CN116440263A

CN116440263A - anti-CTLA-4 antibody pharmaceutical composition and application thereof

Info

Publication number: CN116440263A
Application number: CN202310071664.6A
Authority: CN
Inventors: 刘沛想; 刘洪川; 李宝贤; 刘辉; 姚盛; 冯辉
Original assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Current assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Priority date: 2022-01-17
Filing date: 2023-01-17
Publication date: 2023-07-18
Also published as: WO2023134771A1

Abstract

The invention provides a stable anti-CTLA-4 antibody pharmaceutical composition and application thereof. The pharmaceutical composition comprises a buffer and an anti-CTLA-4 antibody or antigen-binding fragment thereof; the concentration of the anti-CTLA-4 antibody or antigen binding fragment thereof is about 1-100 mg/mL, and the antibody preparation obtained by developing can be used for intravenous injection by selecting a proper buffer system and pH and optimizing a stabilizer and a surfactant, and the components are interacted and cooperated, so that a storage environment suitable for long-term storage is provided for the anti-CTLA-4 monoclonal antibody, the degradation, aggregation or precipitation of the antibody caused in the long-term storage or transportation process of the preparation can be avoided, the long-term storage of the antibody in the environment can be realized, the activity and purity of the antibody can be ensured to be kept stable during the storage, and the drug effect of a biological drug is ensured.

Description

anti-CTLA-4 antibody pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of therapeutic pharmaceutical compositions, in particular to an anti-CTLA-4 antibody pharmaceutical composition and application thereof.

Background

Cytotoxic T lymphocyte-associated protein 4 (CTLA 4 or CTLA-4, cytoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a transmembrane protein encoded by the CTLA-4 gene, which is located on chromosome 2q33 of human. CTLA-4 is a member of the immunoglobulin superfamily, consisting of extracellular V, transmembrane and cytoplasmic domains. CTLA-4 shares homology with the T cell surface co-stimulatory molecule receptor CD28, both competing for binding to its ligands B7-1 (CD 80) and B7-2 (CD 86), which are expressed predominantly on the antigen presenting cell surface. CTLA-4 binds with higher affinity to CD80 and CD86 than CD28 and is therefore able to compete and block CD28 mediated activation. CTLA-4 is typically expressed on the surface of regulatory T cells (tregs) and normal T cells in the activated state. After being combined with B7 molecules, the CTLA-4 can inhibit the activation of T cells, participate in the negative regulation of immune response, act as an immune check point and down regulate the immune response, so that the CTLA-4 plays a very important role in the immune regulation.

Activation of T cells requires stimulation of two signals, the first signal being from an antigen peptide-MHC complex that specifically binds to the surface of an Antigen Presenting Cell (APC) at the T Cell Receptor (TCR), and the second signal pathway requiring participation of a co-stimulatory molecule (e.g., CD 28) that, upon binding of CD28 to B7-1/B7-2 (CD 80/CD 86), further activates the T cell, promoting its maturation and proliferation. Current studies indicate that CTLA-4 down-regulates T cell function during the immune response by: first, CTLA-4 can competitively block transmission of CD28 and CD80/86 co-stimulatory signals through its high affinity with CD80/CD86, thereby inhibiting proliferation of T cells and reducing secretion of IL-2. Second, CTLA-4 can reduce T cell activation involving CD28 by reducing the expression level of CD80/CD86 on Antigen Presenting Cells (APCs) or by removing CD80/CD86 molecules from the surface of Antigen Presenting Cells (APCs) by trans endocytosis. Third, CTLA-4 can inhibit TCR signaling by mediating dendritic cell binding to CD80/CD86 and inducing expression of tryptophan-degrading enzyme IDO. In addition, CTLA-4 can also induce production of regulatory cytokines by recruiting inhibitory molecules to bind to immune synapses, thereby inhibiting APC and TCR signaling.

Blockade of CTLA-4 has been demonstrated in many studies to induce tumor regression. The anti-CTLA-4 antibody can effectively and specifically inhibit cellular and humoral immune responses in vivo and in vitro, has remarkable treatment effect on transplant rejection and various autoimmune diseases, and has low toxic and side effects.

Although the monoclonal drugs Ipilimumab (bezels) and Tremelimumab (aslicarpa) for CTLA-4 are currently available for some cancer treatments and are being tested for other anti-cancer indications, there remains a need for novel anti-CTLA-4 antibodies that include improved activity compared to known antibodies in various respects.

However, antibody drugs have a large molecular weight and a complex structure, and are easily degraded, polymerized, or subjected to undesired chemical modification, and thus are unstable. In order to make antibodies suitable for production and administration, and to maintain stability during storage and subsequent use, and to exert better effects, stable formulation studies of antibody drugs are particularly important.

Disclosure of Invention

The pharmaceutical composition of the invention is a high stability pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4. Particularly, the antibody preparation developed by selecting a proper buffer system and pH, optimizing a stabilizer and a surfactant can be used for intravenous injection, and the components interact and cooperate with each other to provide a storage environment suitable for long-term storage for the anti-CTLA-4 antibody or antigen binding fragment thereof, so that the degradation, aggregation or precipitation of the antibody caused by the preparation in the long-term storage or transportation process can be avoided, the antibody can be stored for a long time in the environment, the activity and purity of the antibody can be ensured to be kept stable during the storage period, and the drug effect of the biological drug is ensured.

The invention provides a pharmaceutical composition comprising: (1) a buffer; (2) an anti-CTLA-4 antibody or antigen-binding fragment thereof.

In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as described above:

LCDR1：RASQNVGTYVA(SEQ ID NO:1)；

LCDR2：STSYRYS(SEQ ID NO:2)；

LCDR3：HQYDTYPLT(SEQ ID NO:3)；

HCDR1：SGYYWN(SEQ ID NO:4)；

HCDR2: YIGYDGSNX1YNPSLKX2, wherein X1 is N or Y, X2 is S or N;

HCDR3: x3YYSGYFDS, X3 is D or N.

In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof described above comprises:

LCDR1 with the amino acid sequence shown as SEQ ID NO. 1;

LCDR2 with the amino acid sequence shown in SEQ ID NO. 2;

LCDR3 with the amino acid sequence shown in SEQ ID NO. 3;

HCDR1 with the amino acid sequence shown in SEQ ID NO. 4;

HCDR2 with amino acid sequence shown as SEQ ID NO 5 or 7 or 9;

the amino acid sequence is HCDR3 shown in SEQ ID NO. 6 or 8.

(1) The amino acid sequences are respectively shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences are respectively shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; or (b)

(2) The amino acid sequences are respectively shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences are respectively shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 4, SEQ ID NO. 7 and SEQ ID NO. 8; or (b)

(3) The amino acid sequences are shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, and the amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 4, SEQ ID NO. 9 and SEQ ID NO. 6 respectively.

In some embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, preferably humanized antibodies or antigen-binding fragments thereof.

(1) A light chain variable region with an amino acid sequence shown as SEQ ID NO. 10, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 11; or (b)

(2) A light chain variable region with an amino acid sequence shown as SEQ ID NO. 12, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 11; or (b)

(3) A light chain variable region with an amino acid sequence shown as SEQ ID NO. 13, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 14; or (b)

(4) A light chain variable region with an amino acid sequence shown as SEQ ID NO. 13, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 15.

In some embodiments, the anti-CTLA-4 antibody described above comprises:

(1) The amino acid sequence of the light chain is shown as SEQ ID NO. 16, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 17.

(2) The amino acid sequence of the light chain is shown as SEQ ID NO. 18, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 19.

(3) The amino acid sequence of the light chain is shown as SEQ ID NO. 20, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 21.

(4) The amino acid sequence of the light chain is shown as SEQ ID NO. 22, and the amino acid sequence of the heavy chain is shown as SEQ ID NO. 23.

In some embodiments, the concentration of anti-CTLA-4 antibodies or antigen-binding fragments thereof in the above pharmaceutical compositions is about 1-100 mg/mL, preferably about 1-50 mg/mL, more preferably about 5-30 mg/mL; more preferably, the anti-CTLA-4 antibody or antigen-binding fragment thereof has a concentration of about 1mg/mL,5mg/mL,7mg/mL,9mg/mL,10mg/mL,11mg/mL,15mg/mL,20mg/mL,25mg/mL,30mg/mL,40mg/mL,50mg/mL,60mg/mL,70mg/mL,80mg/mL,90mg/mL,100mg/mL or any two values within these ranges as the end point, preferably about 9mg/mL,10mg/mL,11mg/mL or 15mg/mL.

In some embodiments, the pH of the pharmaceutical composition is about 4.5 to 6.5, preferably about 5.0 to 6.0, more preferably about 5.3 to 5.7, and the pH of the pharmaceutical composition is in the range of about 4.5,5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5 or any two of these ranges as endpoints, preferably about 5.4,5.5 or 5.6, by way of non-limiting example.

In some embodiments, the osmotic pressure of the above pharmaceutical composition is about 250 to 350mOsm/kg.

In some embodiments, the buffer is selected from one or more of an acetate buffer, a citrate buffer, a phosphate buffer, and a histidine buffer; preferably, the buffer is an acetate buffer.

In some embodiments, the buffer is an acetate buffer, preferably the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer.

In some embodiments, the acetate buffer is an acetate-sodium acetate buffer. In some embodiments, the acetic acid-sodium acetate buffer is made from about 1 to 20mM acetic acid and about 1 to 20mM sodium acetate. In some embodiments, the acetic acid-sodium acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 1:2 to about 1:3. In some embodiments, the acetic acid-sodium acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 1:5 to about 1:8. In some embodiments, the acetic acid-sodium acetate buffer is: an acetate buffer at a pH of about 5.0 made from about 6.5mM acetic acid and about 13.5mM sodium acetate. In some embodiments, the acetic acid-sodium acetate buffer is: an acetate buffer having a pH of about 5.0 to 6.0 made from about 2 to 4mM acetic acid and about 16 to 18mM sodium acetate; preferably an acetate buffer at a pH of about 5.5 made from about 3mM acetic acid and about 17mM sodium acetate.

In some embodiments, the histidine buffer is selected from a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer, preferably a histidine-histidine hydrochloride buffer. In some embodiments, the histidine-histidine hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made from about 1 to 20mM L-histidine and about 1 to 20mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of about 1:1 to about 1:4. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of about 1:1. In some embodiments, the histidine buffer is made up of histidine and histidine hydrochloride in a molar ratio of about 1:3. In some embodiments, the histidine formulation is: histidine buffer at a pH of about 5.5 made from about 4.5mM L-histidine and about 15.5mM L-histidine monohydrochloride. In some embodiments, the histidine formulation is: histidine buffer at a pH of about 6.0 made from about 10mM histidine and about 10mM histidine hydrochloride.

In some embodiments, the buffer is a citric acid buffer, preferably the citric acid buffer is a citric acid-sodium citrate buffer. In some embodiments, the citrate buffer is made from about 1-20 mM citric acid and about 1-20 mM sodium citrate. In some embodiments, the citric acid buffer is made from citric acid and sodium citrate in a molar ratio of about 1:1 to 1:4. In some embodiments, the citrate buffer is: a citric acid buffer having a pH of about 6.5 made from about 5.0mM citric acid and about 15.0mM sodium citrate. In some embodiments, the citrate buffer is: a citric acid buffer having a pH of about 6.0 made from about 10mM citric acid and about 10mM sodium citrate.

In some embodiments, the buffer is a phosphate buffer, preferably the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. In some embodiments, the phosphate buffer is made from about 1 to 20mM disodium hydrogen phosphate and about 1 to 20mM sodium dihydrogen phosphate. In some embodiments, the phosphate buffer is made from disodium hydrogen phosphate and sodium dihydrogen phosphate in a molar ratio of about 1:1 to 1:4. In some embodiments, the phosphate buffer is: phosphate buffer at a pH of about 7.0 made from about 10mM disodium hydrogen phosphate and about 10mM sodium dihydrogen phosphate.

In some embodiments, the concentration of the buffer is about 5 to 100mM, preferably about 10 to 50mM, and more preferably about 10 to 30mM; preferably about 15 to 25mM, and the concentration of the above-mentioned buffer is, in a non-limiting example, about 10mM,15mM,20mM,25mM,30mM,40mM,45mM,50mM or any two values within these ranges as the end point, preferably about 15mM,20mM or 25mM.

In some embodiments, the pH of the buffer is from about 4.5 to about 6.5, preferably from about 5.0 to about 6.0, more preferably from about 5.3 to about 5.7, and the pH of the buffer is in the range of about 5.0,5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3,6.4,6.5 or any two of these ranges, as an endpoint, and preferably is about 5.4,5.5 or 5.6. In some embodiments, the above pharmaceutical composition further comprises a stabilizer selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose, and trehalose; preferably, the arginine salt is arginine hydrochloride. In some embodiments, the concentration of the stabilizer is about 10 to 400mM, preferably about 100 to 300mM, preferably about 130 to 280mM, preferably about 200 to 260mM, and the concentration of the stabilizer is, by way of non-limiting example, about 130mM,135mM,140mM,150mM,160mM,170mM,180mM,190mM,200mM,210mM,220mM,228mM,230mM,240mM,250mM,260mM, or any two values within these ranges, preferably about 210mM,220mM,228mM, or 230mM.

In some embodiments, the stabilizer is mannitol at a concentration of about 130 to 280 mM; or the stabilizer is sucrose with the concentration of about 130-280 mM; or a combination of sodium chloride at a concentration of about 20-80 mM and mannitol at a concentration of about 110-170 mM; or a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; in some embodiments, the stabilizer is mannitol at a concentration of about 200 to 260 mM; or the stabilizer is sucrose with the concentration of about 200-260 mM; or a combination of sodium chloride at a concentration of about 30-70 mM and mannitol at a concentration of about 120-160 mM; or a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM.

In some embodiments, the stabilizer is mannitol. In some embodiments, the stabilizer is mannitol at a concentration of about 100 to 300mM, preferably about 130 to 280mM, and preferably about 200 to 260mM, and non-limiting examples of mannitol concentrations are about 200mM,210mM,220mM,230mM,240mM,250mM,260mM, or any two values within these ranges, as endpoints, and preferably about 240mM.

In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizer is sucrose at a concentration of about 100 mM to about 300mM, preferably about 130 mM to about 280mM, and preferably about 200mM to about 260mM, and non-limiting examples of sucrose concentrations are about 200mM,210mM,220mM,228mM,230mM,240mM,250mM, or any two values within these ranges, as endpoints, and preferably about 220mM or about 228mM.

In some embodiments, the stabilizer is a combination of sodium chloride and mannitol. In some embodiments, the above-described stabilizer is a combination of about 30 to 200mM sodium chloride with about 30 to 200mM mannitol, preferably about 20 to 80mM sodium chloride with about 110 to 170mM mannitol, preferably about 30 to 70mM sodium chloride with about 120 to 160mM mannitol, non-limiting examples of the above-described stabilizer are a combination of about 50mM sodium chloride with about 140mM mannitol, or a combination of about 50mM sodium chloride with about 150mM mannitol.

In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the above-described stabilizer is a combination of about 30 to 200mM sodium chloride and about 30 to 200mM sucrose, preferably about 20 to 80mM sodium chloride and about 110 to 170mM sucrose, preferably about 30 to 70mM sodium chloride and about 120 to 160mM sucrose, non-limiting examples of the above-described stabilizer are a combination of about 50mM sodium chloride and about 140mM sucrose, or a combination of about 50mM sodium chloride and about 160mM sucrose.

In some embodiments, the above pharmaceutical composition further comprises a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188.

In some embodiments, the surfactant is selected from polysorbate 80.

In some embodiments, the surfactant is selected from polysorbate 20.

In some embodiments, the surfactant concentration is about 0.001% to about 0.1%, preferably about 0.01% to about 0.05%, calculated as w/v; as non-limiting examples, the concentration of the above surfactant is about 0.01%,0.02%,0.03%,0.04%,0.08%, or any two values within these ranges are formed as endpoints, preferably about 0.02%.

In some embodiments, the pharmaceutical composition comprises, or consists of, the components set forth in any one of (1) to (13), respectively:

(1) (a) about 1-50 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) 130-280 mM mannitol; and (d) about 0.01% to about 0.1% (w/v) polysorbate 80; or (b)

(2) (a) about 1-50 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) 130-280 mM sucrose; and (d) about 0.01% to about 0.1% (w/v) polysorbate 80; or (b)

(3) (a) about 1-50 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) A stabilizer, said stabilizer being a combination of sodium chloride at a concentration of about 20-80 mM and mannitol at a concentration of about 110-170 mM; and (d) about 0.01% to about 0.1% (w/v) polysorbate 80; or (b)

(4) (a) about 1-50 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) A stabilizer, said stabilizer being a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; and (d) about 0.01% to about 0.1% (w/v) polysorbate 80; or (b)

(5) (a) about 5-30 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) 200-260 mM mannitol; and (d) about 0.01% to about 0.05% (w/v) polysorbate 80; or (b)

(6) (a) about 5-30 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) 200-260 mM sucrose; and (d) about 0.01% to about 0.05% (w/v) polysorbate 80; or (b)

(7) (a) about 5-30 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) A stabilizer, said stabilizer being a combination of sodium chloride at a concentration of about 30-70 mM and mannitol at a concentration of about 120-160 mM; and (d) about 0.01% to about 0.05% (w/v) polysorbate 80; or (b)

(8) (a) about 5-30 mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof; (b) about 10-30 mM acetate buffer, pH about 5.0-6.0; (c) A stabilizer, said stabilizer being a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM; and (d) about 0.01% to about 0.05% (w/v) polysorbate 80; or (b)

(9) (a) about 10mg/mL of an anti-CTLA-4 antibody comprising the light chain amino acid sequence as set forth in SEQ ID No. 16 and the heavy chain amino acid sequence as set forth in SEQ ID No. 17; (b) about 20mM acetate buffer, pH about 5.5; (c) about 240mM mannitol; and (d) about 0.02% (w/v) polysorbate 80; or (b)

(10) (a) about 10mg/mL of an anti-CTLA-4 antibody comprising the light chain amino acid sequence as set forth in SEQ ID No. 16 and the heavy chain amino acid sequence as set forth in SEQ ID No. 17; (b) about 20mM acetate buffer, pH about 5.5; (c) about 220mM sucrose; and (d) about 0.02% (w/v) polysorbate 80; or (b)

(11) (a) about 10mg/mL of an anti-CTLA-4 antibody comprising the light chain amino acid sequence as set forth in SEQ ID No. 16 and the heavy chain amino acid sequence as set forth in SEQ ID No. 17; (b) about 20mM acetate buffer, pH about 5.5; (c) about 228mM sucrose; and (d) about 0.02% (w/v) polysorbate 80; or (b)

(12) (a) about 10mg/mL of an anti-CTLA-4 antibody comprising the light chain amino acid sequence as set forth in SEQ ID No. 16 and the heavy chain amino acid sequence as set forth in SEQ ID No. 17; (b) about 20mM acetate buffer, pH about 5.5; (c) A stabilizer, said stabilizer being a combination of sodium chloride at a concentration of about 50mM and mannitol at a concentration of about 140 mM; and (d) about 0.02% (w/v) polysorbate 80; or (b)

(13) (a) about 10mg/mL of an anti-CTLA-4 antibody comprising the light chain amino acid sequence as set forth in SEQ ID No. 16 and the heavy chain amino acid sequence as set forth in SEQ ID No. 17; (b) about 20mM acetate buffer, pH about 5.5; (c) A stabilizer, said stabilizer being a combination of sodium chloride at a concentration of about 50mM and sucrose at a concentration of about 140 mM; and (d) about 0.02% (w/v) polysorbate 80.

In some embodiments, the pharmaceutical composition described in any of the embodiments herein is a liquid formulation or a lyophilized formulation.

In some embodiments, the pharmaceutical composition is a liquid formulation.

In some embodiments, the liquid formulation or lyophilized formulation is stable at 2-8 ℃ for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some embodiments, the liquid formulation or lyophilized formulation is stable at 40 ℃ for at least 7 days, at least 14 days, or at least 28 days.

The present invention also provides an injection comprising a pharmaceutical composition as described in any one of the schemes herein in combination with a sodium chloride solution or a dextrose solution; preferably, the sodium chloride solution concentration is about 0.85-0.9% (w/v); preferably, the glucose solution concentration is about 5 to 25% (w/v), more preferably about 5 to 10% (w/v); preferably, the concentration of the anti-CTLA-4 antibody in the injection is about 0.05-10.5 mg/mL, more preferably about 0.1-5 mg/mL or about 1-5 mg/mL; the pH of the injection is about 4.5 to 6.5, preferably about 5.0 to 6.0, more preferably about 5.3 to 5.7.

In some embodiments, the pharmaceutical composition or injection is administered intravenously.

The invention also provides the use of a pharmaceutical composition or injection as described in any one of the schemes herein in the manufacture of a medicament for the treatment and/or prophylaxis of CTLA-4 mediated diseases or conditions.

The invention also provides a pharmaceutical composition or injection as described in any of the embodiments herein for use in the treatment and/or prevention of CTLA-4 mediated diseases or conditions.

The invention also provides a method of treating and/or preventing a CTLA-4 mediated disease or disorder, comprising administering to a subject in need thereof a pharmaceutical composition or injection as described in any of the schemes herein.

In some embodiments, the disease or condition is cancer.

Drawings

Fig. 1: results of affinity test of anti-CTLA-4 pharmaceutical composition with recombinant human CTLA-4 protein.

Fig. 2: results of inhibition test of hCTLA4 humanized mice transplanted with EMT6 tumor growth by anti-CTLA-4 pharmaceutical composition.

Fig. 3: ELISA method detects the binding of humanized anti-CTLA-4 antibody to huCTLA-4.

Fig. 4: ELISA method detects the ability of humanized anti-CTLA-4 antibody to block binding of huCTLA-4 to CD 80.

Fig. 5: the biological activity of the humanized anti-CTLA-4 antibody is detected by a luciferase method.

Fig. 6: ADCC activity of humanized anti-CTLA-4 antibodies.

Fig. 7: CDC activity of humanized anti-CTLA-4 antibodies.

Fig. 8: inhibition of tumor growth in mice by humanized anti-CTLA-4 antibodies.

Fig. 9: and identifying the epitope by a Fortebio binding experiment.

Fig. 10: inhibition of hCTLA4 humanized mice transplanted MC38 tumor growth by huJS 007-47.

Fig. 11: inhibition of hCDLA 4 humanized mice transplanted H22 tumor growth by huJS 007-47.

Detailed Description

Definition and description

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. All references cited herein, including patents, patent applications, papers, textbooks, and the like, and to the extent that they have not been cited, are hereby incorporated by reference in their entirety. If one or more of the incorporated documents and similar materials differs from or contradicts the present application, including but not limited to the defined terms, term usage, described techniques, and the like, the present application controls.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

The term "pharmaceutical composition" or "formulation" means a mixture comprising one or more antibodies described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present invention are stable upon storage and their stability is independent of lyophilization (or other state-change methods, such as spray drying).

The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).

The term "excipient" refers to an agent that may be added to a formulation to provide desired characteristics (e.g., consistency, increased stability) and/or to regulate osmotic pressure. Examples of common excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of + -20% or + -10% relative to the particular value, including + -5%, + -1% and + -0.1%, as these variations are suitable for carrying out the disclosed methods.

The term "buffer pH of about 4.5 to 6.5" refers to an agent that, by the action of its acid/base conjugated components, renders a solution containing the agent resistant to pH changes. Buffers used in the formulations of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.

Herein, examples of "buffers" that control pH within this range include acetic acid, acetate (e.g., sodium acetate), succinic acid, succinate (e.g., sodium succinate), gluconic acid, histidine, histamine acid salts (e.g., histidine hydrochloride), methionine, citric acid (citric acid), citrate (citrate), phosphate, citrate/phosphate, imidazole, combinations thereof, and other organic acid buffers.

A "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, and the like, such as histidine buffers containing histidine and histidine hydrochloride; histidine buffers of the present invention also include histidine buffers comprising histidine and acetate (e.g., sodium or potassium salts).

"citrate buffer", also known as "citrate buffer", is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. The preferred citrate buffer is a citrate-sodium citrate buffer.

An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer is acetic acid-sodium acetate buffer.

A "succinic acid buffer" is a buffer that includes succinate ions. Examples of succinic acid buffers include sodium succinate, potassium succinate, calcium succinate, magnesium succinate, and the like. The preferred succinate buffer is a succinic acid-sodium succinate buffer.

A "phosphate buffer" is a buffer that includes phosphate ions. Examples of the phosphate buffer include sodium dihydrogen phosphate-disodium hydrogen phosphate, potassium dihydrogen phosphate-dipotassium hydrogen phosphate, and the like. The preferred phosphate buffer is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or salts thereof (e.g., arginine hydrochloride), glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), opioids (opines), alanines, octopine, glycine (strombine) and the N-oxide of Trimethylamine (TMAO), human serum albumin (hsa), bovine Serum Albumin (BSA), α -casein, globulin, α -lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNAase a. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, and the like, may also act to control osmotic pressure. The stabilizer used in the present invention is one or more selected from the group consisting of polyhydric alcohols, amino acids, salts and saccharides. The preferred salts are sodium chloride, the preferred sugars are sucrose and trehalose, and the preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine, glycine, proline, which may be present in their D-and/or L-forms, but typically in the L-form, which may be present in any suitable salt, for example as the hydrochloride salt, such as arginine hydrochloride. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose.

The term "surfactant" generally includes agents that protect proteins such as antibodies from air/solution interface induced stress, solution/surface induced stress to reduce aggregation of the antibodies or minimize the formation of particulates in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamer), sodium Dodecyl Sulfate (SDS). Herein, unless otherwise specified, the terms "concentration of polysorbate 20" and "concentration of polysorbate 80" both refer to the mass volume concentration (w/v), such as "0.02%" in "about 0.02% polysorbate 80" that is, "0.02 g polysorbate 80 in 100mL of liquid".

The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

The term "stable" formulation is a formulation in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. Pharmaceutical formulations may be stable even if the contained antibodies fail to retain 100% of their chemical structure or biological function after storage for a period of time. In some cases, an antibody structure or function that is capable of maintaining about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage for a period of time may also be considered "stable". Various analytical techniques for measuring protein stability are available in the art and reviewed in peptide and protein drug Delivery (Peptide and Protein Drug Delivery) 247-301, major editions of vincent Lee, marcel Dekker, inc., new York, n.y., pubs (1991), and Jones, a. (1993) adv. Drug Delivery rev.10: 29-90 (both incorporated by reference).

After storage of the formulation at a temperature and for a time, the stability of the formulation can be measured by determining the percentage of natural antibodies remaining therein (and other methods). The percentage of native antibodies may be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SEC-HPLC ]), among other methods, "native" refers to unagglomerated and undegraded. In some embodiments, the stability of a protein is determined as the percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In some embodiments, the formulation may be stable for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form at room temperature, about 25-30 ℃ or 40 ℃.

Stability can be measured by determining the percentage of antibodies ("acid forms") that migrate during ion exchange in this more acidic fraction of the antibody ("primary charged form") as well as other methods, where stability is inversely proportional to the percentage of the acid form of the antibody. The percentage of "acidified" antibody may be measured by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]), among other methods. In some embodiments, an acceptable degree of stability means that the antibody in its acidic form is detectable at most about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% after the formulation has been stored at a temperature for a period of time. The time stored prior to measuring stability may be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, the temperature at which the pharmaceutical formulation is allowed to be stored may be any temperature in the range of about-80 ℃ to about 45 ℃, for example, stored at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 2-8 ℃, about 5 ℃, about 25 ℃, or about 40 ℃.

An antibody "retains its physical stability" in the pharmaceutical composition if it exhibits substantially no signs of, for example, aggregation, precipitation, and/or denaturation when visually inspected for color and/or clarity or measured by UV light scattering or by aperture-exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation may proceed to the point that a visible precipitate forms.

Stability, e.g., physical stability, of the formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurements are related to the turbidity of the formulation. Turbidity of a formulation is in part an inherent property of proteins dissolved in solution and is typically measured by nephelometry and measured in Nephelometry Turbidity Units (NTU).

Turbidity levels that vary with, for example, the concentration of one or more components in a solution (e.g., protein and/or salt concentration) are also referred to as the "opacifying" or "opacifying appearance" of a formulation. Turbidity levels can be calculated with reference to standard curves generated using suspensions of known turbidity. The reference standard for determining turbidity levels of pharmaceutical compositions can be based on the "European Pharmacopeia" standard (European Pharmacopeia (European Pharmacopoeia), fourth edition, "European Committee for pharmaceutical quality" (Directorate for the Quality of Medicine of the Council of Europe) (EDQM), strasbourg, france). A clear solution is defined as a solution having a turbidity lower than or equal to the turbidity of a reference suspension according to the european pharmacopoeia standard having a turbidity of about 3. Nephelometric turbidity measurements can detect Rayleigh scattering in the absence of associative or non-ideal effects, which typically vary linearly with concentration. Other methods for assessing physical stability are known in the art.

An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed, for example, by detecting or quantifying the chemically altered form of the antibody. Chemical changes may include dimensional changes (e.g., scissoring) that can be assessed using, for example, aperture exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

An antibody in a pharmaceutical composition "retains its biological activity" in the pharmaceutical composition if the antibody is biologically active for its intended purpose. For example, a formulation of the invention can be considered stable if after storage of the formulation at isothermal temperatures, e.g., 5 ℃, 25 ℃, 45 ℃ for a period of time (e.g., 1 to 12 months), the formulation contains an anti-CTLA-4 antibody that binds CTLA-4 with an affinity of at least 90%, 95% or more of the binding affinity of the antibody prior to said storage. Binding affinity can also be determined using, for example, ELISA or plasma resonance techniques.

In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody in a pharmacological sense refers to an amount that is effective in the prevention or treatment or alleviation of the symptoms of a disorder that an antibody may effectively treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from onset of a disease or promotes regression of a disease as evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease asymptomatic periods, or prevention of injury or disability caused by pain in the disease. The ability of a drug to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by assaying the activity of the agent in an in vitro assay. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of drug that inhibits the progression or recurrence of a disease when administered alone or in combination with other therapeutic drugs to a subject at risk of developing or a subject with recurrence of the disease.

The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects/patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a particular embodiment of the invention, the subject is a human.

The terms "administering," "administering," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those of skill in the art. Routes of administration of anti-CTLA-4 antibodies include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as injection or infusion. By "parenteral administration" is meant administration other than enteral or topical administration, typically by injection, including, but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation.

anti-CTLA-4 antibodies

The term "antibody" as used herein is to be understood as including intact antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to human CTLA-4 or an epitope thereof. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

The term "isolated antibody" refers to a purified state of the bound compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth media. The term "isolated" does not mean that such materials are completely absent or that water, buffer or salt are absent unless they are present in amounts that would significantly interfere with the experimental or therapeutic use of the binding compounds described herein.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The term "murine antibody" or "hybridoma antibody" is in the present disclosure a monoclonal antibody against human CTLA-4 prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with CTLA-4 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.

The term "chimeric antibody" is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domain is obtained from an antibody ("parent antibody") of a rodent or the like, while the constant domain sequence is obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject as compared to the parent rodent antibody.

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

The term "full length antibody" or "whole antibody molecule" refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (about 25kDa in full length) being interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. VH and VL regions can be further subdivided into Complementarity Determining Regions (CDRs) of high variability and regions spaced apart by more conserved regions called Framework Regions (FR). Each VH or VL region consists of, in order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of immunoglobulins to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

The term "CDR" refers to the complementarity determining region within an antibody variable sequence. There are 3 CDRs in each of the heavy and light chain variable regions, which are designated HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each of the heavy and light chain variable regions. The exact boundaries of these CDRs are defined differently for different systems.

The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia (Chothia et al (1989) Nature 342:877-883, al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", journal of Molecular Biology,273,927-948 (1997) based on Kabat (Kabat et al Sequences of Proteins of Immunological Interest, 4 th edition, u.s. Device of Health and Human Services, national Institutes of Health (1987), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (1999Nucleic Acids Research,27,209-212) based on topology of the CDR loops, and North definition based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures.

As used herein, an "antigen-binding fragment" includes a fragment of an antibody or derivative thereof, typically comprising at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments; a diabody; a linear antibody; single chain antibody molecules, such as scFv; nanobodies (nanobodies) and multispecific antibodies formed from antibody fragments. When the binding activity of an antibody is expressed on a molar basis, the binding fragment or derivative thereof generally retains at least 10% of the antigen binding activity of the parent antibody. Preferably, the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that an antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functional conservative variants" of the antibody).

The anti-CTLA-4 antibodies or antigen-binding fragments thereof described herein include any of the anti-CTLA-4 antibodies or antigen-binding fragments thereof described in the patent applications Ser. No. CN202010708105.8 and PCT/CN2021/107707, the disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the present invention include those derived from the humanized anti-CTLA-4 antibodies described in CN202010708105.8, huJS007-47, huJS007-48, huJS007-79 and huJS007-106, the amino acid sequences of which are shown in Table A.

Table a: amino acid sequences of portions of 4 humanized anti-CTLA-4 antibodies (KABAT scheme)

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively, as follows:

LCDR1：RASQNVGTYVA(SEQ ID NO:1)；

LCDR2：STSYRYS(SEQ ID NO:2)；

LCDR3：HQYDTYPLT(SEQ ID NO:3)；

HCDR1：SGYYWN(SEQ ID NO:4)；

HCDR2: YIGYDGSNX1YNPSLKX2, wherein X1 is N or Y, X2 is S or N;

HCDR3: x3YYSGYFDS, X3 is D or N.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise:

LCDR1 with the amino acid sequence shown as SEQ ID NO. 1;

LCDR2 with the amino acid sequence shown in SEQ ID NO. 2;

LCDR3 with the amino acid sequence shown in SEQ ID NO. 3;

HCDR1 with the amino acid sequence shown in SEQ ID NO. 4;

HCDR2 with amino acid sequence shown as SEQ ID NO 5 or 7 or 9;

the amino acid sequence is HCDR3 shown in SEQ ID NO. 6 or 8.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO. 4, SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO. 4, SEQ ID NO. 9 and SEQ ID NO. 6, respectively.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention are selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, preferably humanized antibodies or antigen-binding fragments thereof.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise a light chain variable region as set forth in SEQ ID NO. 10, and a heavy chain variable region as set forth in SEQ ID NO. 11.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise a light chain variable region as set forth in SEQ ID NO. 12, and a heavy chain variable region as set forth in SEQ ID NO. 11.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise a light chain variable region as set forth in SEQ ID NO. 13, and a heavy chain variable region as set forth in SEQ ID NO. 14.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention comprise a light chain variable region as set forth in SEQ ID NO. 13, and a heavy chain variable region as set forth in SEQ ID NO. 15.

In some embodiments, the anti-CTLA-4 antibodies used in the methods and compositions of the invention comprise a light chain amino acid sequence as set forth in SEQ ID NO. 16, and a heavy chain amino acid sequence as set forth in SEQ ID NO. 17.

In some embodiments, the anti-CTLA-4 antibodies used in the methods and compositions of the invention comprise a light chain amino acid sequence as set forth in SEQ ID NO. 18, and a heavy chain amino acid sequence as set forth in SEQ ID NO. 19.

In some embodiments, the anti-CTLA-4 antibodies used in the methods and compositions of the invention comprise a light chain amino acid sequence as set forth in SEQ ID NO. 20, and a heavy chain amino acid sequence as set forth in SEQ ID NO. 21.

In some embodiments, the anti-CTLA-4 antibodies used in the methods and compositions of the invention comprise a light chain amino acid sequence as set forth in SEQ ID NO. 22, and a heavy chain amino acid sequence as set forth in SEQ ID NO. 23.

In some embodiments, the anti-CTLA-4 antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention are humanized or chimeric antibodies and can include human constant regions. In some embodiments, the constant region is selected from the group consisting of human IgG1, igG2, igG3, and IgG4 constant regions; preferably, the anti-CTLA-4 antibodies or antigen-binding fragments thereof suitable for use in the methods and compositions described herein comprise a heavy chain constant region of human IgG1 or IgG4 isotype.

In some embodiments, the invention provides a method for preparing an anti-CTLA-4 antibody or antigen-binding fragment thereof as described herein, comprising expressing the antibody or antigen-binding fragment thereof in a host cell described herein under conditions suitable for expression of the antibody or antigen-binding fragment thereof, and recovering the expressed antibody or antigen-binding fragment thereof from the host cell.

The present invention provides mammalian host cells for expressing the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, chinese Hamster Ovary (CHO) cells, NS0, SP2/0 cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells, a549 cells, 293T cells, and many other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.

In one embodiment, the invention provides a method of producing an anti-CTLA-4 antibody, wherein the method comprises, upon introduction of an expression vector into a mammalian host cell, producing the antibody by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably secretion of the antibody into the medium in which the host cell is grown. Standard protein purification methods can be used to recover antibodies from the culture medium.

Antibodies expressed by different cell lines or in transgenic animals are likely to have different glycosylation from each other. However, all antibodies encoded by or comprising the amino acid sequences provided herein are part of the invention, regardless of the glycosylation of the antibody. Also, in certain embodiments, nonfucosylated antibodies are advantageous because they generally have greater efficacy than their fucosylated counterparts in vitro and in vivo, and are unlikely to be immunogenic because their sugar structures are normal components of natural human serum IgG.

Pharmaceutical preparation

The anti-CTLA-4 antibodies or antigen-binding fragments thereof in the pharmaceutical compositions of the invention are as described in any of the embodiments of the "anti-CTLA-4 antibodies" section of the application.

For example, the anti-CTLA-4 antibodies or antigen-binding fragments thereof in the pharmaceutical compositions of the invention comprise LCDR1, LCDR2 and LCDR3 having amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and HCDR1, HCDR2 and HCDR3 having amino acid sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively. Preferably, the anti-CTLA-4 antibody or antigen-binding fragment thereof is selected from murine antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, humanized antibodies or antigen-binding fragments thereof, preferably humanized antibodies or antigen-binding fragments thereof. Preferably, the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises the light chain variable region shown as SEQ ID NO. 10 and the heavy chain variable region shown as SEQ ID NO. 11. More preferably, the anti-CTLA-4 antibody comprises the light chain amino acid sequence shown in SEQ ID NO. 16 and the heavy chain amino acid sequence shown in SEQ ID NO. 17.

The concentration of the anti-CTLA-4 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the invention is about 1 to 100mg/mL, preferably about 1 to 50mg/mL, more preferably about 5 to 30mg/mL.

The pharmaceutical composition of the present invention has a pH of about 4.5 to 6.5, preferably about 5.0 to 6.0, more preferably about 5.3 to 5.7.

The buffer solution in the pharmaceutical composition is one or more selected from acetic acid buffer solution, citric acid buffer solution, phosphate buffer solution and histidine buffer solution; preferably, the buffer is an acetate buffer. Preferably, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer.

Preferably, the concentration of buffer is about 5 to 100mM, preferably about 10 to 50mM, preferably about 10 to 30mM; preferably about 15 to 25mM. Preferably, the pH of the buffer is about 4.5 to 6.5, preferably about 5.0 to 6.0, preferably about 5.3 to 5.7.

Accordingly, the pharmaceutical composition of the present invention may contain: an acetate buffer having a pH of about 5.0 to about 6.0 at a concentration of about 10 mM to about 30mM in the pharmaceutical composition; and about 1 to 50mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof of any of the preceding embodiments, particularly humanized antibody huJS007-47 or antigen-binding fragment thereof described herein.

In some embodiments, the pharmaceutical composition of the present invention further comprises a stabilizer selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose, and trehalose; preferably, the arginine salt is arginine hydrochloride. Preferably, the concentration of the stabilizer is about 10 to 400mM, preferably about 100 to 300mM, preferably about 130 to 280mM, preferably about 200 to 260mM. Preferably, the stabilizer is mannitol at a concentration of about 130 to 280 mM; or the stabilizer is sucrose with the concentration of about 130-280 mM; or a combination of sodium chloride at a concentration of about 20-80 mM and mannitol at a concentration of about 110-170 mM; or a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; in some embodiments, the stabilizer is mannitol at a concentration of about 200 to 260 mM; or the stabilizer is sucrose with the concentration of about 200-260 mM; or a combination of sodium chloride at a concentration of about 30-70 mM and mannitol at a concentration of about 120-160 mM; or a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM.

Accordingly, the pharmaceutical composition of the present invention may contain: an acetate buffer having a pH of about 5.0 to about 6.0 at a concentration of about 10 mM to about 30mM in the pharmaceutical composition; and about 1 to 50mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof of any of the preceding embodiments, particularly the humanized antibody huJS007-47 or antigen-binding fragment thereof described herein; and about 130-280 mM of a stabilizer, preferably selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose and trehalose, preferably, the arginine salts are arginine hydrochloride. Preferably, the stabilizer is sucrose at a concentration of about 200 to 260 mM; or a combination of sodium chloride at a concentration of about 30-70 mM and mannitol at a concentration of about 120-160 mM; or a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM.

In some embodiments, the above pharmaceutical composition further comprises a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188. Preferably, the surfactant concentration is about 0.001% to 0.1%, preferably about 0.01% to 0.05%, calculated as w/v.

Accordingly, the pharmaceutical composition of the present invention may contain: an acetate buffer having a pH of about 5.0 to about 6.0 at a concentration of about 10 mM to about 30mM in the pharmaceutical composition; and about 1 to 50mg/mL of the anti-CTLA-4 antibody or antigen-binding fragment thereof of any of the preceding embodiments, particularly the humanized antibody huJS007-47 or antigen-binding fragment thereof described herein; and about 130-280 mM of a stabilizer, preferably selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose and trehalose, preferably, the arginine salts are arginine hydrochloride. Preferably, the stabilizer is sucrose at a concentration of about 200 to 260 mM; or a combination of sodium chloride at a concentration of about 30-70 mM and mannitol at a concentration of about 120-160 mM; or a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM; and about 0.01% to about 0.1% polysorbate 80 in w/v.

The pharmaceutical composition of the invention is a liquid preparation or a freeze-dried preparation.

Medical uses and methods

In the present invention, CTLA-4 mediated diseases refer to diseases in which CTLA-4 is involved in the occurrence and development of the disease, including but not limited to cancer.

"cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are benign and malignant cancers, and dormant tumors or micrometastases. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, carcinoma of the stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer or carcinoma of the kidney, nasopharyngeal carcinoma, esophageal squamous cell carcinoma, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade/follicular non-hodgkin's lymphoma (NHL), small Lymphocytic (SL) NHL, medium-grade/follicular NHL, medium-grade diffuse NHL, advanced immunoblastic NHL, advanced lymphocytic NHL, high-grade small non-nucleated NHL, deposition cell NHL, deposition disease (window) and lymphoblastic), lymphoblastic leukemia (Waldenstrom's) and lymphomatosis (Waldenstrom's), and leukemia (mel's), and leukemia (lymphomas) associated with chronic leukemia, such as vascular leukemia, lymphomatosis (lymphomatosis), and leukemia (lymphomas).

Examples

The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The invention has been described in detail herein, with particular embodiments thereof also disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention, and that any such changes, equivalents, modifications, etc. are intended to be included within the scope of the invention. The methods and materials used in the examples are, unless otherwise indicated, conventional in the art.

Detection method used in examples

(1) Appearance of

Visual inspection was used to detect appearance. The illumination intensity of the clarity detector is ensured to be kept between 1000lx and 1500 lx. The sample is kept at the same level of the eye and gently shaken or inverted to avoid air bubbles. Visual inspection was performed in front of black and white background, respectively. The results were recorded in terms of color, opalescence and visible foreign matter.

(2) Protein content

Protein concentration was measured using a UV spectrophotometer (Thermofisher, BIO MATE 3S). The percent extinction coefficient (E1%) was set at 1.5367 (mg/ml) ^-1 cm ^-1 . Using BIO MATE 3S instrument, washing cuvette with ultra-pure water three times, adding 150 μl of ultra-pure water into the cuvette, click measuring, and performing blank correction with ultra-pure water, wherein absorbance of blank sample is not more than + -0.003. The diluted solution is diluted to 0.3mg/ml to 0.7mg/ml by ultrapure water, the final volume of each part is not lower than 900 mu L, and the single dilution multiple is not more than 10 times. 2 solutions were assayed in parallel for each sample, each solution being assayed in duplicate 3 times; each solution was measured by rinsing the cuvette with 150. Mu.L of solution 2 times before taking 150. Mu.L of solution. And calculating the concentration of the corresponding sample through the extinction coefficient and the OD value.

(3) SEC-HPLC purity

SEC-HPLC purity was measured by HPLC (Waters e2695 instrument) equipped with a SEC column (TOSOH G4000sxl SEC 7.8mm ID. Times.30 cm,8 μm). The mobile phase composition was 50mM Tris, 150mM NaCl,pH 7.4. And carrying out quantitative analysis on the result by adopting peak area normalization. The peak area percentages of the monomer, the polymer and the fragment were calculated, respectively, with the peak area percentage of the monomer as the purity of the sample and the peak area percentage of the polymer and the fragment as the content of the polymer and the fragment. The chromatographic parameters are shown in Table 1 below.

Table 1: SEC-HPLC chromatographic parameters

Chromatographic conditions	Chromatographic parameters
		Detector wavelength	280nm
Temperature of automatic sampler	25±3℃
		Column temperature	5±3℃
Flow rate	0.50mL/min
		Sample injection volume	25μL
Mode of operation	Isocratic elution
		Elution time	30.00min

(4) Purity of R-CE-SDS

The purity detection of the antibody preparation by the reduction CE-SDS electrophoresis method uses a high-voltage direct current electric field as a driving force and uses a capillary as a separation channel. The pre-filled gel can form molecular sieve in capillary, the sodium dodecyl sulfate can eliminate the charge effect of different protein molecules, the reducing agent beta-mercaptoethanol can cut disulfide bonds in samples, and samples with different molecular sizes can move at different speeds in the capillary, so that separation can be performed. Diluting the Sample to 1mg/mL with a loading Buffer (SDS-MW Sample Buffer); 95. Mu.L of loading Buffer (SDS-MW Sample Buffer) was added to 5. Mu.L of beta-mercaptoethanol, and the mixture was vortexed and homogenized to prepare a blank. Taking 95 mu L of a test sample (1 mg/mL), adding 5 mu L of beta-mercaptoethanol, centrifuging at a room temperature of 3000rpm for 30sec, incubating at 70+/-2 ℃ for 15+/-2 min, cooling to the room temperature, centrifuging at a room temperature of 6000rpm for 1min, and detecting with a capillary electrophoresis apparatus (Maurice apparatus) provided with a CE-SDS cartridge for 40 min. And calculating the purity of the Heavy Chain (HC), the non-glycosylated heavy chain (NGHC) and the Light Chain (LC), wherein the sum of the purity and the non-glycosylated heavy chain (NGHC) and the Light Chain (LC) is the purity of the sample.

(5) Purity of NR-CE-SDS

The purity detection of the antibody preparation by a non-reducing CE-SDS electrophoresis method uses a high-voltage direct-current electric field as a driving force and uses a capillary as a separation channel. The pre-filled gel forms molecular sieves in the capillaries, and samples with different molecular sizes and different moving speeds in the capillaries are separated by treating the samples with sodium dodecyl sulfate to eliminate the charge effect of different protein molecules. The alkylating reagent is added into the sample solution, so that the component diffusion can be effectively reduced, the obtained peak is sharp, the separation efficiency is high, and the sample can be ensured to be kept in a non-reduction state. Diluting the Sample to 1mg/mL with a loading Buffer (SDS-MW Sample Buffer); taking 95 mu L of loading Buffer (SDS-MW Sample Buffer), adding 5 mu L of 0.8M iodoacetamide solution, and vortex mixing uniformly to serve as blank control; the sample (1 mg/mL) was taken as 95. Mu.L, 5. Mu.L of 0.8M iodoacetamide solution was added, centrifuged at 3000rpm at room temperature for 30sec, incubated at 70.+ -. 2 ℃ for 5.+ -. 1min, cooled to room temperature, and centrifuged at 6000rpm at room temperature for 1min, and detected by using a capillary electrophoresis apparatus (Maurice apparatus) equipped with a CE-SDS cartridge.

(6) CEX-HPLC purity

Charge heterogeneity was detected using cation exchange chromatography (CEX-HPLC). After dilution of the mobile phase a mixture, the samples were tested using HPLC. The parameters of the detection method are shown in the following table. The peak area normalization method is adopted to calculate the percentages of the main peak, the acid peak and the alkaline peak, and if the automatic integration is adopted and a reasonable integration result cannot be obtained, the manual integration is adopted. The detailed chromatographic parameters are shown in Table 2 below.

Table 2: CEX-HPLC chromatographic parameters

(7) Cell Activity

The first day of the experiment, huJS007-47 antibody was diluted to 80 μg/ml (4 Xanalysis concentration), 1.8-fold gradient diluted, 10 concentrations, with huJS007-47 assay buffer (RPMI 1640Medium+1% FBS). CTLA-4Fc Protein concentration was diluted to 8. Mu.g/ml (4 Xanalysis concentration) for further use. GS-J1/CD28 cell density was adjusted to 2.0X10 ⁶ cells/ml and PHA was added to a concentration of 20. Mu.g/ml (4 Xanalysis concentration) for further use. GS-C1/CD80 cell density was adjusted to 1.0X10 ⁶ cell/ml was ready for use. The huJS007-47 antibody, CTLA-4Fc Protein, GS-J1/CD28 cells and GS-C1/CD80 cells were added to a 96-well round bottom plate at 50. Mu.l/well, and incubated in a 5.0% carbon dioxide incubator at 37.0℃for 24h, respectively. The next day 16. Mu.l of culture supernatant was pipetted into HTRF 96well low volume plate per well, then 4. Mu.l of mixed antibody (Cisbio Human IL2 kits) was added, incubated at room temperature for 18.+ -. 2h away from light, and finally data analysis was performed using a multifunctional microplate reader (Tecan, M1000 Pro) and GraphPad Prism software. The calculation formula is as follows: ratio 665/620=od 665nm/OD620nm 10000.

(8) Binding Activity

Combined ELISA (Binding ELISA) experiment: using a microplate reader (Thermo Scientific, multiskan Go), plates were coated with a fixed concentration of His-tagged CTLA4 antigen (1.0. Mu.g/mL), after blocking with 2% BSA, gradient diluted antibodies (1. Mu.g/mL starting, 2.5-fold gradient diluted 12 total) were added, and Anti-Human IgG (Fc Specific) -Peroxidase antibody produced in goat (Sigma, A0170) was diluted 5000 as detection antibody, followed by development with 0.1mg/mL TMB, and finally reaction was stopped with 2M HCl and plates were read at 450nm/620 nm. EC50 s were fitted using a four-parameter logistic regression (4 PL) model.

(9) Sub-visible particle detection

The sub-visible particle detection was performed using a particle detector (MFI 5100). Because the sample is a high-concentration sample, the sample needs to be diluted to a certain extent before loading, the diluted sample is gently and fully mixed to avoid bubbles, 1.3mL of the sample is sampled into an upper sample plate by a pyrogen-free gun head in an ultra-clean bench, the upper sample plate is tightly covered by clean tinfoil paper, the loading position is input into the instrument after the sample is moved to a working plate corresponding to the instrument from the ultra-clean bench, a detection sequence is operated, and sub-visible particles are photographed and counted by a miniature camera when the sample flows through a flow cell.

Abbreviations in the following examples: "h" means hours, "W" means weeks, "M" means months, "C" means the number of freeze-thawing cycles, "FT" means freeze-thawing cycles, "SH" means shaking, "ST" means stirring, "RT" means room temperature, "rpm" means revolutions per minute, "T0" means initial testing of the prescription sample before it was lofted.

Example 1: buffer system and stabilizer preliminary screening experiment

In liquid pharmaceutical compositions, the buffer system closely affects the stability of the antibody, and each antibody with unique physicochemical properties has the most suitable type of buffer. This example is directed to the initial screening of the optimal buffer system and stabilizer to provide the anti-CTLA-4 antibodies disclosed herein with optimal stability for clinical use.

1.1 Experimental procedure

This example was performed with antibody huJS 007-47. Sample use Millipore Pellicon3 cm 88 ² UF/DF ultrafiltration concentration was performed on the membrane to a concentration of about 21mg/mL, the samples were dialyzed into the corresponding formulations shown in Table 3, the final concentration was adjusted to about 20mg/mL, and polysorbate 80 at the corresponding concentration was added. Aseptically filling the sample into 2R penicillin bottles at an ultra clean bench, and carrying out stability lofting and detection at a concentration of 2.0 mL/bottle.

Table 3: prescription information in a first round of prescription screening-buffer System and stabilizer Primary screening experiments

1.2 experimental results

1.2.1 appearance and concentration results

According to the results of table 4, no significant change in protein content occurred for all samples under accelerated or long term conditions.

According to the results in table 5, no visible foreign matter was found for all samples at T0, no opalescence was evident. After being placed for 4 weeks under the acceleration condition, the appearance of all samples is not changed obviously; after standing for 8 weeks under long term conditions, no significant change in appearance occurred in all other samples except for the protein precipitation of FS 1-4.

Table 4: first round of prescription screening-protein content data

Table 5: first round prescription screening-appearance data

1.2.2SEC purity results

According to the SEC purity results in Table 6, after 4 weeks of standing under accelerated conditions, 7 prescription polymers and fragments increased, prescription FS1-7 decreased in purity significantly, and prescription FS1-1, FS1-3 and FS1-5 performed relatively better; after 12 weeks of standing under long term conditions, no significant decrease in SEC purity occurred for all prescriptions.

Table 6: first round prescription screening-SEC-HPLC data

1.2.3R-CE-SDS purity results

According to the results of the purity of R-CE-SDS in Table 7, the purity of FS1-7 was significantly reduced by the 4-week-allowed-time prescription and the other group was not significantly different from each other by the 4-week-allowed-time treatment, and all the samples were not significantly changed by the long-term-allowed-time treatment.

Table 7: first round of prescription screening-R-CE-SDS data

1.2.4NR-CE-SDS purity results

According to the NR-CE-SDS purity results in Table 8, all samples showed a decrease in purity after 4 weeks of standing under accelerated conditions, and the prescriptions FS1-7 were significantly decreased, the other groups were not greatly different, and the prescriptions FS1-4, FS1-5, FS1-6, and FS1-7 showed a decrease in purity after 4 weeks of standing under long term conditions.

Table 8: first round of prescription screening-NR-CE-SDS data

Note that: HHL refers to an antibody fragment having two heavy chains and one light chain.

1.2.5CEX-HPLC purity results

According to the CEX-HPLC purity results in Table 9, all samples exhibited an increase in the main peak purity decrease by acid-base peaks after 4 weeks of standing under accelerated conditions, with four prescriptions FS1-1, FS1-3, FS1-5, and FS1-6 having slower main peak purity decreases and prescriptions FS1-7 having the worst stability; after standing for 8 weeks under long-term conditions, no significant reduction in purity occurred for all samples.

Table 9: first round prescription screening-CEX-HPLC data

1.2.6 binding Activity results

According to the binding activity results in table 10, no decrease in activity occurred for all formulations after 4 weeks of accelerated or long term standing.

Table 10: first round of prescription screening-binding Activity data

1.2.7 cell Activity results

According to the cellular activity results in table 11, no decrease in activity occurred for all prescriptions after 4 weeks of accelerated or long term storage.

Table 11: first round of prescription screening-cell Activity data

1.2.8 sub-visible particle results

According to the sub-visible particle results in table 12, the particle count for all the prescription samples did not increase significantly after 4 weeks of standing under accelerated conditions; the significant increase in particles for formulas FS1-4 was consistent with appearance of protein precipitation with no significant increase in particle count for all other formulas after standing at 8W for extended periods of time.

Table 12: first round prescription screening-sub-visible particle data

1.3 first round prescription screening conclusion

From the results of protein content, binding activity, no significant differences occurred for all prescriptions. From the appearance point of view, the appearance of the prescription FS1-4 shows protein precipitation for 8 weeks; from the SEC-HPLC results, the increase in the polymers of prescription FS1-7 was evident; the main peaks of formulas FS1-2, FS1-4 and FS1-7 showed a significant decrease in purity from the CEX-HPLC results; the purity of the prescribed FS1-7 was significantly reduced from the results of NR-CE-SDS and R-CE-SDS.

As described above, the acetate buffer (FS 1-3) and the citrate buffer (FS 1-5) perform relatively well. Thus, 20mM acetate buffer (pH 5.5) and 20mM citric acid buffer (pH 6.0) were selected as buffer systems for the next round of screening experiments.

Example 2: stabilizer and surfactant screening experiments

To further explore the effect of different adjuvants on antibody stability, we selected different stabilizers and surfactants for comparative testing. The effect of the above-mentioned different excipients on stability was examined at a concentration of 10mg/mL for antibody huJS007-47 in a pH5.5, 20mM acetate buffer system and a pH6.0, 20mM citrate buffer system.

2.1 Experimental procedure

This example was performed with antibody huJS 007-47. Sample use Millipore Pellicon 3.0.11 m ² UF/DF ultrafiltration concentration was performed on the membrane to a concentration of about 10mg/mL, the samples were dialyzed into the corresponding formulations shown in Table 13, the final concentration was adjusted to about 10mg/mL, and polysorbate 80 at the corresponding concentration was added. Aseptically filling the sample into 2R penicillin bottles at an ultra clean bench, and carrying out stability lofting and detection at a concentration of 2.0 mL/bottle.

Table 13: prescription information in a second round of prescription screening-stabilizer and surfactant screening experiments

The note "/" indicates none.

2.2 experimental results

2.2.1 appearance and concentration results

According to the results in table 14, no significant change in protein content occurred for all samples after 4 weeks of standing under accelerated and long term conditions.

According to the results in table 15, no visible foreign matter was found for all samples at T0, no opalescence was evident. After the mixture is placed for 4 weeks under the acceleration condition, protein precipitation appears on the appearance of two groups of formulas of FS2-5 and FS2-12, and the appearance of the other formulas is normal. No visible foreign matter was found apparent after 3 months of standing under long term conditions.

Table 14: second round of prescription screening-protein content data

Table 15: second round prescription screening-appearance data

/>

2.2.2SEC-HPLC purity results

According to the SEC purity results in Table 16, all samples showed an increase in aggregates and fragments when placed under accelerated conditions of 4W, and the slower decrease rates of formulas FS2-3 and FS2-4 were better, with no significant decrease in monomer purity for all samples when placed under long term conditions of 3M.

Table 16: second round of prescription screening-SEC-HPLC data

/>

2.2.3R-CE-SDS purity results

According to the purity results of R-CE-SDS in Table 17, all samples showed a decrease in purity after 4 weeks of standing under accelerated conditions, and the difference in purity between groups was not large.

Table 17: second round of prescription screening-R-CE-SDS data

/>

2.2.4NR-CE-SDS purity results

According to the NR-CE-SDS purity results in Table 18, all samples showed a decrease in purity after 4 weeks of standing under accelerated conditions, and the difference in purity between groups was not large.

Table 18: second round of prescription screening-NR-CE-SDS data

2.2.5CEX-HPLC purity results

According to the CEX-HPLC purity results in table 19, no significant changes in purity occurred for all samples over a period of 4 weeks of standing under long term conditions. After 4 weeks of standing under accelerated conditions, all samples showed a decrease in the purity of the main peak, with significant increases in the acid-base peaks of formulas FS2-2, FS2-5, FS2-6 and FS2-10, but no significant inter-group differences.

Table 19: second round of prescription screening-CEX-HPLC data

/>

2.2.6 Results of cellular Activity

According to the cell activity results in table 20, no significant change in cell activity results occurred in all samples after 4 weeks of standing under accelerated or long term conditions.

Table 20: second round of prescription screening-cell Activity data

/>

2.2.7 binding Activity results

According to the binding in table 21, no significant change in binding activity results occurred for all samples after 4 weeks of standing under accelerated or long term conditions.

Table 21: second round of prescription screening-binding Activity data

/>

2.2.8 sub-visible particle results

According to the sub-visible particle results in Table 22, all samples showed increased size particles with best performance of FS2-4 after 4 weeks of standing under accelerated conditions; no obvious particle change after 4 weeks of standing under long-term conditions.

Table 22: second round of prescription screening-sub-visible particle data

/>

2.3 second round prescription screening conclusion

No significant differences were seen for all prescriptions from the results of protein content, NR-CE-SDS, R-CE-SDS purity, cellular activity and binding activity. From the appearance result, the stability of the sample containing the auxiliary material trehalose dihydrate in the prescription is poor, and the content of tween 20 and tween 80 is increased, so that the solubility can be improved. From SEC-HPLC and sub-visible particle results, it can be obtained that sucrose containing the auxiliary material has better stability in the acetic acid buffer system. As a result, 20mM acetate buffer (FS 2-4) pH5.5 contained 220mM sucrose, which performed optimally.

Example 3: influence factor experiment

3.1 Experimental procedure

According to the second round of acceleration experiment result, prescriptions of four prescriptions of FS2-3, FS2-4, FS2-7 and FS2-8 are shown to be good in stability, four candidate prescriptions are selected for carrying out influence factor experiments, and the embodiment is carried out by using an antibody huJS 007-47. The specific scheme is shown in Table 23.

Table 23: experimental protocol for influencing factors

3.2 experimental results

Table 24: summarizing influence factor detection results

Conclusion of influence factor experiment: in the four prescriptions, after being inspected by the influence factors such as stirring, freeze thawing, shaking and the like, SEC-HPLC, appearance and sub-visible particle results have no obvious difference, and all show good stability.

Example 4: compatibility stability experiment

Experimental procedure

Prescription FS2-4 was selected to examine the stability of the 0.9% sodium chloride injection and the 5% dextrose injection after dilution, this example was performed with antibody huJS 007-47. The medium was diluted to different concentrations (Table 25) and placed in an incubator at 25℃for 24h and tested as follows.

Table 25: compatibility scheme

Table 26: summarizing compatibility detection results

Note that: sample No. FS2-4-Glu represents dilution with 5% dextrose injection, and sample No. FS2-4-NaCl represents dilution with 0.9% sodium chloride injection.

Compatibility experimental conclusion: when 0.9% sodium chloride injection is used as a diluting solvent, the stability is good in the concentration range of 0.1mg/ml to 5 mg/ml. When 5% glucose injection is used as a solvent, the diluted sample has fragment increase in the SEC-HPLC result at the low concentration of 0.1mg/ml, and has good stability in the concentration range of 1.0mg/ml to 5.0 mg/ml.

In summary, we examined different buffer systems, different pH conditions, different antibody concentrations and different auxiliary material compositions, the target pH range was controlled at 5.0-6.0, the osmotic pressure range was 250-350 mOsm/kg, and the optimal formulation was selected: about 20mM sodium acetate buffer (pH about 5.5), about 220mM sucrose and about 0.02% polysorbate 80; to achieve an osmotic pressure of around 300mOsm/kg, the sucrose concentration may be about 228mM.

Example 5: antigen affinity of anti-CTLA-4 pharmaceutical compositions

40 μg/mL of goat anti-human Fc fragment antibody (Jackson ImmunoResearch) was coupled to a CM5 chip (GE medical life sciences) using a T200 type molecular interaction analyzer Biacore, surface plasmon resonance technologyLife science, goods number: BR 100530), then capturing an anti-CTLA-4 pharmaceutical composition (formulated according to the prescription FS2-4, antibody huJS007-47 concentration 0.5 μg/mL), then injecting a gradient diluted His-tagged recombinant human CTLA-4 antigen protein (made by the monarch), and detecting a binding signal. CTLA-4 antigen protein was diluted in gradient at 24nM, 12nM, 6nM, 3nM, 1.5nM, 0.75nM, where 24nM is the repeat. Kinetic model analysis of Biacore T200 Evaluation Software 3.0 using Biacore analysis software, fitting to obtain affinity K _D Values.

The experimental results are shown in Table 27 and FIG. 1, the anti-CTLA-4 pharmaceutical composition has higher affinity with human CTLA-4 protein, K _D The numerical value is 2.06E-10M.

Table 27: anti-CTLA-4 pharmaceutical compositions and recombinant human CTLA-4 protein affinity results table

Antigen name	k _a (1/Ms)	k _d (1/s)	K _D (M)
				Recombinant human CTLA-4 protein	8.18E+05	1.69E-04	2.06E-10

Example 6: inhibition of hCTLA4 humanized mice transplanted EMT6 tumor growth by anti-CTLA-4 pharmaceutical composition

Mouse breast cancer EMT6 cells (0.5X10) ⁶ ) (ATCC CRL-2755) inoculationTo the right back subcutaneous of the hCTLA-4 humanized mice (Shanghai south mode biotechnology limited). When the tumor size is about 99mm ³ At this time, the mice were randomly divided into 5 groups of 6 mice each. Mice were intraperitoneally injected with 10mg/kg of anti-KLH hIgG1, 3 or 10mg/kg of anti-CTLA-4 pharmaceutical composition (formulated according to the formulation FS2-4, antibody huJS 007-47) twice a week for a total of 5 times. As shown in FIG. 2, on day 24 after tumor inoculation, 10mg/kg of the anti-CTLA-4 pharmaceutical composition significantly inhibited tumor growth compared with anti-KLH hIgG1, with a TGI value of 96.4% (p<0.05). In addition, animals are well tolerated by the anti-CTLA-4 pharmaceutical composition and mice gain weight gradually during the study period. Wherein the tumor inhibition ratio TGI% (TGI% = [1- (Ti-T0)/(Vi-V0)) ]X 100%) (Ti: tumor volume mean, T0, of treatment group on day i of dosing: tumor volume mean on day 0 of dosing for the treatment group; vi: tumor volume mean, V0, of negative control group on day i of dosing: tumor volume mean of negative control group at day 0 of dosing).

Example 7: biological Activity of pharmaceutical compositions of anti-CTLA-4 antibodies

The humanized anti-CTLA-4 antibodies were tested for their ability to bind to huCTLA-4, block the binding of huCTLA-4 to CD80/CD86, and antagonize the biological activity of huCTLA-4. The humanized anti-CTLA-4 antibodies of this example were formulated according to the formulation of prescription FS2-4, and the experimental methods and results are shown below.

7.1ELISA method for detecting binding of humanized anti-CTLA-4 antibody to huCTLA-4

HX1 hCDLA 4 his was diluted to 1.0. Mu.g/mL with PBS (Hyclone), and the ELISA plate was added at 100. Mu.l/well, and the mixture was left to stand in a 37℃incubator for coating for 90min; washing the plate; 200 μl/well of 2% BSA was added to the plate and incubated in a 37℃incubator for 90min; washing the plate. Humanized anti-CTLA-4 antibodies and control antibody Iplilimumab (made by Mild, see patent WO2001014424A2 and CN 1371416A) were diluted to 1000ng/ml with dilution (2% BSA) at a multiple of no more than 10 fold each time. Then, the humanized antibody and the control antibody were diluted in sequence on a sample dilution plate with a 2.5-fold gradient. All humanized antibody and control antibody solutions are added into an ELISA plate at a rate of 100 μl/well, and incubated in a constant temperature incubator at 37deg.C for 60min; washing the plate; HRP-conjugated goat anti-human antibody IgG (Fc specific) (Sigma, cat# A0170) was diluted 5000-fold with 2% BSA, and 100. Mu.l/well of the ELISA plate was added and incubated in a 37℃incubator for 60min; washing the plate; adding a developing solution TMB,100 μl/hole, avoiding bubbles, and developing for 15min at 37deg.C in dark; finally, 2M hydrochloric acid solution is added to stop the reaction, 100 μl/hole is filled, air bubbles are avoided, and the reading of the enzyme labeling instrument (wavelength: 450/620 nm) is completed within 10 min. FIG. 3 is a graph of the relative binding activity of humanized anti-CTLA-4 antibodies versus control antibodies.

As shown in FIG. 3, the humanized anti-CTLA-4 antibodies have good binding to huCTLA-4, comparable to or better than the control antibody, iilimiumab.

7.2ELISA method for detecting the ability of humanized anti-CTLA-4 antibody to block binding of huCTLA-4 to CD80

HX1 hCTLA4-his was diluted to 1.0. Mu.g/mL with PBS (Hyclone), and an ELISA plate was added at 100. Mu.l/well, and incubated at 37℃for 90min at rest in an incubator; washing the plate; 200 μl/well of 2% BSA was added to the plate and incubated in a 37℃incubator for 90min; washing the plate; the MX2 hCD80 Fc (made by the monarch) was diluted to 5.0. Mu.g/mL with 2% BSA and the antibody was diluted at this concentration of MX2 hCD80 Fc. Humanized anti-CTLA-4 antibodies and control antibody Iplilimumab (made by Mild, see patent WO2001014424A2 and CN 1371416A) were diluted to 100 μg/ml at a dilution factor of not more than 10 fold each time. Then, the humanized antibody and the control antibody were diluted in sequence on a sample dilution plate with a 2.5-fold gradient. All humanized antibody and control antibody solutions are added into an ELISA plate at a rate of 100 μl/well, and incubated in a constant temperature incubator at 37deg.C for 90min; washing the plate; HRP-conjugated goat anti-mouse antibody IgG (Fc specificity) was diluted 5000-fold with 2% BSA, and 100. Mu.l/well of the ELISA plate was added and incubated in a 37℃incubator for 60min; washing the plate; adding a developing solution TMB,100 μl/hole, avoiding bubbles, and developing for 15min at 37deg.C in dark; finally, 2M hydrochloric acid solution is added to stop the reaction, 100 μl/hole is filled, air bubbles are avoided, and the reading of the enzyme labeling instrument (wavelength: 450/620 nm) is completed within 10 min. FIG. 4 is a graph of the relative inhibitory activity of humanized anti-CTLA-4 antibodies versus control antibodies.

As shown in FIG. 4, the humanized anti-CTLA-4 antibody has good ability to block binding of huCTLA-4 to CD80, which is comparable to or better than the control antibody Iilimiumab.

7.3 luciferase assay for the biological Activity of humanized anti-CTLA-4 antibodies against huCTLA-4

The humanized anti-CTLA-4 antibodies huJS007-46, 47, 48, 49, 55, 56, 73, 79, 82, 88, 100 and 106 were selected for biological activity analysis based on the binding activity, blocking binding activity and degree of humanization described above.

Jurkat cells expressing huCTLA-4 were plated at 6X 10 per well ⁴ Cell plating was performed with 3X 10 cells per well ⁴ The T cell activation activity was determined by luciferase assay after incubation of Raji APC cells and various concentrations of humanized anti-CTLA-4 antibody or control antibody (Iilimiumab, available from Italiumab, see patent WO2001014424A2 and CN 1371416A) for 6 hours.

As shown in FIG. 5, 12 humanized anti-CTLA-4 antibodies have high biological activity and EC ₅₀ All significantly better than the control antibody Ipilimumab.

Example 8: ADCC Activity of humanized anti-CTLA-4 antibodies

293T-CTLA4 cells were labeled with CFSE and peripheral blood mononuclear cells (PBMC 2144896) and humanized anti-CTLA-4 antibodies or control antibodies (Ipilimumab, made commercially available, see patent WO2001014424A2 and CN 1371416A) at a Target cell to effector ratio of 1:25 were added and incubated overnight. Cells were stained with Propidium Iodide (PI) and analyzed by flow cytometry. ADCC Killing (ADCC Killing,%) was expressed as a percentage of dead target cells (PI and CFSE positive) to total target cells (CFSE positive). The humanized anti-CTLA-4 antibodies of this example are formulated according to the formulation of prescription FS 2-4.

As shown in fig. 6 (the two panels are merely for convenience of illustration), each of the 12 humanized anti-CTLA-4 antibodies has ADCC activity, wherein the humanized anti-CTLA-4 antibodies 46, 47, 48, 73, 79 and 106 have ADCC activity comparable to or better than that of the control antibodies, particularly antibodies 46 and 48.

Example 9: CDC Activity of humanized anti-CTLA-4 antibodies

293T-CTLA4 cells were activated with 12 humanized anti-CTLA-4 antibodies or control antibodies (Iplilimumab, made by monarch, see patent WO2001014424A2 and CN 1371416A) at different concentrations (0.8-100. Mu.g/mL) at 37℃for 15 min, wherein the humanized anti-CTLA-4 antibodies of this example were formulated according to the formulation of prescription FS2-4, then human serum complements of different dilution gradients (1:5, 1:10, 1:20) were added and incubated for 1 hour. After the end of the culture, the cells were stained with Propidium Iodide (PI) and analyzed by BD FACSCalibur flow cytometer. CDC Killing (CDC Killing,%) is expressed as the percentage of PI positive target cells to total target cells. The results are depicted in FIG. 7, which shows that the 12 humanized anti-CTLA-4 antibodies have no or negligible CDC activity.

Example 10: inhibition of mouse tumor growth by anti-CTLA-4 antibody pharmaceutical compositions

50 female B-hCTLA4 humanized mice (Baioci) of 6-8 weeks old were taken, and the colon cancer cells MC38 WT cells (Shanghai Shunhan Biotech Co., ltd.) were cultured at 1X 10 ⁶ Right subcutaneous of vaccinated mice at a concentration of 0.1mL until tumor growth was about 138mm ³ The mice were randomly grouped according to tumor volume, 6 mice per group, 6 total groups, respectively:

g1 KLH IgG1 (0.3 mg/kg) negative control group,

G2 Iplilimumab (0.3 mg/kg) positive control group,

G3 huJS007-47 (0.3 mg/kg) treatment group,

G4 huJS007-48 (0.3 mg/kg) treatment group,

G5 huJS007-79 (0.3 mg/kg) treatment group, and

g6 huJS007-106 (0.3 mg/kg) treatment group.

The humanized anti-CTLA-4 antibodies of this example are formulated according to the formulation of prescription FS 2-4.

All groups were given by intraperitoneal injection at a dose of 0.3mg/kg and at a concentration of 0.03mg/ml. The administration was 2 times per week, 5 times continuously, and the experiment was ended 3 days after the last administration. Tumor volume and body weight were measured 2 times per week and mice body weight and tumor volume were recorded. Mice were euthanized at the end of the experiment, and the relative tumor inhibition ratio TGI% = (1- (Ti-T0)/(Vi-V0)) ×100% was calculated. Ti: tumor volume mean values of treatment group and positive control group at day i of administration; t0: tumor volume mean on day 0 of dosing for treatment and positive control groups; vi: tumor volume mean of negative control group at day i of administration; v0: tumor volume mean of negative control group at day 0 of administration.

As shown in FIG. 8, the average tumor volume of KLH IgG1 negative control group was 975.+ -.115 mm on day 25 after tumor cell inoculation of mice ³ The mean tumor volume of the positive control group of Iplilimumab (made by Junzhen, see patent WO2001014424A2 and CN 1371416A) was 824.+ -.267 mm ³ Relative tumor inhibition was 18.1% compared to KLH IgG 1; the average tumor volumes of the treatment group of huJS007-47, the treatment group of huJS007-48, the treatment group of huJS007-79 and the treatment group of huJS007-106 are 229+/-85 mm respectively ³ 、313±197mm ³ 、550±229mm ³ And 472+ -125 mm ³ Compared with KLH IgG1, the relative tumor inhibition rates are 89.2%, 79.1%, 50.9% and 60.1%, respectively, which shows that the humanized anti-CTLA-4 antibody can inhibit the growth of B-hCDLA 4 humanized mouse MC38-WT cell subcutaneous transplantation tumor in vivo and is obviously superior to the control antibody Ipilimumab.

Example 11: identification of epitope by Fortebio binding experiment

Full length antibodies, i.e., 2.7 μg/mL of humanized anti-CTLA-4 antibody huJS007-47 and 2 μg/mL of control antibody (Ipilimumab, available from Mild, see patent WO2001014424A2 and CN 1371416A), were first captured separately using a Protein A probe (Fortebio). The probe was then immersed in a 55nM human CTLA (huCTLA) antigen solution to bind the full length antibody to the antigen. Finally, the probe was immersed in 600nM Fab solution, including humanized Fab huJS00-47 and control Fab (Ipilimumab), and tested for antigen binding to Fab.

As shown in fig. 9, after the full length antibody huJS007-47 binds to the antigen huCTLA-4, huCTLA-4 can continue to bind to control Fab (Ipilimumab), but according to the Fortebio binding assay, huJS007-47 binds to a different epitope of huCTLA-4 than Ipilimumab.

Example 12: inhibition of hCTLA4 humanized mouse transplanted MC38 tumor growth by huJS007-47 pharmaceutical composition

Female hCTLA4 humanized mice (Bai Osai Jiangsu Gene Biotechnology Co., ltd.) of 6-8 weeks old were taken from the right backSubcutaneous inoculation of the sections 1X 10 ⁶ Mouse colon cancer cell MC38 cell (Hirudan Biotechnology Co., ltd.) (0.1 ml/cell-containing medium RMPI1640 (Gibco)). The tumor volume to be averaged was about 119mm ³ At this time, 40 animals were selected and randomly divided into 5 groups of 8 animals each according to tumor volume. Respectively is

Anti-KLH hIgG1 negative control, 1mg/kg;

iiliimumab positive control group, 1mg/kg;

huJS007-47 treatment group, 0.1mg/kg;

huJS007-47 treatment group, 0.3mg/kg;

huJS007-47 treatment group, 1mg/kg;

All groups were given by intraperitoneal injection, 2 times per week, 6 times continuously, and the experiment was ended 3 days after the last administration. Tumor volume and body weight were measured 2 times per week and mice body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and tumor inhibition TGI% (TGI% = [1- (Ti-T0)/(Vi-V0) ]x100%) was calculated. (Ti: mean tumor volume of the treatment group on day i of administration, T0: mean tumor volume of the treatment group on day 0 of administration; vi: mean tumor volume of the negative control group on day i of administration, V0: mean tumor volume of the negative control group on day 0 of administration).

As shown in FIG. 10, at day 21 after the start of the administration, the average tumor volume of the anti-KLH hIgG1 negative control group at a dose of 1mg/kg was 1116.+ -. 106mm ³ . The average tumor volume of the positive control group of Iplilimumab (made by Mild, see patent WO2001014424A2 and CN 1371416A) at a dose of 1mg/kg was 255.+ -.88 mm ³ TGI% was 86.36%. huJS007-47 at doses of 0.1,0.3 and 1mg/kg, the average tumor volumes were 736.+ -.203 mm, respectively ³ ，47±12mm ³ ，33±15mm ³ TGI% is 38.11%, 107.22% and 108.63%, respectively. The huJS007-47 shows that the volume increase of the MC38 tumor transplanted by the hTLA 4 humanized mice is obviously inhibited at the dosages of 0.1,0.3 and 1mg/kg, and the huJS007-47 shows good dosage effect and the huJS-47 has the same dosage condition(1 mg/kg), huJS007-47 has significantly better tumor-inhibiting effect than Ipilimumab.

Example 13: inhibition of hCDLA 4 humanized mice transplanted H22 tumor growth by huJS007-47 pharmaceutical composition

Female hCTLA4 humanized mice (Shanghai south mode Biotech Co., ltd.) of 6-8 weeks old were inoculated subcutaneously on the right back with 1X 10 ⁶ Mouse liver cancer cell H22 cells (Shanghai Norobu Biotechnology Co., ltd., cat# C01-FV) (0.1 ml/cell-containing medium RMPI1640 (Gibco))). The tumor volume to be averaged was about 119mm ³ At this time, 35 animals were selected and randomly divided into 5 groups of 7 animals each based on tumor volume. Respectively is

Anti-KLH hIgG1 negative control, 0.3mg/kg;

iiliimumab positive control group, 0.1mg/kg;

huJS007-47 treatment group, 0.03mg/kg;

huJS007-47 treatment group, 0.1mg/kg;

huJS007-47 treatment group, 0.3mg/kg;

All groups were given by intraperitoneal injection, 2 times per week, 6 times continuously, and the experiment was ended 3 days after the last administration. Tumor volume and body weight were measured 2 times per week and mice body weight and tumor volume were recorded. At the end of the experiment, mice were euthanized and tumor inhibition TGI was calculated in the same manner as in example 15.

As shown in FIG. 11, at day 21 after the start of the administration, the average tumor volume of the anti-KLH hIgG1 negative control group at a dose of 0.3mg/kg was 2304.+ -. 402mm ³ . The average tumor volume of the positive control group of Iplilimumab (made by Mild, see patent WO2001014424A2 and CN 1371416A) at a dose of 0.1mg/kg was 837.+ -.397 mm ³ TGI% is 67.14%. The huJS007-47 had average tumor volumes of 1674+ -508 mm at 0.03,0.1 and 0.3mg/kg, respectively ³ ，466±171mm ³ ，271±155mm ³ TGI% was 28.83%, 84.12% and 93.04%, respectively. Indicating that huJS007-47 is at a dosage of 0.1 and 0.3mg/kgUnder the condition of the equal dose (0.1 mg/kg), the tumor volume increase of the transplanted H22 of the hTLA 4 humanized mice is obviously inhibited, and the huJS007-47 tumor inhibition effect is obviously better than that of the Iilimiumab.

Sequence listing

SEQ ID NO:1 RASQNVGTYVA

SEQ ID NO:2 STSYRYS

SEQ ID NO:3 HQYDTYPLT

SEQ ID NO:4 SGYYWN

SEQ ID NO:5 YIGYDGSNNYNPSLKS

SEQ ID NO:6 DYYSGYFDS

SEQ ID NO:7 YIGYDGSNYYNPSLKS

SEQ ID NO:8 NYYSGYFDS

SEQ ID NO:9 YIGYDGSNNYNPSLKN

SEQ ID NO:10

DIQMTQSPSSLSASVGDRVTITCRASQNVGTYVAWYQQKPGKVPKPLIYSTSYRYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYFCHQYDTYPLTFGAGTKLELK

SEQ ID NO:11

QVQLQESGPGLVKPSQTLSLTCTVSAYSITSGYYWNWIRQHPGKGLEWIGYIGYDGSNNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARDYYSGYFDSWGQGTTLTVSS

SEQ ID NO:12

DIQMTQSPSSLSASVGDRVTITCRASQNVGTYVAWYQQKPGKAPKPLIYSTSYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCHQYDTYPLTFGAGTKLELK

SEQ ID NO:13

EIVMTQSPATLSVSPGERATLSCRASQNVGTYVAWYQQKPGQAPRPLIYSTSYRYSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCHQYDTYPLTFGQGTKLEIK

SEQ ID NO:14

QVQLQESGPGLVKPSQTLSLTCAVSGYSISSGYYWNWIRQPPGKGLEWIGYIGYDGSNYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAVDTAVYYCARNYYSGYFDSWGQGTTVTVSS

SEQ ID NO:15

QVQLQESGPGLVKPSQTLSLTCAVSGYSITSGYYWNWIRQPPGKGLEWIGYIGYDGSNNYNPSLKNRVTMSVDTSKNQFSLKLSSVTAVDTAVYYCARDYYSGYFDSWGQGTTVTVSS

SEQ ID NO:16

DIQMTQSPSSLSASVGDRVTITCRASQNVGTYVAWYQQKPGKVPKPLIYSTSYRYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYFCHQYDTYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:17

QVQLQESGPGLVKPSQTLSLTCTVSAYSITSGYYWNWIRQHPGKGLEWIGYIGYDGSNNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARDYYSGYFDSWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:18

DIQMTQSPSSLSASVGDRVTITCRASQNVGTYVAWYQQKPGKAPKPLIYSTSYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCHQYDTYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:19

SEQ ID NO:20

EIVMTQSPATLSVSPGERATLSCRASQNVGTYVAWYQQKPGQAPRPLIYSTSYRYSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCHQYDTYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:21

QVQLQESGPGLVKPSQTLSLTCAVSGYSISSGYYWNWIRQPPGKGLEWIGYIGYDGSNYYNPSLKSRVTMSVDTSKNQFSLKLSSVTAVDTAVYYCARNYYSGYFDSWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:22

SEQ ID NO:23

QVQLQESGPGLVKPSQTLSLTCAVSGYSITSGYYWNWIRQPPGKGLEWIGYIGYDGSNNYNPSLKNRVTMSVDTSKNQFSLKLSSVTAVDTAVYYCARDYYSGYFDSWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. A pharmaceutical composition comprising:

(1) A buffer; and

(2) An anti-CTLA-4 antibody or antigen-binding fragment thereof;

wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 as follows:

LCDR1：RASQNVGTYVA(SEQ ID NO:1)；

LCDR2：STSYRYS(SEQ ID NO:2)；

LCDR3：HQYDTYPLT(SEQ ID NO:3)；

HCDR1：SGYYWN(SEQ ID NO:4)；

HCDR2: YIGYDGSNX1YNPSLKX2, wherein X1 is N or Y, X2 is S or N;

HCDR3: x3YYSGYFDS, X3 is D or N;

preferably, the concentration of the anti-CTLA-4 antibody or antigen-binding fragment thereof is about 1 to 100mg/mL, preferably about 1 to 50mg/mL, more preferably about 5 to 30mg/mL;

preferably, the pH of the pharmaceutical composition is about 4.5 to 6.5, preferably about 5.0 to 6.0;

preferably, the pharmaceutical composition has an osmotic pressure of about 250 to 350mOsm/kg;

preferably, the pharmaceutical composition is administered by intravenous injection.

2. The pharmaceutical composition of claim 1, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises:

LCDR1 with the amino acid sequence shown as SEQ ID NO. 1;

LCDR2 with the amino acid sequence shown in SEQ ID NO. 2;

LCDR3 with the amino acid sequence shown in SEQ ID NO. 3;

HCDR1 with the amino acid sequence shown in SEQ ID NO. 4;

HCDR2 with amino acid sequence shown as SEQ ID NO 5 or 7 or 9;

HCDR3 with amino acid sequence shown in SEQ ID NO. 6 or 8;

preferably, the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises:

(3) The amino acid sequences are respectively shown as LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences are respectively shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 4, SEQ ID NO. 9 and SEQ ID NO. 6;

preferably, the anti-CTLA-4 antibody or antigen-binding fragment thereof is selected from murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof, preferably humanized antibody or antigen-binding fragment thereof.

3. The pharmaceutical composition of claim 1 or 2, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises:

(4) A light chain variable region with an amino acid sequence shown as SEQ ID NO. 13, and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 15;

preferably, the anti-CTLA-4 antibody comprises:

(1) A light chain amino acid sequence with an amino acid sequence shown as SEQ ID NO. 16, and a heavy chain amino acid sequence with an amino acid sequence shown as SEQ ID NO. 17;

(2) A light chain amino acid sequence with an amino acid sequence shown as SEQ ID NO. 18, and a heavy chain amino acid sequence with an amino acid sequence shown as SEQ ID NO. 19;

(3) A light chain amino acid sequence with an amino acid sequence shown as SEQ ID NO. 20, and a heavy chain amino acid sequence with an amino acid sequence shown as SEQ ID NO. 21; or (b)

4. The pharmaceutical composition of any one of claims 1-3, wherein the buffer is selected from one or more of an acetate buffer, a citrate buffer, a phosphate buffer, and a histidine buffer; preferably, the buffer is an acetate buffer; preferably, the acetic acid buffer is acetic acid-sodium acetate buffer or acetic acid-potassium acetate buffer, preferably acetic acid-sodium acetate buffer; preferably, the concentration of the buffer is about 5 to 100mM, preferably about 10 to 50mM, preferably about 10 to 30mM; preferably, the pH of the buffer is about 4.5 to 6.5, preferably about 5.0 to 6.0.

5. The pharmaceutical composition of any one of claims 1-4, wherein the pharmaceutical composition further comprises a stabilizer selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose, and trehalose; preferably, the arginine salt is arginine hydrochloride; preferably, the concentration of the stabilizing agent is about 100 to 300mM, preferably about 130 to 280mM, preferably about 200 to 260mM.

6. The pharmaceutical composition of claim 5, wherein the stabilizer is mannitol at a concentration of about 130-280 mM; or the stabilizer is sucrose at a concentration of about 130 to 280 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and mannitol at a concentration of about 110-170 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 20-80 mM and sucrose at a concentration of about 110-170 mM; preferably, the stabilizing agent is mannitol at a concentration of about 200 to 260 mM; or the stabilizer is sucrose at a concentration of about 200 to 260 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 30-70 mM and mannitol at a concentration of about 120-160 mM; or the stabilizer is a combination of sodium chloride at a concentration of about 30-70 mM and sucrose at a concentration of about 120-160 mM.

7. The pharmaceutical composition of any one of claims 1-6, wherein the pharmaceutical composition further comprises a surfactant selected from one or more of polysorbate 80, polysorbate 20, and poloxamer 188; preferably, the surfactant concentration is about 0.01% to 0.1%, more preferably about 0.01% to 0.05%, calculated as w/v.

8. The pharmaceutical composition according to any one of claims 1 to 7, comprising the components as set forth in any one of the following (1) to (13), respectively:

9. An injection comprising the pharmaceutical composition of any one of claims 1-8 and a sodium chloride solution or a dextrose solution; preferably, the sodium chloride solution concentration is about 0.85-0.9% (w/v); preferably, the glucose solution concentration is about 5 to 25% (w/v), preferably about 5 to 10% (w/v); preferably, the concentration of the anti-CTLA-4 antibody in the injection is about 0.05-10.5 mg/mL, more preferably about 0.1-5 mg/mL or about 1-5 mg/mL; preferably, the pH of the injection is about 4.5 to 6.5, more preferably about 5.0 to 6.0.

10. Use of a pharmaceutical composition according to any one of claims 1-8 or an injection according to claim 9 in the manufacture of a medicament for the treatment and/or prevention of CTLA-4 mediated diseases or conditions; preferably, the disease or disorder is cancer.