213> Synthesized sequence

<220>

<221> source

<223> /note="description of the synthesized sequence:

synthetic polypeptide"

<400> 69

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 70

<211> 447

<212>PRT

<213> Synthesized sequence

<220>

<221> source

<223> / note="description of the synthesized sequence:

synthetic polypeptide""

<400> 70

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

1 5 10 15

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

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 71

<211> 11

<212>PRT

<213> Synthesized sequence

<220>

<221> source

<223> / note="description of the synthesized sequence:

synthetic peptide""

<400> 71

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Ala

1 5 10

<210> 72

<211> 7

<212>PRT

<213> Synthesized sequence

<220>

<221> source

<223> /note="description of the synthesized sequence:

synthetic peptide"

<400> 72

Asp Ala Ser Asn Arg Ala Thr

15

<210> 73

<211> 9

<212>PRT

<213> Synthesized sequence

<220>

<221> source

<223> /note="description of the synthesized sequence:

synthetic peptide"

<400> 73

Gln Gln Arg Ser Asn Trp Pro Leu Thr

15

<210> 74

<211> 5

<212>PRT

<213> Synthesized sequence

<220>

<221> source

<223> /note="description of the synthesized sequence:

synthetic peptide"

"

<400> 74

Asp Tyr Tyr Trp Asn

15

<210> 75

<211> 16

<212>PRT

<213> Synthesized sequence

<220>

<221> source

<223> /note="description of the synthesized sequence:

synthetic peptide"

"

<400> 75

Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 76

<211> 12

<212>PRT

<213> Synthesized sequence

<220>

<221> source

<223> / note="description of the synthesized sequence:

synthetic peptide"

<400> 76

Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro

1 5 10

<---

Claims (14)

1. Stable pharmaceutical composition containing: 18-22 mg/ml anti-LAG3 antibody; 4-7 mg/ml anti-PD-1 antibody; 50-90 mg/ml sucrose; 0.05-1.0 mg/ml polysorbate 80; 3-30 mM histidine buffer at pH 5.0-6.0; 40-100 mM L-arginine or a pharmaceutically acceptable salt thereof; and 5-15 mM L-methionine; wherein the anti-LAG3 antibody comprises a light chain sequence of SEQ ID NO: 35 and a heavy chain sequence comprising a heavy chain variable region sequence of SEQ ID NO: 58 and an IgG4 constant region, and the anti-PD-1 antibody comprises a heavy chain sequence of SEQ ID NO: 10 and light chain sequence SEQ ID NO: 5. 2. Pharmaceutical composition according to claim 1, containing: 18-20 mg/ml anti-LAG3 antibody; 4-7 mg/ml anti-PD-1 antibody; 50-60 mg/ml sucrose; 0.05-1.0 mg/ml polysorbate 80; 8-12 mM histidine buffer at pH 5.0-6.0; 40-70 mM L-arginine or a pharmaceutically acceptable salt thereof; and 5-15 mM L-methionine. 3. Pharmaceutical composition according to any one of paragraphs. 1 and 2, containing 5-10 mM L-methionine. 4. Pharmaceutical composition according to claim 1, containing: 18.75 mg/ml anti-LAG3 antibody; 6.25 mg/ml anti-PD-1 antibody; 55 mg/ml sucrose; 0.2 mg/ml polysorbate 80; 10 mM histidine buffer at pH 5.8; 52.5 mM L-arginine or a pharmaceutically acceptable salt thereof; and 7.5 mM L-methionine. 5. Pharmaceutical composition according to claim 1, containing: 20 mg/ml anti-LAG3 antibody; 5 mg/ml anti-PD-1 antibody; 54 mg/ml sucrose; 0.2 mg/ml polysorbate 80; 10 mM histidine buffer at pH 5.8; 56 mM L-arginine or a pharmaceutically acceptable salt thereof; and 8 mM L-methionine. 6. Pharmaceutical composition according to claim 1, containing: 20.83 mg/ml anti-LAG3 antibody; 4.17 mg/ml anti-PD-1 antibody; 53 mg/ml sucrose; 0.2 mg/ml polysorbate 80; 10 mM histidine buffer at pH 5.8; 58.3 mM L-arginine or a pharmaceutically acceptable salt thereof; and 8.3 mM L-methionine. 7. Pharmaceutical composition according to claim 1, containing: 18.02 mg/ml anti-LAG3 antibody; 4.505 mg/ml anti-PD-1 antibody; 50 mg/ml sucrose; 0.2 mg/ml polysorbate 80; 10 mM histidine buffer at pH 5.8; 70 mM L-arginine or a pharmaceutically acceptable salt thereof; and 10 mM L-methionine. 8. Stable pharmaceutical composition containing: 20 mg/ml anti-LAG3 antibody; 50-90 mg/ml sucrose; 0.05-1.0 mg/ml polysorbate 80; 5-20 mM L-histidine buffer at pH 5.0-6.0; 40-100 mM L-arginine or a pharmaceutically acceptable salt thereof; 5-15 mM L-methionine; and 5 mg/ml anti-PD-1 antibody, wherein the anti-LAG3 antibody comprises a light chain sequence of SEQ ID NO: 35 and a heavy chain sequence comprising a heavy chain variable region comprising SEQ ID NO: 58, and an IgG4 constant region, and The anti-PD-1 antibody contains the heavy chain sequence SEQ ID NO: 10 and the light chain sequence SEQ ID NO: 5. 9. Pharmaceutical composition according to claim 8, containing: 20 mg/ml anti-LAG3 antibody; 5 mg/ml anti-PD-1 antibody; 50-60 mg/ml sucrose; 0.05-1.0 mg/ml polysorbate 80; 8-12 mM L-histidine buffer at pH 5.0-6.0; and 40-70 mM L-arginine or a pharmaceutically acceptable salt thereof; and 5-10 mM L-methionine. 10. Pharmaceutical composition according to claim 8, containing: 20 mg/ml anti-LAG3 antibody; 50 mg/ml sucrose; 0.2 mg/ml polysorbate 80; 10 mM L-histidine buffer at pH 5.8-6.0; 70 mM L-arginine or L-arginine-HCl; 10 mM L-methionine; and 5 mg/ml anti-PD-1 antibody. 11. Pharmaceutical composition according to any one of claims 1 to 10, which is a liquid composition. 12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the anti-LAG3 antibody contains the light chain sequence SEQ ID NO: 35 and the heavy chain sequence SEQ ID NO: 57. 13. The pharmaceutical composition according to any one of claims 1 to 12, where the anti-LAG3 antibody consists of two heavy and two light chains and the anti-PD-1 antibody is pembrolizumab. 14. The pharmaceutical composition according to any one of claims 1-3 and 11-13, where the molar ratio of anti-LAG3 antibody to anti-PD-1 antibody is 4:1.