WO2023217234A1 - Liquid antibody composition and use thereof - Google Patents

Abstract

Disclosed in the present invention are a liquid antibody composition and the use thereof. The liquid antibody composition comprises a bispecific antibody targeting PD-L1 and CD47, an acidic buffer solution, a saccharide stabilizer and a polysorbate surfactant, and has a pH of 5.5-6.5, preferably 5.5-6.0. The composition has a good stability under the conditions of a high-concentration bispecific antibody.

Description

Liquid antibody compositions and their applications

priority information

This application requests the priority and rights of the patent application with patent application number 202210511286.4, which was submitted to the State Intellectual Property Office of China on May 11, 2022, and the full text of which is incorporated herein by reference.

Technical field

The present invention relates to the field of medicine, and in particular, to a pharmaceutical composition comprising a bispecific antibody targeting PD-L1 and CD47, especially a stable high-concentration liquid antibody composition, and the therapeutic use of the composition.

Background technique

As biological macromolecules, bispecific antibodies have complex structures. During the production and storage processes, physical changes such as aggregation, denaturation, and precipitation, and chemical changes such as isomerization, deamidation, and oxidation will occur. These changes can affect the safety and effectiveness of the product.

Thus, a stable pharmaceutical composition formulation remains to be developed.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to propose a pharmaceutical composition of a bispecific antibody that specifically binds PD-L1 and CD47, which composition has good stability under high-concentration bispecific antibody conditions.

CD47 is an integrin-related protein that is widely expressed on the cell surface. It can interact with signal regulatory protein α (SIRPα), thrombospondin and integrins to mediate a series of reactions such as apoptosis, proliferation, and immunity. Research has found that CD47 is a "self" signal that represents "don't eat me." CD47 can bind to the signal regulatory protein (SIRPα) on the surface of macrophages, thereby inhibiting the phagocytosis of macrophages. Since CD47 is widely expressed on a variety of tumor cells, tumor cells can escape phagocytosis by macrophages through the CD47-SIRPα signaling pathway. Therefore, CD47 antibodies can be used to block the binding of CD47 and SIRPα to activate macrophages to phagocytose tumor cells. effect.

PD-L1 (programmed cell death-ligand 1) is a type I transmembrane protein with a size of 40 kDa. PD-L1 can interact with the receptor PD-1 on T cells, thereby inhibiting the activation of T cells, causing T cell apoptosis, and plays an important role in negative regulation of immune responses. However, tumor cells also express PD-L1 and induce T cell apoptosis. Therefore, PD-L1 antibodies can be used to bind to PD-L1 of tumor cells, block their binding to T cells, activate the immune system, and discover and eliminate tumor cells.

Humanized anti-CD47/PD-L1 bispecific antibodies can not only activate T cells to identify and kill tumor cells by blocking PD-1/PD-L1, but also promote macrophage by blocking the binding of CD47/SIRPα. cells phagocytose tumor cells and exert anti- tumor effects. The inventor conducted extensive research and experiments on the preparation of this antibody, and obtained the liquid antibody composition of the embodiment of the present invention. The composition has a simple formula, a stable protein system, and is convenient for large-scale production, storage, and transportation.

Thus, according to one aspect of the invention, the invention provides a liquid antibody composition. According to an embodiment of the present invention, the composition includes: a bispecific antibody targeting PD-L1 and CD47; an acidic buffer; a stabilizer, the stabilizer being a sugar or a polyol; a polysorbate surfactant ; And the pH is 5.5-6.5, preferably 5.5-6.0, wherein the bispecific antibody targeting PD-L1 and CD47 includes two heavy chains and two light chains, wherein the first heavy chain can The variable region is the heavy chain variable region of the anti-CD47 monoclonal antibody, which can form a CD47 binding site with the light chain variable region. The second heavy chain variable region is the heavy chain variable region of the anti-PD-L1 monoclonal antibody. , which can form a PD-L1 binding site with the light chain variable region; wherein the sequences of the light chain variable regions of the two light chains are the same, and the light chain variable region is based on anti-CD47 monoclonal The light chain of the antibody and the light chain of the anti-PD-L1 monoclonal antibody are obtained, and the first variable region binding site formed by the light chain variable region and the first heavy chain variable region specifically binds to CD47 Binds, and the second variable region binding site formed by matching the light chain variable region and the second heavy chain variable region specifically binds to PD-L1.

The liquid antibody composition according to the embodiment of the present invention has a simple formula and rich sources, and is a commonly used auxiliary material for biological preparations. Moreover, the formula protein system of the liquid antibody composition according to the embodiment of the present invention is stable and can effectively prevent protein aggregation, degradation, oxidation and Denaturation, etc., thereby maintaining the biological activity of its active ingredients, low cost, and convenient for large-scale production, storage and transportation.

In addition, the liquid antibody composition according to the above embodiments of the present invention may also have the following additional technical features:

According to an embodiment of the present invention, the concentration of the bispecific antibody targeting PD-L1 and CD47 is 10-100 mg/ml, preferably, 30-80 mg/ml.

According to an embodiment of the present invention, the acidic buffer is selected from at least one of a citrate buffer, an acetate buffer and a histidine buffer, preferably, a histidine buffer.

According to an embodiment of the present invention, the concentration of the acidic buffer is 5-25mM, preferably 10-20mM.

According to an embodiment of the present invention, the sugar is sucrose or trehalose, preferably sucrose, and the polyol is mannitol or sorbitol.

According to an embodiment of the present invention, the concentration of the carbohydrate stabilizer is 3%-9% (w/v), preferably, 5% (w/v).

According to an embodiment of the present invention, the surfactant is polysorbate 20 or polysorbate 80.

According to an embodiment of the present invention, the concentration of the surfactant is 0.02%-0.06% (w/v), preferably, 0.02%-0.04% (w/v).

According to an embodiment of the present invention, the first heavy chain variable region includes: heavy chain CDR1, including X ₁ YX ₂ MX ₃ , where X ₁ is selected from N and S, X ₂ is selected from V and A, and X ₃ is selected from From H, S; heavy chain CDR2, including YINPX ₄ NX ₅ X ₆ IKYNEKFX ₇ G, where _{X 4} is selected from Y, G, _{X 5} is selected from D, E, , Q; heavy chain CDR3, including EGDFYANYGRLGFX ₈ Y, _where X ₈ is selected from A _{, D; and, light chain CDR1, including RASQDIX 9 NYLN} , wherein Among them _, X10 is _selected from H, Q _, S; _light chain _CDR3 includes QQGX ₁ 1X ₁₂ Y, K, S, E, N, X ₁₄ is selected from Y, R.

According to an embodiment of the present invention, the heavy chain CDR1 has the sequence shown in SEQ ID NO: 31, 32, 33 or 45; the heavy chain CDR2 has SEQ ID NO: 34, 35, 36, 37, 38 or 39 The sequence shown; the heavy chain CDR3 has the sequence shown in SEQ ID NO: 40 or 41.

According to an embodiment of the present invention, the first heavy chain includes the amino acid sequence shown in SEQ ID NO: 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 .

According to an embodiment of the present invention, the second heavy chain variable region includes CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43 and CDR3 shown in SEQ ID NO:44.

According to an embodiment of the present invention, the light chain has the sequence shown in SEQ ID NO: 1, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

According to an embodiment of the present invention, the Fc segment of the first heavy chain includes the H315R mutation, the Fc segment of the second heavy chain includes the K319E mutation, and the amino acid mutation site of the Fc segment uses Kabat's Eu numbering system.

According to an embodiment of the invention, the composition includes: 10-80mg/ml of the bispecific antibody targeting PD-L1 and CD47; 5-25mM of the histidine buffer; 3%-9% (w /v) the sucrose; 0.02%-0.06% (w/v) the polysorbate 20, and the pH is 5.5-6.0.

According to an embodiment of the present invention, the composition includes; 30 mg/ml of the bispecific antibody targeting PD-L1 and CD47; 20 mM histidine; 5% sucrose; 0.03% polysorbate 20; and pH is 5.5 -6.0.

According to another aspect of the invention, the invention provides a solid antibody composition. According to an embodiment of the present invention, the solid antibody composition is obtained by solidifying the aforementioned liquid antibody composition.

According to an embodiment of the present invention, the solidification is a freeze-drying process.

According to yet another aspect of the invention, a delivery device is provided. According to an embodiment of the present invention, the delivery device includes the aforementioned liquid antibody composition or the aforementioned solid antibody composition.

According to yet another aspect of the present invention, the present invention provides the use of the aforementioned liquid antibody composition or the aforementioned solid antibody composition in the preparation of anti-tumor drugs.

According to embodiments of the present invention, the tumors include melanoma, lung cancer, renal cancer, Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, acute and chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma tumors, bladder cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, kidney cancer and multiple myeloma.

According to yet another aspect of the invention, the invention provides methods of treating a subject suffering from a tumor. According to an embodiment of the present invention, the method includes administering to the subject a therapeutically effective amount of a composition, which is the aforementioned liquid antibody composition or the aforementioned solid antibody composition.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Detailed ways

Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. described below with reference to the accompanying drawings The embodiments are illustrative and are only used to explain the present invention and should not be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise stated, the meaning of "plurality" is two or more.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below:

As used herein, the term "antibody" is used in its broadest sense to refer to proteins containing an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to tribodies, intact antibodies, and antigen-binding fragments of antibodies .

The term "lyophilized composition" refers to a composition obtained or obtainable by freeze-drying a liquid composition. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

A "stable" antibody composition formulation is one in which the antibody in the formulation retains an acceptable degree of physical and/or chemical stability after storage under specified conditions. Although the antibodies contained in the antibody composition formulation may not maintain 100% of their chemical structure after storage for a specified time, they typically maintain about 92%, about 95%, about 96%, about 97%, about An antibody composition formulation is considered "stable" if it is 98% or about 99% of the structure or function of the antibody. In some specific embodiments, the anti-CD47/PD-L1 bispecific antibody protein compositions of the embodiments of the invention exhibit low to undetectable antibody aggregation or degradation during manufacturing, preparation, transportation and long-term storage or Chemically modified, resulting in little or even no loss of biological activity of the anti-CD47/PD-L1 bispecific antibody protein, exhibiting a high degree of stability. In some embodiments, the anti-CD47/PD-L1 bispecific antibody protein compositions of embodiments of the invention substantially retain their physical and chemical stability after storage. Preferably, the liquid composition of the present invention is stable at room temperature or at 40°C for at least 1 month, and/or at 2-8°C for at least 24 months.

A variety of analytical techniques are known in the art for determining protein stability, see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at selected temperatures and selected storage times. For example, storage time may be selected based on the expected shelf life of the formulation. Alternatively, accelerated stability testing can be used. In some embodiments, stability testing is performed by subjecting the antibody preparation to various stress tests. These tests may represent extreme conditions that the formulated antibody preparation may be exposed to during manufacturing, storage, or transportation, or may represent conditions that may accelerate the instability of the antibodies in the antibody preparation during non-manufacturing, storage, or transportation. For example, the formulated anti-CD47/PD-L1 bispecific antibody protein formulation can be filled into glass vials to test the antibody stability under high temperature stress.

I. Antibody Compositions

The invention provides stable liquid antibody compositions comprising (i) anti-CD47/PD-L1 bispecific antibody protein, (ii) buffer, (iii) stabilizer, and (iv) surfactant, optionally , further comprising (v) other excipients, the pH of the antibody preparation is about 5.5-6.5. In a preferred embodiment, the liquid antibody composition of the invention is in the form of an injection.

(i) Anti-CD47/PD-L1 bispecific antibody protein

The "anti-CD47/PD-L1 bispecific antibody protein" in the antibody composition of the present invention is a diabody. The first heavy chain variable region is derived from the heavy chain variable region of the anti-CD47 monoclonal antibody. It forms a CD47 binding site with the light chain variable region. The second heavy chain variable region is derived from the heavy chain variable region of the anti-PD-L1 monoclonal antibody, which can form a PD-L1 binding site with the light chain variable region. ; Wherein, the sequences of the light chain variable regions of the two light chains are the same. The anti-CD47/PD-L1 bispecific antibody protein is capable of binding to CD47 with an affinity constant of at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 and more preferably about 10 ⁹ M ^-1 or greater. , and is capable of binding to PD-L1 with an affinity constant of at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 , and more preferably about 10 ⁹ M ^-1 or greater, such that the antibody can be used as a dual Therapeutic and/or preventive agents that specifically target CD47 molecules and PD-L1 molecules.

For the VH/VL pair that specifically binds to PD-L1 or CD47, it includes 6 VH/VL pairs derived from any anti-PD-L1 antibody reported in the prior art and anti-PD-L1 antibody VH/VL pairs developed in the future. CDR or a sequence that has one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions) from one or more of the 6 CDRs; or Comprising 6 CDRs derived from any anti-CD47 antibody reported in the prior art and anti-CD47 antibody VH/VL pairs developed in the future or sharing one, two, three CDRs with one or more of the 6 CDRs Sequences with one, four, five, six or more amino acid changes (e.g., amino acid substitutions or deletions).

In one embodiment, the VH/VL pair that specifically binds CD47 on the first polypeptide chain and the second polypeptide chain of the anti-CD47/PD-L1 bispecific antibody protein comprises a VH/VL pair derived from Chinese Patent Application No. 202111340715.8 Reported anti-CD47 antibody, which has VH CDR1 represented by _{X 1} YX ₂ MX ₃ , _wherein X ₁ is selected from N and S, X ₂ is selected from V and A, _and X ₆ IKYNEKFX ₇ VH CDR2 shown in G, where X ₄ is selected from Y, G, X ₅ is selected from D, E, X ₆ is selected from G _, A _, and _{VH CDR3 , where _ And the} VL _{CDR3 shown in QQGX 1} X ₁₄ is selected from Y, R, or has one, two, three, four, five, six or more amino acid changes (e.g., amino acid substitutions) with one or more of the 6 CDRs or missing) sequence.

The term "CDR" or "complementarity determining region" or "CDR region" (used interchangeably herein with hypervariable region "HVR"), is the region of amino acids in an antibody variable region that is primarily responsible for binding to an antigenic epitope. The CDRs of the heavy and light chains are often referred to as CDR1, CDR2, and CDR3 and are numbered sequentially starting from the N-terminus. Various protocols for determining the CDR sequence of a given VH or VL or VHH amino acid sequence are known in the art. For example, Kabat complementarity determining regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ( 1991)). Chothia refers to the position of the structural ring (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chot-hia structural loops, and is used by Oxford Molecular's AbM antibody modeling software. "Contact" HVR is based on the analysis of available complex crystal structures. HVR can also be determined based on having the same Kabat number position as a reference CDR sequence (eg, the exemplary CDRs disclosed herein).

In one embodiment, the first heavy chain variable region of the anti-CD47/PD-L1 bispecific antibody protein is derived from anti-CD47 A heavy chain variable region of a monoclonal antibody capable of forming a CD47 binding site with a light chain variable region, the first heavy chain comprising a heavy chain variable derived from SEQ ID NO: 5 of the anti-CD47 antibody hz140-Hm region sequence, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the heavy chain variable region sequence The sequence, for example, the heavy chain variable region sequence shown in 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19.

In one embodiment, the second heavy chain variable region of the anti-CD47/PD-L1 bispecific antibody protein is derived from the heavy chain variable region of the anti-PD-L1 monoclonal antibody, which is capable of forming with the light chain variable region PD-L1 binding site, the second heavy chain comprising the heavy chain variable region sequence derived from SEQ ID NO: 2 of an anti-PD-L1 antibody, or sharing at least 90%, Sequences with 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

In one embodiment, the sequences of the light chain variable regions of the two light chains of the anti-CD47/PD-L1 bispecific antibody protein are identical, and the light chain variable regions are based on an anti-CD47 monoclonal antibody The light chain and the light chain of the anti-PD-L1 monoclonal antibody are obtained, and the first variable region binding site formed by the light chain variable region and the first heavy chain variable region specifically binds to CD47 , and the second variable region binding site formed by matching the light chain variable region and the second heavy chain variable region specifically binds to PD-L1, and the light chain includes SEQ ID NO: 1, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, or substantially identical thereto (e.g., at least 80%, 85%, 90%, 92%, 95%, 97 %, 98%, 99% or more identical) sequences.

As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino-acid-by-amino acid basis within a comparison window. "Percent sequence identity" can be calculated by comparing two optimally aligned sequences in a comparison window and determining the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in both sequences. ) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) Number of positions to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiply the result by 100 to generate percent sequence identity. Optimal alignment for determining percent sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximal alignment within the full-length sequences being compared or within the sequence region of interest.

The bispecific antibodies prepared in the embodiments of the present invention have double binding arms of natural antibodies, and each binding arm has a complete Fab structure, which avoids the introduction of receptor chains (such as CD47 receptor SIRPα) and other non-antibodies into the antibody molecules. The components cause structural instability, functional interference and other shortcomings. Furthermore, through the embodiments of the present invention, on the basis of mutation transformation of the common light chain variable region and heavy chain variable region, the binding ability of the two binding arms is controlled, for example, the first antigen/antigen epitope is prepared Bispecific antibodies with high binding capacity and moderate binding capacity for the second antigen/antigen epitope.

According to the difference in the degree of influence of the six CDRs in the antigen-binding site on the antigen-binding activity in the prior art, that is, the effect of the three CDRs of the heavy chain is greater than that of the three CDRs of the light chain, and the effect of CDR3 is greater than that of CDR2 and CDR1. Combined with anti-PD-L1 mono Based on the similarity in the light chain sequence structure of cloned antibody hz182 and anti-CD47 monoclonal antibody hz140, the inventors designed an anti-human PD-L1/CD47 bispecific antibody with the same light chain. The design of a common light chain not only simplifies the recombinant expression method, but also completely eliminates the problem of light and heavy chain mismatch during the recombinant expression of bispecific antibodies, significantly simplifies the production process, and improves product qualification rate and uniformity. In addition, "knobs-into-holes" mutations can also be introduced in the Fc segment to enhance the accuracy of pairing the Fc segment of the bispecific antibody heavy chain and avoid dimerization of homologous heavy chains.

During the design and preparation process of bispecific antibodies, limited amino acid mutations were carried out in the heavy chain of the anti-CD47 antibody, so that both the heavy chain variable region and the light chain variable region in the anti-CD47 binding arm of the recombinant antibody were initially the same as those of anti-CD47. Monoclonal antibodies have slight structural differences. By introducing slight differences in sequence structure into the above-mentioned anti-CD47 antigen-binding site, the bispecific antibody has moderate anti-CD47 activity, which not only maintains the binding activity of the bispecific antibody to CD47 on the surface of tumor cells, but also enhances the anti-CD47 activity. The tumor inhibitory effect of PD-L1 activity; it also significantly reduces the binding to CD47 on the surface of red blood cells, which not only reduces or eliminates undesirable consequences such as hemolysis and red blood cell agglutination, but also reduces the amount of systemic administration.

In a preferred embodiment, the anti-CD47/PD-L1 bispecific antibody protein of the embodiment of the present invention is the recombinant anti-CD47/PD-L1 bispecific antibody protein disclosed in the Chinese application with application number: 202111340715.8, which has The first polypeptide chain shown in SEQ ID NO:1, the second polypeptide chain shown in SEQ ID NO:5, and the third polypeptide chain shown in SEQ ID NO:10. In one embodiment, the anti-CD47/PD-L1 bispecific antibody protein is produced by recombinant expression in eukaryotic cells such as HEK293 cells or CHO cells and is purified.

(ii)Buffer

Buffers are agents that maintain the pH of a solution within an acceptable range. In some embodiments, acidic buffers used in the formulations of the invention can control the pH of the formulations of the invention to a pH range of about 5.5-6.5, for example, a pH of about 6.0. In some specific embodiments, antibody compositions of the invention have a pH of about 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5.

In some embodiments, the buffer used in the formulations of the present embodiments is selected from the group consisting of citrate buffer, acetate buffer, and histidine buffer, and combinations thereof. In one embodiment, the concentration of the buffer in the liquid antibody composition of the present invention is about 5-25mM. In one embodiment, the concentration of the buffer in the liquid antibody composition of the invention is about 10-20mM, for example, about 10, 12, 15, 18 and 20mM.

In one embodiment, the buffer used in the compositions of the invention is about 10 mM histidine buffer.

(iii)Stabilizer

Suitable stabilizers for use in the present invention may be sugars or polyols. Sugars used as stabilizers include, but are not limited to, sucrose and trehalose. Polyols used as stabilizers include, but are not limited to, mannitol and sorbitol. In some embodiments, the stabilizer is present in the liquid composition of the invention at about 3%-9% (w/v), more preferably at about 4%-7% (w/v), for example, about 4.5% %, 5.0%, 5.5%, 6.0%, 6.5% concentration exists.

In one embodiment, the liquid composition of the present invention contains sucrose as a stabilizer. The amount of sucrose in the liquid composition of the present invention may be about 4%-7% (w/v), preferably about 4.5%-6% (w/v) (for example, about 4.5, 4.8, 5.0, 5.2, 5.4 , 5.6, 5.8 and 6.0% (w/v)).

(iv) Surfactant

As used herein, the term "surfactant" refers to organic substances with an amphiphilic structure; that is, they are composed of groups of opposite solubility tendencies, typically oil-soluble hydrocarbon chains and water-soluble ionic groups. group.

In one embodiment, the surfactant in the liquid composition of the present embodiments is a nonionic surfactant. Specifically, the nonionic surfactant in the composition of the embodiment of the present invention is polysorbate, such as polysorbate, such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-60. Sorbitate-40, etc. In a preferred embodiment, the liquid composition of the present invention contains polysorbate-20 or polysorbate-80 as surfactant.

The amount of surfactant contained in the antibody compositions of the invention may vary depending on the specific intended properties of the formulation, the particular environment, and the specific purpose for which the formulation is used. In preferred embodiments, the formulation may contain about 0.1-1 mg/ml, preferably about 0.2-0.8 mg/ml, such as about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml polysorbate surfactants (e.g., polysorbate-80).

(v)Other excipients

The antibody liquid composition of the present invention may or may not contain other excipients.

In one embodiment, the antibody liquid composition of the invention contains a metal chelator (eg, EDTA or a salt thereof) as an excipient. In another embodiment, the antibody liquid composition of the invention does not contain a metal chelator (eg, EDTA or a salt thereof). In one embodiment, the antibody liquid composition of the invention adding a metal chelating agent (e.g., EDTA or a salt thereof) has a higher stability.

For other reasons, other excipients may also be used in the compositions of the present embodiments. Such excipients include, for example, flavoring agents, antimicrobial agents, sweeteners, antistatic agents, antioxidants, gelatin, and the like. These and other known pharmaceutical excipients and/or additives suitable for use in the compositions of the present invention are well known in the art and are, for example, listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, Rowe et al. , American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st Edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)."

II. Preparation of Formulation

The present invention provides stable composition formulations comprising anti-CD47/PD-L1 bispecific antibody proteins. Anti-CD47/PD-L1 bispecific antibody proteins for use in the compositions of the invention can be prepared using techniques known in the art for producing antibodies. For example, anti-CD47/PD-L1 bispecific antibody proteins can be produced recombinantly. In a preferred embodiment, the anti-CD47/PD-L1 bispecific antibody protein of the present invention is prepared by recombinant expression in eukaryotic cells, for example, as described in the Chinese application with application number 202111340715.8, recombinant preparation of anti-CD47 /PD-L1 bispecific antibody protein.

The use of antibodies as active ingredients in drugs is now widespread. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for puri-fication of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599–613.) describe the use of ion exchange chromatography (anionic IEX and/or cationic CEX chromatography) after the protein A capture step. ) antibody three-column purification method. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies, Biotechnology and Bioengineering 101 (2008) 553–566) describe a two-column purification method in which a weak partitioning anion exchange resin is used after Protein A affinity chromatography.

Generally, recombinantly produced antibodies can be purified using conventional purification methods to provide sufficient reproducibility and Drug substances of moderate purity are used in the formulation of antibody preparations. For example, after the antibodies are secreted from the recombinant expression cells into the culture medium, the supernatant from the expression system can be concentrated using commercially available protein concentration filters such as Amicon's ultrafiltration devices. The antibody can then be purified using methods such as chromatography, dialysis, and affinity purification. Protein A is adapted as an affinity ligand for the purification of IgG1, IgG2 and IgG4 type antibodies. Other antibody purification methods, such as ion exchange chromatography, may also be used. After obtaining an antibody of sufficient purity, a composition containing the antibody can be prepared according to methods known in the art.

For example, the following steps can be used for preparation: (1) After the fermentation, the fermentation broth is centrifuged and clarified to remove impurities such as cells to obtain the supernatant; (2) affinity chromatography (for example, specific for IgG1, IgG2 and IgG4 type antibodies Affinity Protein A column) to capture antibodies; (3) Virus inactivation; (4) Refining and purification (CEX cation exchange chromatography can generally be used) to remove impurities in the protein; (4) Virus filtration (to increase the virus titer For example, reduce by more than 4log10); (5) Ultrafiltration/diafiltration (can be used to replace the protein in a preparation buffer that is conducive to its stability and concentrate it to a suitable concentration for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48–57.

III. Methods of analysis of compositions

During the storage process of antibody compositions, antibodies may undergo aggregation, degradation or chemical modification, resulting in antibody heterogeneity (including size heterogeneity and charge heterogeneity), aggregates and fragments, etc., thereby affecting the quality of the antibody composition. quality. Therefore, it is necessary to monitor the stability of the antibody composition.

Various methods are known in the art for testing the stability of antibody compositions. For example, the purity of the antibody composition and the aggregation level of the antibody can be analyzed by methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC; capillary isoelectric focusing electrophoresis (cIEF), imaging capillary, etc. Electrofocusing electrophoresis (iCIEF) and ion exchange chromatography (IEX), etc., are used to analyze charge variants in antibody compositions. In addition, the stability of the composition can be quickly judged by visually inspecting the appearance of the preparation. Changes in turbidity of formulations can also be detected using the OD _350nm method, which can give information on the amount of soluble and insoluble aggregates. In addition, ultraviolet spectrophotometry (UV method) can be used to detect changes in protein content in the preparation.

The non-reducing CE-SDS method is a method for determining the purity of monoclonal antibodies using a capillary tube as a separation channel. In CE-SDS, protein migration is driven by surface charge induced by SDS binding, which is proportional to the protein's molecular weight. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on the size or hydrodynamic radius of the molecules can be achieved in a capillary molecular sieve gel matrix. This method has been widely used to monitor the purity of denatured intact antibodies. Generally, in the non-reducing CE-SDS method, the test sample is mixed with SDS sample buffer and iodoacetamide. Afterwards, the mixture can be incubated at 68-72°C for about 10-15 minutes, cooled to room temperature and the centrifuged supernatant is used for analysis. Use a UV detector to detect protein migration and obtain an electrophoresis spectrum. The purity of the antibody composition can be calculated as the percentage of the peak area of the main IgG peak to the sum of all peak areas. For further description of the CE-SDS method, see, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

Size-exclusion high-performance liquid chromatography, or SEC-HPLC, is another important method for monoclonal antibody standards and quality control. This method mainly separates molecules based on their size or hydrodynamic radius differences. By SEC-HPLC, antibodies can be separated into three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the main peak area on the chromatogram to the sum of all peak areas. Compare. Through the SEC-HPLC method, the percentage of antibody monomers in the composition product can be measured, giving information on the content of soluble aggregates and shear products. For further description of the SEC-HPLC method, see, for example, J.Pharm.Scien., 83:1645-1650, (1994); Pharm.Res., 11:485 (1994); J.Pharm.Bio.Anal. , 15:1928(1997); J.Pharm.Bio.Anal., 14:1133-1140(1986). In addition, see, for example, R. Yang et al., High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx. doi.org/10.1016/j.jpba.2015.02.032; and Alexandre Goyon et al., Protocols for the analytical characterization of therapeutic monoclonal antibodies. I–Non-denaturing chromatographic techniques, Journal of Chromatography, http://dx.doi.org /10.1016/j.jchromb.2017.05.010.

Imaging capillary isoelectric focusing electrophoresis (iCIEF) can be used to analyze the charge heterogeneity of monoclonal antibodies. This method can provide a quantitative distribution of charge variants. iCIEF achieves the purpose of molecular separation based on the charge difference (apparent pI value) of molecules in the pH gradient. In iCIEF, the separation column is typically a short capillary (e.g., 5 cm long, 100 μm i.d. silica capillary), proteins are focused in the capillary column at high voltage, and focusing is monitored by a full-column imaging detection system operating at 280 nM. Real-time online monitoring. An advantage of this technique is that various charge variants of the antibody sample can be recorded simultaneously via this full-column detection system. Generally, in icIEF, the sample is mixed with urea and icIEF buffer containing methylcellulose, pi molecular weight standards, and ampholytes. Then, you can use an iCIEF column such as an iCIEF column assembled by ProtionSimple on an iCIEF analyzer such as the iCE280 analyzer (Protein Simple, Santa Clara, CA). After the sample is focused for a certain period of time, the absorbance at 280 nm is measured to obtain the focused mAb charge variant. spectrum. In the iCEIF spectrum, the protein-related peaks eluted before the main peak (i.e., the main component) are classified as acidic components; conversely, the protein-related peaks eluted after the main peak are classified as basic components. The relative amounts of the main components, acidic components and basic components can be expressed as a percentage of the total peak area. For further description of iCIEF, see, for example, Salas-Solano O et al., Robustness of iCIEF methodology for the analysis of monoclonal antibodies:an interlaboratory study, J Sep Sci.2012Nov; 35(22):3124-9.doi:10.1002/ jssc.201200633.Epub 2012Oct 15; and Dada OO et al., Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation, Electrophoresis.2015 Nov; 36(21-22):2695-27 02.doi: 10.1002/elps.201500219.Epub 2015 Sep 18.

Charge variants of antibodies in antibody compositions can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC). In this assay, peaks eluting from the CEX-HPLC column with a retention time earlier than the main peak are labeled as “acidic peaks”, while those eluting from the CEX-HPLC column with a later retention time than the main peak The peak is labeled "Basic Peak".

Accelerated stability studies can be used to examine the stability properties of a product and facilitate the screening of stable pharmaceutical formulations. For example, accelerated stability studies can be conducted by subjecting composition samples to elevated temperatures, such as about 40°C ± 2°C and 25°C ± 2°C. Detection indicators can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reducing CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).

Additionally, antibodies can be tested for efficacy or biological activity. For example, the ability of the antibody in the composition to bind to its antigen molecules (CD47 molecule and PD-L1 molecule) can be detected. Those skilled in the art know that various methods can be used to quantify the specific binding of antibodies to antigens, such as immunoassay tests, ELISA, etc.

The anti-CD47/PD-L1 bispecific antibody protein composition of the embodiment of the present invention is stable. In one embodiment, after storage at about 25°C, 37°C, 40°C, or 45°C for at least 1 month or 2 months, for example, after 1 month at 40°C ± 2°C, the antibody combination of the invention The purity of the anti-CD47/PD-L1 bispecific antibody protein in the substance is at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater, as determined by size exclusion chromatography Or measured by non-reducing CS-SDS. In one embodiment, after storage at about 25°C, 37°C, 40°C, or 45°C for at least 1 month or 2 months, for example, after 1 month of storage at 40°C ± 2°C, the embodiments of the present invention At least 50%, preferably at least 55%, of the anti-CD47/PD-L1 bispecific antibody protein in the antibody composition is in the non-basic and non-acidic form (i.e., the major peak or major charge form), as determined by the IEC-HPLC method .

IV. Use of the composition

The antibody composition of the invention comprising an anti-CD47/PD-L1 bispecific antibody protein can be used to treat, prevent or delay various diseases related to the SIRPα/CD47 signaling pathway and/or to the PD1/PD-L1 signaling pathway. Pathway-related diseases or conditions. "Diseases or disorders related to the SIRPα/CD47 signaling pathway" and/or "diseases or disorders related to the PD1/PD-L1 signaling pathway" herein refer to the anti-CD47/PD-L1 bispecific antibodies of the present invention that can be used A disease or condition that the protein composition treats (eg, ameliorates) or prevents. Any disease or condition that can benefit from treatment with the antibody compositions of the invention is suitable for use in the present invention.

In one aspect, the composition of the invention comprising an anti-CD47/PD-L1 bispecific antibody protein can be used to prevent or treat various tumors in a subject, including but not limited to melanoma, lung cancer, renal cancer, Hodgkin's disease Lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, acute and chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, bladder cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, kidney cancer and multiple Myeloma.

The present invention also provides the use of the composition of the present invention in the preparation of a medicament, wherein the medicament is used to deliver an anti-CD47/PD-L1 bispecific antibody protein to a mammal, or for treating, preventing or ameliorating the above-mentioned diseases and conditions. one or more of them. Preferably, the mammal is a human.

The antibody compositions of the invention can be administered to a subject or patient in a variety of ways. For example, administration can be by infusion or by syringe. Accordingly, in one aspect, the invention provides a delivery device (eg, a syringe) comprising an antibody composition of the invention (eg, a prefilled syringe). The patient will receive an effective amount of the anti-CD47/PD-L1 bispecific antibody protein as the primary active ingredient, i.e., an amount sufficient to treat, ameliorate or prevent the disease or condition of interest.

Therapeutic effects may include reduction of physical symptoms. The optimal effective amount and concentration of antibody for use in any particular subject will depend on a variety of factors, including the patient's age, weight, health and/or sex, the nature and extent of the disease, the activity of the specific antibody, the body's response to its clearance, and also any possible other treatments administered in combination with the antibody preparation. For a particular case, the effective amount delivered can be determined within the discretion of the clinician. Depending on the indication to be treated, an effective dose may be from about 0.005 mg/kg to about 50 mg/kg of body weight, or from about 0.1 mg/kg to about 20 mg/kg of body weight. In this regard, the use of known antibody-based drugs can provide some guidance. Dosage may be a single dose regimen or a multiple dose regimen.

The present invention will be described below with reference to specific examples. It should be noted that these examples are only illustrative and should not be construed as limitations of the present invention.

The solutions of the present invention will be explained below with reference to examples. Those skilled in the art will understand that the following embodiments They are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially, for example, from Sigma.

Example 1: Preparation and purification of anti-PD-L1/CD47 bispecific antibodies

According to 202111340715.8, the anti-CD47/PD-L1 bispecific antibody 2MW1531-m7 was recombinantly expressed and purified in eukaryotic cells. The anti-CD47/PD-L1 bispecific antibody consists of anti-PD-L1 monoclonal antibody and anti-CD47 monoclonal antibody. The polypeptide chain of each antibody has the following amino acid sequence from the N-terminus to the C-terminus:

Anti-PD-L1 monoclonal antibody:

(The light chain amino acid sequence is SEQ ID NO.1, and the heavy chain variable region amino acid sequence is SEQ ID NO.2)

Anti-CD47 monoclonal antibody:

(The light chain/heavy chain variable region sequence is SEQ ID NO.3/SEQ ID NO.5)

The anti-CD47/PD-L1 bispecific antibody: (heavy chain variable region sequence: SEQ ID NO.19/SEQ ID NO.2, heavy chain constant region sequence: SEQ ID NO.4/SEQ ID NO.6, The common light chain sequence is SEQ ID NO. 26).

Example 2: Antibody composition screening experiment

The antibodies prepared in Example 1 were prepared into different compositions, and the composition formulas were screened, as follows:

1. Buffer system and pH screening:

The antibody is replaced into the target buffer. The antibody concentration is 30mg/ml. After sterile filtration, it is packed and placed at 4°C and 40°C for stability testing. Samples are taken for testing at 0 days, 7 days, and 14 days. The testing items include SEC ,IEC.

Buffer composition:

Combined with the SEC method, the changes in the main peak content of the antibodies of the embodiments of the present invention under the SEC method and the IEC method at 40°C were investigated to determine the optimal buffer system and pH suitable for the double antibody. The results are shown in Table 1, starting from the experiment 0 days, 40 The contents of the SEC main peak and IEC main peak were placed for 7 and 14 days to calculate the decline rate (%/day).

Table 1. Investigation results of SEC main peak and IEC main peak of high temperature stability of different buffer systems

from error! Reference source not found. It can be seen that under the condition of pH 5.5-6.5 of the histidine buffer system, the decline rate of the main peaks of SEC and IEC is slightly better than that of the citric acid and acetic acid systems. Therefore, the histidine buffer system was selected for the next step of screening.

2. Protein concentration screening:

After selecting the histidine buffer system for the next step of screening, the impact of different protein concentrations on stability was further investigated. The antibody samples prepared in Example 1 were replaced with different compositions, placed at 40°C for accelerated stability experiments, and samples were taken for SEC detection at 0 days, 7 days, 14 days, and 28 days. The decline rate (%/day) of the main peak was calculated based on the SEC main peak content on the 0th day after the start of the experiment and at 40 degrees for 7 days, 14 days and 28 days. The results are shown in Table 2.

Table 2. Results of SEC main peak investigation on high temperature stability at different protein concentrations

As can be seen from Table 2, as the concentration increases, the decrease rate of the SEC main peak content tends to increase. In view of this result, future clinical medication plans will be comprehensively considered, and further investigation will be conducted around the concentration of 30mg/ml.

3. Buffer salt concentration screening:

The antibody samples prepared in Example 1 were replaced into buffers of different concentrations, and then placed at 40°C for accelerated stability experiments, and samples were taken for IEC detection at 0 days, 7 days, 14 days, and 28 days. The decline rate (%/day) of the main peak was calculated based on the IEC main peak content on the 0th day after the experiment and at 40 degrees for 7 days, 14 days and 28 days. The results are shown in Table 3.

Table 3. Results of IEC main peak investigation on high temperature stability at different buffer concentrations

Table 3 shows that the increase in buffer concentration does not have much impact on the decrease rate of the IEC main peak of the antibody. At the same time, in order to maintain the buffering capacity of the buffer, 10mM was selected as the final buffer concentration.

4. Screening of protective agents:

After the above conditions are determined, further research and comparison on adding protective agents are conducted to select excipient additives that can stabilize the antibody. The results are as follows:

As shown in Table 4 and Table 5, the results show that among different protective agents, there is no significant difference between the decline rate of the main SEC peak and the decline rate of the main IEC peak. Therefore, sucrose, which is commonly used in biological drugs, was selected as the additive.

Table 4. SEC main peak investigation results of high temperature stability of different protective agents

Table 5. Results of IEC main peak investigation of high temperature stability of different protective agents

5. Screening of protective agent concentration:

Using sucrose as a protective agent, the effects of different concentrations of sucrose on the antibodies prepared in Example 1 were examined. The results are as follows:

As shown in Table 6 and Table 7, the results show that the protein in different sucrose concentrations is relatively stable. Based on the principle of adding as little excipients as possible and In order to fully protect the protein, 5% sucrose was selected as the protective agent for the antibody.

Table 6. SEC main peak investigation results of high temperature stability with different protective agent concentrations

Table 7. Results of IEC main peak investigation of high temperature stability of different protective agents

6. Surfactant screening:

The antibody sample prepared in Example 1 was replaced into a buffer solution of 10 mM histidine and 5% sucrose, with a protein concentration of 30 mg/ml, and polysorbate 20 and 80 at different final concentrations were added to the replaced sample. The prepared samples were repeatedly frozen and thawed once, 3 times and 5 times and then the insoluble particles in the samples were measured.

Table 8. Results of insoluble particles in freeze-thaw samples of polysorbate 20 with different proportions

Table 9. Results of insoluble particles in freeze-thaw samples of polysorbate 80 in different proportions

The experimental results are shown in Table 8 and

As shown in Table 9, the results show that the sample without Tween has a significant increase in insoluble particles, while the sample with Tween has no significant increase in insoluble particles. There is no significant difference in the results of insoluble particles with different Tween contents. Therefore, the commonly used surfactant polysorbate 20 is selected as the surfactant of the antibody; in order to fully inhibit the generation of insoluble particles, a concentration of polysorbate 20 of 0.03% is preferred.

Example 3: Compound prescription verification experiment

The prescription verification test of the compounds of the embodiments of the present invention includes a freeze-thaw stability test, a 10°C shaking stability test and a light test. The CD47/PD-L1 bispecific antibody prepared in Example 1 was investigated in the prescription (10mM histidine, Stability in 5% sucrose, 0.03% polysorbate 20, pH 5.8±0.3).

1. Oscillation stability test:

The antibody composition was subjected to a vibration stability test, shaken at 10°C for 5 days, and the antibody monomer content (SEC) and charge isomer main peak content (IEC) were examined. The results are summarized in

Table 10.

Table 10. Examination results of physical and chemical properties of antibody compositions on vibration stability

2. Freeze-thaw stability test:

The antibody composition was repeatedly frozen and thawed 1, 3, and 5 times, and the antibody monomer content (SEC) and charge isomer main peak content (IEC) were examined. The results are summarized in Table 11.

Table 11. Examination results of physical and chemical properties of antibody compositions freeze-thaw stability

3. Light stability test:

The antibody composition was exposed to light at 4500lx±500lx for 5 days and 10 days, and the same sample was placed in a packaging box as a light-proof control. Focus on investigating the 6MW3211 monomer content (SEC) and charge isomer main peak content (IEC). The results are summarized in Table 12.

Table 12. Examination results of physical and chemical properties of light stability of antibody compositions

The above results show that under the determined composition formula of the embodiment of the present invention, shaking for 5 days and freezing and thawing 5 times have no significant effect on the physical and chemical properties of the antibody protein. After 10 days of illumination, the main peak of IEC decreased significantly, indicating that the CD47/PD-L1 bispecific antibody should be stored under light-proof conditions. Moreover, the above results illustrate that under the composition prescription of the embodiment of the present invention, the CD47/PD-L1 bispecific antibody Antibodies are relatively stable.

Example 4: Protein concentration improvement experiment

The increase in protein concentration will provide convenience in actual use. After conducting verification experiments on low-concentration prescriptions, in order to facilitate clinical application, in this example, an experiment on increasing protein concentration was attempted, and the protein concentration was increased to 80 mg/ml. In this example The CD47/PD-L1 bispecific antibody prepared in Example 1 was used to conduct the test.

The histidine concentration in the low-concentration antibody composition prescription is increased to 20mM, and the rest remains unchanged, that is, 20mM histidine, 5% sucrose, 0.03% polysorbate 20, pH 5.8±0.3, as the high-concentration composition prescription, Conduct freeze-thaw stability test, 10℃ vibration stability test, light test and 40℃ high temperature stability test.

1. Oscillation stability test:

The high-concentration preparation composition was subjected to a shaking stability test and shaken at 10°C for 5 days. Focus on the CD47/PD-L1 bispecific antibody monomer content (SEC) and charge isomer main peak content (IEC). The results are summarized in Table 13.

Table 13. Investigation results of physical and chemical properties of high concentration oscillation stability of 6MW3211

2. Freeze-thaw stability test:

The high-concentration preparation composition was repeatedly frozen and thawed 10 times, and the CD47/PD-L1 bispecific antibody monomer content (SEC) and charge isomer main peak content (IEC) were examined. The results are summarized in Table 14.

Table 14. Investigation results of physical and chemical properties of high concentration freeze-thaw stability of 6MW3211

3. Light stability test:

High-concentration formulation compositions were subjected to light stability testing. Place the sample under the conditions of 4500lx±500lx for 14 days (the total illumination of the light source is not less than 1.2×10 ⁶ lux·hr, and the energy of the near-UV lamp is not less than 200W·hr/m ² ), and at the same time, the same sample is placed in the packaging box as a light-protected control. Focus on investigating the CD47/PD-L1 bispecific antibody monomer content (SEC) and electron The main peak content of charge isomer (IEC), the results are summarized in Table 15.

Table 15. Investigation results of physical and chemical properties of 6MW3211 at high concentration and light stability

4. 40℃ high temperature stability test:

The high-concentration preparation composition was subjected to a high-temperature stability test at 40°C. The sample composition was placed at 40°C to examine 7D, 14D and 1M, and the CD47/PD-L1 bispecific antibody monomer content (SEC) and charge isomer main peak content (IEC) were examined. The results are summarized in Table 16.

Table 16. Investigation results of physical and chemical properties of 6MW3211 at high concentration and high temperature

The above results show that shaking for 5 days and freezing and thawing 10 times have no significant effect on the high-concentration CD47/PD-L1 bispecific antibody composition. After 14 days of illumination, the main peak of IEC decreased slightly, indicating that high concentrations of CD47/PD-L1 bispecific antibodies should be stored in dark conditions. After one month of high-temperature inspection at 40°C, the main peak of SEC decreased slightly and the main peak of IEC decreased significantly. The change trend is in line with expectations. According to the above results, it can be explained that under the formulation of this composition, the CD47/PD-L1 bispecific antibody is more stable at high concentrations.

In summary, by investigating different buffer systems, different pH conditions, different antibody concentrations, and different compositions of protective agents and surfactants, the stability of humanized anti-CD47/PD-L1 bispecific antibodies was explored and studied. A water injection composition formula is obtained, which contains composition formulas with two protein concentrations.

In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the invention. The scope of the invention is defined by the claims and their equivalents.

Claims (23)

1. A liquid antibody composition, characterized by comprising:

   Bispecific antibodies targeting PD-L1 and CD47;

   acidic buffer;

   Stabilizer, the stabilizer is sugar or polyol;

   Polysorbate surfactants;

   And the pH is 5.5-6.5, preferably 5.5-6.0,

   in,

   The bispecific antibody targeting PD-L1 and CD47 includes two heavy chains and two light chains, wherein the first heavy chain variable region is the heavy chain variable region of an anti-CD47 monoclonal antibody, which can be combined with The light chain variable region forms a CD47 binding site, and the second heavy chain variable region is the heavy chain variable region of an anti-PD-L1 monoclonal antibody, which can form a PD-L1 binding site with the light chain variable region; where , the sequences of the light chain variable regions of the two light chains are the same, and the light chain variable region is obtained based on the light chain of the anti-CD47 monoclonal antibody and the light chain of the anti-PD-L1 monoclonal antibody, And the first variable region binding site formed by the light chain variable region and the first heavy chain variable region specifically binds to CD47, and the light chain variable region and the second heavy chain can The second variable region binding site formed by matching the variable regions specifically binds to PD-L1.
2. The composition according to claim 1, wherein the concentration of the bispecific antibody targeting PD-L1 and CD47 is 10-100 mg/ml, preferably 30-80 mg/ml.
3. The composition according to claim 1, wherein the acidic buffer is selected from at least one of citric acid buffer, acetate buffer and histidine buffer, preferably, histidine buffer. .
4. The composition according to claim 1, characterized in that the concentration of the acidic buffer is 5-25mM, preferably 10-20mM.
5. The composition according to claim 1, wherein the sugar is sucrose or trehalose, preferably sucrose, and the polyol is mannitol or sorbitol.
6. The composition according to claim 1, characterized in that the concentration of the carbohydrate stabilizer is 3%-9% (w/v), preferably 5% (w/v).
7. The composition according to claim 1, wherein the surfactant is polysorbate-20, polysorbate-80, polysorbate-60 or polysorbate-40, preferably polysorbate. Ester-20 or polysorbate-80.
8. The composition according to claim 1, characterized in that the concentration of the surfactant is 0.02%-0.06% (w/v), preferably 0.02%-0.04% (w/v).
9. The composition according to claim 1, wherein the first heavy chain variable region includes:

   Heavy chain CDR1 includes X ₁ YX ₂ MX ₃ , where X ₁ is selected from N and S, X ₂ is selected from V and A, and X ₃ is selected from H and S;

   Heavy chain CDR2, including YINPX ₄ NX ₅ X ₆ IKYNEKFX ₇ G, _where X ₄ is selected from Y and G, X ₅ is selected from D and E, X ₆ is selected from G and A, and

   Heavy chain CDR3 includes EGDFYANYGRLGFX ₈ Y, where X ₈ is selected from A and D.
10. The composition according to claim 9, wherein the heavy chain CDR1 has the sequence shown in SEQ ID NO: 31, 32, 33 or 45;

    The heavy chain CDR2 has the sequence shown in SEQ ID NO: 34, 35, 36, 37, 38 or 39;

    The heavy chain CDR3 has the sequence shown in SEQ ID NO: 40 or 41.
11. The composition according to claim 9, wherein the first heavy chain includes SEQ ID NO: 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 Or the amino acid sequence shown in 19.
12. The composition according to claim 9, wherein the second heavy chain variable region includes the CDR1 shown in SEQ ID NO:42, the CDR2 shown in SEQ ID NO:43 and the CDR2 shown in SEQ ID NO:44. CDR3 shown.
13. The composition according to claim 9, characterized in that the light chain has the sequence shown in SEQ ID NO: 1, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 .
14. The composition according to claim 9, wherein the Fc segment of the first heavy chain includes the H315R mutation, the Fc segment of the second heavy chain includes the K319E mutation, and Kabat is used for the amino acid mutation site of the Fc segment. Eu numbering system.
15. The composition according to claim 1, characterized in that it includes:

    The bispecific antibody targeting PD-L1 and CD47 at 10-80 mg/ml;

    5-25mM histidine buffer;

    3%-9% (w/v) the sucrose;

    0.02%-0.06% (w/v) of the polysorbate 20,

    And the pH is 5.5-6.0.
16. The composition according to claim 1, characterized in that it includes;

    30 mg/ml of the bispecific antibody targeting PD-L1 and CD47;

    10mM histidine;

    5% sucrose;

    0.03% polysorbate 20;

    And the pH is 5.5-6.0,

    Optionally, the composition includes:

    80 mg/ml of the bispecific antibody targeting PD-L1 and CD47;

    20mM histidine;

    5% sucrose;

    0.03% polysorbate 20;

    And the pH is 5.8±0.3.
17. A solid antibody composition, characterized in that the solid antibody composition is obtained by solidifying the liquid antibody composition according to any one of claims 1 to 16.
18. The solid antibody composition according to claim 17, wherein the solidification is freeze-drying.
19. A delivery device, characterized by comprising the liquid antibody composition according to any one of claims 1-16 or the solid antibody composition according to claim 17.
20. Use of the liquid antibody composition according to any one of claims 1 to 16 or the solid antibody composition according to claim 17 in the preparation of anti-tumor drugs.
21. The use according to claim 20, wherein the tumors include melanoma, lung cancer, renal cancer, Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, acute and chronic myeloid leukemia, and acute lymphoblastic leukemia. , non-Hodgkin lymphoma, bladder cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, kidney cancer and multiple myeloma.
22. A method of treating a subject suffering from a tumor, comprising administering to the subject a therapeutically effective amount of a composition, the composition being the liquid antibody composition of any one of claims 1-16 Or the solid antibody composition according to claim 17.
23. The method of claim 22, wherein the tumors include melanoma, lung cancer, renal cancer, Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, acute and chronic myeloid leukemia, and acute lymphoblastic leukemia. , non-Hodgkin lymphoma, bladder cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, kidney cancer and multiple myeloma.