Anti-ctla-4 antibody composition

<u style="single">Cross-reference between related patents and patent applications</u> This application is filed on March 8, 2005, United States Provisional Patent Application Serial No. 60 / 659,766; United States Provisional Patent Application Serial No. 60 / 728,165 filed on October 19, 2005; 2005. United States Provisional Patent Application Serial No. 60 / 752,712 filed on 20 December; Claims the benefits of United States Provisional Patent Application Serial No. 60 / 762,456 filed on January 26, 2006. All of these provisional patent applications are incorporated herein by reference in their entirety.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a member of the protein immunoglobulin (Ig) superfamily. CTLA-4 down-regulates T cell activation and maintains immunological homeostasis. Inhibition of CTLA-4 (eg, by using CTLA-4 antibody) has been shown to increase the effectiveness of cancer immunotherapy in model animals.

Antibodies that bind to CTLA-4 and suppress its activity have been reported in the literature. For example, US Pat. No. 6,682,736, assigned to Pfizer and Abgenics, reports several human monoclonal antibodies to CTLA-4. Among them is an anti-CTLA-4 antibody having a heavy chain amino acid sequence and a light chain amino acid sequence of antibody 11.2.1 (now known as Ticilimumab®). The hybridoma cell line that produces antibody 11.2.1 was deposited under ATCC registration number PTA-5169. Another monoclonal antibody is reported in US Pat. No. 5,977,318, assigned to Bristol-Myers Squibb. The antibody blocks CTLA-4 from binding to the B7 antigen by recognizing the extracellular domain of CTLA-4 and binding to that extracellular domain. US Application Publication No. 20050201994, assigned to Medarex, reports several human sequence antibodies against CTLA-4. Among them is what is now called ipilimumab®.

One possible method of administering such a CTLA-4 antibody is parenteral administration. For example, US Pat. No. 6,682,736 reports an intravenous anti-CTLA-4 antibody formulation. This formulation is a pH 5.5 sterile solution containing anti-CTLA-4 antibody, 20 mM sodium acetate, 0.2 mg / ml polysorbate 80 and 140 mM sodium chloride.

Like other protein preparations, CTLA-4 antibody preparations may cause the antibody to chemically and physically decompose in the preparation over time. In general, CTLA-4 antibody preparations need to exhibit chemically and physically acceptable stability under expected storage times and conditions of use. That is, the CTLA-4 antibody preparation needs to have a sufficient commercial life and maintain its biological activity. Due to the limited time and resources required to make CTLA-4 antibody products, formulations that reduce product loss are desirable. Therefore, this application discloses a novel CTLA-4 antibody preparation having improved chemical and / or physical stability as compared with the conventional CTLA-4 antibody preparation disclosed in the literature.

<p> According to one feature of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A liquid pharmaceutical composition is provided that further comprises at least one antibody that binds to human CTLA-4; a chelating agent.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is an IgG2 antibody is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is a human antibody is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is human V._H3-33 V using germline genes_HCompositions comprising an amino acid sequence are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is human V._HA V containing the human FR1, FR2, and FR3 sequences that utilize the 3-33 germline gene family and are functionally linked in-frame with the CDR1, CDR2, and CDR3 sequences._HCompositions having an amino acid sequence are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is an isolated antibody is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is a recombinant antibody is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody specifically binds to a conformational epitope on a human CTLA-4 polypeptide. Compositions are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 A composition that further comprises at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody has a heavy chain amino acid sequence that is at least 95% identical in sequence to SEQ ID NO: 2. And a composition comprising the light chain amino acid sequence which is at least 95% identical in sequence to SEQ ID NO: 4 are also provided.</p><p> According to the present invention, in addition to the amino acid sequence which matches at least 90% with the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence which matches at least 90% with the light chain amino acid sequence shown in SEQ ID NO: 4 is provided according to the present invention. A composition that further comprises at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody has a heavy chain amino acid sequence that is at least 99% identical in sequence to SEQ ID NO: 2. And a composition comprising the light chain amino acid sequence which is at least 99% identical in sequence to SEQ ID NO: 4 are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , At least one antibody that binds to human CTLA-4; a composition comprising a chelating agent, wherein the antibody comprises a heavy chain amino acid sequence containing the variable region of SEQ ID NO: 2 and a variable of SEQ ID NO: 4. Compositions comprising a light chain amino acid sequence comprising a region are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody comprises a heavy chain variable region amino acid sequence comprising SEQ ID NO: 5 and a light chain comprising SEQ ID NO: 6. Compositions comprising a chain variable region amino acid sequence are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody comprises a heavy chain amino acid sequence comprising SEQ ID NO: 2 and a light chain amino acid comprising SEQ ID NO: 4. Compositions comprising sequences are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent; the absence of lysine at the C-terminus of the heavy chain of the antibody is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% with the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% with the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody has a heavy chain amino acid sequence and a light chain amino acid sequence of antibody 11.2.1. Compositions comprising CTLA-4 antibody are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, the antibody produced by the hybridoma cell line 11.2.1.4 deposited under ATCC registration number PTA-5169. Compositions having the same heavy and light chain amino acid sequences as the antibodies are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is thicilimumab is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , With at least one antibody that binds to human CTLA-4; a chelating agent was included, and the chelating agent choice was aminopolycarboxylic acid, hydroxyaminocarboxylic acid, EDTA salt and EDTA derivatives, N-substituted. Compositions made from the group consisting of glycine, deferroxamine derivatives, and mixtures thereof are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , With at least one antibody that binds to human CTLA-4; a chelating agent is included, and the choice of chelating agent is ethylenediaminetetraacetic acid, diethylenetriaminetetraacetic acid 5, nitrilotriacetic acid, N-2-acetamide-2- Iminodiacetic acid, bis (aminoethyl) glycol ether, N, N, N', N'-tetraacetic acid, trans-diaminocyclohexanetetraacetic acid, glutamate, aspartic acid, N-hydroxyethyliminodiacetic acid, N, N-bis -Hydroxyethyl glycine, N- (trishydroxymethylmethyl) 10 glycine, glycylglycine, 2- (2-amino-2-oxoctyl) aminoethanesulfonic acid, deferroxamine, deferroxamine mesylate, dipotassium edetate, disodium edetate Tate, edetate calcium disodium, sodium edetate, trisodium edetate, potassium edetate, citrate, sodium citrate, anhydrous citrate, trisodium citrate dihydrate, niacinamide, sodium desoxycholate, Also provided are compositions made from the group consisting of mixtures thereof.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. Also provided is a composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the chelating agent is EDTA.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and further comprising a buffer is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , With at least one amino acid that binds to human CTLA-4; a composition that further contains a buffer in addition to the chelating agent, the buffer of which is selected as acetate, succinate, gluconate. , Citrate, histidine, acetic acid, phosphate, phosphate, ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, hydrogen carbonate, carboxylic acid, succinic acid, sodium benzoate Compositions made from the group consisting of benzoic acid, gluconate, editate, maleate, tris, and mixtures thereof are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , With at least one antibody that binds to human CTLA-4; in addition to the chelating agent, a composition further comprising a buffer, wherein the buffer contains histidine, is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. Also provided is a composition that further contains histidine in addition to the chelating agent, with at least one antibody that binds to human CTLA-4; the histidine contains L-histidine or D-histidine. Will be done.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , With at least one antibody that binds to human CTLA-4; in addition to the chelating agent, a composition further comprising histidine, wherein the histidine contains L-histidine, is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the concentration of the antibody is in the range of about 0.1 to about 200 mg / ml is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the concentration of the antibody is about 20 mg / ml is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , At least one antibody that binds to human CTLA-4; a chelating agent, and at least one component selected from the group consisting of tensioning agents, surfactants, and buffers. Compositions containing a shaping agent are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least two components selected from the group consisting of tensioning agents, surfactants, and buffers. Compositions containing a shaping agent are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and further comprising a tonic, a surfactant, and a buffer. Will be done.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, which further comprises a tonic, an antioxidant, a surfactant, and a buffer. Compositions are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , At least one antibody that binds to human CTLA-4; a chelating agent, and at least one component selected from the group consisting of tensioning agents, surfactants, and buffers. Compositions are also provided in which the formant is included and the tonic contains saccharides.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one additive selected from the group consisting of tonics, surfactants, buffers. Excipients are included and the tonics are fructose, glucose, mannose, sorbose, xylose, lactose, maltose, sucrose, dextran, purulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble glucan, and these. Compositions containing at least one excipient selected from the group consisting of mixtures are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one modification selected from the group consisting of tonics, surfactants, buffers. Compositions are also provided in which the agent is included and the tonic contains a polyol.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one additive selected from the group consisting of tensioning agents, surfactants, and buffers. Formulations are included and the choice of polyol is from the group consisting of mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, propylene glycol, polyethylene glycol, inositol, and mixtures thereof. Compositions made are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one addition selected from the group consisting of tonics, surfactants, buffers. Compositions are also provided in which the shaping agent is included and the tonic contains a non-reducing sugar.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one additive selected from the group consisting of tonics, surfactants, and buffers. Also provided is a composition comprising a formant in which the tonic contains a non-reducing sugar containing sucrose, trehalose, and at least one excipient selected from the group consisting of mixtures thereof.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one additive selected from the group consisting of tonics, surfactants, buffers. Also provided is a composition comprising a formant, the tonic containing a non-reducing sugar and the non-reducing sugar being trehalose.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one additive selected from the group consisting of tonics, surfactants, and buffers. Formulations are included and the choice of surfactants is polysorbate, poroxamer, triton, sodium dodecyl sulfate, sodium laurel sulfate, octyl glycoside sodium, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-. Sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleil-betaine, myristyl-betaine, cetyl-betaine, lauroamidepropyl-betaine, cocamidopropyl-betaine, linoleamidepropyl-betaine, myrista Midpropyl-betaine, palmidopropyl-betaine, isostearamidepropyl-betaine, myristamidepropyl-dimethylamine, palmidopropyl-dimethylamine, isostearamidepropyl-dimethylamine, sodium cocoylmethyltaurine, oleylmethyltaurine disodium, dihydroxy Also provided are compositions made up of the group consisting of propyl PEG5 linoleum ammonium chloride, polyethylene glycol, propylene glycol, and mixtures thereof.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one addition selected from the group consisting of tensioning agents, surfactants, and buffers. A formant is included and the choice of surfactant is of a group consisting of 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and mixtures thereof. Compositions made from the inside are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one additive selected from the group consisting of tonics, surfactants, buffers. Also provided is a composition that comprises a shaping agent and the surfactant is polysorbate 80.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and at least one addition selected from the group consisting of tonics, surfactants, buffers. Compositions are also provided in which the formant is included and the buffer contains histidine.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and further comprising polysolvate 80 is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, and further comprising trehalose is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; the composition also comprises a histidine, trehalose, polysolvate 80, and EDTA.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a pH of about 5.0 to about. A composition of 6.5 is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a histidine concentration of approximately 1 mM. Compositions of ~ about 50 mM are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a histidine concentration of approximately 20 mM. The composition is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a concentration of polysorbate 80 approximately. Compositions ranging from 0.01 mg / ml to about 10 mg / ml are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a concentration of polysorbate 80 approximately. A composition of 0.2 mg / ml is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a concentration of EDTA of about 0.001. Compositions ranging from mg / ml to about 10 mg / ml are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a concentration of EDTA of about 0.1. Compositions in mg / ml are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a concentration of trehalose of approximately 10 mg. Compositions ranging from / ml to about 100 mg / ml are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one antibody that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with a concentration of trehalose of approximately 84 mg. A composition of / ml is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one amino acid that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with approximately 0.1 mg / mg of amino acid. ml ~ about 100 mg / ml; EDTA about 0.001 mg / ml ~ about 1.0 mg / ml; histidine about 1 mM ~ about 50 mM; polysolvate 80 about 0.01 mg / ml ~ about 10 mg / ml; trehalose about 10 mg / ml ~ Compositions containing about 100 mg / ml are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition containing at least one amino acid that binds to human CTLA-4; this composition contains histidine, trehalose, polysorbate 80, and EDTA, with approximately 20 mg / ml of amino acid. Compositions containing about 0.1 mg / ml of EDTA; about 20 mM of histidine; about 0.2 mg / ml of polysorbate 80; about 84 mg / ml of trehalose are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. Also provided is a composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is stable at a temperature of about 5 ° C for at least about 26 weeks.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. Also provided is a composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is stable at a temperature of about 25 ° C for at least about 26 weeks.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. Also provided is a composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is stable at a temperature of about 40 ° C. for at least about 26 weeks.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is stable throughout at least one cycle of freezing and thawing the composition. Things are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, wherein the antibody is stable throughout at least 6 cycles of freezing and thawing the composition. Things are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent of the aggregate chromatogram when the composition is stored at a temperature of about 40 ° C for about 24 weeks. Reduction of peak area compared to peak area of aggregate chromatogram when the same composition was stored at a temperature of about 40 ° C for a period of about 24 weeks, except that it did not contain a chelating agent. Compositions of at least about 2% are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, of an aggregate chromatogram when the composition is stored at a temperature of about 40 ° C for about 24 weeks. Reduction of peak area compared to peak area of aggregate chromatogram when the same composition was stored at a temperature of about 40 ° C for a period of about 24 weeks, except that it did not contain a chelating agent. Also provided is a composition in which SE-HPLC is included in the chromatographic separation operation, which is at least about 2%.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, of the aggregate chromatogram when the composition is stored at a temperature of about 40 ° C for about 24 weeks. Decrease in peak area compared to peak area in aggregate chromatogram when the same composition was stored at a temperature of about 40 ° C for a period of about 24 weeks, except that it did not contain a chelating agent. Is at least about 2%, and compositions are also provided that use ultraviolet detection to measure the amount of aggregated antibody.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent of the aggregate chromatogram when the composition is stored at a temperature of about 40 ° C for about 24 weeks. Reduction of peak area compared to peak area of aggregate chromatogram when the same composition was stored at a temperature of about 40 ° C for a period of about 24 weeks, except that it did not contain a chelating agent. Is at least about 2%, and compositions are also provided in which ultraviolet detection is performed at 214 nanometers.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent of the aggregate chromatogram when the composition is stored at a temperature of about 40 ° C for about 24 weeks. Reduction of peak area compared to peak area of aggregate chromatogram when the same composition was stored at a temperature of about 40 ° C for a period of about 24 weeks, except that it did not contain a chelating agent. Also provided is a composition in which the composition remains substantially colorless and transparent after storage of the composition at a temperature of about 40 ° C. for a period of about 24 weeks.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, of an aggregate chromatogram when the composition is stored at a temperature of about 40 ° C for about 24 weeks. Reduction of peak area compared to peak area of aggregate chromatogram when the same composition was stored at a temperature of about 40 ° C for a period of about 24 weeks, except that it did not contain a chelating agent. Was measured by reverse phase HPLC separation after digestion with an enzyme called lysyl endopeptidase when the composition was stored at a temperature of about 40 ° C for a period of about 24 weeks. Also provided is a composition in which the total oxidation rate of the methionine residue at amino acid position 432 is reduced by 2.2% or more compared to the antibody in the composition containing no chelating agent.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , A composition comprising at least one antibody that binds to human CTLA-4; a chelating agent, of an aggregate chromatogram when the composition is stored at a temperature of about 40 ° C for about 24 weeks. Reduction of peak area compared to peak area of aggregate chromatogram when the same composition was stored at a temperature of about 40 ° C for a period of about 24 weeks, except that it did not contain a chelating agent. Was measured by reverse phase HPLC separation after digestion with an enzyme called lysyl endopeptidase when the composition was stored at a temperature of about 40 ° C for a period of about 24 weeks. Also provided is a composition in which the total oxidation rate of the methionine residue at amino acid position 256 is reduced by 4.2% or more compared to the antibody in the composition containing no chelating agent.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 90% with the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 90% with the light chain amino acid sequence shown in SEQ ID NO: 4 is the main component. Also provided is a composition comprising at least one antibody that binds to human CTLA-4; a chelating agent.</p><p> According to the present invention, a human CTLA comprises an amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2 and an amino acid sequence that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4. Compositions comprising at least one antibody that binds -4 and a chelating agent are also provided.</p><p> INDUSTRIAL APPLICABILITY According to the present invention, in order to prepare a stable liquid pharmaceutical composition, a method comprising mixing a monoclonal anti-CTLA-4 antibody with a pharmacologically acceptable chelating agent in an amount reducing the instability of the antibody. The peak area of the aggregate chromatogram when the composition containing the monoclonal anti-CTLA-4 antibody and the chelating agent was stored at a temperature of about 40 ° C for about 24 weeks was not included in the chelating agent. Also provided is a method in which the reduction is at least about 2% when compared to the peak area of the aggregate chromatogram when the same composition is stored at a temperature of about 40 ° C for a period of about 24 weeks, except that the same composition is stored for a period of about 24 weeks. To.</p><p> INDUSTRIAL APPLICABILITY According to the present invention, in order to stabilize a monoclonal anti-CTLA-4 antibody in a liquid pharmaceutical composition, a method comprising an operation of forming a liquid composition containing the antibody and a pharmacologically acceptable chelating agent. When the composition was stored at a temperature of about 40 ° C for about 24 weeks, the peak area of the aggregate chromatogram of this stable liquid pharmaceutical composition was the same except that it did not contain a chelating agent. Also provided is a method in which the reduction when compared to the peak area of the aggregate chromatogram when the composition is stored at a temperature of about 40 ° C. for a period of about 24 weeks is at least about 2%.</p><p> The present invention is a method of treating a neoplastic disease in a subject, the liquid pharmaceutical composition comprising a therapeutically effective amount of a monoclonal anti-CTLA-4 antibody ticilimumab; a pharmacologically acceptable chelating agent. Methods including the operation of administering a substance are also provided.</p><p> The present invention is a method of treating neoplastic disease in a subject, the liquid pharmaceutical composition comprising a therapeutically effective amount of a monoclonal anti-CTLA-4 antibody ticilimumab; a pharmacologically acceptable chelating agent. Methods including the operation of intravenously administering a substance are also provided.</p><p> The present invention is a method of treating neoplastic disease in a subject, the liquid pharmaceutical composition comprising a therapeutically effective amount of a monoclonal anti-CTLA-4 antibody ticilimumab; a pharmacologically acceptable chelating agent. Also provided are methods that include the operation of administering a substance and the subject in need of treatment for a neoplastic disease.</p><p> The present invention is a method of treating a neoplastic disease in a subject, the liquid pharmaceutical composition comprising a therapeutically effective amount of a monoclonal anti-CTLA-4 antibody thicilimumab; a pharmacologically acceptable chelating agent. The newly formed diseases include brain tumor, squamous cell carcinoma, bladder cancer, gastric cancer, pancreatic cancer, breast cancer, head cancer, neck cancer, esophageal cancer, and prostate. A cancer method selected from the group consisting of cancer, colorectal cancer, lung cancer, kidney cancer, ovarian cancer, female genital cancer, and thyroid cancer is also provided.</p><p> According to the present invention, in order to prepare a liquid composition of a stable antibody, a first container containing a solution of the monoclonal anti-CTLA-4 antibody ticilimumab and a second container containing a pharmacologically acceptable chelating agent are contained. A kit with and is also provided.</p><p> The present invention also provides a manufacturing apparatus having a container holding a mixture of at least one anti-CTLA-4 antibody having a heavy chain amino acid sequence and a light chain amino acid sequence of thicilimumab and a chelating agent.</p><p> According to the present invention, a liquid pharmaceutical composition comprising a monoclonal anti-CTLA-4 antibody and a pharmacologically acceptable chelating agent, wherein the antibody has a molar concentration in the range of about 0.0006 mmol to about 1.35 mmol and chelate. Liquid pharmaceutical compositions are also provided in which the molar concentration of the agent ranges from about 0.003 mmol to about 50 mmol and the molar ratio of antibody to chelating agent is from about 0.00001 to about 450.</p><p> According to the present invention, a liquid pharmaceutical composition comprising a monoclonal anti-CTLA-4 antibody and a pharmacologically acceptable chelating agent, wherein the molar concentration of the antibody ranges from about 0.0006 mmol to about 1.35 mmol and chelate. The molar concentration of the agent ranges from about 0.003 mmol to about 50 mmol, the molar ratio of the antibody to the chelating agent is about 0.00001 to about 450, and the antibody is a monoclonal having a heavy chain amino acid sequence and a light chain amino acid sequence of thicilimumab. Liquid pharmaceutical compositions comprising anti-CTLA-4 antibodies are also provided.</p><p> According to the present invention, a liquid pharmaceutical composition comprising a monoclonal anti-CTLA-4 antibody and a pharmacologically acceptable chelating agent, wherein the antibody has a molar concentration in the range of about 0.0006 mmol to about 1.35 mmol and chelate. Liquid pharmaceutical compositions are also provided in which the molar concentration of the agent ranges from about 0.003 mmol to about 50 mmol and the molar ratio of antibody to chelating agent is from about 0.0001 to about 100.</p><p> According to the present invention, a liquid pharmaceutical composition comprising a monoclonal anti-CTLA-4 antibody and a pharmacologically acceptable chelating agent, wherein the antibody has a molar concentration in the range of about 0.0006 mmol to about 1.35 mmol and chelate. Liquid pharmaceutical compositions are also provided in which the molar concentration of the agent ranges from about 0.003 mmol to about 50 mmol and the molar ratio of antibody to chelating agent is from about 0.001 to about 10.</p><p> According to the present invention, a liquid pharmaceutical composition comprising a monoclonal anti-CTLA-4 antibody and a pharmacologically acceptable chelating agent, wherein the antibody has a molar concentration in the range of about 0.0006 mmol to about 1.35 mmol and chelate. Liquid pharmaceutical compositions are also provided in which the molar concentration of the agent ranges from about 0.003 mmol to about 50 mmol and the molar ratio of antibody to chelating agent is from about 0.1 to about 1.</p><p> According to the present invention, a liquid pharmaceutical composition comprising a monoclonal anti-CTLA-4 antibody and a pharmacologically acceptable chelating agent, wherein the antibody has a molar concentration in the range of about 0.0006 mmol to about 1.35 mmol and chelate. Liquid pharmaceutical compositions are also provided in which the molar concentration of the agent ranges from about 0.003 mmol to about 50 mmol and the molar ratio of antibody to chelating agent is about 0.5.</p><p> The present invention also provides a liquid pharmaceutical composition comprising at least one human monoclonal anti-CTLA-4 antibody that binds to human CTLA-4 and a pharmacologically acceptable chelating agent.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. Also provided are pharmaceutical compositions that include at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, and the concentration of the antibody is at least about 10 mg / ml.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , With at least one antibody that binds to human CTLA-4; a pharmaceutical composition containing pharmacologically acceptable excipients and antibody concentrations ranging from about 10 mg / ml to about 25 mg / ml. Things are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. , With at least one antibody that binds to human CTLA-4; a pharmaceutical composition containing pharmacologically acceptable excipients and antibody concentrations ranging from about 10 mg / ml to about 200 mg / ml. Things are also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. Also provided are pharmaceutical compositions that include at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, and the concentration of the antibody is about 20 mg / ml.</p><p> According to the present invention, with at least one chelating agent; an amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, and an amino acid that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4. A composition comprising at least one antibody comprising a sequence, wherein the antibody binds to human CTLA-4, is also provided.</p><p> According to the present invention, with at least one chelating agent; an amino acid sequence that matches at least 90% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, and an amino acid that matches at least 90% of the light chain amino acid sequence shown in SEQ ID NO: 4. A composition comprising at least one antibody comprising a sequence, a monoclonal IgG2 anti-CTLA-4 antibody that binds to human CTLA-4 and has a heavy chain amino acid sequence and a light chain amino acid sequence of thicilimumab. Compositions containing the above are also provided.</p><p> The present invention also provides a method for preparing a liquid pharmaceutical composition comprising the operation of mixing a solution of at least one anti-CTLA-4 antibody having a heavy chain amino acid sequence and a light chain amino acid sequence of thicilimumab with at least one chelating agent. To.</p><p> INDUSTRIAL APPLICABILITY According to the present invention, a method for treating a neoplastic disease in a subject, and an amount effective for treating at least one anti-CTLA-4 antibody having a heavy chain amino acid sequence and a light chain amino acid sequence of thicilimumab; Also provided are methods that include the operation of administering a liquid pharmaceutical composition comprising a physically acceptable chelating agent.</p><p> According to the present invention, in order to prepare a liquid composition of a stabilized antibody, a first container containing a solution of at least one monoclonal anti-CTLA-4 antibody having a heavy chain amino acid sequence and a light chain amino acid sequence of thicilimumab, and a first container. A kit with a second container containing a pharmacologically acceptable chelating agent is also provided.</p><p> According to the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 is further included. Also provided are liquid pharmaceutical compositions that contain at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, and the concentration of the antibody is at least about 10 mg / ml. ..</p>

<figref num="1">It is a bar graph which shows the result of having examined the ratio (%) of aggregates contained in the test preparation by size exclusion chromatography (SEC) when various test preparations were stored at 40 ° C. for up to 7 weeks.</figref><figref num="2">A bar graph showing the results of reducing the proportion of total (hydrolyzed) impurities formed in the test formulations when various test formulations were stored at 40 ° C for up to 7 weeks by reducing SDS-PAGE (rSDS-PAGE). Is.</figref><figref num="3">It is a line graph which shows the result of having examined the ratio (%) of aggregates contained in the test preparation by SEC when various test preparations were stored under the acceleration condition of 40 ° C for up to 24 weeks.</figref><figref num="4">A line graph showing the results of rSDS-PAGE examination of the proportion of total (hydrolyzed) impurities formed in the test formulations when various test formulations were stored under accelerated conditions of 40 ° C for up to 24 weeks. is there.</figref><figref num="5">It is a line graph which shows the result of having examined the ratio (%) of aggregates contained in the test preparation by SEC when various test preparations were stored under the acceleration condition of 40 ° C for up to 24 weeks.</figref><figref num="6">A line graph showing the results of rSDS-PAGE examination of the proportion of total (hydrolyzed) impurities formed in the test formulations when various test formulations were stored under accelerated conditions of 40 ° C for up to 24 weeks. is there.</figref><figref num="7">A bar graph showing the SEC's assessment of the percentage of aggregates in the test formulations when the various test formulations were stored for up to 24 weeks under accelerated conditions of 40 ° C, as a function of EDTA levels. Is.</figref><figref num="8">When various test preparations were stored under accelerated conditions of 40 ° C for up to 24 weeks, the proportion of total (hydrolyzed) impurities formed in the test preparations was examined by rSDS-PAGE and found to be the level of EDTA. It is a bar graph shown as a function.</figref><figref num="9">It is a line graph which shows the result of having examined the ratio (%) of the aggregates contained in the test preparation when various test preparations were stored under the acceleration condition of 40 ° C for up to 13 weeks by SEC.</figref><figref num="10">A line graph showing the results of rSDS-PAGE examination of the proportion of total (hydrolyzed) impurities formed in the test formulations when various test formulations were stored under accelerated conditions of 40 ° C for up to 13 weeks. is there.</figref><figref num="11-1">Figures 11A through 11D show the nucleotide and amino acid sequences of the anti-CTLA-4 antibody 11.2.1 (now known as ticilimumab). FIG. 11A is the full-length nucleotide sequence of the heavy chain of anti-CTLA-4 antibody 11.2.1 (SEQ ID NO: 1). FIG. 11B shows the full-length amino acid sequence of the heavy chain of anti-CTLA-4 antibody 11.2.1 (SEQ ID NO: 2), and the amino acid sequence of the heavy chain variable region of anti-CTLA-4 antibody 11.2.1 (SEQ ID NO: 5). [] Is shown in parentheses. The amino acid sequence of each heavy chain CDR of anti-CTLA-4 antibody 11.2.1 is underlined. The CDR sequences are as follows: CDR1: GFTFSSYGMH (SEQ ID NO: 7); CDR2: VIWYDGSNKYYADSV (SEQ ID NO: 8); CDR3: DPRGATLYYYYYGMDV (SEQ ID NO: 9).</figref><figref num="11-2">FIG. 11C is the full-length nucleotide sequence of the light chain of anti-CTLA-4 antibody 11.2.1 (SEQ ID NO: 3). FIG. 11D shows the full-length amino acid sequence of the light chain of anti-CTLA-4 antibody 11.2.1 (SEQ ID NO: 4) and the amino acid sequence of the light chain variable region of anti-CTLA-4 antibody 11.2.1 (SEQ ID NO: 6). [] Is shown in parentheses. Each CDR sequence is as follows: CDR1: RASQSINSYLD (SEQ ID NO: 10); CDR2: AASSSLQS (SEQ ID NO: 11); CDR3: QQYYSTPFT (SEQ ID NO: 12).</figref>

Unless otherwise specified, the methods and techniques of the present invention are generally practiced according to methods well known in the art. The methods and techniques are described in various general and more specific references cited and addressed throughout this specification. For example, Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, NY, 1989; Ausbel et al., "Latest Protocols in Molecular Biology", See Green Publishing Associates, 1992; Harlow and Lane, Antibodies: Laboratory Manual, Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, NY, 1990. Enzymatic reactions and purification techniques are performed as is commonly done in the prior art or as described herein according to the manufacturer's instructions. The terms and laboratory procedures and techniques used in analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein are well known and commonly used in prior art. Standard techniques are used in chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and treatment of subjects.

<u style="single">Definition:</u> To make the following detailed explanation easier for the reader to understand, it is defined as follows.

As used herein, the term "formulation" or "composition" as used in the context of an anti-CTLA-4 antibody means a combination of that antibody with a pharmacologically acceptable excipient containing a chelating agent. To do. For example, the formulation of the present invention has improved product life and / or stability as compared to conventional formulations.

As used herein, the term "antibody" means a complete antibody, or antigen binding site that competes with that complete antibody for specific binding. For general literature, see Basic Immunology, Chapter 7 (Paul, W., 2nd Edition, Raven Publishing, NY, 1989). Antigen binding sites can be created by recombinant DNA technology, or by enzymatic or chemical cleavage of the complete antibody. In some embodiments, the antigen binding site is a Fab fragment, a Fab'fragment, F (ab').₂Contains fragments, Fd fragments, Fv fragments, dAb fragments, complementarity determining region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, bispecific antibodies, or at least a portion of an antibody. Examples thereof include polypeptides to which a specific antigen can bind to the presence. Both mature light and heavy chain variable regions have regions FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminus to the C-terminus. The assignment of amino acids to each region is defined by Kabat (National Institute of Health, Bethesda, Maryland, 1987 and 1991), Chothia and Lesk, J. Mol. Biol., Vol. 196, pp. 901-917. , 1987 Definition, Chothia et al., Nature, Vol. 342, pp. 878-883, 1989.

As used herein, the term "polypeptide" includes natural or artificial proteins, fragments of proteins, polypeptide analogs of protein sequences. The polypeptide may be a monomer or a polymer.

In this specification, the Fd fragment is V_HDomain and C_HIt means an antibody fragment consisting of one domain. Fv fragment is a single arm V of one antibody_LDomain and V_HConsisting of domains, the dAb fragment (Ward et al., Nature, Vol. 341, pp. 544-546, 1989) is a V._HConsists of domains.

The expression "or its antigenic binding", when used with an "antibody", lacks the amino and / or carboxy terminus, or the remaining amino acid sequence coincides with the corresponding position in the natural sequence. Means a polypeptide that is present. In some embodiments, the length of the fragment is at least 5, 6, 8 or 10 amino acids. In another embodiment, the length of the fragment is one of at least 14, at least 20, at least 50, at least 70, 80, 90, 100, 150, 200 amino acids.

As used herein, the term "monoclonal antibody" means an antibody obtained from a substantially uniform population of antibodies. That is, the individual antibodies included in the population are the same, except that there are spontaneous mutations that may be present in small amounts and that the C-terminal lysine is deficient. Monoclonal antibodies are highly specific and go to a single antigenic site. Moreover, each monoclonal antibody generally contains a variety of antibodies and is directed towards a single determinant on the antibody, unlike conventional (polyclonal) antibody preparations that are directed to a variety of determinants (epitopes). The modifier "monoclonal" is characterized by the fact that the antibody is derived from a substantially uniform population of antibodies, and it is not necessary to consider that the antibody needs to be made in any particular way. It means that it does not become. For example, the monoclonal antibody used in the present invention is made by the hybridoma method first described by Kohler et al., Nature, Vol. 256, p. 495, 1975, or the recombinant DNA method (see, eg, United States Patent No. 4,816,567). Can be made by. "Monoclonal antibodies" are described, for example, in Clackson et al., Nature, Vol. 352, pp. 624-628, 1991 and Marks et al., J. Mol. Biol., Vol. 222, pp. 581-597, 1991. The method can also be used to isolate from the phage antibody library.

As used herein, the expression "isolated antibody" or "purified antibody" means one of four antibodies, depending on their source or origin: (1) Accompanying in their original state. Antibodies without natural ingredients, (2) antibodies that do not contain other proteins from the same species, (3) antibodies that are expressed in cells from different species, (4) antibodies that do not occur in nature. Therefore, chemically synthesized antibodies or antibodies synthesized in a cell line different from the original source cell are isolated and purified from the components associated with the natural antibody. By isolating and purifying the antibody using a protein purification method well known in the prior art, it is possible to substantially eliminate the components associated with the natural state. Specific examples of the isolated / purified antibody include anti-CTLA-4 antibody affinity-purified using CTLA-4, anti-CTLA-4 antibody synthesized by a hybridoma or other cell line in vitro, and transgenic mouse. There are human anti-CTLA-4 antibodies derived from.

Specific examples of isolated / purified antibodies include anti-CTLA-4 antibodies affinity-purified with CTLA-4, anti-CTLA-4 antibodies synthesized in vitro by hybridomas or other cell lines, and transgenic antibodies. There are human anti-CTLA-4 antibodies derived from mice. Thus, in a preferred embodiment, the anti-CTLA-4 antibody is at least about 95% pure (w / w, weight of anti-CTLA-4 antibody / weight of components other than pharmacologically acceptable excipients). In yet another embodiment, the anti-CTLA-4 antibody has a purity of about 95% w / w to about 99.5% w / w.

Antibodies are in a "substantially pure," "substantially uniform," and "substantially purified" state when at least about 60-75% of the sample is a single type of antibody. The antibody may be a monomer or a polymer. Substantially pure antibodies may generally contain about 50%, 60%, 70%, 80%, 90% w / w per antibody sample, but more commonly about 95%. It is more preferred that it is more than 99% pure. The purity or uniformity of an antibody can be known by a number of methods well known in the art. For example, a polyacrylamide gel electrophoresis of an antibody sample is performed and then the gel is stained with a well-known dye in the prior art to visualize a single polypeptide band. For some purposes, higher resolution can be achieved using HPLC or other means well known in the art for purification.

As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention include, for example, in CDRs (particularly CDR3) by amino acid residues not encoded by human germline immunoglobulin sequences (eg, by random mutagenesis or site-specific mutagenesis in vitro). Introduced mutations, or mutations introduced by somatic mutations in vivo) may be included. However, herein, the term "human antibody" does not include antibodies in which CDR sequences from germline of another species of mammal (eg, mouse) are transplanted onto human framework sequences. Shall be.

In this specification, the term "recombinant human antibody" is used to refer to all human antibodies prepared, expressed, created, and isolated by recombinant techniques (eg, antibodies expressed by transfecting a host cell with a recombinant expression vector (recombinant). Antibodies isolated from combinatorial human antibody libraries, antibodies isolated from transgenic animals (eg mice) into which human immunoglobulin genes have been introduced (eg Taylor, LD et al., 1992, Nucl. Acids Res., Vol. 20), 6287-6295), antibodies prepared, expressed, created, isolated by any other means, including splicing human immunoglobulin gene sequences into other DNA sequences). Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences, but in some embodiments such recombinant human antibodies suddenly occur in vitro. V of the recombinant antibody because it undergoes mutagenesis (or, when using a transgenic animal into which a human Ig sequence has been introduced, somatic cell mutagenesis in vivo)_HDomain and V_LThe amino acid sequence of the domain is V of the human germline._HArray and V_LAlthough derived from and associated with such sequences, they may not be naturally present in the in vivo human antibody germline repertoire.

As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably to mean a nucleotide in the form of a polymer that is at least 10 bases in length. The nucleotide in that case is either a ribonucleotide or a deoxynucleotide, or a modified form of either of these nucleotides. The term includes single-strand and double-strand forms. Unless otherwise stated, the "polynucleotide" or "nucleic acid" sequence also includes its complement. Therefore, when referring to a nucleic acid having a specific sequence, it should be understood that a complementary strand having a sequence complementary thereto is also included.

As used herein, the expression "isolated polynucleotide" or "isolated nucleic acid" means a genomic polynucleotide, a cDNA polynucleotide, or a synthesized polynucleotide, or any combination thereof. An "isolated polynucleotide" has one of three properties, depending on its origin or origin: (1) the whole or all of the polynucleotides found with that "isolated polynucleotide" in nature. It is not partly associated, (2) functionally linked to a polynucleotide that is not naturally linked, and (3) does not occur as part of a larger sequence in nature.

As used herein, "naturally occurring nucleotides" include deoxyribonucleotides. As used herein, the term "modified nucleotide" includes nucleotides having modified saccharides, substituted saccharides, and the like. In this specification, the term "oligonucleotide binding" is used to refer to oligonucleotides such as phosphorothioate, phosphorodithioate, phosphoroserenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladate, and phosphoramidate. Includes binding. For example, La Planche et al., Nucl. Acids Res., Vol. 14, p. 9081, 1986; Stec et al., J. Am. Chem. Soc., Vol. 106, p. 6077, 1984; Stein et al., Nucl. Acids Res. , Vol. 16, pp. 3209, 1988; Zon et al., Anti-Cancer Drug Design, Vol. 6, pp. 539, 1991; Zon et al., Oligonucleotides and Analogs: Practical Approach, pp. 87-108 (F) .. Eckstein, Oxford University Press, Oxford, United Kingdom, 1991); United States Patent No. 5,151,510; Uhlman and Peyman, Chemical Reviews, Vol. 90, p. 543, 1990. The disclosure contents of these documents are incorporated in this specification for reference. If desired, the oligonucleotide can also include a label for detection.

Functionally linked sequences include expression-regulated sequences that are contiguous with the gene of interest and expressions that act in trans, that is, act at distant positions to control the gene of interest. Both control sequences are included. As used herein, the term "expression control sequence" means a polynucleotide sequence required for the expression or processing of a coding sequence that is contiguous with that sequence. Expression control sequences include appropriate transcription initiation sequences, transcription termination sequences, promoter sequences, enhancer sequences; effective RNA processing signals (eg splicing signals and polyadenylation signals); sequences that stabilize cytoplasmic mRNA. A sequence that increases translation efficiency (ie, a Kozak consensus sequence); a sequence that increases protein stability; a sequence that increases protein secretion, if desired. The nature of such control sequences depends on what the host organism is. In prokaryotes, such control sequences generally include promoters, ribosome binding sites, transcription termination sequences, etc., and in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "regulatory sequence" shall include at a minimum all elements essential for expression and processing. The term may also include additional elements that are preferably present (eg, leader sequence, fusion partner sequence).

As used herein, the term "vector" means a nucleic acid molecule capable of carrying another nucleic acid linked to that nucleic acid. In some embodiments, the vector is a plasmid. That is, it is a circular double-stranded DNA loop to which additional DNA segments can be linked inside. In some embodiments, the vector is a viral vector, in which additional DNA segments can be ligated into the genome of the virus. In some embodiments, the vector can replicate autonomously in the host cell into which the vector has been introduced (eg, a bacterial vector having a bacterial origin of replication or an episomal mammalian vector). In another embodiment, the vector (eg, a non-episome mammalian vector) is replicated with the host's genome because it can be integrated with the host's genome when introduced into the host cell. In addition, certain vectors can direct the expression of genes that are functionally linked to the vector. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

As used herein, the term "recombinant host" (or simply "host") means a cell into which a recombinant expression vector has been introduced. It should be understood that "recombinant host cell" and "host cell" include not only the cell of a particular subject, but also the progeny of that cell. Offspring may not actually be exactly the same as the parent cell, as subsequent generations may undergo certain changes due to mutations or environmental influences. However, such offspring are also included within the scope of the term "host cell" as used herein.

As used herein, the phrase "specifically capable of binding" means that an antibody binds to an antigen with a dissociation constant of 1 μM or less. This dissociation constant is preferably 1 nM or less, and most preferably 10 pM or less.

As used herein, the expression "selectively hybridize" means detectable and specific binding. The polynucleotides, oligonucleotides, and fragments thereof of the present invention selectively bind to nucleic acid strands under hybridization and washing conditions that minimize the amount of detectable binding to non-specific nucleic acids. The "very stringent" conditions can be utilized to achieve the conditions of selective hybridization known in the art and described herein. An example of "very harsh" conditions is when a polynucleotide is incubated with other polynucleotides, a hybridization buffer (6 x SSPE or SSC, 50% formamide, 5 x Denhardt solution, 0.5% SDS, 100 μg / ml. In (consisting of denatured and fragmented salmon sperm DNA), one polynucleotide was fixed to a solid surface such as a membrane at 42 ° C for 12 to 16 hours, and then a washing buffer solution (1 ×). It consists of SSC and 0.5% SDS) and is washed twice at 55 ° C. See also Sambrook et al., Supra, pp. 9.50-9.55.

The term "% sequence match" in the context of nucleic acid sequences means the percentage of residues that match when the first sequence is side-by-side and compared to best match the second sequence. .. The length for comparing sequence matches can be at least about 9 nucleotides. This length is generally at least about 18, more generally at least about 24, typically at least about 28, more typically at least about 32, preferably at least about 36, 48 or more. is there. A number of different algorithms that can be used to measure nucleotide sequence matching are known in the art. For example, polynucleotide sequences can be compared using FASTA, Gap, or Best Fit. These are programs included in Wisconsin Package Version 10.0 (Genetics Computer Group (GCG), Madison, Wisconsin). FASTA (which includes programs such as FASTA2 and FASTA3) provides alignment and% sequence matching for the regions that most overlap between the question and search sequences (Pearson, Methods Enzymol., No. 1). 183, 63-98, 1990; Pearson, Methods Mol. Biol., 132, 185-219, 2000; Pearson, Methods Enzymol., 266, 227-258, 1996; Pearson , J. Mol. Biol., Vol. 276, pp. 71-84, 1998). Unless otherwise noted, use the default parameters of individual programs or algorithms. For example,% sequence matching between nucleic acid sequences can be done by using FASTA with the default parameters (word size 6 and NOPAM factor for scoring matrix factors), or GCG version 6.

When referring to a "polynucleotide" or "nucleic acid" sequence, its complement is also included, unless otherwise noted. Therefore, when referring to a nucleic acid having a particular sequence, it should be understood that a complementary strand having a sequence complementary thereto is also included therein.

When referring to a nucleic acid or fragment thereof, the expression "substantial similarity" or "substantial sequence similarity" refers to another nucleic acid (or its complementary strand) with the appropriate inclusion or deletion of nucleotides. When optimal alignment is achieved, nucleotide sequence matching is at least about 85% of nucleotide bases as measured using any well-known sequence matching algorithm (eg FASTA, BLAST, Gap, etc. above). It means that there is. Sequence matching is preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, 99%.

The terms "substantial match," "percent match," and "% match" are used when applied to a polypeptide, using a program such as gap or best fit with the default values provided to that program for gap weights. This means that the sequence match is at least 70%, 75%, and 80% when optimal alignment is achieved with the two peptide sequences. Sequence matching is preferably at least 90% or 95%, more preferably at least 96%, 97%, 98%, 99%. In some embodiments, the mismatched residue positions are the difference in conserved amino acid substitutions. A "conserved amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue having a side chain group R with similar chemical properties (eg, charge or hydrophobicity). In general, conserved amino acid substitutions do not substantially alter protein function. If the difference between two or more amino acid sequences is a conserved substitution difference, the% sequence match may be adjusted upwards to correct for the conservative nature of the substitution. Such adjustment methods are well known to those of skill in the art. For example, Pearson, Methods Mol. See Biol., Vol. 243, pp. 307-331, 1994. Specific examples of a group of amino acids with side chains with similar chemical properties are: 1) aliphatic side chains: glycine, alanine, valine, leucine, isoleucine; 2) aliphatic-hydroxyl side chains: serine, threonine; 3 ) Amino acid-containing side chains: aspartic acid, glutamine; 4) Aromatic side chains: phenylalanine, tyrosine, tryptophan; 5) Basic side chains: lysine, arginine, histidine; 6) Acidic side chains: aspartic acid, glutamic acid; 7) Sulfur Containing side chains: There are cysteine and methionine. Conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and aspartic-glutamine.

Sequence matching of polypeptides is generally examined using sequence analysis software. Protein analysis software matches sequences with indicators of similarity assigned to various substitutions, deletions, and other modifications (eg, conserved amino acid substitutions). For example, GCG includes programs such as "gap" and "best fit" that, by using them with the default parameters specified in the program, are closely related to each other (eg, different species). Reveal sequence homology or sequence matching between (polypeptides) homologous to each other, or between wild-type proteins and their mutations. See, for example, GCG version 6.1. Polypeptide sequences can also be compared to each other using FASTA as the default or recommended parameter values. See GCG version 6.1 (University of Wisconsin, Wisconsin). FASTA (eg FASTA2 and FASTA3) provides alignment and% sequence matching of the most overlapping regions between the question and search sequences (Pearson, Methods). Enzymol., Vol. 183, pp. 63-98, 1990; Pearson, Methods Mol. Biol., Vol. 132, pp. 185-219, 2000). Another preferred algorithm for comparing the sequences of the present invention with a database containing a large number of sequences from different organisms is a computer program called BLAST (particularly blastp or tblastn), which is provided to that program. Used with default parameters. See, for example, Altschul et al., J. Mol. Biol., Vol. 215, pp. 403-410, 1990; Altschul et al., Nucleic Acids Res., Vol. 25, pp. 3389-3402, 1997. The length of the polypeptide sequence for comparing homology is generally about 16 amino acid residues. The number of amino acid residues is generally at least about 20, more generally at least about 24, typically at least about 28, and more preferably about 35 or more. When searching a database containing sequences from many different organisms, it is preferable to compare amino acid sequences.

"Therapeutically effective amount" means an amount effective to achieve the desired therapeutic effect (eg, treatment or prevention of neoplastic disease) with respect to the required dose and duration. Note that the dosage depends on the degree of the disease to be alleviated. In addition, for individual subjects, the specific dosing regimen should be adjusted over time according to individual needs and the professional judgment of the person who controls or supervises the administration of the composition. It should be understood that the dosage ranges described herein are exemplary only and are not intended to limit the scope or use of the compositions of the present invention. Similarly, a therapeutically effective amount of antibody or antibody moiety is desirable for an individual's disease status, age, gender, body weight, and the ability of the antibody or antibody moiety to elicit the desired response to the individual, the antibody composition. It may vary depending on factors such as the route of administration. A therapeutically effective amount is also such that some toxic or detrimental effect of the antibody or antibody portion does not exceed the therapeutically effective effect.

Although the term "subject" for therapeutic purposes includes any subject herein, it is preferred that the subject is a subject in need of treatment for a neoplastic disease. For prophylactic purposes, the subject is any subject, but is preferably the subject at risk of developing neoplastic disease or the subject prone to neoplastic disease. The term "object" shall include a variety of organisms (eg, prokaryotes and eukaryotes). Specific examples of subjects are mammals (eg, humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, non-human transgenic animals). In a particular embodiment of the invention, the subject is a human.

In this specification, "neoplasty" and "neoplastic disease" are both used interchangeably to mean new cell proliferation resulting from non-response to normal growth control (eg, "neoplastic" cell proliferation). .. Neoplasia is also used herein interchangeably with "cancer," and in the present invention, cancer is a subtype of neoplasia. As used herein, the term "neoplastic disease" also includes other cellular abnormalities (eg, hyperplasia, metaplasia, dysplasia). The terms neoplasia, metaplasia, dysplasia, and hyperplasia are used interchangeably herein and generally refer to cells with abnormal cell proliferation.

As used herein, the term "treatment" means both therapeutic and prophylactic treatment. The purpose of prophylactic treatment is to prevent (reduce) the targeted pathological condition or disease, or to slow the progression of the targeted pathological condition or disease. Those in need of treatment include those who already have the disease and those who are prone to or should be prevented from the disease.

When introducing an element of the present invention or a preferred embodiment thereof, the articles "one" and "that" shall mean that there is one or more of the elements. The terms "contain", "contain", "contain", "contain", and "have" are comprehensive, and that the terms may have additional elements other than those listed. It shall mean.

<u style="single">Anti-CTLA-4 antibody:</u> INDUSTRIAL APPLICABILITY According to the present invention, the stability of a given anti-CTLA-4 antibody described herein is mixed with the anti-CTLA-4 antibody with a pharmacologically acceptable chelating agent such as ethylenediaminetetraacetic acid (EDTA). It was found that it could be improved by doing so.

Without being bound by theory, the presence of chelators in the compositions of the invention will result in anti-CTLA-4 antibody aggregation, fragmentation, oxidation, freezing / thawing instability, discoloration, and deamidation. Since the occurrence of one or more is reduced, it is considered to be useful for improving the stability of the antibody polypeptide. The present invention includes anti-CTLA-4 antibody preparations with improved chemical and / or physical stability as compared to previously known anti-CTLA-4 antibody compositions.

Therefore, according to one feature of the present invention, there is provided a composition comprising a pharmacologically acceptable chelating agent (eg, EDTA) and a monoclonal anti-CTLA-4 antibody or antigen binding portion thereof. Yet another feature is that this anti-CTLA-4 antibody liquid composition comprising a chelating agent comprises additional pharmacologically acceptable excipients such as buffers, antioxidants, tonics, surfactants, as well as these. One or more excipients selected from the mixture of

The present invention provides novel formulations for anti-CTLA-4 antibodies. As used herein, the term "anti-CTLA-4 antibody" is isolated from a cytotoxic T lymphocyte-related protein 4 ("CTLA-4") polypeptide that may be present in any animal or from any animal. Means any antibody that can bind to any portion of a CTLA-4 polypeptide that can, or any portion thereof. In some embodiments, the CTLA-4 polypeptide is a human CTLA-4 polypeptide.

Anti-CTLA-4 antibody suitable for use in the present invention can be selected from polyclonal antibody or monoclonal antibody. According to one feature, the monoclonal anti-CTLA-4 antibody can be a mouse chimeric humanized antibody or a human antibody. In yet another embodiment, the monoclonal anti-CTLA-4 antibody is a human monoclonal anti-CTLA-4 antibody.

In some embodiments, the anti-CTLA described in United States Patent No. 6,682,736, filed December 23, 1999 and granted to Hanson et al., As an anti-CTLA-4 antibody suitable for use in the present invention. -4 Antibodies and their preparation methods. In another embodiment, as an anti-CTLA-4 antibody suitable for use in the present invention, a monoclonal anti antibody having a heavy chain amino acid sequence and a light chain amino acid sequence of the antibody 11.2.1 described in US Pat. No. 6,682,736. CTLA-4 antibody can be mentioned. In another embodiment, anti-CTLA-4 antibodies suitable for use in the present invention include monoclonal anti-CTLA-4 antibodies having heavy and light chain amino acid sequences of the antibodies thicilimumab and ipilimumab. In another embodiment, an anti-CTLA-4 antibody suitable for use in the present invention includes a monoclonal anti-CTLA-4 antibody having a heavy chain amino acid sequence and a light chain amino acid sequence of an antibody called ticilimumab.

As used herein, the numbered antibodies have the same heavy and light chain amino acid sequences as the monoclonal antibodies obtained from the same numbered hybridomas. For example, the monoclonal antibody 11.2.1 has the same amino acid sequences of the heavy chain and the light chain as those obtained from the hybridoma 11.2.1. Therefore, antibody 11.2.1 includes the heavy chain and light chain amino acid sequences shown in SEQ ID NOs: 2 and 4, the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 6. Includes the antibody ticylimumab® with. Antibodies lacking lysine at the end of the heavy chain are also included in antibody 11.2.1. This is because it is natural that the lysine is lost at a certain rate during the production of the antibody.

In addition, such anti-CTLA-4 antibodies can be selected based on differences in amino acid sequences in the constant region of the heavy chain. For example, anti-CTLA-4 antibodies can be selected from the IgG class with "γ" type heavy chains. Classes and subclasses of anti-CTLA-4 antibodies can be identified by any method known in the art. In general, certain antibody classes and subclasses can be identified using antibodies specific for a particular class and subclass of antibody. Such antibodies are commercially available. Classes and subclasses can be identified by ELISA, Western blot, or other methods. Alternatively, classes and subclasses sequence all or part of the constant region of the heavy and / or light chain of an antibody and obtain the resulting amino acid sequence as an amino acid sequence already known for the various classes and subclasses of immunoglobulin. It can be clarified by comparing with.

The anti-CTLA-4 antibody can be an IgG molecule, an IgM molecule, an IgE molecule, an IgA molecule, or an IgD molecule. In yet another embodiment, the anti-CTLA-4 antibody is IgG and is a subclass of IgG1, IgG2, IgG3, IgG4. However, it will turn out that killing CTLA-4 expressing cells is generally undesirable. Instead, it is generally desirable to alleviate the downregulation of T cells by simply blocking CTLA-4 from binding to the corresponding ligand. One of the major mechanisms by which antibodies kill cells is through complement fixation and participation in the CDC. The constant region of the antibody plays an important role in the ability of the antibody to immobilize complement and participate in the CDC. Therefore, in general, antibody isotypes are selected depending on their ability to immobilize complement. In the case of the present invention, it is generally not preferable to use an antibody that kills cells as described above. There are many isotypes of antibodies that allow complement fixation and participation in the CDC. For example, mouse IgM, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgM, human IgG1, human IgG3 and the like. Conversely, preferred isotypes that are unable to fix complement and participate in CDC are human IgG2, human IgG4, and the like. In addition to the different heavy chain sequences, each IgG antibody differs between subclasses in the number of disulfide bonds and the length of the hinge region. For example, the IgG2 subclass is distinctly different from other subclasses in some respects. Subclass IgG2 and IgG4 are known to have four disulfide bonds in the hinge region, whereas IgG1 has two disulfide bonds and IgG3 has 11 disulfide bonds. Another difference with IgG2 antibodies is their reduced ability to cross the placenta and their inability to bind to lymphocyte Fc receptors. Thus, in some embodiments, the anti-CTLA-4 antibody is subclass IgG2 or IgG4. Another preference

In another embodiment, suitable anti-CTLA-4 antibodies can be selected based on differences in heavy chain amino acid sequences. For example, the anti-CTLA-4 antibody of the present invention is human V._HGermline gene V_H1, V_H2, V_H3, V_H4, V_HIt is possible to have a γ-type heavy chain utilizing any of 5. In some embodiments, the anti-CTLA-4 antibody is a human V_H3 Use germline genes. In yet another embodiment, the anti-CTLA-4 antibody is a human V_HThe 3 germline genes and the heavy chain variable region of human DP-50 or DP-46 are utilized, and in another embodiment, the anti-CTLA-4 antibody utilizes the human DP-50 heavy chain variable region. DP-50 gene is V_HAlso called a 3-33 family gene. DP-46 gene is V_HAlso called 3-30.3 family gene. In yet another embodiment, the anti-CTLA-4 antibody is human D selected from D1-26, DIR4, DIR3._HUtilizing the gene, in another embodiment, the anti-CTLA-4 antibody is D1-26 human D._HUtilize genes. In yet another embodiment, the anti-CTLA-4 antibody is J_H4 and J_H Human J selected from 6_HUtilizing the gene, in another embodiment, the anti-CTLA-4 antibody is J_H 6 Human J_HUtilize genes.

In yet another embodiment, the anti-CTLA-4 antibody can be selected based on the difference in the amino acid sequence of the light chain. For example, a suitable anti-CTLA-4 antibody can include a λ light chain or a κ light chain. However, in some embodiments, the anti-CTLA-4 antibody of the invention comprises a kappa light chain. In some embodiments where the anti-CTLA-4 antibody comprises a kappa light chain, the polynucleotide encoding the variable region of the light chain is V._κL5 gene, O12 gene, L2 gene, B3 gene, L15 gene, A27 gene, and human J_κ1 gene, J_κ2 genes, J_κ3 genes, J_κ4 genes, J_κContains any of the 5 genes. In some embodiments where the antibody comprises a kappa light chain, the light chain variable region (V)_L) Is a partially human V_κO12 gene or V_κA27 gene and human J_κ3 genes or J_κIt is encoded by 4 genes. In a particular embodiment of the invention, the light chain variable region is human V._κO12 gene / J_κIt is encoded by 3 genes.

In addition, the antibody is V_H3-30 genes or V_HIt can include a heavy chain amino acid sequence containing a human CDR amino acid sequence derived from a 3-33 gene or a gene having a conserved substitution or somatic mutation therein. V_HThe 3-33 gene is thought to encode FR1 to FR3 in the heavy chain variable region of the antibody molecule. Therefore, the present invention includes an antibody that shares at least 85% with the sequence of FR1 to FR3 of the antibody thicilimumab. The degree of commonality is preferably at least 90%, more preferably at least 91%, even more preferably at least 94%, even more preferably at least 95%, and at least 97%. More preferably, it is more preferably at least 98%, even more preferably at least 99%, and most preferably 100%.

Antibodies can further include CDR regions in the light chains derived from the A27 or O27 genes, or can include the CDR regions of the antibody thicilimumab.

In another embodiment of the invention, the antibody blocks the binding of CTLA-4 to one or both of B7-1 and B7-2. The antibody can block the binding to B7-1, IC₅₀Is preferably about 100 nM or less. I c₅₀Is more preferably about 10 nM or less (for example, about 5 nM or less), further preferably about 2 nM or less, and even more preferably 1 nM or less, for example. Similarly, the antibody can block binding to B7-2 and IC₅₀Is less than about 100 nM. I c₅₀Is more preferably about 10 nM or less, for example, about 5 nM or less, more preferably about 2 nM or less, and even more preferably about 1 nM or less.

Furthermore, in another embodiment, the anti-CTLA-4 antibody has a binding affinity for CTLA-4 of about 10.^-8That is all. Binding affinity is about 10^-9More preferably, about 10^-10The above is more preferable, about 10^-11The above is more preferable.

Anti-CTLA-4 antibodies include antibodies that compete for binding with antibodies that have the heavy and light chain amino acid sequences of the antibody ticilimumab. In addition, anti-CTLA-4 antibodies can compete for binding with the antibody ticilimumab.

In another embodiment, the anti-CTLA-4 antibody is a heavy and light chain sequence of the antibody ticilimumab, and / or a heavy chain variable sequence and a light chain variable sequence, and / or a heavy chain CDR sequence and a light chain CDR sequence. It is preferable to cross-competition with the antibody having. For example, an anti-CTLA-4 antibody can bind to an epitope to which an antibody having heavy and light chain amino acid sequences and / or variable sequences and / or CDR sequences of the antibody thicilimumab binds. In another embodiment, the anti-CTLA-4 antibody cross-competites with an antibody having a heavy and light chain sequence of MDX-D010, or an antigen binding sequence.

In another embodiment, an anti-antibody having a heavy chain CDR-1, CDR-2, CDR-3 amino acid sequence and a light chain CDR-1, CDR-2, CDR-3 amino acid sequence. The present invention is carried out using a CTLA-4 antibody or an anti-CTLA-4 antibody having a sequence altered from its CDR sequence. Note that changes from the CDR sequence are conserved or unconserved substitutions, and conserved changes are substitutions by other non-polar residues of non-polar residues, other polarities of polarly charged residues. Substitutions selected from the group consisting of substitutions with uncharged residues, substitutions with other polar charged residues, and substitutions with structurally similar residues, and unconserved substitutions are polar charged residues. Is selected from the group consisting of substitution with polar uncharged residues, substitution of non-polar residues with polar residues, additions, and deletions.

In yet another embodiment of the invention, the anti-CTLA-4 antibody has less than 10 or less than 7 or less than 5 or less amino acid differences from germline sequences in the framework or CDR regions. Less than three. In another embodiment, the anti-CTLA-4 antibody has less than 5 amino acid differences in the framework regions and less than 10 in the CDR regions. In one preferred embodiment, the anti-CTLA-4 antibody has less than 3 amino acid differences in the framework regions and less than 7 in the CDR regions. In a preferred embodiment, the changes in the framework regions are conservative changes and the changes in the CDR regions are somatic mutations.

It is even more preferred that the anti-CTLA-4 antibody is 100% sequence-matched or sequence-similar to the heavy and light chains of the antibody ticilimumab, or separately to the heavy and light chains.

In another embodiment, the anti-CTLA-4 antibody is germline V._κA27, germline V_κO12, germline DP50 (V)_HThe sequence of the 3-33 locus allele) and the entire full-length sequence of heavy and light chains, or separately the entire heavy or light chain, have at least 80% sequence match or sequence similarity. This percentage is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 94%, even more preferably at least 95%, at least 99%. It is even more preferable to have. Even more preferably, the anti-CTLA-4 antibody is 100% sequence-matched or sequence-similar to the germline DP50 heavy chain sequence and / or the germline A27 or germline O12 light chain sequence. That is.

In one embodiment, the anti-CTLA-4 antibody is antibody 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, thicilimumab, 11.6.1, 11.7.1. , 12.3.1.1, 12.9.1.1, the sequence of ipilimumab and the sequences of the variable regions of heavy and light chains, or the sequences of the variable regions of heavy and light chains separately, and at least 80% of the sequences (eg, amino acid sequences). One or both of the nucleic acid sequences) match or sequence similarity. This percentage is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 94%, even more preferably at least 95%, at least 99%. It is even more preferable to have. Even more preferred are anti-CTLA-4 antibodies, antibodies 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, thicilimumab, 11.6.1, 11.7. 100% sequence match or sequence similarity with the variable region sequences of the heavy and light chains of the antibody selected from 1, 12.3.1.1, 12.9.1.1, ipilimumab, or separately with the heavy or light chains. It is that you are.

In another embodiment, the anti-CTLA-4 antibody has a germline DP50 (V) with a heavy chain variable sequence._HHeavy chain variable sequence of 3-33 locus allele) or germline V_κA27 or germline V_κAt least 80% sequence-matched or sequence-similar to the light chain variable sequence of O12. This percentage is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 94%, even more preferably at least 95%, at least 99%. It is even more preferable to have. Even more preferably, the heavy chain sequence of the anti-CTLA-4 antibody is 100% sequence-matched or sequence-similar to the germline DP50 sequence or the germline A27 or germline O12 light chain sequence. That is.

In one embodiment of the invention, the anti-CTLA-4 antibody has a sequence from FR1 to FR4 of one or both of the heavy chain and the light chain that is at least 80% sequenced with the sequence of the FR1 to FR4 region of the antibody thicilimumab. Or it is sequence-like. This ratio is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99%. Even more preferred is that the anti-CTLA-4 antibody is 100% sequence-matched or sequence-similar to the antibody thicilimumab over the entire sequence from one or both of the heavy and light chains FR1 to FR4.

In another embodiment of the invention, the anti-CTLA-4 antibody has a heavy chain sequence from FR1 to FR3 that is at least 80% sequence-matched or sequence-similar to the sequence in the FR1 to FR3 regions of germline DP50. Is. This ratio is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99%, at about 100%. Most preferably.

In yet another embodiment of the invention, the anti-CTLA-4 antibody has a germline V sequence from FR1 to FR4 of the light chain._κA27 or germline V_κAt least 80% sequence match or sequence similarity to the sequences in the FR1 to FR4 regions of O12. This ratio is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99%, at about 100%. Most preferably.

In one embodiment of the invention, the anti-CTLA-4 antibody is at least 80% sequenced or sequenced with the sequence of CDR-1, CDR-2, CDR-3 of one or both of the heavy and light chains of the antibody thicilimumab. It is similar. This ratio is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and even more preferably at least 99%. Even more preferably, the anti-CTLA-4 antibody is 100% sequence-matched or sequence-similar throughout the sequence of CDR-1, CDR-2, CDR-3 of one or both of the heavy and light chains of the antibody ticilimumab. That is.

In another embodiment of the invention, the anti-CTLA-4 antibody has a heavy chain CDR-1 and CDR-2 sequence that is at least 80% of the germline DP50 CDR-1 and CDR-2 sequence. Sequence match or sequence similarity. This ratio is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99%, at about 100%. Most preferably.

In yet another embodiment of the invention, the anti-CTLA-4 antibody has a germline V sequence of light chain CDR-1, CDR-2, CDR-3._κA27 or germline V_κAt least 80% sequence match or sequence similarity to the sequences of CDR-1, CDR-2, and CDR-3 of O12. This ratio is more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 99%, at about 100%. Most preferably.

In one embodiment, the anti-CTLA-4 antibody is an antibody known as thicilimumab.

Table 1 shows the heavy and light chain human germline genes derived for the monoclonal anti-CTLA-4 antibody 11.2.1 (ie, ticilimumab).

<tables num="1"><img file="JP2012167120A_D0001.tif" /></tables>

Some anti-CTLA-4 antibodies according to the present invention were produced by making extensive use of the DP-50 heavy chain variable region. DP-50 gene is V_HIt is also called the 3-33 family gene. In Xenomouse®, there are 30 or more different functional heavy chain variable genes used to make antibodies. The bias therefore indicates a preferred binding motif for antibody-antigen interactions with respect to both antigen-binding and functional activity properties.

In some embodiments, the antibody is a single chain antibody (scFv), V._LDomain and V_HTwo domains can form a single protein chain because the domains are paired through a synthetic linker to form a monovalent molecule. Bird et al., Science, Vol. 242, pp. 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 5879-5883, 1988. In some embodiments, the antibody is a bispecific antibody. That is, the antibody is a divalent antibody, V_HDomain and V_LThe domains are expressed on a single polypeptide strand, but the two domains are complementary to another strand because a linker is used that is too short to pair the two domains on the same strand. It has to be paired with the domain, resulting in two antigen-binding sites. See, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 6444-6448, 1993; Poljak RJ et al., Structure, Volume 2, pp. 1121-1123, 1994. In some embodiments, one or more CDRs from the antibodies of the invention are covalently or non-covalently incorporated into one molecule into an immunoadhesive that specifically binds that molecule to CTLA-4. can do. In such embodiments, the CDRs are incorporated as part of a larger polypeptide chain, covalently linked to another polypeptide chain, or incorporated by non-covalent binding.

In another embodiment, the anti-CTLA-4 antibody has at least 100-fold greater selectivity (specificity) for CTLA-4 than for any other polypeptide. In some embodiments, the anti-CTLA-4 antibody does not bind specifically to detect any protein other than CTLA-4. The selectivity of anti-CTLA-4 antibodies to CTLA-4 can be investigated using methods well known in the art, as described herein. For example, Western blot, FACS, ELISA, RIA can be used to examine selectivity. For example, in some embodiments, the monoclonal anti-CTLA-4 antibody can specifically bind CTLA-4.

In some embodiments, lysine is absent at the C-terminus of the heavy chain of the anti-CTLA-4 antibody according to the invention. According to certain features of the invention, anti-CTLA-4 antibodies are generally free of signal polypeptides. This is because signal polypeptides are generally removed during post-translational modifications. In various embodiments of the invention, one or both of the heavy and light chains of the anti-CTLA-4 antibody comprises the signal sequence (or part thereof). In another embodiment of the invention, neither the heavy chain nor the light chain of the anti-CTLA-4 antibody contains a signal sequence.

Table 2 lists the nucleic acids encoding the heavy and light chain variable regions and the sequence identifiers (SEQ ID NOs) of the corresponding expected amino acid sequences for the monoclonal anti-CTLA-4 antibody 11.2.1.

<tables num="2"><img file="JP2012167120A_D0002.tif" /></tables>

In some embodiments, the nucleic acid molecule is the V of the monoclonal antibody 11.2.1 (SEQ ID NO: 4)._LIt contains a nucleotide sequence encoding an amino acid sequence, or a part thereof. In some embodiments, the portion comprises at least a CDR2 region. In some embodiments, the nucleic acid encodes the amino acid sequence of the light chain CDR of this antibody. In some embodiments, the portion is a contiguous portion comprising CDR1 to CDR3. According to certain characteristics, the light chain CDR1 amino acid sequence is shown by SEQ ID NO: 10, the light chain CDR2 amino acid sequence is shown by SEQ ID NO: 11, and the light chain CDR3 amino acid sequence is shown by SEQ ID NO: 12.

In another embodiment, the nucleic acid molecule is V of SEQ ID NO: 4._LV that matches at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% of the amino acid sequence_LIt encodes an amino acid sequence. In another embodiment, the nucleic acid molecule comprises, or is part of, a nucleotide sequence encoding the light chain amino acid sequence of SEQ ID NO: 4. The nucleic acid molecule of the present invention contains a nucleic acid that hybridizes to the nucleic acid sequence encoding the light chain amino acid sequence of SEQ ID NO: 4, for example, under very severe conditions as described herein.

In yet another embodiment, the nucleic acid molecule is V of antibody 11.2.1._HA nucleotide sequence encoding at least a portion of the amino acid sequence (SEQ ID NO: 2), or a sequence containing an amino acid mutation conserved in that sequence, and / or a total of 3 or less conserved sequences. Contains a nucleotide sequence that encodes at least part of a sequence that has no amino acid substitutions. In various embodiments, the sequence comprises one or more CDR regions (preferably CDR3 regions), all three CDR regions, contiguous portions containing CDR1 to CDR3, V._HYou are coding one of the entire domains. According to one feature, the heavy chain CDR1 amino acid sequence is shown by SEQ ID NO: 7, the heavy chain CDR2 amino acid sequence is shown by SEQ ID NO: 8, and the heavy chain CDR3 amino acid sequence is shown by SEQ ID NO: 9.

In some embodiments, the nucleic acid molecule is V of SEQ ID NO: 2._HV that matches at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% of the amino acid sequence_HIt encodes an amino acid sequence. In yet another embodiment, the nucleic acid molecule comprises, or is part of, a nucleotide sequence encoding the heavy chain amino acid sequence of SEQ ID NO: 2. The nucleic acid molecule of the present invention, for example, the upper and under very severe conditions, such as reporting, including a nucleic acid encoding the heavy chain amino acid sequence a nucleic acid sequence hybridizing SEQ ID NO: 2.

Due to certain features of the invention, human V_H3-33 V using germline genes_HA liquid pharmaceutical composition comprising at least one isolated human antibody comprising an amino acid sequence and binding to CTLA-4 and a pharmacologically acceptable excipient containing a chelating agent is provided. ..

Another feature of the invention is a liquid pharmaceutical composition comprising at least one isolated human antibody that binds to CTLA-4, wherein the antibody has a heavy chain amino acid sequence that is at least 90% identical to SEQ ID NO: 2. , A liquid pharmaceutical composition comprising a light chain amino acid sequence that matches at least 90% of SEQ ID NO: 4 is provided.

According to another feature, a liquid pharmaceutical composition comprising at least one isolated human antibody that binds to CTLA-4, wherein the antibody has a heavy chain amino acid sequence that is at least 95% identical to SEQ ID NO: 2 and SEQ ID NO: A liquid pharmaceutical composition comprising 4 and a light chain amino acid sequence that matches at least 95% is provided.

According to another feature, a liquid pharmaceutical composition comprising at least one isolated human antibody that binds to CTLA-4, wherein the antibody has a heavy chain amino acid sequence that is at least 99% identical to SEQ ID NO: 2 and SEQ ID NO: A liquid pharmaceutical composition comprising 4 and a light chain amino acid sequence that matches at least 99% is provided.

According to yet another feature, the anti-CTLA-4 antibody comprises a heavy chain amino acid sequence containing the variable region of SEQ ID NO: 2 and a light chain amino acid sequence containing the variable region of SEQ ID NO: 4. According to yet another feature, the anti-CTLA-4 antibody comprises a heavy chain amino acid sequence comprising SEQ ID NO: 5 and a light chain amino acid sequence comprising SEQ ID NO: 6. According to yet another feature, the anti-CTLA-4 antibody comprises a heavy chain amino acid sequence comprising SEQ ID NO: 2 and a light chain amino acid sequence comprising SEQ ID NO: 4. According to yet another feature, the anti-CTLA-4 antibody is a human V_HA V containing the human FR1, FR2, and FR3 sequences that utilize the 3-33 gene family and are functionally linked in-frame with the CDR1, CDR2, and CDR3 sequences._HContains the amino acid sequence.

In one embodiment, the anti-CTLA-4 antibody is thicilimumab (also known as CP-675,206) and comprises the heavy and light chain amino acid sequences of the antibody thicilimumab.

In one embodiment of the invention, the anti-CTLA-4 antibody specifically binds to a conformational epitope on human CTLA-4. In another embodiment, the anti-CTLA-4 antibody blocks the growth of human tumors after being administered to a subject.

<u style="single">Preparation of monoclonal anti-CTLA-4 antibody preparations:</u> Anti-CTLA-4 antibodies are generally formulated as pharmaceutical compositions for parenteral administration to a subject. In one embodiment, the pharmaceutical composition is a liquid composition. In another embodiment, the pharmaceutical composition is a liquid composition.

The compositions of the invention combine one or more monoclonal anti-CTLA-4 antibodies of the invention with pharmacologically acceptable excipients, including histidines and / or chelators. Includes. The liquid formulations of the invention combine one or more monoclonal anti-CTLA-4 antibodies of the invention with pharmacologically acceptable excipients, including histidines and / or chelators. Includes.

The term "pharmaceutical composition" means a preparation in a form such that the biological activity of the active ingredient can have an effect. Pharmacologically acceptable excipients (vehicles, additives) can be safely administered to a subject to ensure that an effective amount of the active ingredient used is given to the subject (ie, safe). It is an excipient. As used herein, the term "excipient" or "base" means an inert substance and is commonly used as a diluent, vehicle, preservative, binder, stabilizer for a drug. As used herein, the term "diluent" means a pharmacologically acceptable (safe and non-toxic to human administration) solvent to help prepare the liquid formulations described herein. .. Specific examples of the diluent include sterilized water and bacteriostatic water for injection (BWFI).

Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody and a pharmacologically acceptable chelating agent. Another embodiment of the present invention relates to a liquid pharmaceutical composition comprising an anti-CTLA-4 antibody and EDTA. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody and DTPA.

Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent, and a pharmacologically acceptable buffer. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent, and histidine. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, EDTA and histidine. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, DTPA and histidine.

Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent, and a pharmacologically acceptable tonicity agent. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent, and trehalose. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, EDTA and trehalose. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, DTPA and trehalose.

Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent, and a pharmacologically acceptable surfactant. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, EDTA, and a pharmacologically acceptable surfactant. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, DTPA and a pharmacologically acceptable surfactant. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent selected from the group consisting of EDTA and DTPA, and polysorbate 80.

Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, a pharmacologically acceptable buffer and a pharmacologically acceptable surfactant. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, histidine and a pharmacologically acceptable surfactant. Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody, histidine and polysorbate 80.

Another embodiment of the invention comprises an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent, a pharmacologically acceptable buffer solution, and a pharmacologically acceptable surfactant. With respect to the composition comprising.

Another embodiment of the invention comprises an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent, a pharmacologically acceptable buffer and a pharmacologically acceptable tonicity agent. Regarding the composition.

Another embodiment of the invention comprises an anti-CTLA-4 antibody, a pharmacologically acceptable chelating agent, a pharmacologically acceptable buffer solution, and a pharmacologically acceptable surfactant. , With respect to a composition comprising a pharmacologically acceptable tonic.

Another embodiment of the invention relates to a composition comprising an anti-CTLA-4 antibody and histidine.

As the anti-CTLA-4 antibody present in the composition, those already described herein are possible. In one embodiment, the composition is V shown in SEQ ID NO: 4._LV that matches 90%, 95%, or 99% of the amino acid sequence_LIn addition to the amino acid sequence, V shown in SEQ ID NO: 2_HV that matches 90%, 95%, or 99% of the amino acid sequence_HIt contains an anti-CTLA-4 antibody that further contains an amino acid sequence. In another embodiment, the composition comprises a monoclonal anti-CTLA-4 antibody 11.2.1 as an anti-CTLA-4 antibody.

As the anti-CTLA-4 antibody present in the liquid pharmaceutical composition, those already described herein are possible. In one embodiment, the liquid pharmaceutical composition is V_LV that matches 90%, 95%, or 99% of the amino acid sequence_LIn addition to the amino acid sequence, V shown in SEQ ID NO: 2_HV that matches 90%, 95%, or 99% of the amino acid sequence_HIt contains an anti-CTLA-4 antibody that further contains an amino acid sequence. In another embodiment, the liquid pharmaceutical composition comprises a monoclonal anti-CTLA-4 antibody 11.2.1 as an anti-CTLA-4 antibody.

The concentration of anti-CTLA-4 antibody contained in the liquid pharmaceutical composition of the present invention is generally at least about 0.1 mg / ml or more, at least about 1.0 mg / ml or more, at least about 10 mg / ml or more, and at least about 50 mg / ml per milliliter. Above, at least about 100 mg / ml or more, at least about 200 mg / ml or more. In some embodiments, the concentration of anti-CTLA-4 antibody is generally about 0.1 mg / ml to about 200 mg / ml, about 0.5 mg / ml to about 100 mg / ml, about 1 mg / ml to about 70 mg / ml, About 2.0 mg / ml ~ about 65 mg / ml, about 5.0 mg / ml ~ about 50 mg / ml, about 10 mg / ml ~ about 35 mg / ml, about 15 mg / ml ~ about 25 mg / ml, about 20 mg / ml is there. In one embodiment, the concentration of anti-CTLA-4 antibody contained in the liquid pharmaceutical composition ranges from about 50 mg / ml to about 100 mg / ml. In some embodiments, the concentration of antibody can be increased if the composition is to be administered subcutaneously.

As used herein, the term "chelating agent" generally means an excipient capable of forming at least one bond (eg, covalent bond, ionic bond, etc.) with a metal ion. Chelating agents are commonly used as stabilizers in selected liquid compositions and are multivalent ligands capable of forming complexes with species that may promote destabilization. Compounds that can act as chelating agents often have electron-rich functional groups. Suitable electron-rich functional groups include carboxylic acid groups, hydroxy groups, amino groups and the like. When these groups are arranged in an aminopolycarboxylic acid, a hydroxypolycarboxylic acid, a hydroxyaminocarboxylic acid, etc., a portion capable of binding to a metal is created.

Increased antibody stability is primarily due to the ability of the chelating agent to form bonds with metal ions, but the present invention is not intended to be limited to chelating agents. Therefore, the present invention is not intended to be limited to the specific mechanism by which a chelating agent stabilizes the formulation of the present invention, and the excipient referred to herein as a chelating agent is a chelating agent that forms a bond with a metal ion. Properties that increase the stability of the antibody may be realized through a mechanism that is completely independent of ability.

Suitable chelating agents for use in the present invention include aminopolycarboxylic acid, hydroxyaminocarboxylic acid, N-substituted glycine, 2- (2-amino-2-oxoctyl) aminoethanesulfonic acid (BES), deferroxamine ( DEF), citric acid, niacinamide, desoxycholate and the like. Specific examples of suitable aminopolycarboxylic acids include ethylenediaminetetraacetic acid (EDTA), diethylenetriaminetetraacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2-acetamide-2-iminodiacetic acid (ADA), and bis. (Aminoethyl) glycol ether, N, N, N', N'-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamate, aspartic acid and the like. Specific examples of suitable hydroxyaminocarboxylic acids include N-hydroxyethyliminodiacetic acid (HIMDA), N, N-bis-hydroxyethylglycine (bicine), N- (trishydroxymethylmethyl) 10 glycine (tricine), etc. There is. An example of a suitable N-substituted glycine is glycylglycine. An example of a suitable desoxycholate salt is sodium desoxycholate. A mixture of two or more chelating agents is also included in the present invention.

The chelating agent used in the present invention is, where possible, in the form of a free acid or free base of the compound (referred to herein as "EDTA" or "edetate", both used interchangeably), or corresponding. It can be present in the form of a salt (eg, a corresponding acid- or base-added salt, specifically disodium editate, etc.). Suitable acid addition salts include, for example, alkali metal salts (eg sodium or potassium salts), alkaline earth metal salts (eg calcium salts), and salts should be prepared using other weakly bound metal ions. Can be done. As is known in the art, the nature of the salt and the number of charges to be neutralized depend on the number of carboxyl groups present and the pH at which the stabilizing chelating agent is supplied. As is known in the prior art, chelating agents have varying intensities of binding to specific targeted ions. More specifically, suitable salts of EDTA include dipotassium edetate, disodium edetate, edetate calcium disodium, sodium edetate, trisodium edetate, potassium edetate, and deferoxamine (DEF). A suitable salt of) is deferroxamine mesylate (DFM).

The chelating agent used in the present invention can exist as an anhydride, solvate, hydrate of the compound, or as a corresponding salt. When the chelating agent is in the form of a solvate or hydrate, it can exist in various solvated or hydrated states (eg, anhydrous, hydrate, dihydrate, trihydrate). Form of thing). More specifically, a suitable hydrate of EDTA is EDTA disodium dihydrate, and suitable forms of citric acid include anhydrous citric acid, citric acid monohydrate, trisodium citrate. There are dihydrates and the like.

Suitable chelating agents for use in the antibody compositions of the present invention include O available in combination with metal ions in solution.₂And the one that makes the metal ion unable to react. Then, the production of hydroxyl groups that freely react with the antibody and decompose the antibody is minimized or blocked. Chelating agents reduce the formation of reduced oxygen species and / or the formation of acidic species (eg, deamidation) and / or reduce antibody fragmentation in the compositions of the invention. Can be done. In yet another embodiment, the chelating agent can reduce or prevent antibody aggregation in the compositions described herein. Such chelating agents can reduce or prevent the degradation of formulated antibodies without protection by the chelating agent.

When referring to the concentration of the chelating agent, the indicated concentration represents the molar concentration of the free acid or free base form of the chelating agent. For example, the concentration of chelating agent in a given liquid pharmaceutical composition is generally from about 0.01 micromol to about 50 mmol, from about 1 micromol to about 10.0 mmol, from about 15 micromol to about 5.0 mmol, from about 0.01 mmol to about 1.0. Millimole, in the range of about 0.03 mmol to about 0.5 mmol. In some embodiments, the concentration of chelating agent in the liquid pharmaceutical composition is about 0.01 mmol, 0.02 mmol, 0.027 mmol, 0.03 mmol, about 0.04 mmol, about 0.05 mmol, about 0.06 mmol, about 0.07 mmol, about 0.10. It can be any of mmol, about 0.20 mmol, about 0.26 mmol, about 0.27 mmol, about 0.30 mmol, about 0.31 mmol, about 0.34 mmol, about 0.40 mmol, about 0.50 mmol, about 1.0 mmol. In some embodiments, the chelating agent concentration is one of about 0.027 mmol, about 0.05 mmol, about 0.13 mmol, or about 0.27 mmol. In one embodiment, the concentration of chelating agent is about 0.05 mmol. In another embodiment, the concentration of chelating agent is about 0.13 mmol.

Unless otherwise stated, the concentrations described herein are those under ambient conditions (ie 25 ° C and atmospheric pressure). An intermediate range of the above concentrations for the chelating agent is also a part of the present invention. For example, a range using any combination of the above values as the upper limit and / or the lower limit is also included in the present invention.

In one embodiment, the chelating agent is selected from among EDTA, DTPA, DFM, and mixtures thereof. In another embodiment, the chelating agent is DFM. In another embodiment, the chelating agent is EDTA. In another embodiment, the chelating agent is DTPA. In another embodiment, the liquid pharmaceutical composition generally contains EDTA at about 0.01 micromol to about 50 mmol, about 1 micromol to about 20.0 mmol, about 15 micromoles to about 10.0 mmol, about 0.01 mmol to about 5.0 mmol. , Includes an amount in the range of about 0.03 mmol to about 1 mmol. In some embodiments, the concentration of EDTA in the liquid pharmaceutical composition is about 0.01 mmol, 0.02 mmol, 0.027 mmol, 0.03 mmol, about 0.04 mmol, about 0.05 mmol, about 0.06 mmol, about 0.07 mmol, about 0.10 mmol. , About 0.20 mmol, about 0.26 mmol, about 0.27 mmol, about 0.30 mmol, about 0.31 mmol, about 0.34 mmol, about 0.40 mmol, about 0.50 mmol, about 1.0 mmol. In some embodiments, the concentration of EDTA is either about 0.027 mmol, about 0.05 mmol, about 0.13 mmol, or about 0.27 mmol. In one embodiment, the concentration of EDTA is about 0.05 mmol. In another embodiment, the concentration of EDTA is about 0.13 mmol. In another embodiment, the liquid pharmaceutical composition comprises an amount of about 0.27 mmol of EDTA.

As already pointed out, the compositions of the present invention may optionally include a pharmacologically acceptable buffer in addition to the chelating agent. As used herein, the term "buffer" means a composition added to allow a liquid antibody formulation to resist changes in pH. In some embodiments, the addition of a buffer allows the liquid antibody formulation to resist changes in pH by the action of the acid-base conjugate component of the buffer.

For example, buffered formulations can be prepared by adding appropriate amounts of L-histidine-HCl (L-histidine-hydrochloride) and L-histidine to reach the desired pH. However, in another embodiment, the addition of a buffer allows the liquid antibody formulation to resist changes in pH by the action of its acid-base conjugate component. To give a second example, buffered formulations can be prepared by adding appropriate amounts of acid (eg, hydrochloric acid) and L-histidine to reach the desired pH.

Specific examples of suitable buffers include acetate (eg, sodium acetate), succinate (sodium succinate), gluconate, citrate (eg), and other organic acid buffers (eg, amino acids (histidine)). Etc.), acetic acid, phosphate, phosphate, ascorbate, tartrate, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, carboxylic acid, hydrogen carbonate, succinic acid, sodium benzoate and benzoate , Gluconate, edetate (EDTA), acetate, maleate, tris, and mixtures thereof). In one embodiment, the buffer is acetate.

In another embodiment, the buffer is histidine. Histidine as a starting material used in the preparation of the compositions of the present invention can exist in various forms. Histidine is, for example, in the form of enantiomers (eg L-enantiomers or D-enantiomers), racemic morphology, free acid or free base morphology, salt morphology (eg monohydrochloride, dihydrochloride, hydrobromid, etc. Sulfates, acetates), solvates, hydrates (eg monohydrates), and anhydrides are possible. The purity of the histidine base and / or histidine salt used in the preparation of the composition can generally be at least about 98%, at least about 99%, and at least about 99.5%. As used herein, the term "purity" in the context of histidine means the chemical purity of histidine as understood in the art. For example, see the description in "Merck Index", 13th edition, O'Neil et al. (Merck, 2001).

When referring to the concentration of the buffer, the indicated concentration represents the molar concentration of the free acid or free base form of the buffer. For example, the concentration of buffer in a given liquid pharmaceutical composition is generally from about 0.1 mmol (mM) to about 100 mM. In one embodiment, the buffer concentration is from about 1 mM to about 50 mM. In another embodiment, the buffer concentration is from about 5 mM to about 30 mM. In various embodiments, the buffer concentration is about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM. , About 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM. In one embodiment, the concentration of histidine in the pharmaceutical composition is about 10 mM. In another embodiment, the pharmaceutical composition comprises about 10 mM L-histidine (in the form of a base). In another embodiment, the concentration of histidine in the pharmaceutical composition is about 20 mM. In another embodiment, the pharmaceutical composition comprises about 20 mM L-histidine (in the form of a base). An intermediate range of the above concentrations of histidine shall also be part of the present invention. For example, a range using any combination of the above values as the upper limit and / or the lower limit is also included in the present invention.

Generally, buffers are used to maintain pH in liquid pharmaceutical compositions at acceptable levels (pH levels can affect antibody stability). Liquid pharmaceutical compositions generally maintain a pH in the range of about 4 to about 8, about 4.5 to about 7, about 5.0 to about 6.5, and about 5.3 to about 6.3 with buffer. An intermediate range of pH ranges listed here is also considered to be part of the present invention. A range using, for example, any other combination listed herein as the upper and / or lower limits shall also be included in the present invention. In one embodiment, a buffer is used to maintain the pH of the liquid pharmaceutical composition at about 5.5. In another embodiment, a buffer is used to maintain the pH of the liquid pharmaceutical composition at about 6.0.

As already pointed out, the compositions of the present invention may optionally further include a pharmacologically acceptable tonic in addition to the chelating agent. As used herein, the term "tensioning agent" means an excipient capable of regulating the osmotic pressure of a liquid antibody formulation. In some embodiments, the tonic agent adjusts the osmotic pressure of the liquid antibody formulation to isotonic so that the antibody formulation is biologically compatible with the cells of the body tissue of interest. In yet another embodiment, the "tensioning agent" can contribute to improving the stability of all the anti-CTLA-4 antibodies described herein. An "isotonic" formulation is a composition that has substantially the same osmotic pressure as human blood. Isotonic preparations generally have an osmotic pressure of about 250-350 mOsm. The term "hypotonic" is used to describe formulations with lower osmotic pressure than human blood, whereas the term "hypertonic" refers to formulations with higher osmotic pressure than human blood. Used for. Isotonicity can be measured, for example, using a vapor pressure or freezing point type osmotic pressure gauge.

The tonic used in the preparation of the compositions of the present invention can be present in various forms. The tonic is, for example, in the form of an enantiomer (eg, L-enantiomer or D-enantiomer), or racemic form; isomers (eg, α or β, including αα, ββ, αβ, βα. ); Free acid or free base form; Hydrate form (eg monohydrate); Anhydrous form is possible.

In one embodiment, the tonic is saccharide. As used herein, the term "saccharide" means a group of molecules that are derivatives of polyhydric alcohols. Saccharides are commonly referred to as carbohydrates and can contain different amounts of sugar units (eg monosaccharides, disaccharides, polysaccharides). Saccharides suitable for use as tensioning agents in the present invention include fructose, glucose, mannose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble glucan, etc. And saccharides selected from the group consisting of mixtures thereof.

In another embodiment, the tonic is a polyol. As used herein, the term "polypoly" means an excipient having a large number of hydroxyl groups, such as sugars (reducing sugars, non-reducing sugars), sugar alcohols, sugar acids and the like. In one embodiment, the polyol has a molecular weight of less than about 600 kD (eg, in the range of about 120 to about 400 kD). "Reducing sugar" means a sugar containing a hemiacetal group capable of reducing metal ions and covalently reacting with lysine and other amino groups in proteins. "Non-reducing sugar" means a sugar that does not have these properties of a reducing sugar. Suitable polyols for use as tonics in the present invention include mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, propylene glycol, polyethylene glycol, inositol, and mixtures thereof. Examples thereof include polyols selected from. In one embodiment, the tonic is a non-reducing sugar selected from the group consisting of trehalose, sucrose, and mixtures thereof.

In one embodiment, the tonic is mannitol. In another embodiment, the tonic is D-mannitol. In another embodiment, the tonic agent is trehalose. In another embodiment, the tonic is αα-trehalose dihydrate. In another embodiment, the tonic is sucrose.

In one embodiment, the concentration of the tonic in the liquid pharmaceutical composition ranges from about 1 mmol to about 600 mmol, about 1 mmol to about 400 mmol, about 1 mmol to about 300 mmol, and about 200 mmol to about 275 mmol. is there. In another embodiment the tonic is mannitol, which is present in the liquid pharmaceutical composition at a concentration of about 247 mmol. In another embodiment the tonic is trehalose, which is present in the liquid pharmaceutical composition at a concentration of about 222 mmol. In another embodiment the tonic is trehalose, which is present in the liquid pharmaceutical composition at a concentration of about 238 mmol. In another embodiment the tonic is sucrose, which is present in the liquid pharmaceutical composition at a concentration of about 263 mmol.

In one embodiment, the concentration of the tonic in the liquid pharmaceutical composition ranges from about 1 mg / ml to about 300 mg / ml, about 1 mg / ml to about 200 mg / ml, and about 50 mg / ml to about 150 mg / ml. In another embodiment the tonic is mannitol, which is present in the liquid pharmaceutical composition at a concentration of about 45 mg / ml. In another embodiment the tonic is trehalose, which is present in the liquid pharmaceutical composition at a concentration of about 84 mg / ml. In another embodiment the tonic is trehalose, which is present in the liquid pharmaceutical composition at a concentration of about 90 mg / ml. In another embodiment the tonic is sucrose, which is present in the liquid pharmaceutical composition at a concentration of about 90 mg / ml.

In one embodiment, the tonic is a salt (eg, sodium chloride). In one embodiment, when the tonic is a salt, the concentration of the salt in the liquid pharmaceutical composition is from about 1 mg / ml to about 20 mg / ml. In another embodiment the tonic is sodium chloride and the concentration of sodium chloride in the liquid pharmaceutical composition is about 8.18 mg / ml.

An intermediate range of the above concentrations for the tonic is also part of the present invention. For example, a range using any combination of the above values as the upper limit and / or the lower limit is also included in the present invention.

An intermediate range of the above concentrations for the tonic is also part of the present invention. For example, a range using any combination of the above values as the upper limit and / or the lower limit is also included in the present invention.

As already pointed out, the compositions of the present invention may optionally further include a pharmacologically acceptable surfactant in addition to the chelating agent. As used herein, the term "surfactant" means an excipient capable of varying the surface tension of a liquid antibody formulation. In some embodiments, the surfactant reduces the surface tension of the liquid antibody formulation. In yet another embodiment, the "surfactant" can contribute to improving the stability of any of the anti-CTLA-4 antibodies described herein. For example, surfactants can reduce the aggregation of the formulated antibody and / or minimize the particles formed in the formulation and / or reduce adsorption. Surfactants can also improve the stability of the antibody during and after the freeze / thaw cycle.

Suitable surfactants include polysolvate surfactants, poroxamers (eg poroxamar 18 and poroxamer 407), triton surfactants (eg Triton X-100®), polysolvate surfactants (eg Tween 20®). ), Tween 80®), sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosin, myristyl-sarcosin, Linoleil-Sarkosine, Stearyl-Sarcosine, Linoleyl-Betaine, Myristyl-Betaine, Cetyl-Betaine, Lauroamidepropyl-Betaine, Cocamidopropyl-Betaine, Linolamidepropyl-Betaine, Myristamidepropyl-Betaine, Palmidopropyl-Betaine Stealamide propyl-betaine, myristamide propyl-dimethylamine, palmidopropyl-dimethylamine, isostearamide propyl-dimethylamine, cocoyl methyl taurine sodium, oleyl methyl taurine disodium, dihydroxypropyl PEG5 linoleum ammonium chloride, polyethylene glycol, propylene glycol , As well as mixtures thereof.

In one embodiment, the surfactant is selected from at least a group consisting of 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, and mixtures thereof. It is a polysorbate-based surfactant containing one excipient. In another embodiment, the liquid pharmaceutical composition comprises polysorbate 80.

When the surfactant is present in the composition, the concentration of the surfactant is about 0.01 mg / ml to about 10 mg / ml, about 0.05 mg / ml to about 5.0 mg / ml, about 0.1 mg / ml. The range of ~ about 1.0 mg / ml and about 0.2 mg / ml ~ about 0.7 mg / ml is possible. In another embodiment, the surfactant is present in an amount of about 0.2 mg / ml. In another embodiment, the surfactant is present in an amount of about 0.5 mg / ml. In one embodiment, the liquid pharmaceutical composition comprises about 0.2 mg / ml of polysorbate 80. In another embodiment, the liquid pharmaceutical composition comprises about 0.4 mg / ml of polysorbate 80. In another embodiment, the liquid pharmaceutical composition comprises about 0.5 mg / ml of polysorbate 80.

An intermediate range of the above concentrations for surfactants shall also be part of the present invention. For example, a range using any combination of the above values as the upper limit and / or the lower limit is also included in the present invention.

In addition to the chelating agent, the compositions of the present invention may further include a pharmacologically acceptable antioxidant. Suitable antioxidants include methionine, sodium thiosulfate, catalase, platinum and the like. For example, a liquid pharmaceutical composition can contain methionine at a concentration in the range of 1 mM to about 100 mM, especially at a concentration of about 27 mM.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 Includes a composition comprising at least one antibody that binds to human CTLA-4; a chelating agent.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 Also included is a liquid pharmaceutical composition comprising at least one antibody that binds to human CTLA-4; a chelating agent.

One embodiment of the invention includes a composition comprising at least one monoclonal anti-CTLA-4 antibody that binds to human CTLA-4; a chelating agent.

One embodiment of the invention includes a liquid pharmaceutical composition comprising at least one monoclonal anti-CTLA-4 antibody that binds to human CTLA-4; a chelating agent.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A composition comprising at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, wherein the concentration of the antibody is at least about 10 mg / ml, at least about about. Contains compositions that are either 15 mg / ml, at least about 20 mg / ml, or at least about 25 mg / ml.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A liquid pharmaceutical composition comprising at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, wherein the concentration of the amino acid is from about 10 mg / ml to about. Includes liquid pharmaceutical compositions in the range of 200 mg / ml.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A composition comprising at least one antibody that binds to human CTLA-4 and a pharmacologically acceptable excipient, wherein the concentration of the amino acid is from about 15 mg / ml to about 200 mg /. Compositions in the range of ml are included.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A composition comprising at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, wherein the concentration of the amino acid is from about 20 mg / ml to about 200 mg /. Compositions in the range of ml are included.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A composition comprising at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, wherein the concentration of the amino acid is from about 50 mg / ml to about 200 mg /. Compositions in the range of ml are included.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A liquid pharmaceutical composition comprising at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, wherein the concentration of the amino acid is from about 100 mg / ml to about. Includes liquid pharmaceutical compositions in the range of 200 mg / ml.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A composition comprising at least one antibody that binds to human CTLA-4 and a pharmacologically acceptable excipient, wherein the concentration of the amino acid is from about 10 mg / ml to about 25 mg /. Compositions in the range of ml are included.

In one embodiment of the present invention, in addition to the amino acid sequence that matches at least 95% of the heavy chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence that matches at least 95% of the light chain amino acid sequence shown in SEQ ID NO: 4 A composition comprising at least one antibody that binds to human CTLA-4; a pharmacologically acceptable excipient, wherein the concentration of the amino acid is about 20 mg / ml. Is included.

In one embodiment, the liquid pharmaceutical composition comprises from about 0.01 mg / ml to about 200 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; from about 0.3 micromol to about 50 mmol of chelating agent.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 100 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; from about 3 micromoles to about 5.0 mmol of chelating agent.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 100 mg / ml of the monoclonal anti-CTLA-4 antibody thicilimumab; about 0.27 mmol of the chelating agent.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 100 mg / ml of the monoclonal anti-CTLA-4 antibody thicilimumab; from about 0.3 micromol to about 50 mmol of EDTA.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 100 mg / ml of the monoclonal anti-CTLA-4 antibody thicilimumab; from about 3 micromoles to about 10.0 mmol of EDTA.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 100 mg / ml of the monoclonal anti-CTLA-4 antibody thicilimumab; from about 0.1 mmol to about 1.0 mmol of EDTA.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 100 mg / ml of the monoclonal anti-CTLA-4 antibody thicilimumab; about 0.27 mmol of EDTA.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 100 mg / ml of the monoclonal anti-CTLA-4 antibody thicilimumab; from about 3 micromoles to about 5.0 mmol of DTPA.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 100 mg / ml of the monoclonal anti-CTLA-4 antibody ticilimumab; from about 3 micromoles to about 5.0 mmol of deferoxamine.

In one embodiment, the liquid pharmaceutical composition comprises from about 0.01 mg / ml to about 200 mg / ml of the monoclonal anti-CTLA-4 antibody thicilimumab; from about 1 mM to about 100 mM histidine.

In another embodiment, the liquid pharmaceutical composition is with about 0.1 mg / ml to about 200 mg / ml of monoclonal anti-CTLA-4 antibody histidine mab; with about 3 micromoles to about 5.0 mmol of chelating agent; about 1 mM to about. Contains 100 mM histidine.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 200 mg / ml with the monoclonal anti-CTLA-4 antibody ticilimumab; with about 3 micromoles to about 5.0 mmol of chelating agent; from about 10 mmol to. Contains about 400 mmol of trehalose.

In another embodiment, the liquid pharmaceutical composition comprises from about 0.1 mg / ml to about 200 mg / ml with the monoclonal anti-CTLA-4 antibody thycylimumab; with about 3 micromoles to about 5.0 mmol of chelating agent; from about 10 mmol to. It contains about 400 mmol of trehalose; about 1 mM to about 100 mM histidine.

In another embodiment, the liquid pharmaceutical composition is with about 0.1 mg / ml to about 200 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; with about 3 micromoles to about 5.0 mmol of chelating agent; about 10 mmol to. It contains about 400 mmol of trehalose; about 1 mM to about 100 mM histidine; about 0.005 mmol to about 10 mmol of polysorbate 80.

In another embodiment, the liquid pharmaceutical composition is with about 0.1 mg / ml to about 200 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; about 3 micromoles to about 5.0 mmol of EDTA; about 10 mmol to about. It contains 400 mmol of tonic and; about 1 mM to about 100 mM buffer; and about 0.005 to about 10 mmol of surfactant.

In another embodiment, the liquid pharmaceutical composition is with about 0.1 mg / ml to about 200 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; about 3 micromoles to about 5.0 mmol of EDTA; about 10 mmol to about. It contains 400 mmol of tonic and; about 1 mM to about 100 mM histidine; and about 0.005 mmol to about 10 mmol of surfactant.

In another embodiment, the liquid pharmaceutical composition is with about 0.1 mg / ml to about 200 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; about 3 micromoles to about 5.0 mmol of EDTA; about 10 mmol to about. It contains 400 mmol of trehalose; about 1 mM to about 100 mM histidine; and about 0.005 mmol to about 10 mmol of surfactant.

According to some features of the invention, the liquid anti-CTLA-4 antibody composition is with about 0.1 mg / ml to about 200 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; with about 1 mM to about 100 mM histidine; It contains about 0.005 mmol to about 10 mmol of polysorbate 80; about 3 micromoles to about 5.0 mmol of EDTA; and about 10 mmol to about 400 mmol of trehalose.

According to another feature of the invention, the liquid anti-CTLA-4 antibody composition is with about 1.0 mg / ml to about 100 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; with about 10 mM to about 50 mM histidine; about. It contains 0.01 mmol to about 1.0 mmol of polysorbate 80; about 3 micromoles to about 5.0 mmol of EDTA; and about 100 mmol to about 300 mmol of trehalose.

According to another feature of the invention, the liquid anti-CTLA-4 antibody composition is with about 10 mg / ml to about 50 mg / ml of monoclonal anti-CTLA-4 antibody ticilimumab; with about 10 mM to about 30 mM histidine; about 0.05. It contains from mmol to about 0.5 mmol of polysorbate 80; about 0.1 mmol to about 1 mmol of EDTA; and about 200 mmol to about 250 mmol of trehalose.

According to another feature of the invention, the liquid anti-CTLA-4 antibody composition is with about 20 mg / ml monoclonal anti-CTLA-4 antibody ticilimumab; with about 20 mM histidine; with about 0.15 mmol of polysorbate 80; about 0.27. It contains mmol of EDTA and about 222 mmol of trehalose.

Another embodiment of the invention is a stable liquid pharmaceutical composition comprising an anti-CTLA-4 antibody and a pharmacologically acceptable chelating agent, wherein the antibody has a molar concentration of from about 0.0006 mmol to about. The molar concentration of the chelating agent is in the range of 1.35 mmol, the molar concentration of the chelating agent is in the range of about 0.003 mmol to about 50 mmol, and the molar ratio of the antibody to the chelating agent is about 0.00001 to about 450, about 0.0001 to about 100, and about 0.005 to about 50. , About 0.001 to about 10, about 0.01 to about 5, about 0.1 to about 1, about 0.5, relating to a stable liquid pharmaceutical composition.

Another embodiment of the present invention is a stable liquid pharmaceutical composition comprising thicilimumab and a pharmacologically acceptable chelating agent, wherein the molar concentration of thicilimumab ranges from about 0.0006 mmol to about 1.35 mmol. The molar concentration of the chelating agent is in the range of about 0.003 mmol to about 50 mmol, and the molar ratio of thicilimumab to the chelating agent is about 0.00001 to about 450, about 0.0001 to about 100, about 0.005 to about 50, and about 0.001 to. It relates to a stable liquid pharmaceutical composition that is any of about 10, about 0.01 to about 5, about 0.1 to about 1, and about 0.5.

Another embodiment of the present invention is a stable liquid pharmaceutical composition comprising thicilimumab, a pharmacologically acceptable chelating agent and histidine, wherein the molar concentration of thicilimumab is from about 0.0006 mmol to about 1.35. It is in the range of mmol, the molar concentration of the chelating agent is in the range of about 0.003 mmol to about 50 mmol, the molar concentration of histidine is in the range of about 1 mmol to about 100 mmol, and the molar ratio of thicilimumab to the chelating agent is about. It relates to a stable liquid pharmaceutical composition that is any of 0.00001 to about 450, about 0.0001 to about 100, about 0.005 to about 50, about 0.001 to about 10, about 0.01 to about 5, about 0.1 to about 1, and about 0.5.

Another embodiment of the present invention is a stable liquid pharmaceutical composition comprising thicilimumab, a pharmacologically acceptable chelating agent and histidine, wherein the molar concentration of thicilimumab is from about 0.0006 mmol to about 1.35. It is in the range of mmol, the molar concentration of the chelating agent is in the range of about 0.003 mmol to about 50 mmol, the molar concentration of histidine is in the range of about 10 mmol to about 50 mmol, and the molar ratio of thicilimumab to the chelating agent is about. It relates to a stable liquid pharmaceutical composition that is any of 0.0001 to about 100, about 0.005 to about 50, about 0.001 to about 10, about 0.01 to about 5, about 0.1 to about 1, and about 0.5.

Another embodiment of the present invention is a stable liquid pharmaceutical composition comprising thicilimumab, a pharmacologically acceptable chelating agent and histidine, wherein the molar concentration of thicilimumab is from about 0.0006 mmol to about 1.35. It is in the range of mmol, the molar concentration of the chelating agent is in the range of about 0.003 mmol to about 50 mmol, the molar concentration of histidine is in the range of about 10 mmol to about 30 mmol, and the molar ratio of thicilimumab to the chelating agent is about. It relates to a stable liquid pharmaceutical composition that is any of 0.005 to about 50, about 0.001 to about 10, about 0.01 to about 5, about 0.1 to about 1, and about 0.5.

Another embodiment of the present invention is a stable liquid pharmaceutical composition comprising thicilimumab, a pharmacologically acceptable chelating agent and histidine, wherein the molar concentration of thicilimumab is from about 0.0006 mmol to about 1.35. It is in the range of mmol, the molar concentration of the chelating agent is in the range of about 0.003 mmol to about 50 mmol, the molar concentration of histidine is in the range of about 10 mmol to about 30 mmol, and the molar ratio of thicilimumab to the chelating agent is about. It relates to a stable liquid pharmaceutical composition that is any of about 0.001 to about 10, about 0.01 to about 5, about 0.1 to about 1, and about 0.5.

Another embodiment of the present invention is a stable liquid pharmaceutical composition comprising thicilimumab, a pharmacologically acceptable chelating agent and histidine, wherein the molar concentration of thicilimumab is from about 0.0006 mmol to about 1.35. The molar concentration of the chelating agent is in the range of about 0.003 mmol to about 50 mmol, the molar concentration of histidine is about 20 mmol, and the molar ratio of thicilimumab to the chelating agent is about 0.001 to about 10, about 10. For stable liquid pharmaceutical compositions that are any of 0.01 to about 5, about 0.1 to about 1, and about 0.5.

<u style="single">Method for producing anti-CTLA-4 antibody and antibody-producing cell line:</u> The antibody of the present invention can be prepared using transgenic mice. This transgenic mouse contains an important part of the inserted human antibody-producing genome, but is internally prevented from producing mouse antibodies. Thus, such mice can produce human immunoglobulin molecules and antibodies, but not mouse immunoglobulin molecules and antibodies. The technology for that purpose will be described below.

It is possible to produce transgenic animals (eg, mice) that can produce a complete repertoire of human antibodies when immunized without the production of endogenous immunoglobulins. However, one embodiment of the production of transgenic mice and the antibodies obtained from those mice is disclosed, in particular, in United States Patent No. 6,682,736, granted to Hanson et al. Using such a technique, an antibody that binds to CTLA-4 and a hybridoma that produces the antibody can be prepared.

Human antibodies avoid some of the problems associated with antibodies that have variable and / or constant regions in mice or rats. The presence of proteins from mice or rats can result in rapid excretion of the antibody and an immune response to the antibody in subjects administered with such antibody.

For example, a chimeric germline mutant mouse with an antibody heavy chain binding region (J)_HIt has been reported that homozygous deletion of the gene) completely blocks the production of endogenous antibodies. Transferring a human germline immunoglobulin gene array to such germline mutant mice would produce human antibodies when challenged with an antigen (eg CTLA-4). For example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 2551, 1993; Jakobovits et al., Nature, Vol. 362, pp. 255-258, 1993; Bruggermann et al., Year in Immuno., No. See Volume 7, page 33, 1993; Duchosal et al., Nature, Volume 355, page 258, 1992. Human antibodies can also be from phage presentation libraries (Hoogenboom et al., J. Mol. Biol., Vol. 227, p. 381, 1991; Marks et al., J. Mol. Biol., Vol. 222, 581-597, 1991; Vaughan et al., Nature Biotech, 14, 309, 1996).

In some embodiments, the human anti-CTLA-4 antibody can be produced by immunizing a non-human transgenic animal (eg, Xenomouse®). Since the genome of this mouse contains the human immunoglobulin gene, this recombinant mouse produces human antibodies. Xenomouse® is a genetically engineered mouse strain that does not produce mouse antibodies because it contains large fragments of the heavy and light chain loci of human immunoglobulin. Xenomouse® produces an adult-like human repertoire of all human antibodies to generate antigen-specific human antibodies. In some embodiments, Xenomouse® is a germline configuration yeast artificial chromosome (YAC) fragment of the human heavy and κ light chain loci with a base size of 1 mega. By introduction, it has about 80% of the human antibody V gene repertoire. In another embodiment, Xenomouse® further comprises almost all loci of the λ light chain. For example, Green et al., Nature Genetics, Vol. 7, pp. 13-21, 1994; United States Patents 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364, 6,162,963. See No. 6,150,584. WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00/09560, See also WO 00/037504.

In some embodiments, the non-human animal comprising the human immunoglobulin gene is an animal having a human immunoglobulin "minigene". In the minigene method, the Ig locus is mimicked by including individual genes from the exogenous Ig locus. Therefore one or more Vs_HGene, one or more D_HA gene, one or more IH genes, a μ constant region, and a second constant region (preferably a γ constant region) are formed in the structure and inserted into the animal. This method is, in particular, United States Patents 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205. It is described in No. 5,721,367, No. 5,789,215, and No. 5,643,763.

Thus, in some embodiments, human antibodies can be produced by immunizing a non-human animal containing the heavy and light chain loci of human immunoglobulin in its genome with the CTLA-4 antigen. ..

In some embodiments, the CTLA-4 antigen is isolated CTLA-4 and / or purified CTLA-4. In a preferred embodiment, the CTLA-4 antigen is human CTLA-4. In some embodiments, the CTLA-4 antigen is a fragment of CTLA-4. In some embodiments, the fragment of CTLA-4 comprises at least one epitope of CTLA-4. In another embodiment, the CTLA-4 antigen is a cell that expresses or overexpresses CTLA-4 or an immunogenic fragment thereof on the surface. In yet another embodiment, the CTLA-4 antigen is a CTLA-4 fusion protein. CTLA-4 can be purified from natural sources using known methods.

In a preferred embodiment, the non-human animal is Xenomouse® (Abgenics, Fremont, CA). Another non-human animal that can be used is a transgenic mouse produced by Madarex (Princeton, NJ).

Immunization of animals can be performed by any method known in the art. See, for example, Harlow and Lane, Antibodies: Laboratory Manual, Cold Spring Harbor Publishing, NY, 1990. Methods of immunizing non-human animals (eg, mice, rats, ovis, goats, pigs, cows, horses) are well known in the art. See, for example, Harlow and Lane, the above literature, and US Pat. No. 5,994,619. In a preferred embodiment, the CTLA-4 antigen is administered with an adjuvant to promote an immune response. Specific examples of the adjuvant include complete Freund adjuvant, incomplete Freund adjuvant, RIBI (muramyl dipeptide), ISCOM (immunostimulatory complex) and the like. Such an adjuvant prevents the polypeptide from being rapidly dispersed by being blocked in a local sediment, or the host secretes chemotactic factors for macrophages and other components of the immune system. Can contain substances that promote. When administering a polypeptide, the immunization schedule allows the polypeptide to be administered more than once over a period of several weeks.

After immunizing an animal with CTLA-4 antigen, antibodies and / or antibody-producing cells can be obtained from the animal. In some embodiments, the animal is bleeding or euthanized to obtain serum containing anti-CTLA-4 antibody. This serum can be used as is obtained from an animal, an immunoglobulin fraction can be obtained from the serum, or an anti-CTLA-4 antibody can be purified from the serum.

In some embodiments, an immortalized cell line that produces antibodies is prepared from cells isolated from immunized animals. After immunizing the animal, it is euthanized to immortalize the lymph nodes and / or spleen B cells. As a method for immortalizing a cell, a method of transfecting the cell with an oncogene, a method of infecting the cell with a cancer virus, a method of culturing the cell under conditions in which an immortalized cell can be selected, and a carcinogenic compound. Alternatively, there are methods of exposure to mutagenesis compounds, fusion of the cells with immortalized cells (eg, myeloma cells), inactivation of tumor suppressor genes, and the like. See, for example, Harlow and Lane, above. In a preferred embodiment, the immunized animal is a non-human animal expressing a human immunoglobulin gene, fusing spleen B cells with myeloma cells from the same species as the non-human animal. In one more preferred embodiment, the immunized animal is Xenomouse® and the myeloma cell line is non-secretory mouse myeloma. In one more preferred embodiment, the myeloma cell line is P3-X63-AG8-653. When a fusion with a myeloma cell is used, it is preferable that the myeloma cell does not secrete an immunoglobulin polypeptide (non-secretory cell line). Immortalized cells are screened with CTLA-4, a portion thereof, or CTLA-4 expressing cells. In a preferred embodiment, the initial screening is performed utilizing a solid phase enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay. An example of ELISA screening is described in WO 00/37504.

Anti-CTLA-4 antibody-producing cells (eg, hybridomas) with desirable properties (eg, vigorous proliferation, high antibody production, and desirable properties of antibodies described in detail below) are selected, cloned, and further screened. Hybridomas can be grown in vivo in animals of the same genotype or animals without an immune system (eg, nude mice), or in in vitro cell cultures. Methods of selecting, cloning and propagating hybridomas are well known to those of skill in the art.

The antibody of the present invention can be recombinantly expressed in a cell line other than the hybridoma cell line. Nucleic acid sequences encoding cDNA or genomic clones for a particular antibody can be utilized to transform suitable mammalian or non-mammalian host cells.

The present invention also includes nucleic acid molecules encoding anti-CTLA-4 antibodies. In some embodiments, the heavy and light chains of the anti-CTLA-4 antibody are encoded by different nucleic acid molecules. In another embodiment, the same nucleic acid molecule encodes the heavy and light chains of the anti-CTLA-4 antibody. In one embodiment, the nucleic acid encodes the anti-CTLA-4 antibody of the invention.

Nucleic acid molecules encoding the entire heavy or light chain of anti-CTLA-4 antibodies, or parts thereof, can be isolated from any source that produces such antibodies. In various embodiments, the nucleic acid molecule is isolated from B cells isolated from anti-CTLA-4 immunized animals or simply from immortalized cells derived from anti-CTLA-4 antibody-expressing B cells. Release. Methods of isolating the mRNA encoding the antibody are well known in the prior art. See, for example, Sambrook et al., Molecular Cloning, 3rd Edition, Volume 3, 1989. The mRNA can be used to make cDNA and perform polymerase chain reaction (PCR) or cDNA cloning of antibody genes. In a preferred embodiment, the nucleic acid molecule is isolated from a hybridoma having human immunoglobulin-producing cells from a non-human transgenic animal as one of the fusion partners. In a more preferred embodiment, human immunoglobulin-producing cells are isolated from Xenomouse®. In another embodiment, the human immunoglobulin-producing cells are from non-human non-mouse transgenic animals as described above. In another embodiment, nucleic acids are isolated from non-human non-transgenic animals. Nucleic acid molecules isolated from non-human animals can be used, for example, in humanized antibodies.

In some embodiments, the nucleic acid encoding the heavy chain of the anti-CTLA-4 antibody of the invention is in-frame bound to a nucleotide sequence encoding a heavy chain constant region from any source. V of the present invention_HIt can include a nucleotide sequence that encodes a domain. Similarly, the nucleic acid molecule encoding the light chain of the anti-CTLA-4 antibody of the invention is in-frame bound to a nucleotide sequence encoding the light chain constant region from any source. V_LIt can include a nucleotide sequence that encodes a domain.

According to yet another feature of the present invention, heavy chains (V)_H) And light chain (V)_LThe nucleic acid molecule encoding the variable region of) is "converted" to a full-length antibody gene. In one embodiment, V_HDomain or V_LThe nucleic acid molecule encoding the domain is placed in the heavy chain constant region (C)._H) Or light chain constant region (C)_L) Are converted into full-length antibody genes by inserting them into the expression vectors already encoded. At that time, V_HThe segment is C inside the vector_HFunctionally linked to the segment, V_LThe segment is C inside the vector_LMake a functional link with the segment. In another embodiment, standard methods of molecular biology are utilized, V._HDomain and / or V_LThe nucleic acid molecule encoding the domain, C_HDomain and / or C_LIt is converted to a full-length antibody gene by linking (eg, linking) with a nucleic acid molecule encoding a domain. Nucleic acid sequences of genes in the immunoglobulin constant regions of human heavy and light chains are known in the art. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publishing, 91-3242, 1991. The nucleic acid molecule encoding the full-length heavy and / or light chain can then be expressed from the cell into which the nucleic acid molecule has been introduced to isolate the anti-CTLA-4 antibody.

The present invention also provides a vector containing a nucleic acid molecule encoding a heavy chain of the anti-CTLA-4 antibody of the present invention or an antigen-binding portion thereof. The present invention also provides a vector containing a nucleic acid molecule encoding the light chain of such an antibody or its antigenic binding moiety. The invention further provides vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes of such nucleic acids.

In some embodiments, the DNA encoding part or all of the light and heavy chains obtained as described above is introduced into an expression vector to transfer the gene to the required expression control sequence (eg, a transcription control sequence). , Translation control sequence) to express the anti-CTLA-4 antibody of the present invention or its antigen-binding portion. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (eg, cauliflower mosaic virus, tobacco mosaic virus), cosmid, YAC, and episomes derived from EBV. The antibody gene is ligated to the vector so that the transcriptional and translational regulatory sequences within the vector perform the expected function in the regulation of transcription and translation of the antibody gene. For the expression vector and expression control sequence, select one that is compatible with the expression host cell to be used. The light and heavy chain genes of an antibody can be inserted into separate vectors. In one preferred embodiment, both genes are inserted into the same vector. The antibody gene is inserted into the expression vector by standard methods (eg, ligation of the vector with complementary restriction sites on fragments of the antibody gene, or blunt-ended ligation in the absence of regulatory sites).

Convenient vector is a fully functional human C_HImmunoglobulin sequence or human C_LIt encodes an immunoglobulin sequence and has an appropriate restriction site as described above._HArray or V_LIt is a vector that facilitates expression by inserting a sequence. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site in front of the human C domain, resulting in human C._HSplicing also occurs in the splice region that occurs inside the exon. Polyadenylation and transcription termination occur at the original chromosomal site downstream of the coding region. Recombinant expression vectors can also encode signal peptides that facilitate the secretion of antibody chains from host cells. The antibody chain gene can be cloned into a vector and the signal peptide can be linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a protein that is not an immunoglobulin).

In addition to the antibody chain gene, the recombinant expression vector of the present invention comprises a regulatory sequence that regulates the expression of the antibody chain gene in a host cell. Those skilled in the art will appreciate that the design of the expression vector (including the selection of regulatory sequences) may vary depending on factors such as the choice of host cell to transform and the level of expression of the desired protein. Let's go. Preferred regulatory sequences for expression in mammalian host cells include viral elements that express high amounts of protein in mammalian cells (eg, promoters and / or enhancers derived from retroviruses (eg, retrovirus LTRs), cytomegalo. Virus (CMV) -derived promoters and / or enhancers (CMV promoters / enhancers, etc.), Simian virus 40 (SV40) -derived promoters and / or enhancers (SV40 promoters / enhancers, etc.), Adenovirus-derived promoters and / or enhancers (such as adenovirus-derived promoters / / or enhancers) Examples include adenovirus major late promoters (AdMLPs), polyoma-derived promoters and / or enhancers) and strong mammalian promoters (eg, the original immunoglobulin promoters, actin promoters). Virus regulatory elements and their sequences. See, for example, United States Patents No. 5,168,062, No. 4,510,245, No. 4,968,615 for a more detailed explanation of the above. For example, see United States Patent No. 6,517,529 (the contents of which are incorporated herein by reference). Polypeptides in bacterial or fungal cells (eg, yeast cells). The method of expressing the promoter is also well known in the prior art.

In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector of the present invention can contain additional sequences such as a sequence that regulates vector replication in a host cell (eg, an origin of replication) or a selectable marker gene. Selectable marker genes facilitate the selection of vector-introduced host cells (see, eg, US Pat. Nos. 4,399,216, 4,634,665, 5,179,017). For example, a selectable marker gene confers a vector-introduced host cell with resistance to a drug (eg, G418, hygromycin, methotrexate). Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (used for selection / amplification with methotrexate in DHFR host cells), the neomycin resistance gene (for G418 selection), the glutamate synthase gene, and the like.

A nucleic acid molecule encoding an anti-CTLA-4 antibody and a vector containing the nucleic acid molecule can be used to transform suitable mammalian, plant, bacterial, or yeast host cells. The antibodies of the invention are obtained by making transgenic mammals or transgenic plants with respect to the heavy and light chain sequences of the immunoglobulins of interest and causing the animals or plants to produce the antibodies in a recoverable form. ..

Transformation is any known method of introducing a polynucleotide into a host cell (eg, wrapping the polynucleotide in a virus (or viral vector) and transforming the host cell with the virus (or vector)). , Or by a transfection method known in the art (eg, United States Patents No. 4,399,216, No. 4,912,040, No. 4,740,461, No. 4,959,455 have specific examples). Which transformation method is used depends on what the transforming host is. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the prior art, and specific examples include dextran-mediated transfection methods, calcium phosphate precipitation methods, polybrene-mediated transfection methods, and protoplast fusion. Methods include methods such as electroporation, particle implantation, polynucleotide encapsulation in liposomes, peptide conjugates, transfection, and microinjection of DNA directly into the nucleus.

Mammalian cell lines that can be used as hosts for expression are well known in the art, and specific examples include a large number of immortalized cell lines available from the American Standard Culture Collection (ATCC). For example, Chinese hamster ovary (CHO) cells, NS0 cells, healer cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2), and many others. It is a cell line. Non-mammalian cells (eg bacteria, yeast, insects, plants) can also be used to express recombinant antibodies. To prevent non-human glycosylation from altering immunogenic and / or pharmacokinetic function and / or effector function, antibody C_HIt is preferable to induce site-specific mutations in the two domains to eliminate glycosylation. The expression method is selected by examining which system has the highest expression level and which system produces an antibody having constitutive CTLA-4 binding properties.

In addition, expression of the antibody of the invention (or other portion from that antibody) by the producing cell line can be increased in a number of ways. For example, a system that expresses glutamine synthetase and the DHFR gene is a common method of increasing expression under certain conditions. Highly expressed cell clones can be identified using conventional methods (eg, dilution cloning method or microdroplet method). All or part of the glutamine synthetase system is described in European Patents 0 216 846, 0 256 055, 0 323 997 and European Patent Application 89303964.4.

When it comes to production in transgenic mammals, it is possible to generate antibodies in the milk of goats, cows, or other mammals and recover the antibodies from the milk. See, for example, United States Patents No. 5,827,690, No. 5,756,687, No. 5,750,172, No. 5,741,957.

The anti-CTLA-4 antibody expressed in the above cell lines can be purified and / or recovered from the relevant cell material. Antibodies may be present in whole cells, cell lysates, partially purified forms, or substantially pure forms. Purified by standard methods (eg alkali / SDS treatment, column chromatography or other methods well known in the art) and other cellular components or other contaminants (eg nucleic acids or proteins of other cells). ) Is removed. See Ausbel, F. et al., "Latest Protocols in Molecular Biology," Green Publishing &amp; Wily Interscience, New York, 1987.

In the present invention, the anti-CTLA-4 antibodies of the present invention expressed in various cell lines or transgenic animals may have different glycosylation patterns from each other. However, all anti-CTLA-4 antibodies encoded by the nucleic acids and amino acids described herein are considered to be part of the invention regardless of their glycosylation patterns, modifications or deletions. Therefore, for the purposes of the present invention, the anti-CTLA-4 antibody may or may not be glycosylated. If the anti-CTLA-4 antibody is glycosylated, it can take any possible glycosylation pattern. In addition, each heavy chain of one antibody may have the same glycosylation pattern, or the two heavy chains may have different glycosylation patterns. To prevent non-human glycosylation from altering immunogenic and / or pharmacokinetic function and / or effector function, antibody C_HIt is also included in the present invention to induce site-designated mutations in two domains to eliminate glycosylation.

As used herein, the term "glycosylation" means a pattern of hydrocarbon units that covalently bind to an antibody. When we say that the anti-CTLA-4 antibodies described herein have a particular glycosylation pattern, it means that most of the mentioned anti-CTLA-4 antibodies have that particular glycosylation pattern. As another feature, when the anti-CTLA-4 antibody described herein is said to have a particular glycosylation pattern, it is 50%, 75%, 90%, 95% of the mentioned anti-CTLA-4 antibody. It means that 99% or more, or 100%, has that particular glycosylation pattern.

Anti-CTLA-4 antibodies of the invention also include those with different glycosylation patterns (eg, insertion of glycosylation sites or deletion of any glycosylation sites by deletion, insertion, or substitution of appropriate amino acid residues). ).

Glycosylation of polypeptides is generally N- or O-linked. Glycosylation of antibody polypeptides is generally N-linked and forms a two-antenna structure. N-bonding means that the hydrocarbon moiety attaches to the side chain of the asparagine residue. The asparagine-X-serine and asparagine-X-threonine tripeptide sequences (where X is any amino acid other than proline) are sequences that recognize that the hydrocarbon moiety has been attached to the side chain of asparagine by an enzyme. Therefore, the presence of either of these tripeptide sequences in the antibody creates a potential glycosylation site.

The three different structures of the two-antenna glycans are indicated by "G0", "G1" and "G2" which have 0, 1 and 2 terminal galactose residues on the non-reducing end of the glycan, respectively. See Jefferis et al., Biochem. J., Vol. 268, pp. 529-537, 1990. In some cases, the glycan structure can also include a fucose residue attached to N-acetylglucosamine, which is covalently attached to an amino acid called asparagine (eg, position 297) in the antibody. In the presence of fucose (F), the name of the two-antenna glycan changes to "G0F", "G1F", "G2F" depending on the number of terminal galactose residues. Teillaud, Expert Opin. Biol. See Ther., Volume 5 (Supplement 1), pages S15-S27, 2005. In addition, if the antibody has both of the two heavy chains, the glycan name is repeated for each of the two heavy chains. The "G0F, G0F" glycoform is a chemical species in which G0 glycans are attached to both heavy chains, and each G0 glycan has a fucose (F) residue bound to N-acetylglucosamine. In the "G0F, G1F" glycoform, G0 glycan is attached to one of the heavy chains, G1 glycan is attached to the other heavy chain, and each of G0 glycan and G1 glycan is bound to N-acetylglucosamine (F). ) A chemical species with residues.

In some embodiments, the anti-CTLA-4 antibody was selected from the group consisting of "G0F, G0F", "G0F, G1F", "G1F, G1F", "G1F, G2F", and mixtures thereof. It has a glycosylation pattern. In another embodiment, the anti-CTLA-4 antibody has a glycosylation pattern of "G0F, G1F" in more than 50% of the antibodies produced. In another embodiment, the anti-CTLA-4 antibody has a glycosylation pattern of "G0F, G0F" in less than 50% of the antibodies produced. For example, in one embodiment, the anti-CTLA-4 antibody 11.2.1 described herein has a glycosylation pattern of "G0F, G0F" or "G0F, G1F". In some embodiments, the anti-CTLA-4 antibody (11.2.1) is produced as a mixture of different glycosylation patterns. For example, in one antibody (11.2.1) sample, there is an antibody (11.2.1) mixture in which the glycosylation pattern "G0F, G1F" and the glycosylation pattern "G0F, G0F" have a ratio of about 3: 2. Will do.

<u style="single">Route of administration and dosage:</u> The compositions of the present invention can be in solution (eg, solutions for injection or infusion). The preferred form depends on the planned dosage form and what is being treated. A typical preferred composition is in the form of a solution for injection or infusion (eg, a composition similar to that used for human passive immunity). Preferred dosage forms are parenteral administration (eg, intravenous administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intrasternal administration) or infusion in the form of bactericidal injections or oily suspensions. As will be appreciated by those skilled in the art, the route of administration and / or morphology will vary depending on the desired outcome. In a preferred embodiment, the antibody is administered intravenously by infusion or injection. In another preferred embodiment, the antibody is injected intramuscularly or subcutaneously.

Therapeutic compositions are generally sterile and stable under manufacturing and storage conditions.

The composition can be formulated as a solution, microemulsion, dispersion or liposome. Sterilization injection solutions can be prepared by incorporating the required amount of anti-CTLA-4 antibody into a suitable diluent with one or more of the above components after sterilization (eg, filtration sterilization) if necessary. Generally, the dispersion is prepared by incorporating the active compound into a sterilizing vehicle containing a basic dispersion medium and other necessary components selected from the above components. Such suspensions can be formulated by appropriately dispersing suitable wetting or suspending agents, or other acceptable bases, in a manner known in the art. The bactericidal injection formulation can be a bactericidal injection solution using a non-toxic parenteral acceptable diluent or solvent, a bactericidal injection suspension (eg, a solution using 1,3-butanediol). Acceptable vehicles and solvents that can be used include water, Ringer's solution, isotonic sodium chloride solution and the like. Furthermore, sterilizing non-volatile oils are commonly used as solvents or suspension media. For this purpose, inert non-volatile oils (eg synthetic monoglycerides and diglycerides) can be used. In addition, n-3 polyunsaturated fatty acids can also be used in the preparation of injections.

In the case of pasteurized powders for the preparation of pasteurized injection solutions, preferred preparation methods are vacuum drying, freeze drying, from the pre-sterilized and filtered solution, with the active ingredient and any other desired ingredient. A powder consisting of is obtained. Proper fluidity of the solution is maintained by using a coating such as lecithin, by ensuring the required particle size (in the case of dispersion), by using a surfactant. can do.

For long-term absorption of the injectable composition, the composition may contain a slowing agent (eg, monostearate or gelatin) or a long-term absorption form of the composition (eg, depot, liposome, etc.). Polymer microspheres, polymer gels, implants).

Other methods of administration of the antibodies described herein include skin patches that release the drug directly into the skin of the subject. Such patches may contain the antibodies of the invention in buffered solutions, dissolved and / or dispersed in adhesives, and dispersed in polymers. Can include.

Yet another method of administration of the antibodies described herein is ophthalmic droplets for the eye.

The antibody can be administered once, but more preferably multiple doses. For example, the antibody can be administered once daily to once every 6 months or longer. Administration is scheduled, for example, 3 times a day, 2 times a day, once a day, once every 2 days, once every 3 days, once a week, once every 2 weeks, 1 month It can be administered once every two months, once every three months, and once every six months.

The antibody can also be administered continuously through a minipump. The antibody can be administered to a tumor site or an inflamed site of the body, or to a tumor site or an inflamed site of the body, or to a site away from the tumor site or an inflamed site of the body. Antibodies may be administered once, or at least twice, or at least for the duration of treatment, alleviation, or cure of the disease. Antibodies can generally be administered as long as the tumor is present. Unless the tumor or cancer has stopped growing due to antibodies, the weight or volume of the tumor or cancer has decreased, or the inflamed area of the body has healed. The antibody will generally be administered as part of the pharmaceutical composition described above.

The composition of the present invention may contain a therapeutically effective amount or a prophylactically effective amount of the antibody or antigen-binding portion of the present invention. When preparing this composition, the therapeutically effective amount of anti-CTLA-4 antibody present in the composition is, for example, the desired dose and method of administration, the nature and extent of the disease to be treated, the age of the subject and It can be decided in consideration of the physique.

To exemplify the dose range of the pharmaceutical composition of the invention to be administered to a subject, milligrams (mg) of anti-CTLA-4 antibody administered per kilogram (kg) of body weight of the subject, from about 0.01 mg / kg to about 200 mg / kg. ), Approximately 0.1 mg / kg to approximately 100 mg / kg, approximately 1.0 mg / kg to approximately 50 mg / kg, approximately 5.0 mg / kg to approximately 20 mg / kg, and approximately 15 mg / kg. For the present invention, the average body weight when the subject is a human is about 70 kg.

An intermediate range of any doses described herein, such as about 0.01 mg / kg to 199 mg / kg, shall also be part of the present invention. For example, a range using any combination of the above values as the upper limit and / or the lower limit is also included in the present invention.

The dosing regimen should be adjusted to give the subject the desired optimal response (eg, therapeutic response, prophylactic response) by administering to the subject in several divided doses over time, or depending on the treatment and situational constraints. The dose can be gradually reduced or increased. Parenteral compositions are particularly desirable in the form of unit doses for ease of administration and constant dose.

As used herein, the form of a unit dose means a physically separated unit that is suitable as a unit dose for the mammal to be treated. Each unit contains a predetermined amount of activating compound calculated to produce the desired therapeutic effect and the required pharmaceutical base. The unit dose form specifications according to the invention address (a) the unique properties of the anti-CTLA-4 antibody or parts thereof and the specific therapeutic or prophylactic effect to be achieved, as well as (b) individual susceptibility. Therefore, it depends on the limitations inherent in the prior art in converting such antibodies into compounds.

The liquid formulations of the present invention can be in the form of unit doses. For example, a unit dose in a vial can contain 1 to 1000 milliliters (ml) of anti-CTLA-4 antibody at various concentrations. In another embodiment, the unit dose in the vial is about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml of anti-CTLA-4 antibody of various concentrations. , 40ml, 50ml, 100ml can be included. If necessary, a bactericidal diluent can be added to each vial to adjust the preparation to the desired concentration. The liquid formulations of the present invention can also be in the form of unit doses in sterilized bags or containers. This form is suitable for connecting to an intravenous administration line or catheter.

<u style="single">Evaluation of stability:</u> The present invention includes a stable pharmaceutical composition comprising the anti-CTLA-4 antibody described herein and a pharmacologically acceptable chelating agent. Stable compositions are resistant, for example, to maintaining the appearance and stability of the product, or to changes in the appearance and stability of the product (eg, the potential for physical and chemical degradation to reduce biological activity). It is desirable to have. Various analytical methods and indicators for measuring protein stability have been reported in the literature, and many such methods and indicators are described in Vincent Lee, "Drug Delivery of Peptides and Proteins" (Marcel Decker). Company, New York, NY, 1991), pp. 247-301, and Jones, A., Drug Delivery Rev., Vol. 10, pp. 29-90, 1993. In general, the liquid compositions of the present invention have improved stability when stored for periods of low temperature and / or after one or more freezing / thawing cycles.

In one embodiment, the chelating agent is contained when the composition is stored at a temperature of about 2 ° C to about 8 ° C for at least 12 months, preferably at least about 18 months, more preferably at least about 24 months. It is more stable than the same composition stored under the same conditions for the same period, except that it is not contained.

In another embodiment, the composition chelate when stored at a temperature of about 25 ° C to about 30 ° C for at least 3 months, preferably at least about 6 months, more preferably at least about 12 months. It is more stable than the same composition stored under the same conditions for the same period of time, except that it contains no agent.

In another embodiment, the composition comprises a chelating agent when stored at a temperature of about 40 ° C. for at least 1 month, preferably at least about 2 months, more preferably at least about 3 months. It is more stable than the same composition stored under the same conditions for the same period, except that it is absent.

In this specification, the term freeze / thaw cycle refers to the use of a liquid antibody sample after cryopreservation, so that the sample remains in a liquid state for a sufficient period of time after freezing the sample at 0 ° C or below. It means a method of returning to a recovery temperature and returning to a freezing temperature (preferably 0 ° C or lower). As used herein, the term "freeze storage" means to freeze a previously liquid antibody sample at a temperature of 0 ° C or lower, preferably -20 ° C or lower to maintain that condition.

In one embodiment, the compositions of the invention have at least one freeze / thaw cycle, preferably at least two freeze / thaw cycles, more preferably at least three freeze / thaw cycles, and even more preferably at least four. After a freezing / thawing cycle, more preferably at least 5 freezing / thawing cycles, and even more preferably at least 6 freezing / thawing cycles, the same composition except that it does not contain a chelating agent. It is more stable than those under the same freezing / thawing conditions.

In another embodiment, the composition satisfies two or more of the conditions shown below. (a) The composition contains a chelating agent when stored at a temperature of about 2 ° C to about 8 ° C for at least 12 months, preferably at least about 18 months, more preferably at least about 24 months. It is more stable than the same composition stored under the same conditions for the same period, except that it is not. (b) The composition contains a chelating agent when stored at a temperature of about 25 ° C to about 30 ° C for at least 3 months, preferably at least about 6 months, more preferably at least about 12 months. It is more stable than the same composition stored under the same conditions for the same period, except that it is not. (c) The composition is free of chelating agents when stored at a temperature of about 40 ° C. for at least 1 month, preferably at least about 2 months, more preferably at least about 3 months. It is more stable than the same composition stored under the same conditions for the same period; (d) The composition comprises at least one freezing / thawing cycle, preferably at least two freezing / thawing cycles, more preferably at least three freezing / thawing cycles, and even more preferably at least four freezing / thawing cycles. The same composition under the same freezing / thawing conditions, except that it does not contain a chelating agent, even more preferably after at least 5 freezing / thawing cycles, and even more preferably at least 6 freezing / thawing cycles. It is more stable than the one that was made.

In another embodiment, the composition meets three or more of the conditions set forth immediately above.

For the purposes of the present invention, for example, antibody aggregation and / or antibody fragmentation and / or discoloration of the composition can be used as indicators of composition stabilization. In general, the liquid pharmaceutical compositions of the present invention have the same composition under the same conditions when subjected to one or more of the above storage conditions or freezing / thawing conditions, except that they do not contain a chelating agent. At least one of antibody aggregation, antibody fragmentation, and discoloration of the composition is less than that of the one.

Agglutination of proteins in liquid pharmaceutical compositions can be measured by a variety of methods known in the art. As a method, there is gel filtration chromatography that separates proteins based on their molecular weight. A "gel" is a matrix of water and a polymer (eg, agarose or polymerized acrylamide). The present invention also includes the use of gel filtration HPLC (High Performance Liquid Chromatography). Another known method for measuring aggregation is cation exchange chromatography. This method is a general liquid chromatography method called ion exchange chromatography using an anion column. The cations exchanged in the present invention are from protein molecules. Since polyvalent protein aggregates may have a charge that is several times the pure charge of the single-stranded antigen-binding protein, the aggregate is retained more strongly to separate it from the single-stranded molecule. Can be done. A preferred cation exchanger is a polyaspartic acid column. The monomer protein can then be easily identified from the aggregate. However, those skilled in the art will appreciate that the aggregate assay of the present invention is not limited to a particular type of chromatographic column, provided that the two forms of the protein can be separated.

Fragments of proteins contained in liquid pharmaceutical compositions can be measured by a variety of methods known in the art. Such methods include, for example, size exclusion chromatography, ultraviolet detection (eg, detection at 214 nanometers), SDS-PAGE, matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Fragmentation of proteins and altered charge (which occurs, for example, as a result of deamidation) can be investigated, for example, by ion exchange chromatography or isoelectric focusing (IEF).

Discoloration of the composition is generally visible by visually observing the composition itself. Liquid pharmaceutical compositions of the present invention containing a chelating agent generally have less discoloration (eg, pink or yellow) of the composition as compared to the same composition except that it does not contain a chelating agent. And / or the clarity of the composition (eg, turbidity and / or cloudiness, and / or particle formation) is maintained. In the present invention, the term "discoloration" refers to a change in color (eg, from colorless and transparent to pink or yellow) and a change in transparency (eg, from colorless and transparent to turbid and / or cloudy, and / or with particles). Means both. Discoloration of the composition can generally be examined by other methods. It is examined, for example, by UV detection at 214 nanometers and / or by comparing compositions with and without chelating agents to standard color scales. See PhEr5.0, 2005 Monograph 2.2.2.

In one embodiment, antibody aggregation is examined after at least one of the following states. (a) The composition is stored at a temperature of about 2 ° C to about 8 ° C for at least 12 months, preferably at least about 18 months, more preferably at least about 24 months; (b) The composition is stored at a temperature of about 25 ° C to about 30 ° C for at least 3 months, preferably at least about 6 months, more preferably at least about 12 months; (c) The composition is stored at a temperature of about 40 ° C. for at least 1 month, preferably at least about 2 months, more preferably at least about 3 months; (d) The composition has at least one freeze / thaw cycle, preferably at least two freeze / thaw cycles, more preferably at least three freeze / thaw cycles, and even more preferably at least four freeze / thaw cycles. , More preferably at least 5 freeze / thaw cycles, and even more preferably at least 6 freeze / thaw cycles. The aggregated antibody is then chromatographically separated from the monomer (eg, utilizing HPLC) and the resulting chromatogram reveals the degree of aggregation. The peak area of aggregates on the chromatogram in the stable liquid pharmaceutical composition of the present invention is generally less than about 6%, less than about 5%, less than about 4%, about 3 of the total peak area on the chromatogram. Less than%, less than about 2%, less than about 1.5%. In a special example of this method of measuring aggregation, the composition is stored at 40 ° C. for 24 weeks, then separated by chromatography using SE-HPLC, and UV detection at 214 nanometers is performed. Using this method, antibody aggregation was measured in Example 11. In Example 11, for example, formulation number 37 (containing chelating agent) had a peak area of aggregates on the chromatogram of about 1.1%, whereas formulation number 26 (without chelating agent) The peak area of the aggregate on the chromatogram was about 6.4%.

In general, the peak area of agglomerates on a chromatogram in the stable liquid pharmaceutical composition of the present invention and the peak of agglomerates in a chromatogram under the same conditions of the same composition except that it does not contain a chelating agent. The area difference is at least about 2%, at least about 3%, at least about 4%, or at least about 4.5%. For example, formulation No. 37 (the area of the peak of the aggregate on the chromatogram is about 1.1%) and formulation No. 26 (the area of the peak of the aggregate on the chromatogram is about 6.4%) tested in Example 11 as described above. This difference is about 5.3%.

In another embodiment, antibody fragmentation is examined after the composition is in one or more of the states shown below. (a) The composition is stored at a temperature of about 2 ° C to about 8 ° C for at least about 12 months, preferably at least about 18 months, more preferably at least about 24 months; (b) The composition is stored at a temperature of about 25 ° C to about 30 ° C for at least about 3 months, preferably at least about 6 months, more preferably at least about 12 months; (c) The composition is stored at a temperature of about 40 ° C. for at least about 1 month, preferably at least about 2 months, more preferably at least about 3 months; (d) The composition has at least one freeze / thaw cycle, preferably at least two freeze / thaw cycles, more preferably at least three freeze / thaw cycles, and even more preferably at least four freeze / thaw cycles. , More preferably at least 5 freeze / thaw cycles, and even more preferably at least 6 freeze / thaw cycles. The antibody fragments are then separated from the composition by chromatography (eg, utilizing gel filtration) and the degree of fragmentation is determined from the resulting chromatogram. The volume of the band of fragments on the chromatogram in the stable liquid pharmaceutical composition of the present invention is generally less than about 9%, less than about 8%, less than about 7%, about 6 of the total volume of the bands on the chromatogram. Either less than%, less than about 5%, or less than about 4.5%. A special example of this method of measuring fragmentation is that the composition is stored at 40 ° C for 24 weeks and then separated using reduced SDS-PAGE (rSDS-PAGE) for molecular dynamics personal PDQC-. The volume of the band is revealed by scanning with a 90 photographic densitometer or a Bio-Rad GS800 imaging photographic densitometer. Using this method, antibody fragmentation was measured in Example 11. In Example 11, for example, formulation number 37 (containing chelating agent) had a fragment band volume of about 4.5% on the chromatogram, whereas formulation number 26 (without chelating agent) had a chromatogram. The volume of the upper fragment band was about 10.1%.

In general, the difference between the volume of the fragment band in the stable liquid pharmaceutical composition of the present invention and the volume of the fragment band of the same composition under the same conditions except that it does not contain a chelating agent is at least about 2%, at least about about. Either 3%, at least about 4%, or at least about 5%. This difference between formulation number 37 (volume of fragment band on chromatogram is about 4.5%) and formulation number 26 (volume of fragment band on chromatogram is about 10.1%) tested in Example 11 as described above is , About 5.6%.

<u style="single">Treatment:</u> Any type of antibody described herein may be used therapeutically. In a preferred embodiment, the anti-CTLA-4 antibody is a human antibody. In another preferred embodiment, CTLA-4 is of humans and the subject is a human patient. In yet another preferred embodiment, the anti-CTLA-4 antibody is a human IgG2 antibody. Alternatively, the subject can be a mammal expressing the CTLA-4 protein with which the anti-CTLA-4 antibody cross-reacts. For veterinary medicine or as a model animal for human disease, the antibody may be administered to a non-human mammal (eg, a primate) that expresses CTLA-4 with which the antibody cross-reacts. Such model animals are useful in assessing the therapeutic effect of antibodies according to the invention.

The present invention is a method of treating a neoplastic disease in a subject, wherein the subject is an anti-CTLA-4 antibody; a chelating agent alone, or a chelating agent and other excipients (eg, buffers, tonics, surfactants, etc.) Methods are provided that include the operation of administering a liquid pharmaceutical composition comprising a combination of these). In yet another embodiment, the subject is a subject in need of prevention or treatment of a neoplastic disease.

In another embodiment of the invention, a method of treating a neoplastic disease of a subject, the subject to which is an anti-CTLA-4 antibody thicilimumab; a chelating agent alone, or a chelating agent and other excipients (eg,). Methods are provided that include the operation of administering a liquid pharmaceutical composition comprising a pharmacologically acceptable excipient comprising a combination of a buffer, a tonic, a surfactant, a mixture thereof). ..

The terms "neoplasty" and "neoplasty" both refer to benign, premalignant, metastatic, malignant "neoplasms" or tumors. The present invention also includes benign, premalignant, metastatic and malignant neoplasias. The present invention also includes benign, premalignant, metastatic and malignant tumors. Therefore, benign, premalignant, metastatic, malignant neoplasias or tumors are all included in the present invention and can all be referred to as neoplasias, neoplasms, neoplasia-related diseases. Tumors are generally known to be neoplastic masses or "neoplastic" cells. But even a single neoplastic cell is considered to be a neoplasia or neoplasia in the present invention.

Neoplastic diseases that can be treated with the anti-CTLA-4 antibodies of the invention may include cancers of any tissue or organ. For example, bone tumors, brain cancers, lung cancers, squamous cell carcinomas, bladder cancers, stomach cancers, pancreatic cancers, breast cancers, head cancers, neck cancers, liver cancers, kidney cancers, ovarian cancers, and prostate cancers. Cancer, colorectal cancer, esophageal cancer, gynecological cancer (eg cervical cancer and ovarian cancer), nasopharyngeal cancer, thyroid cancer. The term neoplastic disease also includes bone metastases, melanomas, lymphomas, leukemias, and multiple myeloma. In particular, the anti-CTLA-4 antibody preparation of the present invention is useful for the treatment of breast cancer, prostate cancer, colorectal cancer, and lung cancer.

In another embodiment, the methods and compositions of the present invention are used to prevent and treat neoplastic diseases selected from the groups shown below. The group includes terminal melanoma, photokeratosis, adenocarcinoma, glandular cyst cancer, adenomas, familial adenomatous polyposis, familial polyps, colon polyps, polyps, adenocarcinoma, glandular squamous cell carcinoma, glands. Cortical cancer, AIDS-related lymphoma, anal cancer, stellate cell tumor, baltrin adenocarcinoma, basal cell cancer, bile duct cancer, bladder cancer, brain stem gliome, brain tumor, breast cancer, bronchial adenocarcinoma, capillary cancer, cartinoid, cancer, Faropius canal cancer, endometrial cancer, carcinosarcoma, spongy lymphoma, central nervous system lymphoma, encephalocytes, bile duct cancer, chondrosarcoma, choroidal papilloma / cancer, clear cell cancer, skin cancer , Brain tumor, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, cyst adenoma, endometrial sinus tumor, endometrial proliferative disease, endometrial stromal sarcoma, endometrial adenocarcinoma, coat cell cancer, etc. Epithelial cell cancer, esophageal cancer, Ewing sarcoma, extragonadal germ cell tumor, fibroplate hyperplasia, focal nodular hyperplasia, biliary sac cancer, gastrinoma, germ cell tumor, fertility membrane tumor, polymorphic glia Blast cell tumor, gliome, glucagonoma, hemangioblastoma, vascular endothelial tumor, hemanoma, hepatocellular gland type, hepatocellular adenopathy, hepatocellular carcinoma, hodgkin lymphoma, pharyngeal laryngeal cancer, hypothalamic gliome, tract gliome , Insulinoma, intraepithelial neoplasia, interepithelial squamous cell neoplasia, intraocular melanoma, invasive squamous cell carcinoma, large cell carcinoma, islet cell carcinoma, capoic sarcoma, kidney cancer, pharyngeal cancer, smooth muscle tumor, malignant Kuroko melanoma, leukemia-related diseases, lip cancer, oral cancer, liver cancer, lung cancer, lymphoma, malignant mesenteric tumor, malignant thoracic adenoma, myelblastoma, myelin epithelioma, melanoma, meningeal cancer, Mercer Cellular cancer, mesenteric cancer, metastatic cancer, mucocutaneous epidermoid cancer, multiple myeloma / plasma cell neoplasia, mycobacterial sarcoma, myelodystrophy syndrome, myeloid proliferation disease, nasal cavity cancer, sinus cancer, nasopharyngeal , Neuroblastoma, Neuroepithelial line cancer, Nodular melanoma, Neoplasia of the central nervous system (eg, primary CNS lymphoma, spinal tumor, brain stem gliome, pituitary gland type), non-hodgkin lymphoma, swallow cell cancer, Rare glial cell cancer, oral cancer, oropharyngeal cancer,

In one more preferred embodiment, the anti-CTLA-4 antibody is administered to a subject suffering from breast cancer, prostate cancer, lung cancer, colon cancer. In a more preferred embodiment, this method stops the abnormal growth of the cancer, prevents the weight or volume of the cancer from increasing, or reduces the weight or volume of the cancer.

<u style="single">Manufacturing equipment:</u> In another embodiment of the invention, a manufacturing apparatus is provided. This manufacturing apparatus is a liquid containing at least one monoclonal anti-CTLA-4 antibody of the present invention in a preparation containing only a chelating agent or a combination of a chelating agent and another pharmacologically acceptable excipient. It is equipped with a container for storing pharmaceutical preparations. Suitable containers include, for example, bottles, vials, bags, syringes and the like. Containers can be made of a variety of materials (glass and plastic). An example of a container is a 3-20 cc disposable glass vial. Alternatively, in the case of multiple dose formulations, the container may be a 3-100 cc glass vial. The container contains the formulation and can be affixed to the surface of the container or labeled with instructions on the label attached to the container. The manufacturing equipment also includes other materials that are desirable from a product and user perspective (eg, other buffers, diluents, filters, needles, syringes, package attachments with instructions, contraindications, possible side effects). Can be prepared.

According to the present invention, a first container containing a solution of monoclonal anti-CTLA-4 antibody 11.2.1 and a sufficient amount of chelating agent to stabilize the antibody alone or other chelating agents are modified. A kit for preparing a stabilized antibody liquid composition with a second container containing the agent in combination is also provided.

Embodiments of the present invention will be described in the following examples. Other embodiments within the scope of the claims of the invention will be apparent to those skilled in the art from the detailed description or embodiments of the invention disclosed herein. The detailed description and examples are merely examples, and the scope and spirit of the present invention are set forth in the appended claims. In the examples, all% values are weight% unless otherwise noted. One of ordinary skill in the art can convert the weight and / or weight to volume ratios described in the Examples to molars and / or molar concentrations using the molecular weights known for the components described herein. Yeah. The weight (eg, grams) specifically shown herein is a value for the stated volume (eg, buffer, antibody formulation, etc.). Those skilled in the art will appreciate that the weight can be changed proportionally when the volume of the formulation is desired to be a different value.

<p><u style="single">Example 1</u> In this example, the production of a hybridoma cell line that produces an anti-CTLA-4 antibody will be described. At that time, it shall be as described in US Pat. No. 6,682,736 granted to Hanson et al.</p><p> The antibody of the present invention was prepared, selected and examined as follows.</p><p><u style="single">Antigen preparation:</u>Three different immunogens were prepared to immunize Xenomouse®. That is, it was prepared from cells transfected with (i) CTLA-4-IgG1 fusion protein, (ii) CTLA-4 peptide, and (iii) CTLA-4 mutant (Y201V) constitutively expressed on the cell surface. 300.19 Mouse lymphoma cells.</p><p>CTLA-4-IgG1 fusion protein:<u style="single">Expression vector composition</u> The extracellular domain of CTLA-4 from the human thymic cDNA library (Clontech) using primers designed according to published sequences (Eur. J. Immunol., Vol. 18, pp. 1901-1905, 1988). The cDNA encoding the above was amplified by PCR. A directional subcloning of the fragment was performed to obtain the human oncostatin M signal peptide and human IgGγ1 (IgG1) C in pSR5 (Sindbis virus expression plasmid (Invitrogen))._H1 / C_H2 / C_HI put it between 3 domains. This fusion protein does not contain a hinge region, but contains cysteine 120 in the extracellular domain of CTLA-4 to form a covalently bound dimer. The resulting vector was named CTLA-4-IgG1 / pSR5. The complete CTLA-4-IgG1 cDNA sequence within the vector was confirmed for both strands. The amino acid sequence of CTLA-4-Ig protein is shown below. The mature extracellular domain of CD44 was amplified by PCR from a human lymphocyte library (Clontech), subcloned into pSinRep5, and produced the same control protein as IgG1 in the tail.</p><p><chemistry num="1"><img file="JP2012167120A_D0003.tif" /></chemistry></p><p> The mature extracellular domain of CD28 was amplified by PCR from the human lymphocyte library (Clontech) and then subcloned into pCDM8 (J. Immunol., Vol. 151, pp. 5261-5271, 1993) produced a human IgG1 fusion protein containing both the thrombin cleavage region and the hinge region. Marmosets, cynomolgus monkeys, and rhesus monkeys CTLA-4 were cloned from mRNA isolated from PHA-stimulated PBMCs using standard degenerate PCR methods. Sequencing revealed that the amino acid sequences of rhesus monkeys and cynomolgus monkeys differed from the extracellular domain of mature human CTLA-4 in three places (S13N, I17T, L105M, G106S). Marmosets found that the extracellular domain of mature human CTLA-4 differed by 10 amino acids (V21A, V33I, A41T, A51G, 541, S71F, Q75K, T88M, L105M). Using site-specific mutagenesis, point mutations were made in all different amino acids in marmoset CTLA-4, mapping amino acids important for antibody-human CTLA-4-IgG interaction. To map the epitope, human and marmoset CTLA-4-IgG was mutated by matchmaker site-designated mutagenesis (Promega). Cos7 cells were run to produce a transiently transfected IgG fusion protein and purified using standard protein A methods. Immune blotting and BIA core analysis were used to examine the binding of the mutant CTLA-4-IgG protein to the antibody.</p><p><u style="single">Expression / purification of recombinant protein</u> Electroporation of SP6 CTLA-4-IgG1 / pSR5 mRNA and DH-26S helper mRNA transcribed in vitro as described by Invitrogen into baby hamster kidney cells (Gibco) ) To make a recombinant Sindbis virus. After 48 hours, the recombinant virus was recovered and titrated to optimize protein expression in Chinese hamster ovary cells (CHO-K1). CHO-K1 cell culture was heat-inactivated with 10% fetal bovine serum (Gibco), non-essential amino acids (Gibco), 4 mM glutamine (Gibco), and penicillin / streptomycin (Gibco). Was suspended in DMEM / F12 (Gibco) containing 10 mM Hepes (pH 7.5) (Gibco). 1 × 10 cells per ml of CHO-K1 cells to produce CTLA-4-IgG⁷It was resuspended in DMEM / F12 in proportions and incubated with Sindbis virus for 1 hour at room temperature. Next, 1% fetal bovine serum deficient in bovine IgG using Protein A Sepharose (Pharmacia), non-essential amino acids, 4 mM glutamine, 12.5 mM hepes (pH 7.5), and penicillin / streptomycin. 1 × 10 cells per ml of CHO-K1 cells in DMEM / F12 containing⁶Diluted into pieces. Pellet cells 48 hours after infection, collect break-in medium, supplement with break-in medium protease inhibitor tablets (Beringer-Manheim), adjust pH to 7.5, 0.2 μm membrane (0.2 μm membrane) The fusion protein was affinity-purified at a flow rate of 10 ml / min using FPLC (Pharmacia) using a 5 ml Protein A High Trap Column (Pharmacia). The column was washed with 30 times the volume of PBS on the floor and eluted with 0.1 M glycine / HCl (pH 2.8) at 1 ml / min. The fraction (1 ml) was immediately neutralized with Tris (pH 9) to a pH of 7.5. Fractions containing CTLA-4-IgG1 were identified by SDS-PAGE, then concentrated using Centriplus 50 (Amicon) and then placed on a Sepharose 200 column (Pharmacia) at 1 ml / min using PBS as a solvent. Filled. Fractions containing CTLA-4-IgG1 were pooled, sterilized and filtered through a 0.2 μm membrane (Millipore), aliquoted and frozen at -80 ° C. CD44-IgG1 was expressed and purified in the same manner. CD28-IgG1 was purified from conditioned medium from transiently transfected Cos7 cells.</p><p><u style="single">CTLA-4-IgG1 characterization:</u> Purified CTLA-4-IgG1 was transferred as a single band on SDS-PAGE with a colloidal Coomassie dye (Novex). Under non-reducing conditions, CTLA-4-IgG1 was a dimer (100 kDa). When it was treated with 50 mM DTT, it was reduced to a 50 kDa monomer. Amino acid sequencing of a solution of purified CTLA-4-IgG1 confirmed the amino terminus of CTLA-4 (MHVAQPAVVLAS) and cleaved the oncostatin M signal peptide from the mature fusion protein. CTLA-4-IgG1 bound to immobilized B7.1-IgG1 in a concentration-dependent manner, and the binding was blocked by a hamster anti-human anti-CTLA-4 antibody (BNI3: Farmingen). Since sterilized CTLA-4-IgG1 does not contain endotoxin, it was quantified by OD280 with an extinction constant of 1.4. Yields of purified CTLA-4-IgG1 ranged from 0.5 to 3 mg per liter of CHO-K1 cells.</p><p><u style="single">CTLA-4 peptide:</u> The following CTLA-4 peptides were prepared as described below.<chemistry num="2"><img file="JP2012167120A_D0004.tif" /></chemistry></p><p><u style="single">Abbreviation / Material:</u> NMP: N-methylpyrrolidone; TFE: 2,2,2-trifluoroethanol; DCM: dichloromethane; FMOC: fluorenylmethoxycarbonyl. All reagents except TFE (Oldrich Chemical); FMOC-PAL-PEG resin (Perceptive Biosystems) were supplied by PerkinElmer. For amino acids that require side chain protecting groups, Fmoc-Arg (PMC) -OH; Fmoc-Asn (Trt) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc- Glu (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-His (Boc) -OH, Fmoc-Lys (BOC) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu)- OH and Fmoc-Tyr (tBu) -OH were used.</p><p><u style="single">Peptide synthesis:</u> Peptides were synthesized on Perkin-Elmer 431A modified for feedback monitoring through UV absorbance at 301 nm (Perkin-Elmer model 759A detector). Peptide sequences were assembled on FMOC-PAL-PEG resin utilizing a conditioned double coupling cycle. Forced double coupling was performed in 10 cycles, 11 cycles, 18 cycles, 19 cycles, 20 cycles, and 28-33 cycles. At the end of each acylation cycle, the resin was washed with a mixture of 50% DCM and 50% TFE, and then the unreacted amino groups were capped with acetic anhydride in NMP. After the 49th cycle was completed, the resin was removed from the reactor and the rest was reacted to the end. Cleavage of the peptide from 415 mg of resin over 6 hours using Reagent K (King et al., International Journal of Protein and Peptide Research, Vol. 36, pp. 255-266, 1990) yielded 186 mg of crude CTLA-4 peptide. Was done.</p><p><u style="single">Peptide characterization:</u> Aliquot 25 mg of crude CTLA-4 peptide 6M guanidine HCl / 100 mM K₂PO₃Dissolve in 5 ml (pH 6.4), 2M guanidine HCl / 100 mM K₂PO₃Pharmacia High Road Superdex 75 at 2 ml / min using (pH 6.4) Elution with a 16/60 column (16 mm x 600 mm, floor volume 120 ml) for 180 minutes yielded 5 ml of fraction. This fraction was analyzed by placing 1.7 μl of this fraction on a NuPAGE layered gel with a MES running buffer and visualizing it with Daiichi silver staining protocol. This fraction was found to have a molecular weight of 12 kDa by comparison with the molecular weight standard. The fractions were combined and stored at 4 ° C. The combined fractions were analyzed by UV and gel electrophoresis. Amino acid sequences were sequenced by absorbing 100 μl of the sample into a prosorb cartridge (absorbed by a PVDF membrane) and washing to remove salt from the buffer. Sequencing was performed with Applied Biosystems 420 Sequencer. The expected N-terminal sequence (MHVAQPAVVLA) was observed. Immunoblotting revealed that this peptide was recognized by the BNI3 anti-human CTLA-4 antibody (Farmingen). 3500 Da aliquot containing 648 μg material to remove salt It was placed in a MWCO dialysis tube and dialyzed against 0.1% TFA / H20 at 4 ° C for 9 days with stirring. The entire contents of the dialysis bag were lyophilized to a powder.</p><p><u style="single">300.19 cells transfected with CTLA-4 (Y201V) peptide antigen:</u> Full-length CTLA-4 cDNA was amplified by PCR from the human thymic cDNA library (Stratagene), subcloned into pIRESneo (Clontech). A matchmaker mutagenesis system (Promega) was used to introduce mutations in CTLA-4 constitutively expressed on the cell surface. The mutation that turns tyrosine Y201 into valine blocks the binding of the adaptin protein AP50, which is important for the rapid internalization of CTLA-4 (Chuang et al., J. et al. Immunol., Vol. 159, pp. 144-151, 1997). 300.19 mouse lymphoma cells without mycoplasma, 10% fetal bovine serum, non-essential amino acids, penicillin / streptomycin, 2 mM glutamine, 12.5 mM hepes (pH 7.5), 25 μM β-mercaptoethanol Cultivated in RPMI-1640 containing. In a 1 ml chamber, cells were electroporated with 20 μg CTLA-4-Y201V / pIRES neo using 200V / 1180μF (Gibco cell polator). After allowing the cells to stand for 10 minutes, 8 ml of pre-warmed complete RPMI medium was added. After 48 hours, 0.5 × 10 cells in complete RPMI medium containing 1 mg / ml G418 (Gibco).⁶Diluted to / ml. Resistant cells were grown and CTLA-4 was expressed on the cell surface using a BNI3 antibody conjugated to phycoerythrin. Highly expressed cells were isolated by aseptic sorting.</p><p><u style="single">Immunization and hybridoma production:</u> Xenomouse® (8-10 weeks old) was immunized. To do so, (i) 1 × 10 in which CTLA-4 was expressed by transfection as described above.⁷300.19 cells are resuspended in phosphate buffered saline (PBS) containing a complete Freund's adjuvant and administered subcutaneously to the base of the tail, or (ii) (a) 10 μg CTLA-4 fusion protein, Alternatively, (b) an emulsion of 10 μg CTLA-4 peptide and complete Freund's adjuvant was subcutaneously administered to the base of the tail. In each case, administration with an incomplete Freund's adjuvant was repeated 3 or 4 times. Four days prior to fusion, mice were given a final injection of immunogen, ie, PBS containing cells. Spleens and / or lymph nodes from immunized mice were fused to the mouse non-secretory myeloma P3 cell line and HAT selected as previously reported (Galfre, G. and Milstein, C., " Preparation of Monoclonal Antibodies: Strategies and Procedures, Methods Enzymol., Vol. 73, pp. 3-46, 1981). Large numbers of hybridomas are all CTLA-4 specific human IgG₂The K antibody was secreted and the hybridoma was recovered.</p><p> The following hybridomas producing anti-CTLA-4 antibodies were deposited on April 29, 2003 in the American Standard Culture Collection (10801 University Boulevard, Manassas, Virginia 20110-2206).</p><p><u style="single">clone</u><u style="single">Subclone</u><u style="single">ATCC deposit number</u> 11.2.1 11.2.1.4 PTA-5169 4.1.1 4.1.1.1 PTA-5166</p><p><u style="single">Example 2</u> This example describes the production of recombinant mammalian cell lines that produce anti-CTLA-4 antibodies.</p><p> The DNA encoding the heavy and light chains of the monoclonal antibody 11.2.1 was cloned from the respective hybridoma cell line 11.2.1, and the DNA sequence was clarified by a method known in the prior art. From the nucleic acid sequence of antibody 11.2.1 and the expected amino acid sequence, the genes used in each antibody chain were clarified.</p><p> Next, the 11.2.1 DNA sequence insert was subcloned into an expression vector. This expression vector was then transfected into mouse myeloma (NS0) host cells to create a variety of primary transfectant cell lines producing anti-CTLA-4 antibodies. Lead cell lines were selected based on the results of growth and productivity analysis. The lead cell line was later subcloned to create a cloned cell line.</p><p> Using this cell line, anti-CTLA-4 antibody was produced in cell culture in a bioreactor containing a cell medium. During production, the medium is supplemented with nutrients. After reaching the recovery criteria, recovery from the bioreactor was performed by filtration alone or by filtration after centrifugation. The clear supernatant was then purified in three stages by chromatography with a protein A affinity column and two ion exchange columns. Low pH inactivation and virus filtration were also performed to remove any potential virus left during the process. The product was concentrated and dialytically filtered into a formulation buffer to make the drug substance.</p><p><u style="single">Example 3</u> Experiments were conducted to investigate the effects of four different buffers on antibody aggregation and fragmentation. In particular, four liquid formulations were prepared containing the anti-CTLA-4 antibody 11.2.1 and having a buffer solution of either acetate, succinate, histidine or EDTA. These formulations were then stored at 40 ° C and antibody aggregation and fragmentation were measured at 0, 2, 5, and 7 weeks.</p><p><u style="single">Preparation of buffer solution:</u> Four buffer solutions were prepared as shown in Table 3. Each solution was first prepared by dissolving a certain amount of buffering chemicals (listed in Table 3) in water (approximately 80% of the target value). The pH of each buffer solution was adjusted to 5.5 by adding a sufficient amount of the acidic solution or basic solution shown in Table 3. After adjusting the pH, more water was added to bring the final concentration of the solution to 20 mM. The buffer concentration was set to 20 mM to ensure that the pH was sufficiently stable at the selected pH of 5.5. The buffer was then sterilized and filtered (pore size 0.22 micron) and placed in a sterilized container for later use.</p><p><tables num="3"><img file="JP2012167120A_D0005.tif" /></tables></p><p> A 1% v / v glacial acetic acid solution was prepared by appropriately diluting glacial acetic acid (99.9%) with water (1 ml-100 ml). A 1 mol (M) sodium hydroxide solution was prepared by dissolving 40 g of solid sodium hydroxide in 1 liter of water. A 5 mol (M) hydrochloric acid solution was prepared by appropriately diluting concentrated hydrochloric acid (37.8%) with water.</p><p><u style="single">Preparation of antibody preparations:</u> The antibody preparations examined are listed in Table 4 below. To prepare each formulation, a certain amount of tonic (indicated in mg / ml as a unit in Table 4) was first added to the buffer solution shown in the table and the solution was stirred until the tonic was dissolved. .. From the purification process described in Example 2, a solution containing 13.2 mg / ml of bulk antibody in 20 mM sodium acetate buffer (pH 5.5) + 140 mM sodium chloride was obtained. The buffer solution of this bulk solution was replaced with the above formulation solution. At that time, the Amicon Ultra 15 MWCO 10K (UFC901024) centrifuge and concentrator attached to the Beckman Coulter Allegra 21R centrifuge was used at 6500 rpm at 5 ° C. Used in. Approximately 8 volume integrals were exchanged and the antibody solution was concentrated to 27-30 mg / ml. Approximately 3-4 ml of formulations 1-18 were prepared. The antibody concentration is determined by the ultraviolet-visible spectroscopy (UV-Vis) method and has an extinction coefficient of 1.43 (mg / ml) at 280 nm.^-1cm^-1It was decided using the value.</p><p> A concentrated solution of 20 mg / ml PS80 was prepared by diluting and dissolving polysorbate 80 (PS80) with the appropriate buffer prepared as described above. Next, an appropriate amount of buffer, antibody, tensioning agent, and water are added to the antibody buffer solution together with PS80 as a 20 mg / ml concentrate, and a preparation containing a monoclonal anti-CTLA-4 antibody solution having a final concentration of 20 mg / ml. Got The formulation corresponds to the compositions in Table 4 shown below.</p><p> For formulation number 2 in Table 4, PEG3350 was added as a 200 mg / ml concentrate at this point.</p><p> The formulation was then filtered through a sterile grade 0.22 micron filter and placed in a vial. A 2 ml type glass vial was filled with 1.5 to 1 ml. The vial is capped with Daikyo Seiko 777-1 Flurotech® coated, crimp-sealed, placed in a stable chamber and upright at 40 ° C for 2 weeks, 5 weeks, 7 weeks. Stored over. The vials were washed and steam sterilized, as did the 13 mm Daikyo Seiko 777-1 serum stopper. The degree of agglutination and fragmentation was immediately analyzed for the two prepared vials.</p><p><tables num="4"><img file="JP2012167120A_D0006.tif" /></tables></p><p><u style="single">Analysis of agglomeration:</u> The antibody preparations in Table 4 were stored at a temperature of 40 ° C. Aggregation of each formulation was analyzed using size exclusion chromatography (SEC) at 0, 2, 5, and 7 weeks. Size exclusion chromatography was performed using a TSK gel G3000SWXL-G2000SWXL column and a 0.2 M sodium phosphate (pH 7.0) mobile phase at a flow rate of 1 ml / min and UV detection at 214 nm. Figure 1 shows the percentage of eluted high molecular weight species (ie, the aggregate of monoclonal anti-CTLA-4 antibody 11.2.1) measured at the time of description for each formulation. The level of aggregation was calculated by integrating the area below the peak for the chromatogram of each formulation, and the integrated value of the area below the peak of the high molecular weight species was displayed as a percentage of the total peak area (Fig. 1). checking). As can be seen from FIG. 1, the composition in which the buffer solution was EDTA had the lowest aggregation level, and then the buffer solution was histidine, acetate, and succinate in order from the lowest.</p><p><u style="single">Fragmentation analysis:</u> As pointed out above, the antibody preparations in Table 4 were stored at a temperature of 40 ° C. Fragmentation of each formulation was also analyzed using rSDS-PAGE at 0, 2, 5, and 7 weeks. Analysis by rSDS-PAGE was performed using NuPAGE 4-12% bis-tris gel and colloidal blue dye (Coomassie). The reduced gel (rSDS-PAGE) was reduced with a NuPAGE® reducing agent. The total amount of impurities to hydrolyze (ie, fragments of monoclonal anti-CTLA-4 antibody 11.2.1) is estimated by scanning with a Molecular Dynamics Personal PDQC-90 photographic densitometer or Bio-Rad GS800 Imaging photographic densitometer. did. FIG. 2 shows the rate of fragmentation measured at the time of description for each formulation. The level of fragmentation was calculated as a percentage of the total band volume (see Figure 2). As can be seen from FIG. 2, the composition in which the buffer solution was EDTA had the lowest fragmentation level, and then the buffer solution was histidine, acetate, and succinate in order from the lowest.</p><p> Graphs in Tables 5 (a) (0 weeks), 5 (b) (2 weeks), 5 (c) (5 weeks), 5 (d) (7 weeks) below, and in Figures 1 and 2. The data of aggregation and fragmentation shown as are shown.</p><p><tables num="5"><img file="JP2012167120A_D0007.tif" /></tables></p><p><tables num="6"><img file="JP2012167120A_D0008.tif" /></tables></p><p><tables num="7"><img file="JP2012167120A_D0009.tif" /></tables></p><p><tables num="8"><img file="JP2012167120A_D0010.tif" /></tables></p><p><u style="single">Example 4</u> Experiments were conducted to examine the ability of various liquid formulations containing the monoclonal anti-CTLA-4 antibody 11.2.1 to withstand multiple freeze / thaw cycles.</p><p> Often, the ability of liquid formulations to withstand multiple freeze / thaw cycles is examined. This is to keep the formulation frozen (take it to another location if desired) and later thaw it to see if it can be used.</p><p> The investigated formulations are shown in Table 6 below. The procedure used to prepare the formulation is the same as described in Example 3. 2.5 ml of each solution was placed in a 5 ml type glass vial, stoppered and sealed. The preparations having the numbers 1 to 4, 7 to 8, 11 to 12, and 15 to 16 are the same as the preparations having the same identification number in Example 3.</p><p><tables num="9"><img file="JP2012167120A_D0011.tif" /></tables></p><p> Each formulation was subjected to 6 freeze / thaw cycles. The first three cycles were performed in a speed-controlled freezer. The latter three cycles were slower cycles and were performed using a large number of water-filled vials to accommodate the heavy heat load in the freezer or refrigerator. In cycles 1, 2 and 3, the vials containing the formulation were placed in a speed-controlled freezer (Plana Cryo 560-16) and experienced the following cycles: formulation at a rate of 0.2 ° C / min- Cool to 70 ° C, maintain -70 ° C for 1.5-3 hours, and thaw the formulation at a rate of 0.3 ° C / min until the temperature reaches 5 ° C. In cycles 4, 5 and 6, these vials and other water-filled vials (one sample vial for each formulation; 17 formulation vials plus a total of approximately 30 vials filled with water. ) Was put in the box. The box was then first placed in a freezer with a temperature of -70 ° C for about 17 hours and then in a refrigerator with a temperature of 2-8 ° C for about 50 hours. Using a thermal probe for recording installed in the box, the average cooling rate of 0.09 ° C / min in the freezing process and the average heating rate of 0.03 ° C / min in the thawing process were measured.</p><p> Each formulation was visually evaluated at the end of each freezing / thawing cycle for particle formation, color change, and turbidity change. Such observations of each formulation by eye were performed in a light box against a black and white background while the formulation was still cold with each thawing. The results are shown in Table 7 (presented below).</p><p><tables num="10"><img file="JP2012167120A_D0012.tif" /></tables></p><p> Formulations containing only sodium chloride (ie, chlorine ions) had more soluble particles after the freeze / thaw cycle than preparations containing any of trehalose, sucrose, or sorbitol. However, when PEG was added to the sodium chloride-containing formulation, the levels of soluble particles measured after the freeze / thaw cycle appeared to be lower than the corresponding PEG-free formulation.</p><p><u style="single">Analysis of agglomeration:</u> In addition, the rate of increase in soluble particles was measured using size exclusion chromatography for each formulation after 6 consecutive freeze / thaw cycles.</p><p> After 6 freeze / thaw cycles, exclusion chromatography was used to analyze the agglomeration of each formulation. Elimination chromatography was performed using a TSK gel G3000SWXL-G2000SWXL column and a 0.2 M sodium phosphate (pH 7.0) mobile phase at a flow rate of 1 ml / min and UV detection at 214 nm. Table 7 shows the percentage of eluted high molecular weight species (ie, agglomerates of monoclonal anti-CTLA-4 antibody 11.2.1) measured at the time of description for each treated formulation. The level of aggregation was calculated by integrating the area below the peak for each formulation chromatogram, and the integral value of the area below the peak of the high molecular weight species was displayed as a percentage of the total peak area (Table 7). checking). As can be seen from Table 7, the composition in which the buffer solution was EDTA had the lowest aggregation level, and then the buffer solution was histidine, acetate, and succinate in order from the lowest.</p><p><u style="single">Example 5</u> Experiments were conducted to investigate the effects of EDTA, methionine, and oxygen deficiency on discoloration and aggregation of liquid preparations containing the monoclonal anti-CTLA-4 antibody 11.2.1. Discoloration and agglutination in such liquid formulations are generally undesirable from the standpoint of one or both of product aesthetics and product integrity.</p><p> Table 8 below shows the treatment methods for the investigated formulations. The general procedure used to prepare the formulation was the same as described in Example 3. In this example, monoclonal anti-CTLA-4 antibody 11.2.1 (5 mg / ml), sodium acetate buffer (20 mM), sodium chloride (8.2 mg / ml), and polysolvate 80 (0.2 mg / ml) were used. A starting formulation containing, pH 5.5, was prepared and added to several 10 ml glass vials with a seal top capable of sterile sampling.</p><p> The starting preparations were subjected to the various treatments shown in Table 8 below. Methionine was added to several vials as shown in Table 8. Two different concentrations of EDTA were added to the other vials. A vial containing EDTA or methionine was selected and nitrogen gas was added to its upper space. In addition, nitrogen gas was injected into the upper space after deflating some of the remaining untreated vials. In addition, some of the remaining vials were left untreated and used as experimental controls.</p><p> The two vials with each treatment shown in Table 8 were stored at 40 ° C for 0 weeks, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 14 weeks, 16 weeks and 18 weeks. One of the two vials stored at each time point was visually inspected and the other vial was aseptically sampled to measure 11.2.1 antibody aggregation levels after storage. The results are shown in Tables 9 and 10.</p><p><tables num="11"><img file="JP2012167120A_D0013.tif" /></tables></p><p><u style="single">Analysis of formulation appearance:</u> For each formulation, particle formation, color change, and turbidity change at 0 week (initial state), 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 14 weeks, 16 weeks, and 18 weeks. I evaluated it visually. Table 9 shows the results of visual observation.</p><p><tables num="12"><img file="JP2012167120A_D0014.tif" /></tables></p><p> The results in Table 9 show that EDTA and / or methionine-free formulations turned pink in vials after storage at 40 ° C for at least 4 weeks. Without being bound by any theory, in one embodiment of the invention, at least some of the color changes may be due to the oxidation process. However, in another embodiment, the color change may be due to a number of other processes unrelated to oxidation.</p><p> The addition of nitrogen gas to the upper space of the vial appeared to be less effective in reducing discoloration than the addition of methionine and / or EDTA.</p><p><u style="single">Analysis of agglomeration:</u> Antibody preparations treated according to Table 8 were stored at a temperature of 40 ° C. Aggregation of each formulation was analyzed using size exclusion chromatography at 0, 2, 6, 8, 10, 10, 14, 16, and 18 weeks. Elimination chromatography was performed using a TSK gel G3000SWXL-G2000SWXL column and a 0.2 M sodium phosphate (pH 7.0) mobile phase at a flow rate of 1 ml / min and UV detection at 214 nm. Table 10 shows the percentage of eluted high molecular weight species (ie, agglomerates of monoclonal anti-CTLA-4 antibody 11.2.1) measured at the time of description for each treated formulation. The level of aggregation was calculated by integrating the area below the peak for each formulation chromatogram, and the integrated value of the area below the peak of the high molecular weight species was displayed as a percentage of the total peak area (Table 10). checking).</p><p><tables num="13"><img file="JP2012167120A_D0015.tif" /></tables></p><p> The results in Table 9 show that EDTA and / or methionine-free formulations begin to turn pink in vials after storage at 40 ° C for at least 4 weeks. As can be seen from Table 10, the preparations treated with EDTA and / or methionine had the lowest aggregation levels, followed by the preparations treated with nitrogen gas, followed by the untreated control preparations.</p><p><u style="single">Example 6</u> Experiments were conducted to investigate the effects of methionine and EDTA on some methionine residues in anti-CTLA-4 antibody 11.2.1 when stored as a liquid formulation.</p><p><u style="single">Analysis of methionine oxidation:</u> Oxidation levels of methionine residues at amino acid positions 256 and 432 of anti-CTLA-4 antibody 11.2.1 after storage at 40 ° C for 8 weeks were measured by lysine-C mapping.</p><p> Glass vials containing Formulation Nos. 26, 29, 33 (Table 8) and treated with Example 5 were aseptically sampled at 8 weeks. The sample was then digested with lysine-C enzyme in Tris buffer (pH 8.0) under standard conditions and then analyzed by reverse phase high performance liquid chromatography. Separation was performed using a Grace Vidac Protein C4 analytical column by gradient elution of water with 0.1% TFA and acetonitrile with 0.085% TFA.</p><p><tables num="14"><img file="JP2012167120A_D0016.tif" /></tables></p><p> The results in Table 11 show that the addition of methionine or EDTA to the 11.2.1 antibody formulation reduces the rate of oxidation at the positions of the above two methionine residues compared to formulations stored without EDTA or methionine. ing.</p><p><u style="single">Example 7</u> An experiment was conducted to examine the oxidation of some tryptophan residues and tyrosine residues in the anti-CTLA-4 antibody 11.2.1.</p><p> The anti-CTLA-4 antibody 11.2.1 formulation, which turns pink over time, was found by ultraviolet / visible spectroscopy (UV-Vis) to have a characteristic absorption peak at 500 nm.</p><p> The procedure used to prepare the formulation is the same as described in Example 3. In this example, a solution containing 5 mg / ml of monoclonal anti-CTLA-4 antibody 11.2.1 in 20 mM sodium acetate buffer, 8.2 mg / ml sodium chloride, and 0.2 mg / ml polysolvate 80 ( When the formulation containing pH 5.5) was stored in two glass vials at 40 ° C for 4 weeks, the formulation turned pink at that time.</p><p> Next, when a solution of one of the vials containing the discolored formulation was subjected to molecular weight (cutoff) filtration, the excipient of the formulation passed through the filtration device, but the antibody remained unpassed. .. The filtered eluate (eg water and excipients) was clear and colorless, but the recovered fraction (eg antibody 11.2.1) remained pink. Therefore, from this filtration experiment, it was found that the discoloration to pink was not caused by the excipient of the preparation but was related to the antibody 11.2.1 itself.</p><p> The second vial, which turned pink, was then digested with trypsin under standard conditions and analyzed by reverse phase high performance liquid chromatography equipped with a mass spectrometer (LC-MS). Separation was performed using a Grace Vidac Protein C4 analytical column by gradient elution of water with 0.1% TFA and acetonitrile with 0.085% TFA. The UV-Vis absorbance of the digested peptide at 500 nm was monitored and the corresponding peptide was identified based on its molecular weight.</p><p> The tryptic digested peptide that correlates with the peak absorbance at 500 nm had the amino acid sequence GLEWVAVIWYDGSNK. Next, when this amino acid sequence GLEWVAVIWYDGSNK was further digested with Asp-N protease under standard conditions, the peak absorbance (UV-Vis) at 500 nm was adjusted to match the peptide digested with Asp-N protease. moved. The amino acid sequence of this peptide was GLEW VAVIWY.</p><p> Therefore, I am not bound by any theory, but at a site where one or both of two tryptophan residues (W) or one tyrosine residue (Y) contained in a protease-digested peptide (GLEWVAVIWY) can be oxidized. It is believed that this is the cause of the antibody 11.2.1 formulation of this example turning pink. In a particular embodiment, one or both of the two tryptophan residues (W) contained in the protease digested peptide (GLEWVAVIWY) are considered to be oxidizable sites and are responsible for the discoloration to pink. there is a possibility.</p><p> However, it is possible that one or more individual discolorations (eg, pink or yellow) seen in the various formulations examined herein may be due to mechanisms other than oxidation.</p><p><u style="single">Example 8</u> Experiments were conducted to investigate the effects of EDTA and DTPA on discoloration, aggregation and fragmentation of anti-CTLA-4 antibody 11.2.1.</p><p> In particular, three types were prepared: a liquid preparation containing antibody 11.2.1 and EDTA, a liquid preparation containing antibody 11.2.1 and DTPA, and a liquid preparation containing antibody 11.2.1 alone and not containing EDTA or DTPA. These preparations were stored at 40 ° C. and antibody discoloration, agglutination, and fragmentation were evaluated at 0, 2, 4, 6, 8, and 10 weeks.</p><p> In this example, a monoclonal anti-CTLA-4 antibody in 20 mM sodium acetate buffer (pH 5.5), 8.2 mg / ml sodium chloride, and 0.2 mg / ml polysolvate 80 as in Example 3. A solution containing 20 mg / ml of 11.2.1 was prepared, divided into several glass vials, and then treated with the addition of EDTA or DTPA. EDTA and DTPA were added as solids to the vial containing the formulation. Immediate analysis of discoloration, aggregation and fragmentation on some vials and some overlapping vials at 40 ° C for 2 weeks, 4 weeks, 6 weeks, 8 weeks, upright Stored for 10 weeks.</p><p> Then, aseptically sampled from treated and untreated vials at 0, 2, 4, 6, 8 and 10 weeks, aggregation and fragmentation of antibody 11.2.1 in the formulation. Level was measured and discoloration was visually observed. The results are shown in Tables 12 and 13.</p><p><u style="single">Analysis of formulation appearance:</u> For each formulation, particle formation, color change, and turbidity changes were visually evaluated at 0 weeks (initial state), 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks. Table 12 shows the results of visual observation.</p><p><tables num="15"><img file="JP2012167120A_D0017.tif" /></tables></p><p> The results in Table 12 show that EDTA or DTPA-free formulations turned pink in vials after storage at 40 ° C for at least 6 weeks.</p><p><u style="single">Analysis of agglomeration:</u> Antibody preparations treated according to Table 12 were stored at a temperature of 40 ° C. Aggregation of each formulation was analyzed using size exclusion chromatography at 0, 2, 6, 8 and 10 weeks. Size exclusion chromatography was performed using a TSK gel G3000SWXL-G2000SWXL column and a 0.2 M sodium phosphate (pH 7.0) mobile phase at a flow rate of 1 ml / min and UV detection at 214 nm. Table 13 shows the percentage of eluted high molecular weight species (ie, aggregates of monoclonal anti-CTLA-4 antibody 11.2.1) measured at the time of description for each treated formulation. The level of aggregation was calculated by integrating the area below the peak for each formulation chromatogram, and the integrated value of the area below the peak of the high molecular weight species was displayed as a percentage of the total peak area (Table 13). checking).</p><p><tables num="16"><img file="JP2012167120A_D0018.tif" /></tables></p><p> As can be seen from Table 13, both EDTA-containing and DTPA-containing preparations had lower levels of aggregation compared to EDTA or DTPA-free preparations.</p><p><u style="single">Example 9</u> An experiment was conducted to investigate the effect of EDTA and nitrogen gas on the stability of anti-CTLA-4 antibody 11.2.1.</p><p> In particular, the effects of EDTA and nitrogen gas on anti-CTLA-4 antibody 11.2.1 were analyzed for discoloration, aggregation, oxidation, fragmentation and formation of charged seeds in formulations containing trehalose and polysorbate 80 as buffer with histidine.</p><p> The investigated formulations are shown in Table 13 below. The procedure used to prepare the formulation is the same as described in Example 10 below. The formulation was stored at 40 ° C and its stability was evaluated at 0, 4, 8, 12, and 24 weeks.</p><p> In this example, a solution containing 20 mg / ml of monoclonal anti-CTLA-4 antibody in 20 mM histidine buffer (pH 5.5), 84 mg / ml trehalose, and 0.2 mg / ml polysorbate 80 was carried out. Prepared as in Example 10. The first part of the formulation was prepared by diluting a concentrated storage solution of the antibody with a storage solution of trehalose and polysorbate 80 so that the anti-CTLA-4 antibody in the composition had a final concentration of 20 mg / ml. The second part of the formulation is 10 mg / ml concentrated Na₂EDTA 2H₂It was prepared in the same manner except that the final concentration was 0.1 mg / ml by the additional step of adding O. Next, 1 ml of the formulation was placed in a 2 ml glass vial. Next, half of the vials containing each formulation were placed in a lyophilizer, the upper space was evacuated and then filled with nitrogen. After filling the vial with nitrogen, the oxygen level was measured and found to be about 1.5-1.6%. On the other hand, in the vial with air in the upper space, oxygen was about 19.7 to 20%.</p><p> Immediate analysis of discoloration, aggregation and fragmentation on some vials and some overlapping vials at 40 ° C for 2 weeks, 4 weeks, 8 weeks and 12 weeks in an upright position. Stored for 24 weeks. At each point in time, two stored vials for one treatment were removed from their respective states and the level of aggregation, fragmentation, oxidation and formation of charged seeds of antibody 11.2.1 in the formulation was measured for discoloration. Observed. The results are shown in Tables 14-18.</p><p><tables num="17"><img file="JP2012167120A_D0019.tif" /></tables></p><p><u style="single">Analysis of formulation appearance:</u> For each formulation, particle formation, color change, and turbidity changes were visually evaluated at 0 weeks (initial state), 4 weeks, 8 weeks, 12 weeks, and 24 weeks. The results of visual observation are shown in Table 14.</p><p><tables num="18"><img file="JP2012167120A_D0020.tif" /></tables></p><p> The results in Table 14 show that the formulations without EDTA or nitrogen gas turned pink after storage at 40 ° C for 4 weeks. The results in Table 14 also show that the preparations that replaced the air in the upper space of the vial with nitrogen also delayed the discoloration to pink until 12 weeks. Both formulations containing EDTA had no visible discoloration at least 24 weeks.</p><p><u style="single">Analysis of agglomeration:</u> Antibody preparations prepared according to Table 13 were stored at 40 ° C. Aggregation of each formulation was analyzed using size exclusion chromatography at 0, 4, 8, 12, and 24 weeks. At each point in time, aseptic sampling was performed from the vial containing the formulation. Size exclusion chromatography was performed using a TSK gel G3000SWXL-G2000SWXL column and a 0.2 M sodium phosphate (pH 7.0) mobile phase at a flow rate of 1 ml / min and UV detection at 214 nm. Table 15 shows the percentage of eluted high molecular weight species (ie, agglomerates of monoclonal anti-CTLA-4 antibody 11.2.1) measured at the time of description for each treated formulation. The level of aggregation was calculated by integrating the area below the peak for each formulation chromatogram, and the integrated value of the area below the peak of the high molecular weight species was displayed as a percentage of the total peak area (Table 15). checking).</p><p><tables num="19"><img file="JP2012167120A_D0021.tif" /></tables></p><p> As can be seen from Table 15, EDTA-containing preparations, nitrogen gas preparations, and EDTA + nitrogen gas preparations have lower aggregation levels over time than preparations that do not contain EDTA and have air in the upper space. It was.</p><p><u style="single">Fragmentation analysis:</u> Antibody preparations prepared according to Table 13 were stored at 40 ° C. At 0, 4, 8, 12, and 24 weeks, reduced SDS-PAGE (rSDS-PAGE) was used to analyze total hydrolyzed impurities (ie, fragmentation) of each formulation. At each point in time, aseptic sampling was performed from the vial containing the formulation, placed on a NuPAGE 4-12% bis-tris gel and stained with a colloidal blue dye (Coomacy). A NuPAGE® reducing agent was used to reduce the gel. The percentage of impurities (ie, fragmentation) in the bands of each sample in the reduced gel was estimated by scanning with a Molecular Dynamics Personal PDQC-90 photographic densitometer or a Bio-Rad GS800 Imaging photographic densitometer. The level of fragmentation was calculated as a percentage of the total band volume (see Table 16).</p><p><tables num="20"><img file="JP2012167120A_D0022.tif" /></tables></p><p> As can be seen from Table 16, EDTA-containing preparations, nitrogen gas preparations, and EDTA + nitrogen gas preparations have higher levels of fragmentation over time than preparations that do not contain EDTA and have air in the upper space. It was low.</p><p><u style="single">Formation of acidic and basic species:</u> Antibody preparations prepared according to Table 13 were stored at 40 ° C. At 0, 4, 12, and 24 weeks, imaging capillary electrophoresis (iCE) was used to analyze the formation of acidic and basic species for each formulation. Imaging Capillary Electrophoresis is a Convergent Biosciences iCE for assessing charge heterogeneity₂₈₀It was carried out using an analyzer. Convergent iCE₂₈₀Is an Imaging Capillary Isoelectric Focusing (IEF) device, which allows the user to obtain an image of a separated sample contained in the capillary.</p><p> At each point in time, aseptic sampling was performed from the vial containing the formulation. The sample was then prepared as a mixture of an amphoteric electrolyte for electrophoresis, methyl cellulose, a marker for calibration, and water. ICE this sample₂₈₀Introduced into and applied high potential / voltage. An IEF assay was performed using a polyacrylamide gel with a pH of 3 to 10.5 prepared by hand with Coomassie Blue. The protein components of the sample were separated based on their relative equipotential point (pI) and position. The relative amount of each separated component was observed with a CCD camera for imaging. The data was then processed and displayed using traditional chromatographic integration software as a reduction in major peaks (ie, formation of acidic and basic species) (see Table 17).</p><p><tables num="21"><img file="JP2012167120A_D0023.tif" /></tables></p><p> As can be seen from Table 17, EDTA-containing, nitrogen-gas, and EDTA + nitrogen-gas preparations have a complete peak level over time, with EDTA-free and air-filled upper space. It was expensive compared to. Therefore, over time, the amount of acidic and basic species formed is higher in formulations without EDTA and / or nitrogen gas in the upper space.</p><p><u style="single">Analysis of Amino Acid Oxidation:</u> Oxidation levels of methionine residues at amino acid positions 256 and 432 of anti-CTLA-4 antibody 11.2.1 after storage at 40 ° C for 12 weeks were measured by lysine-C mapping.</p><p> Vials containing the formulations from Table 13 were aseptically sampled at 12 weeks. Samples were then digested under standard conditions with Lysyl endopeptidase (Lys-C) enzyme Tris buffer (pH 8.0) and analyzed by reverse phase high performance liquid chromatography. Separation was performed using a Grace Vidac Protein C4 analytical column by gradient elution of water with 0.1% TFA and acetonitrile with 0.085% TFA.</p><p><tables num="22"><img file="JP2012167120A_D0024.tif" /></tables></p><p> The results in Table 18 show that when EDTA was added to the antibody 11.2.1 formulation and / or when nitrogen gas was added to the upper space of the vial, the rate at which the above two methionine residues were oxidized was without EDTA and / or. It shows that it was reduced compared to the formulation without nitrogen gas in the upper space.</p><p><u style="single">Example 10</u> Experiments were conducted to compare the effects of anti-CTLA-4 antibody 11.2.1 preparations containing sodium acetate buffer and sodium chloride (ie, chlorine ions) on stability with preparations containing histidine buffer and trehalose.</p><p> In particular, the effect of antibody 11.2.1 on stability was analyzed for discoloration, agglutination and fragmentation.</p><p> The investigated formulations are listed in Table 19 below. The procedure used to prepare the formulation is the same as described in Example 3.</p><p> After preparing the formulation in Table 19 by incorporating a storage solution containing 11.9 mg / ml of antibody 11.2.1 in 20 mM sodium acetate buffer (pH 5.5) and 140 mM sodium chloride, Millipore Love. Pelicon XL PBTK 30K 50cm in scale TFF system²Ultrafiltration / dialysis filtration (UF / DF) steps were performed using membranes. Next, a concentrated solution of antibody 11.2.1 containing antibody 11.2.1 in the range of 35 to 40 mg / ml in 20 mM sodium acetate or 20 mM histidine buffer was prepared.</p><p> A concentrated solution was prepared in which a tonic of 3 times the desired final concentration was contained in sodium acetate buffer or histidine buffer. Concentrated Na in each buffer₂EDTA 2H₂A concentrated solution of polysorbate 80 containing 10 mg / ml of O and 20 mg / ml of polysorbate 80 was prepared. Individual formulations were prepared by appropriately diluting this concentrated solution. The formulation was then filtered through a sterile grade 0.2 μm filter and filled into several vials prepared in two varieties. A 2 ml type glass vial was filled with 1 ml. Vials were capped with Daikyo Seiko 777-1 Flurotech® coated, crimp-sealed, placed in a stable chamber and stored upright at 25 ° C and 40 ° C. Another set of vials was kept at -20 ° C for 4 weeks, and another set was subjected to 4 freezing / thawing cycles as described in Example 4 (water-filled vials). Box containing the). All formulations had a pH of 5.5 and an anti-CTLA-4 antibody 11.2.1 concentration of 20 mg / ml.</p><p> Immediately analyze the levels of discoloration, aggregation and fragmentation of some vials and the other of the two prepared vials at 25 ° C and 40 ° C for 4 or 8 weeks in an upright position. Stored for 12, 18, 24, and 36 weeks. At each point in time, two stored vials for one formulation were removed from their respective states, the level of antibody 11.2.1 aggregation and fragmentation was measured, and discoloration was also observed. The results are shown in Tables 20 to 24 and Figures 3 and 4.</p><p><tables num="23"><img file="JP2012167120A_D0025.tif" /></tables></p><p><u style="single">Analysis of formulation appearance:</u> 1) After the formulation is first mixed, 2) the formulation is frozen at -20 ° C for 4 weeks, then 3) freeze / thaw cycle (as described in Example 4, box with vials filled with water). After experiencing 4 times (from -70 ° C to 5 ° C), each formulation was visually evaluated. For each formulation, particle formation, color change, and turbidity changes were visually evaluated at 0 weeks (initial state), 8 weeks, 12 weeks, and 24 weeks. Particle formation, color change, and turbidity change were evaluated for each formulation and are listed in Table 20 (freeze / thaw), Table 21 (stored at 25 ° C), and Table 22 (stored at 40 ° C). ..</p><p><tables num="24"><img file="JP2012167120A_D0026.tif" /></tables></p><p><tables num="25"><img file="JP2012167120A_D0027.tif" /></tables></p><p><tables num="26"><img file="JP2012167120A_D0028.tif" /></tables></p><p> The results in Tables 20 to 22 show that the antibody 11.2.1 preparation containing EDTA has less discoloration, less turbidity, and less particle formation than the preparation without EDTA. In short, the preparation containing sodium chloride had more discoloration, higher turbidity, and more particle formation than the preparation containing EDTA but not sodium chloride.</p><p><u style="single">Analysis of agglomeration:</u> Antibody preparations prepared according to Table 19 were stored at 25 ° C and 40 ° C. Aggregation of formulations stored at 25 ° C. was analyzed using size exclusion chromatography at 0 weeks (initial state), 4 weeks, 8 weeks, 12 weeks, 24 weeks, and 36 weeks. Aggregation of formulations stored at 40 ° C. was analyzed using size exclusion chromatography at 4, 8, 12, and 24 weeks. Aseptic sampling was performed from the vial containing the formulation at each point in time. Size exclusion chromatography was performed using a TSK gel G3000SWXL-G2000SWXL column and a 0.2 M sodium phosphate (pH 7.0) mobile phase at a flow rate of 1 ml / min and UV detection at 214 nm. Tables 23 (a) and 23 (b) show the percentages of eluted high molecular weight species (ie, monoclonal anti-CTLA-4 antibody 11.2.1 aggregates) measured at the time of description for each treated formulation. It is shown. The level of aggregation was calculated by integrating the area below the peak for each formulation chromatogram, and the integrated value of the area below the peak of the high molecular weight species was displayed as a percentage of the total peak area (Table 23). See (a) and Table 23 (b)).</p><p><tables num="27"><img file="JP2012167120A_D0029.tif" /></tables></p><p> Table 23 (b) below shows the agglomeration data, and Figure 3 shows the data graphically.</p><p><tables num="28"><img file="JP2012167120A_D0030.tif" /></tables></p><p> As can be seen from Tables 23 (a) and 23 (b) and FIG. 3, EDTA-containing formulations do not contain EDTA but are acetate buffer and sodium chloride after storage at 25 ° C and 40 ° C. The level of aggregation was lower than that of the preparation containing. In addition, formulations containing histidine buffer (but not containing EDTA) had less aggregation than formulations that did not contain EDTA but contained acetate buffer and sodium chloride.</p><p><u style="single">Fragmentation analysis:</u> Antibody preparations prepared according to Table 19 were stored at 25 ° C and 40 ° C. Total hydrolyzed impurities (ie fragmentation) of each formulation using reduced SDS-PAGE (rSDS-PAGE) at 0 weeks (initial state), 4 weeks, 8 weeks, 12 weeks, 18 weeks, 36 weeks Was analyzed. At each point in time, aseptic sampling was performed from the vial containing the formulation, placed on a NuPAGE 4-12% bis-tris gel and stained with a colloidal blue dye (Coomacy). A NuPAGE® reducing agent was used to reduce the gel. The percentage of impurities (ie, fragmentation) in the bands of each sample in the reduced gel was estimated by scanning with a Molecular Dynamics Personal PDQC-90 photographic densitometer or a Bio-Rad GS800 Imaging photographic densitometer. The level of fragmentation was calculated as a percentage of the total band volume (see Table 24 (a) and Table 24 (b)).</p><p><tables num="29"><img file="JP2012167120A_D0031.tif" /></tables></p><p> Table 24 (b) below shows the fragmentation data, and Figure 4 shows the data graphically.</p><p><tables num="30"><img file="JP2012167120A_D0032.tif" /></tables></p><p> As can be seen from Tables 24 (a) and 24 (b) and FIG. 4, EDTA-containing formulations do not contain EDTA but are acetate buffer and sodium chloride after storage at 25 ° C and 40 ° C. The level of fragmentation was lower than that of the preparation containing.</p><p><u style="single">Example 11</u> Experiments were conducted to compare the effects of various concentrations of EDTA on the stability of anti-CTLA-4 antibody 11.2.1 preparations. A formulation containing histidine buffer and trehalose, in which part of trehalose was replaced with mannitol, was also tested.</p><p> In particular, the effects of discoloration, agglutination, fragmentation, and oxidation on the stability of antibody 11.2.1 were analyzed.</p><p> The formulations examined are shown in Table 25 below. The procedure used to prepare the formulation is the same as described in Example 10.</p><p> After preparing the formulation in Table 25 by incorporating a storage solution containing 11.9 mg / ml of antibody 11.2.1 in 20 mM sodium acetate buffer (pH 5.5) and 140 mM sodium chloride, Millipore Love. Pelicon XL PBTK 30K 50cm in scale TFF system²Ultrafiltration / dialysis filtration (UF / DF) steps were performed using membranes. Next, a concentrated solution of antibody 11.2.1 containing antibody 11.2.1 in the range of 35 to 40 mg / ml in 20 mM sodium acetate or 20 mM histidine buffer was prepared.</p><p> A concentrated solution was prepared in which a tonic of 3 times the desired final concentration was contained in sodium acetate buffer or histidine buffer. Concentrated Na in each buffer₂EDTA 2H₂A concentrated solution of polysorbate 80 containing 10 mg / ml of O and 20 mg / ml of polysorbate 80 was prepared. Individual formulations were prepared by appropriately diluting this concentrated solution. (Na₂EDTA 2H₂Examination of the concentration of EDTA (as O) was in the range of 0-0.1 mg / ml. The formulation was then filtered through a sterile grade 0.2 μm filter and filled into several vials prepared in two varieties. A 2 ml type glass vial was filled with 1 ml.</p><p> Vials were capped with Daikyo Seiko 777-1 Flurotech® coated, crimp-sealed, placed in a stable chamber and stored upright at 25 ° C and 40 ° C. Another set was subjected to four freeze / thaw cycles as described in Example 10. All formulations had a pH of 5.5 and an anti-CTLA-4 antibody 11.2.1 concentration of 20 mg / ml.</p><p> Immediate analysis of discoloration, aggregation, fragmentation and oxidation levels on some vials and the other of the two prepared vials at 25 ° C and 40 ° C for 4 weeks, 8 Stored for weeks, 13, 18, and 24 weeks. At each point in time, two stored vials for one formulation were removed from their respective states, the level of antibody 11.2.1 aggregation and fragmentation was measured, and discoloration was also observed. The results are shown in Tables 26 to 31 and 5 to 8.</p><p><tables num="31"><img file="JP2012167120A_D0033.tif" /></tables></p><p><u style="single">Analysis of formulation appearance:</u> 1) After the formulation is first mixed, 2) 4 freeze / thaw cycles, then 3) 4 weeks, 8 weeks, 13 weeks, 18 weeks, 24 weeks at 25 ° C and 40 ° C After storage, each formulation was visually evaluated. Particle formation, color change, and turbidity change were evaluated for each formulation and are listed in Tables 26-28.</p><p><tables num="32"><img file="JP2012167120A_D0034.tif" /></tables></p><p><tables num="33"><img file="JP2012167120A_D0035.tif" /></tables></p><p><tables num="34"><img file="JP2012167120A_D0036.tif" /></tables></p><p> The results in Tables 26-28 show that antibody 11.2.1 formulations containing all concentrations of EDTA tested had less discoloration, less turbidity, and less particle formation than formulations without EDTA. ..</p><p> After all, preparations containing sodium chloride were less protected from freezing / thawing cycles than preparations containing EDTA but not sodium chloride. This is evidenced by increased discoloration, increased turbidity, and increased particle formation.</p><p><u style="single">Analysis of agglomeration:</u> Antibody preparations prepared according to Table 25 were stored at 25 ° C and 40 ° C. At 0 week (initial state), 4 weeks, 8 weeks, 13 weeks, 18 weeks, and 24 weeks, size exclusion chromatography was used to analyze agglutination of formulations stored at 25 ° C and 40 ° C. At each point in time, aseptic sampling was performed from the vial containing the formulation. Size exclusion chromatography was performed using a TSK gel G3000SWXL-G2000SWXL column and a 0.2 M sodium phosphate (pH 7.0) mobile phase at a flow rate of 1 ml / min and UV detection at 214 nm. Table 29 (a) shows the percentage of antibody 11.2.1 aggregates measured at the time listed after storage at 25 ° C for each formulation. Table 29 (b) shows the percentage of antibody 11.2.1 aggregates measured after storage at 40 ° C. The level of aggregation was calculated by integrating the area below the peak for each formulation chromatogram, and the integrated value of the area below the peak of the high molecular weight species was displayed as a percentage of the total peak area (Table 29). See (a) and Table 29 (b)).</p><p><tables num="35"><img file="JP2012167120A_D0037.tif" /></tables></p><p> Table 29 (b) below shows the agglomeration data, and Figure 5 shows the data graphically.</p><p><tables num="36"><img file="JP2012167120A_D0038.tif" /></tables></p><p> As can be seen from Tables 29 (a) and 29 (b) and FIG. 5, formulations containing EDTA contain EDTA at all concentrations of EDTA tested after storage at 25 ° C and 40 ° C. The level of aggregation was lower compared to no formulation. FIG. 7 graphs the reduction in aggregation rate for the formulations from Table 25 as a function of EDTA concentration.</p><p><u style="single">Fragmentation analysis:</u> Antibody preparations prepared according to Table 25 were stored at 25 ° C and 40 ° C. Total hydrolyzed impurities (ie fragmentation) of each formulation using reduced SDS-PAGE (rSDS-PAGE) at 0 weeks (initial state), 4 weeks, 8 weeks, 13 weeks, 18 weeks, 24 weeks. Was analyzed. At each point in time, aseptic sampling was performed from the vial containing the formulation, placed on a NuPAGE 4-12% bis-tris gel and stained with a colloidal blue dye (Coomacy). A NuPAGE® reducing agent was used to reduce the gel. The percentage of impurities (ie, fragmentation) in the bands of each sample in the reduced gel was estimated by scanning with a Molecular Dynamics Personal PDQC-90 photographic densitometer or a Bio-Rad GS800 Imaging photographic densitometer. The level of fragmentation was calculated as a percentage of the total band volume (see Tables 30 (a) and 30 (b)).</p><p><tables num="37"><img file="JP2012167120A_D0039.tif" /></tables></p><p> Table 30 (b) below shows the fragmentation data, and Figure 6 shows the data graphically.</p><p><tables num="38"><img file="JP2012167120A_D0040.tif" /></tables></p><p> As can be seen from Tables 30 (a) and 30 (b) and FIG. 6, EDTA-containing formulations do not contain EDTA but are acetate buffer and sodium chloride after storage at 25 ° C and 40 ° C. The level of fragmentation was lower than that of the preparation containing. In addition, formulations containing histidine and trehalose but not EDTA were less fragmented than formulations containing sodium chloride but not EDTA.</p><p> FIG. 8 graphs the reduction in fragmentation rate for the formulations from Table 25 as a function of EDTA concentration.</p><p><u style="single">Analysis of Amino Acid Oxidation:</u> Oxidation levels of several methionine residues at amino acid positions 256 and 432 of anti-CTLA-4 antibody 11.2.1 were measured by the lysine-C mapping method. Vials containing the formulations from Table 25 were aseptically sampled at 18 and 24 weeks. Samples were then digested under standard conditions with Lysyl endopeptidase (Lys-C) enzyme Tris buffer (pH 8.0) and analyzed by reverse phase high performance liquid chromatography. Separation was performed using a Grace Vidac Protein C4 analytical column by gradient elution of water with 0.1% TFA and acetonitrile with 0.085% TFA. The results are shown in Table 31.</p><p><tables num="39"><img file="JP2012167120A_D0041.tif" /></tables></p><p> As can be seen from Table 31, the presence of EDTA in the antibody 11.2.1 formulation reduces the level of methionine oxidation that occurs over time.</p><p><u style="single">Example 12</u> Experiments were conducted to compare the effects of mannitol and sorbitol on the stability of anti-CTLA-4 antibody 11.2.1 preparations. In this example, a histidine-trehalose formulation was tested in which a portion of trehalose was replaced with various concentrations of mannitol and / or sorbitol (Table 32). (Na₂EDTA 2H₂Examination of the concentration of EDTA (as O) was in the range of 0-0.1 mg / ml.</p><p> In particular, the effect of antibody 11.2.1 on stability was analyzed for discoloration, agglutination, fragmentation and oxidation.</p><p> The investigated formulations are listed in Table 32 below. The procedure used to prepare the formulation is the same as described in Example 10.</p><p> After preparing the formulation in Table 32 by incorporating a storage solution containing 11.9 mg / ml of antibody 11.2.1 in 20 mM sodium acetate buffer (pH 5.5) and 140 mM sodium chloride, Millipore Love. Pelicon XL PBTK 30K 50cm in scale TFF system²Ultrafiltration / dialysis filtration (UF / DF) steps were performed using membranes. Next, a concentrated solution of antibody 11.2.1 containing antibody 11.2.1 in the range of 35 to 40 mg / ml in 20 mM sodium acetate or 20 mM histidine buffer was prepared.</p><p> A concentrated solution was prepared in which a tonic of 3 times the desired final concentration was contained in sodium acetate buffer or histidine buffer. Concentrated Na in each buffer₂EDTA 2H₂A concentrated solution of polysorbate 80 containing 10 mg / ml of O and 20 mg / ml of polysorbate 80 was prepared. Individual formulations were prepared by appropriately diluting this concentrated solution. The formulation was then filtered through a sterile grade 0.2 μm filter and filled into several vials prepared in two varieties. A 2 ml type glass vial was filled with 1 ml.</p><p> Vials were capped with Daikyo Seiko 777-1 Flurotech® coated, crimp-sealed, placed in a stable chamber and stored upright at 25 ° C and 40 ° C. Another set was subjected to four freeze / thaw cycles as described in Example 10. All formulations had a pH of 5.5 and an anti-CTLA-4 antibody 11.2.1 concentration of 20 mg / ml.</p><p> Immediate analysis of discoloration, aggregation, fragmentation and oxidation levels on some vials and the other of the two prepared vials at 25 ° C and 40 ° C for 4 weeks, 8 Stored for weeks, 13, 18, and 24 weeks. At each point in time, two stored vials for one formulation were removed from their respective states, the level of antibody 11.2.1 aggregation and fragmentation was measured, and discoloration was also observed. The results are shown in Tables 33 to 37 and 9 to 10.</p><p><tables num="40"><img file="JP2012167120A_D0042.tif" /></tables></p><p><u style="single">Analysis of formulation appearance:</u> 1) First mixing of formulations, then 2) 4 freezing / thawing cycles (from -70 ° C to 5 ° C in a box with vials filled with water, as described in Example 4) After that, 3) each preparation was visually evaluated after storage at 25 ° C and 40 ° C for 8 weeks, 13 weeks, and 24 weeks. Particle formation, color change, and turbidity change were evaluated for each formulation and are listed in Tables 33-35.</p><p><tables num="41"><img file="JP2012167120A_D0043.tif" /></tables></p><p><tables num="42"><img file="JP2012167120A_D0044.tif" /></tables></p><p><tables num="43"><img file="JP2012167120A_D0045.tif" /></tables></p><p> The results in Tables 33-35 show that antibody 11.2.1 preparations containing sodium chloride but not EDTA had less protection from freezing / thawing cycles than preparations containing EDTA but not sodium chloride. This is evidenced by increased discoloration, increased turbidity, and increased particle formation. The results in the table also show that antibody 11.2.1 formulations containing EDTA had less discoloration, less turbidity, and lower levels of particle formation at all concentrations of EDTA tested. Shown.</p><p><u style="single">Analysis of agglomeration:</u> Antibody preparations prepared according to Table 32 were stored at 25 ° C and 40 ° C. At 0 week (initial state), 4 weeks, 8 weeks, 13 weeks, 18 weeks, and 24 weeks, size exclusion chromatography was used to analyze agglutination of formulations stored at 25 ° C and 40 ° C. At each point in time, aseptic sampling was performed from the vial containing the formulation. Size exclusion chromatography was performed using a TSK gel G3000SWXL-G2000SWXL column and a 0.2 M sodium phosphate (pH 7.0) mobile phase at a flow rate of 1 ml / min and UV detection at 214 nm. Table 36 (a) shows the percentage of antibody 11.2.1 aggregates measured at the time listed after storage at 25 ° C for each formulation. Table 36 (b) shows the percentage of antibody 11.2.1 aggregates measured after storage at 40 ° C. The level of aggregation was calculated by integrating the area below the peak for each formulation chromatogram, and the integrated value of the area below the peak of the high molecular weight species was displayed as a percentage of the total peak area (Table 36). See (a) and Table 36 (b)).</p><p><tables num="44"><img file="JP2012167120A_D0046.tif" /></tables></p><p> Table 36 (b) below shows the fragmentation data, and Figure 9 shows the data graphically.</p><p><tables num="45"><img file="JP2012167120A_D0047.tif" /></tables></p><p> As can be seen from Tables 36 (a) and 36 (b) and FIG. 9, formulations containing EDTA contain EDTA at all concentrations of EDTA tested after storage at 25 ° C and 40 ° C. Although not, the level of fragmentation was lower compared to formulations containing acetate buffer and sodium chloride (ie, chlorine ions).</p><p><u style="single">Fragmentation analysis:</u> Antibody preparations prepared according to Table 32 were stored at 25 ° C and 40 ° C. For formulations stored at 25 ° C and 40 ° C using reduced SDS-PAGE (rSDS-PAGE) at 0 weeks (initial state), 4 weeks, 8 weeks, 13 weeks, 18 weeks, 24 weeks. Total hydrolyzed impurities (ie fragmentation) were analyzed. At each point in time, aseptic sampling was performed from the vial containing the formulation, placed on a NuPAGE 4-12% bis-tris gel and stained with a colloidal blue dye (Coomassie). A NuPAGE® reducing agent was used to reduce the gel. The percentage of impurities (ie, fragmentation) in the bands of each sample in the reduced gel was estimated by scanning with a Molecular Dynamics Personal PDQC-90 photographic densitometer or a Bio-Rad GS800 Imaging photographic densitometer. The level of fragmentation was calculated as a percentage of the total band volume (see Table 37 (a) and Table 37 (b)).</p><p><tables num="46"><img file="JP2012167120A_D0048.tif" /></tables></p><p> Table 37 (b) below shows the fragmentation data, and Figure 10 shows the data graphically.</p><p><tables num="47"><img file="JP2012167120A_D0049.tif" /></tables></p><p> As can be seen from Tables 37 (a) and 37 (b) and FIG. 10, EDTA-containing formulations do not contain EDTA but are acetate buffer and sodium chloride after storage at 25 ° C and 40 ° C. The level of fragmentation was lower compared to formulations containing (ie, chloride ions).</p><p><u style="single">Example 13</u> In this example, the anti-CTLA-4 antibody thicilimumab, L-histidine monohydrochloride monohydrate, ethylenediamine tetraacetate disodium dihydrate, αα-trehalose dihydrate, and polysorbate 80 are included. A method for producing a liquid pharmaceutical composition will be described.</p><p> The liquid pharmaceutical composition of the present invention was constructed by obtaining the following components. Its components are the anti-CTLA-4 antibody ticilimumab (obtained from the hybridoma cell line 11.2.1.4 deposited as ATCC registration number PTA-5169 according to Example 1 or recombinantly prepared from the mammalian cell line according to Example 2). Can be), L-histidine monohydrochloride monohydrate (available from Ajinomoto, Inc., Raleigh, NC), L-histidine (available from Ajinomoto, Inc., Raleigh, NC), and ethylenediamine. Disodium tetraacetate dihydrate (available as Titriplex III from Merck KgaA, Darmstadt, Germany) and αα-trehalose dihydrate (Product Number from Ferro Vanstil, Walkiegan, Illinois) Available as T-104-1-MC) and Polysorbate 80 (available as Crillet 4HP from Kuroda, Mill Hall, Pennsylvania).</p><p> First, a storage solution of anti-CTLA-4 antibody thicilimumab, L-histidine monohydrochloride monohydrate, ethylenediamine tetraacetate disodium dihydrate, αα-trehalose dihydrate, and polysorbate 80. A liquid pharmaceutical composition was prepared by preparing several. Prepare 20 mM histidine buffer (pH 5.5) by dissolving 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate and 0.68 mg / ml (4.4 mM) L-histidine in water. To do. 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate, 0.68 mg / ml (4.4 mM) L-histidine, 84 mg / ml (222 mM) αα-trehalose dihydrate, 0.2 A 1 × formulation buffer is prepared by dissolving mg / ml polysorbate 80 and 0.1 mg / ml (0.268 mM) ethylenediamine tetraacetate disodium dihydrate in water. 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate, 0.68 mg / ml (4.4 mM) L-histidine, 168 mg / ml (444 mM) αα-trehalose dihydrate, 0.4 Prepare a 2x formulation buffer by dissolving mg / ml polysorbate 80 and 0.2 mg / ml (0.536 mM) ethylenediamine tetraacetate disodium dihydrate in water. A storage solution of the anti-CTLA-4 antibody ticilimumab was prepared according to Example 2 and 42-55 mg / ml in histidine buffer using ultrafiltration using a type 50 kDa membrane (Biomax PES) (target). Is concentrated to 45 mg / ml).</p><p> To prepare the pharmaceutical composition, place the same amount of anti-CTLA-4 antibody thicilimumab and 2x formulation buffer in a container suitable for tightly mixing the liquid composition. After mixing, a small amount of solution is removed and the extinction coefficient is 1.43 (mg / ml).^-1cm^-1The antibody concentration is determined by ultraviolet-visible spectroscopy (UV-Vis) (the expected range is 21 to 27.5 mg / ml, and the target value is 22.5 mg / ml). Finally, add the calculated appropriate volume of 1 × buffer for formulation and mix to bring the antibody concentration to the target value of 20 mg / ml (range 18-22 mg / ml).</p><p> The pharmaceutical composition is then filtered through a 0.2 μm sterile grade filter and filled into vials. A 20 ml type 1 glass vial was filled with 20 ml. The vial was capped with a stopper coated with Daikyo Seiko 777-1 Flurotech (registered trademark) and crimp-sealed. These glass vials were sterilized and the same was applied to the 20 mm Daikyo Seiko 777-1 serum stopper.</p><p> In each vial unit container, approximately 400 mg of anti-CTLA-4 antibody thisilimumab, 65.4 mg of L-histidine monohydrochloride monohydrate, 13.6 mg of L-histidine, and 2 mg of ethylenediaminetetraacetic acid disodium disodium di It contains a hydrate, 1680 mg of αα-trehalose dihydrate and 4 mg of polysorbate 80.</p><p><u style="single">Example 14</u> In this example, a liquid pharmaceutical composition comprising the anti-CTLA-4 antibody thicilimumab, L-histidine monohydrochloride monohydrate, ethylenediaminetetraacetate disodium, αα-trehalose dihydrate, and polysorbate 80. A promising manufacturing method for a product will be described.</p><p> The liquid pharmaceutical composition of the present invention was constructed by obtaining the following components. Its components are the anti-CTLA-4 antibody thicilimumab (obtained from the hybridoma cell line 11.2.1.4 deposited as ATCC registration number PTA-5169 according to Example 1 or recombinantly prepared from the mammalian cell line according to Example 2). Can be), L-histidine monohydrochloride monohydrate (available from Ajinomoto, Inc., Raleigh, NC), L-histidine (available from Ajinomoto, Inc., Raleigh, NC), and ethylenediamine. Calcium tetraacetate disodium (available as Titriplex III from Sigma-Oldrich, St. Louis, Missouri) and αα-trehalose dihydrate (Fero van Stil, Walkiegan, Illinois, product number T) -104-1-MC available) and Polysorbate 80 (available as Crillet 4HP from Kuroda, Mill Hall, Pennsylvania).</p><p> First, prepare several storage solutions of anti-CTLA-4 antibody thicilimumab, L-histidine monohydrochloride monohydrate, ethylenediaminetetraacetate disodium, αα-trehalose dihydrate, and polysorbate 80. To prepare a liquid pharmaceutical composition. Prepare 20 mM histidine buffer (pH 5.5) by dissolving 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate and 0.68 mg / ml (4.4 mM) L-histidine in water. To do. 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate, 0.68 mg / ml (4.4 mM) L-histidine, 84 mg / ml (222 mM) αα-trehalose dihydrate, 0.2 A 1 x formulation buffer is prepared by dissolving mg / ml polysorbate 80 and 0.1003 mg / ml (0.268 mM) calcium ethylenediamine tetraacetate disodium in water. 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate, 0.68 mg / ml (4.4 mM) L-histidine, 168 mg / ml (444 mM) αα-trehalose dihydrate, 0.4 Prepare a 2x formulation buffer by dissolving mg / ml polysorbate 80 and 0.206 mg / ml (0.536 mM) calcium ethylenediamine tetraacetate disodium in water. A storage solution of the anti-CTLA-4 antibody ticilimumab was prepared according to Example 2 and 42-55 mg / ml in histidine buffer using ultrafiltration using a type 50 kDa membrane (Biomax PES) (target). Is concentrated to 45 mg / ml).</p><p> To prepare the pharmaceutical composition, place the same amount of anti-CTLA-4 antibody thicilimumab and 2x formulation buffer in a container suitable for tightly mixing the liquid composition. After mixing, a small amount of solution is removed and the extinction coefficient is 1.43 (mg / ml).^-1cm^-1The antibody concentration is determined by ultraviolet-visible spectroscopy (UV-Vis) (the expected range is 21 to 27.5 mg / ml, and the target value is 22.5 mg / ml). Finally, add the calculated appropriate volume of 1 × buffer for formulation and mix to bring the antibody concentration to the target value of 20 mg / ml (range 18-22 mg / ml).</p><p> The pharmaceutical composition is then filtered through a 0.2 μm sterile grade filter and filled into vials. A 20 ml type 1 glass vial was filled with 20 ml. The vial was capped with a stopper coated with Daikyo Seiko 777-1 Flurotech (registered trademark) and crimp-sealed. These glass vials were sterilized and the same was applied to the 20 mm Daikyo Seiko 777-1 serum stopper.</p><p> In each vial unit container, approximately 400 mg of anti-CTLA-4 antibody thisilimumab, 65.4 mg of L-histidine monohydrochloride monohydrate, 13.6 mg of L-histidine, and 2.606 mg of ethylenediamine tetraacetate dicalcium di It contains sodium, 1680 mg of αα-trehalose dihydrate and 4 mg of polysorbate 80.</p><p><u style="single">Example 15</u> In this example, a liquid pharmaceutical composition comprising the anti-CTLA-4 antibody thicilimumab, L-histidine monohydrochloride monohydrate, ethylenediaminetetraacetic acid trisodium, αα-trehalose dihydrate, and polysorbate 80. The promising manufacturing method of is described.</p><p> The liquid pharmaceutical composition of the present invention was constructed by obtaining the following components. Its components are the anti-CTLA-4 antibody thicilimumab (obtained from the hybridoma cell line 11.2.1.4 deposited as ATCC registration number PTA-5169 according to Example 1 or recombinantly prepared from the mammalian cell line according to Example 2). Can be), L-histidine monohydrochloride monohydrate (available from Ajinomoto, Inc., Raleigh, NC), L-histidine (available from Ajinomoto, Inc., Raleigh, NC), and ethylenediamine. Trisodium tetraacetate (available as Titriplex III from Sigma-Oldrich, St. Louis, Missouri) and αα-trehalose dihydrate (Fero van Steil, Walkiegan, Illinois, product number T- Available as 104-1-MC) and Polysorbate 80 (available as Crillet 4HP from Kuroda, Mill Hall, Pennsylvania).</p><p> First, prepare several storage solutions of anti-CTLA-4 antibody thicilimumab, L-histidine monohydrochloride monohydrate, ethylenediaminetetraacetic acid trisodium, αα-trehalose dihydrate, and polysorbate 80. Thus, a liquid pharmaceutical composition was prepared. Prepare 20 mM histidine buffer (pH 5.5) by dissolving 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate and 0.68 mg / ml (4.4 mM) L-histidine in water. To do. 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate, 0.68 mg / ml (4.4 mM) L-histidine, 84 mg / ml (222 mM) αα-trehalose dihydrate, 0.2 A 1 x formulation buffer is prepared by dissolving mg / ml polysorbate 80 and 0.096 mg / ml (0.268 mM) trisodium ethylenediamine tetraacetate in water. 3.27 mg / ml (15.6 mM) L-histidine HCl monohydrate, 0.68 mg / ml (4.4 mM) L-histidine, 168 mg / ml (444 mM) αα-trehalose dihydrate, 0.4 Prepare a 2x formulation buffer by dissolving mg / ml polysorbate 80 and 0.192 mg / ml (0.536 mM) trisodium ethylenediamine tetraacetate in water. A storage solution of the anti-CTLA-4 antibody ticilimumab was prepared according to Example 2 and 42-55 mg / ml in histidine buffer using ultrafiltration using a type 50 kDa membrane (Biomax PES) (target). Is concentrated to 45 mg / ml).</p><p> To prepare the pharmaceutical composition, place the same amount of anti-CTLA-4 antibody thicilimumab and 2x formulation buffer in a container suitable for tightly mixing the liquid composition. After mixing, a small amount of solution is removed and the extinction coefficient is 1.43 (mg / ml).^-1cm^-1The antibody concentration is determined by ultraviolet-visible spectroscopy (UV-Vis) (the expected range is 21 to 27.5 mg / ml, and the target value is 22.5 mg / ml). Finally, add the calculated appropriate volume of 1 × buffer for formulation and mix to bring the antibody concentration to the target value of 20 mg / ml (range 18-22 mg / ml).</p><p> The pharmaceutical composition is then filtered through a 0.2 μm sterile grade filter and filled into vials. A 20 ml type 1 glass vial was filled with 20 ml. The vial was capped with a stopper coated with Daikyo Seiko 777-1 Flurotech (registered trademark) and crimp-sealed. These glass vials were sterilized and the same was applied to the 20 mm Daikyo Seiko 777-1 serum stopper.</p><p> In each vial unit container, approximately 400 mg of anti-CTLA-4 antibody thisilimumab, 65.4 mg of L-histidine monohydrochloride monohydrate, 13.6 mg of L-histidine, and 1.92 mg of ethylenediamine tetraacetate trisodium It contains 1680 mg of αα-trehalose dihydrate and 4 mg of polysorbate 80.</p><p><chemistry num="3"><img file="JP2012167120A_D0050.tif" /></chemistry></p><p> The variable region (SEQ ID NO: 5) is shown in parentheses [] and the CDR is underlined. CDR1 is shown with SEQ ID NO: 7, CDR2 is shown with SEQ ID NO: 8, and CDR3 is shown with SEQ ID NO: 9.</p><p><chemistry num="4"><img file="JP2012167120A_D0051.tif" /></chemistry></p><p> The variable region (SEQ ID NO: 6) is shown in parentheses [] and the CDR is underlined. CDR1 is shown by SEQ ID NO: 10, CDR2 is shown by SEQ ID NO: 11, and CDR3 is shown by SEQ ID NO: 12.</p>