Stable anti-CLEVER-1 antibody formulation

Abstract

The present invention relates to stable formulations comprising an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a buffer, and a stabilizer. The present invention further relates to stable formulations of an anti-CLEVER-1 antibody or antigen-binding fragment thereof for use in treating various diseases and disorders.

Description

[Technical Field]

[0001] The present invention relates to stable formulations of anti-CLEVER-1 antibodies or antigen-binding fragments thereof. The present invention further relates to stable formulations of anti-CLEVER-1 antibodies or antigen-binding fragments thereof for use in the treatment of various diseases and disorders. [Background technology]

[0002] Human CLEVER-1 (common lymphatic and vascular endothelial receptor-1) is disclosed in Patent Document 1. Clever-1 is also known as Stabilin-1 or Feel-1. The biology of CLEVER-1 has also been reviewed by Non-Patent Document 1. Clever-1 is expressed in lymphatic endothelial cells, certain vascular endothelial cells, but also in alternatively activated immunosuppressive macrophages, such as tumor-associated macrophages. It has also been previously shown in Patent Document 2 that blocking CLEVER-1 with a specific antibody reduces malignant tumor size and / or growth. It has also been previously shown, for example, in Patent Document 3 that blocking CLEVER-1 shifts alternatively activated macrophages from an immunosuppressive (M2) phenotype to a pro-inflammatory (M1) phenotype.

[0003] Patent Document 1 also discloses that CLEVER-1 mediates the binding of other types of leukocytes, such as monocytes and granulocytes, to HEV-like blood vessels. Therefore, by blocking the interaction between CLEVER-1 and malignant tumor cells, it is possible to control metastasis by preventing malignant cells that bind to CLEVER-1 from being taken up by lymphatic vessels, thereby preventing the spread of malignant tumors to lymph nodes.

[0004] Anti-CLEVER-1 antibodies can inhibit CLEVER-1 expression or bind to CLEVER-1 to block the function of CLEVER-1 or to block the interaction of CLEVER-1 with cells involved in disease pathogenesis.

[0005] Antibody drugs for human use may differ somewhat in the amino acid sequences of their constant domains or in their framework sequences within the variable domains, but they typically differ most dramatically in the complementarity-determining region (CDR) sequences. These differences result in different stabilities in solution due to different responsiveness to excipients in the solution or to the pH of the solution. In addition, changes in the amino acid configuration or changes in one or several amino acid residues can result in different antibody stability and susceptibility to sequence-specific degradation pathways. Furthermore, antibodies for use as pharmaceuticals in human subjects require storage before use, and therefore also require stable formulations suitable for storage without affecting antibody functionality. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 03 / 057130 [Patent Document 2] International Publication No. 2010 / 122217 [Patent Document 3] International Publication No. 2017 / 182705 [Non-patent literature]

[0007] [Non-Patent Document 1] Kzhyshkowska J. (2010), TheScientificWorld-JOURNAL 10, 2039-2053, “Multifunctional receptor Stabilin-1 in homeostasis and disease” Summary of the Invention

[0008] An object of the present invention is to provide a stable formulation of an anti-CLEVER-1 antibody or antigen-binding fragment thereof for pharmaceutical use, wherein the formulation of the anti-CLEVER-1 antibody or antigen-binding fragment thereof is stable during storage and has a shelf life long enough for clinical use.

[0009] Furthermore, it is an object of the present invention to provide a pharmaceutical formulation comprising an anti-CLEVER-1 antibody or an antigen-binding fragment thereof, which has good colloidal and thermodynamic stability of the protein, low aggregation during handling and storage, and low protein denaturation.

[0010] Furthermore, it is an object of the present invention to provide a chemically stable formulation for maintaining the functionality of the complementarity determining region (CDR) sequences of the anti-CLEVER-1 antibody and the efficacy of the anti-CLEVER-1 antibody during storage.

[0011] In order to achieve, inter alia, the above mentioned objects, the present invention is characterized by what is presented in the attached independent claims. Certain preferred embodiments of the invention are set out in the other claims.

[0012] The embodiments and advantages mentioned herein relate, where applicable, to both pharmaceutical formulations and uses according to the invention, although not always specifically mentioned.

[0013] An exemplary stable pharmaceutical formulation according to the present invention comprises: 1 to 100 mg / ml of an anti-CLEVER-1 antibody or an antigen-binding fragment thereof; a buffer of 5-50 mM histidine in combination with 150-400 mM trehalose, proline, or mannitol as a stabilizer, such that the pH of the pharmaceutical formulation is in the range of 5.5-6.5, or 5-50 mM Tris buffer in combination with 100-200 mM sodium chloride as a stabilizer, wherein the pH of the pharmaceutical formulation is in the range of 7.0-7.6; and - containing 0.01 to 0.1% (w / v) of polysorbate as a non-ionic surfactant, The anti-CLEVER-1 antibody or antigen-binding fragment thereof has the following sequence in the complementarity-determining region (CDR) of the heavy chain: CDR1: TSGMGIG (SEQ ID NO: 1), CDR2: HIWWDDDKRYNPALKS (SEQ ID NO: 2), and CDR3: HYGYDPYYAMDY (SEQ ID NO: 3), and The following sequence of the light chain complementarity determining region (CDR): CDR1: TASSSVSSSYLH (SEQ ID NO: 4), CDR2: RTSNLAS (SEQ ID NO: 5), and CDR3: HQYHRSPPT (SEQ ID NO: 6).

[0014] A stable pharmaceutical formulation according to one embodiment of the present invention is a liquid formulation comprising an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a stabilizer, and a histidine buffer at a pH of 5.5 to 6.5. Alternatively, the formulation according to the present invention may be in a lyophilized form prepared by lyophilizing a liquid composition comprising an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a stabilizer, and a histidine buffer at a pH of 5.5 to 6.5. A formulation according to another embodiment of the present invention is a liquid formulation comprising an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a stabilizer, and a Tris buffer at a pH of 7.0 to 7.6. Furthermore, a formulation according to one embodiment of the present invention may be in a lyophilized form prepared by lyophilizing a liquid composition comprising an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a stabilizer, and a Tris buffer at a pH of 7.0 to 7.6. The present invention also relates to aqueous compositions obtained by reconstituting the lyophilized formulation.

[0015] The pharmaceutical preparation according to the invention is suitable for use as an infusion or injection, and is particularly suitable for intravenous administration.

[0016] It has been observed that chemical denaturation appears to be the most important degradation pathway for anti-CLEVER-1 antibodies. Therefore, according to the present invention, chemical degradation, particularly of anti-CLEVER-1 antibodies, is avoided by providing a storage-stable composition. Maintaining antibody stability is also important for maintaining antibody functionality and efficacy, which is now achieved by the formulations according to the present invention. Furthermore, pharmaceutical formulations according to the present invention provide colloidal and thermodynamic stability.

[0017] According to the present invention, anti-CLEVER-1 antibodies or antigen-binding fragments thereof include those capable of specifically binding to human CLEVER-1 or inhibiting CLEVER-1 expression. Anti-CLEVER-1 antibodies or antigen-binding fragments thereof can be used to increase, enhance, stimulate, or upregulate immune responses. The present invention also relates to formulations comprising anti-CLEVER-1 antibodies or antigen-binding fragments thereof for use as pharmaceuticals. Pharmaceutical formulations according to the present invention can be used to remove immunosuppression. Pharmaceutical formulations according to the present invention are suitable for use in the treatment and / or prevention of cancer. Furthermore, pharmaceutical formulations according to the present invention are suitable for use in the treatment of chronic infections and / or acute inflammatory infections that lead to immune exhaustion. Pharmaceutical formulations according to the present invention can also be used as adjuvants for vaccines. Furthermore, pharmaceutical formulations according to the present invention are suitable for use in the treatment of hypercholesterolemia, dyslipidemia, and / or atherosclerotic cardiovascular disease.

[0018] Typically, a method of treatment according to the present invention comprises administering to a patient an effective amount of a pharmaceutical formulation according to the present invention, hi certain embodiments, the effective amount comprises a dose of an anti-CLEVER-1 antibody or antigen-binding fragment thereof in the range of 0.1 to 50 mg / kg, preferably 0.1 to 10 mg / kg. [Brief explanation of the drawings]

[0019] [Figure 1] 1 shows the amino acid sequences of the light and heavy chains of the anti-CLEVER-1 antibody bexmarilimab according to a preferred embodiment of the present invention. [Figure 2]The results of the concentration challenge test at 5°C ± 3°C and 35°C for one week before and after storage, i.e., the antibody concentration, are shown. [Figure 3] The results of the concentration challenge test at 5°C ± 3°C and 35°C for one week before and after storage, i.e., antibody turbidity, are shown. [Figure 4] The results of concentration challenge tests at 5°C ± 3°C and 35°C for one week before and after storage are shown, i.e., antibody aggregate formation. [Figure 5]

[0023] Figure 1 shows the results of a forced degradation study in which formulation variants were exposed to light stress, heat stress with agitation, and freeze / thaw stress. Antibody concentrations were analyzed before the study and after exposure under stress conditions. [Figure 6]

[0023] Figure 1 shows the results of a forced degradation study in which formulation variants were exposed to light stress, heat stress by agitation, and freeze / thaw stress. Antibody turbidity was analyzed before the study and after exposure under stress conditions. [Figure 7]

[0023] Figure 1 shows the results of a forced degradation study in which formulation variants were exposed to light stress, heat stress by agitation, and freeze / thaw stress. The aggregate-forming properties of the antibody were analyzed before the study and after exposure under stress conditions. [Figure 8]

[0023] Figure 1 shows the results of a forced degradation study in which formulation variants were exposed to light stress, heat stress by agitation, and freeze / thaw stress. The charge heterogeneity of the antibody was analyzed before the study and after exposure under stress conditions. [Figure 9]

[0023] Figure 1 shows the results of forced degradation studies in which formulation variants were exposed to light stress, heat stress with agitation, and freeze / thaw stress. Oxidative degradation products were analyzed before the study and after exposure under stress conditions. [Figure 10] Results of accelerated stability studies at 5±3° C., 25° C., and 35° C. are shown, i.e., antibody concentration was analyzed before, during, and after 12 weeks of storage. [Figure 11] Results of accelerated stability studies at 5±3° C., 25° C., and 35° C. are shown, i.e., antibody turbidity was analyzed before, during, and after 12 weeks of storage. [Figure 12]Results of accelerated stability studies at 5±3° C. are presented, i.e., aggregate and fragment content (SE-HPLC) was analyzed before, during, and after 12 weeks of storage. [Figure 13] Results of accelerated stability studies at 25° C. are presented, i.e., aggregate and fragment content (SE-HPLC) was analyzed before, during, and after 12 weeks of storage. [Figure 14] Results of accelerated stability studies at 35° C. are presented, i.e., aggregate and fragment content (SE-HPLC) was analyzed before, during, and after 12 weeks of storage. [Figure 15] Results of accelerated stability studies at 5±3° C., 25° C., and 35° C. are presented, i.e., charge non-uniformity was analyzed before, during, and after 12 weeks of storage. [Figure 16] Results of accelerated stability studies at 5±3° C., 25° C., and 35° C. are presented, i.e., oxidative degradation products were analyzed before, during, and after 12 weeks of storage. [Figure 17] The results of stability tests during and after storage at 5°C ± 3°C for 18 months are shown. [Figure 18] The results of stability tests during and after storage at 5°C ± 3°C for 18 months are shown. [Figure 19] 1 shows baseline and follow-up computed tomography scans of metastatic lesions in patients with best responses in a Phase I / II clinical trial. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure provides stable formulations of biologically active anti-CLEVER-1 antibodies or antigen-binding fragments thereof. The term "biologically active" refers to antibodies or antibody fragments capable of binding to CLEVER-1 and directly or indirectly exerting a biological effect. Pharmaceutical formulations according to the present invention comprise at least a pharmacologically effective amount of an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a buffer, and a stabilizer. Pharmaceutical formulations according to the present invention comprise a histidine buffer or a Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol) buffer. According to a preferred embodiment of the present invention, the pharmaceutical formulation further comprises a surfactant and / or antioxidant to further improve the stability of the formulation.

[0021] As used herein, the term "anti-CLEVER-1 antibody or antigen-binding fragment thereof" refers to any form of antibody or antigen-binding fragment thereof that exhibits the desired biological activity. Anti-CLEVER-1 antibody or antigen-binding fragment thereof refers to antibodies and their fragments, peptides, etc. that can inhibit the expression of CLEVER-1, block the function of CLEVER-1, or bind to CLEVER-1 to block the interaction of CLEVER-1 with cells involved in disease pathogenesis. CLEVER-1 has previously been disclosed in detail in publication WO 03 / 057130. The term "anti-CLEVER-1 antibody or antigen-binding fragment thereof" is used in the broadest sense and should be understood to include monoclonal antibodies, chimeric antibodies, humanized antibodies, or primatized antibodies, as well as antibody fragments and single-chain antibodies (e.g., Fab, Fv), so long as they exhibit the desired biological activity.

[0022] The formulations of the present disclosure include biologically active anti-CLEVER-1 antibodies or antigen-binding fragments thereof. According to one embodiment of the present invention, the anti-CLEVER-1 antibodies or antibody fragments capable of binding to CLEVER-1 are capable of binding to a specific CLEVER-1 epitope and are capable of directly or indirectly exerting a biological effect. According to the present invention, the anti-CLEVER-1 antibodies or antigen-binding fragments thereof have the following sequences in the complementarity-determining regions (CDRs) of the heavy chain: CDR1: TSGMGIG (SEQ ID NO: 1), CDR2: HIWWDDDKRYNPALKS (SEQ ID NO: 2), and CDR3: HYGYDPYYAMDY (SEQ ID NO: 3), and The following sequence of the light chain complementarity determining region (CDR): CDR1: TASSSVSSSYLH (SEQ ID NO: 4), CDR2: RTSNLAS (SEQ ID NO: 5), and CDR3: HQYHRSPPT (SEQ ID NO: 6).

[0023] In one embodiment according to the present invention, the anti-CLEVER-1 antibody is a humanized monoclonal anti-CLEVER-1 antibody. In one embodiment according to the present invention, the anti-CLEVER-1 antibody may be a humanized antibody based on monoclonal antibody 3-372 disclosed in Patent Publication WO03 / 057130. According to one embodiment of the present invention, the anti-CLEVER-1 antibody is a humanized monoclonal anti-CLEVER-1 antibody previously presented in Patent Publication WO2017 / 182705. In one embodiment according to the present invention, the humanized anti-CLEVER-1 antibody comprises human IgG4 heavy chain and kappa light chain constant regions. In one embodiment of the present invention, the humanized anti-CLEVER-1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 10. In one embodiment according to the present invention, the humanized anti-CLEVER-1 antibody comprises constant regions derived from a human immunoglobulin G4 (IgG4) heavy chain and kappa light chain, a heavy chain variable region comprising SEQ ID NO: 9, and a light chain variable region comprising SEQ ID NO: 10. In one embodiment of the invention, the constant regions derived from the human IgG4 heavy chain and κ light chain may contain mutations, where one or more conservative amino acid substitutions are present. According to one embodiment of the invention, the anti-CLEVER-1 antibody or antigen-binding fragment thereof comprises the heavy chain amino acid sequence SEQ ID NO: 7 and the light chain amino acid sequence SEQ ID NO: 8. The amino acid sequences of the heavy chain (SEQ ID NO: 7) and light chain (SEQ ID NO: 8) of the anti-CLEVER-1 antibody according to a preferred embodiment of the invention are also shown in Figure 1.

[0024] In one embodiment of the present invention, the anti-CLEVER-1 antibody is the humanized monoclonal immunoglobulin G4κ antibody bexmarilimab (under the International Nonproprietary Name (INN) disclosed in WHO Drug Information, Vol. 33, No. 4, pp. 814-815 (2019) as the proposed INN and in WHO Drug Information, Vol. 34, No. 3 (2020), pp. 699-700 as the recommended INN), or a bexmarilimab variant, or a bexmarilimab biosimilar. The anti-CLEVER-1 antibody bexmarilimab is an exemplary antibody used in the stable formulations described herein. As used herein, "bexmarilimab" refers to a humanized IgG4 monoclonal antibody having the structure described in WHO Drug Information, Vol. 33, No. 4, pp. 814-815 (2019) and WHO Drug Information, Vol. 34, No. 3 (2020). The humanized IgG4 monoclonal anti-CLEVER-1 antibody bexmalilimab comprises the heavy chain amino acid sequence SEQ ID NO: 7 and the light chain amino acid sequence SEQ ID NO: 8. The anti-CLEVER-1 antibody bexmalilimab comprises the above-described light chain and heavy chain CDRs (SEQ ID NOs: 1 to 6). The sequences shown in SEQ ID NOs: 7 and 8 also include the amino acid sequences corresponding to the heavy and light chain variable regions of the anti-CLEVER-1 antibody bexmalilimab, i.e., SEQ ID NOs: 9 and 10.

[0025] A bexmalilimab biosimilar refers to a biological product approved by a regulatory authority in any country for marketing as a bexmalilimab biosimilar. In one embodiment, a bexmalilimab biosimilar contains a bexmalilimab variant as the drug substance. In one embodiment, a bexmalilimab biosimilar has heavy and light chains with substantially the same amino acid sequences as bexmalilimab. As used herein, a "bexmalilimab variant" refers to an antibody comprising the same heavy and light chain sequences as bexmalilimab (SEQ ID NO: 7 and SEQ ID NO: 8, respectively), except that the variant positions are located within the framework or constant regions, e.g., with one or more conservative amino acid substitutions at positions outside the light chain CDRs and / or one or more conservative amino acid substitutions at positions outside the heavy chain CDRs. In other words, bexmalilimab and a bexmalilimab variant contain the same CDR sequences but differ from each other due to conservative amino acid substitutions at other positions in the full-length light and heavy chain sequences. The bexmarilimab variants are substantially identical to bexmarilimab in terms of binding affinity to CLEVER-1.

[0026] According to one embodiment of the present invention, a cell line producing the anti-CLEVER-1 antibody bexmarilimab (FP-1305) has been deposited under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, on May 27, 2020, and has accession number DSM ACC3361. The deposited embodiment is intended as a single illustration of one aspect of the invention, and the invention should not be limited in scope by the deposited culture, as any culture that is functionally equivalent is within the scope of the invention. The deposit of material herein does not constitute an admission that the written description contained herein is insufficient to enable the practice of any aspect of the invention, including its best mode, nor should it be construed as limiting the scope of the claims to the particular exemplifications it represents.

[0027] According to the present invention, a stable formulation may contain 1 to 100 mg / mL of an anti-CLEVER-1 antibody or antigen-binding fragment thereof. According to embodiments of the present invention, the concentration of the anti-CLEVER-1 antibody or antigen-binding fragment thereof in the stable pharmaceutical formulation may range from 1 to 100 mg / mL or 5 to 100 mg / mL. A pharmaceutical formulation according to one embodiment of the present invention may be a concentrate, which means that it is diluted to a desired concentration before administration. In some embodiments according to the present invention, the pharmaceutical formulation may contain 10 to 100 mg / mL or 20 to 100 mg / mL, preferably 20 to 40 mg / mL, and more preferably 20 to 30 mg / mL of an anti-CLEVER-1 antibody or antigen-binding fragment thereof. In some embodiments according to the present invention, the pharmaceutical formulation according to the present invention may be a concentrate comprising 1 to 100 mg / ml, preferably 10 to 100 mg / ml, more preferably 20 to 40 mg / ml or 20 to 30 mg / ml or about 25 mg / ml, for example 22.5 to 27.5 mg / ml of an anti-CLEVER-1 antibody or antigen-binding fragment thereof.

[0028] The stable pharmaceutical formulation according to the present invention comprises a histidine buffer or a Tris buffer. The term "buffer" encompasses those agents that maintain the solution pH within an acceptable range, i.e., provide sufficient buffering capacity. pH is typically measured at 25°C using a standard glass bulb pH meter. As used herein, a pharmaceutical formulation comprising a "buffer of pH X" refers to a pharmaceutical formulation solution that contains a buffer of pH X, i.e., pH is intended to refer to the pH of the solution.

[0029] A stable formulation according to one embodiment of the present invention comprises a histidine buffer. The pH of the formulation according to the present invention containing a histidine buffer is adjusted to be in the range of 5.5 to 6.5. In one embodiment of the present invention, the pharmaceutical formulation contains a histidine buffer and has a pH of 5.5 to 6.2, preferably 5.8 to 6.2, and more preferably about 6.0. It has been observed that anti-CLEVER-1 antibodies or antigen-binding fragments thereof have the highest colloidal and thermodynamic stability in a weakly acidic histidine buffer system. This pH range is also acceptable for intravenous infusion or bolus injection. According to one embodiment of the present invention, the histidine buffer contains L-histidine. Furthermore, a pharmaceutical formulation according to one embodiment of the present invention may contain a sufficient amount of hydrochloric acid and / or sodium hydroxide together with a histidine buffer, such as an L-histidine buffer, to adjust the pH to a range of about 5.5 to about 6.5 or about 5.5 to about 6.2, preferably about 5.8 to about 6.2, more preferably about 6.0. L-histidine / HCl as a buffer covers a neutral to slightly acidic pH range. In one embodiment according to the present invention, the buffer contains histidine, preferably L-histidine, and HCl (hydrochloric acid), and the pH of the pharmaceutical formulation according to the present invention is 5.5 to 6.5, preferably 5.5 to 6.2 or 5.8 to 6.2.

[0030] According to one embodiment of the present invention, the formulation comprises a 5-50 mM histidine buffer, preferably a 5-20 mM or 5-15 mM histidine buffer. According to one embodiment of the present invention, the formulation comprises about 10 mM histidine buffer, for example, a 5-15 mM, 8-12 mM, 9-11 mM, or 9.5-10.5 mM histidine buffer. According to one embodiment of the present invention, the formulation comprises a sodium chloride-free histidine buffer.

[0031] According to another embodiment of the present invention, the pharmaceutical formulation comprises a Tris (tris(hydroxymethyl)aminomethane) buffer, such as Tris base or Tris hydrochloride, and has a pH of 7.0 to 7.6, or 7.2 to 7.6, or 7.3 to 7.5. In one embodiment according to the present invention, the pharmaceutical formulation comprises a Tris buffer and has a pH of about 7.4. In one embodiment of the present invention, the formulation comprises a 5 to 50 mM Tris buffer, preferably a 5 to 20 mM or 5 to 15 mM Tris buffer. In one embodiment of the present invention, the formulation comprises about 10 mM Tris buffer, for example, a 5 to 15 mM Tris buffer, or an 8 to 12 mM Tris buffer, or an 9 to 11 mM Tris buffer, or an 9.5 to 10.5 mM Tris buffer. Furthermore, the pharmaceutical formulation according to one embodiment of the present invention may comprise a sufficient amount of hydrochloric acid together with the Tris buffer to adjust the pH to a range of about 7.1 to about 7.6, or 7.2 to 7.6, or 7.3 to 7.5, or to about 7.4.

[0032] The pharmaceutical formulation according to the present invention further comprises a stabilizer. The stabilizer is used to further increase colloidal stability and thermodynamic stability. In one embodiment according to the present invention, the formulation comprises trehalose, proline, or mannitol, preferably trehalose or proline, as a stabilizer in combination with a histidine buffer. In a preferred embodiment of the present invention, the stabilizer comprises trehalose, such as trehalose dihydrate, in combination with a histidine buffer. In one embodiment of the present invention, the formulation comprises 150 to 400 mM, preferably 200 to 360 mM, of trehalose, proline, or mannitol as a stabilizer. In one embodiment of the present invention, the stabilizer comprises 150 to 400 mM, preferably 200 to 360 mM, of trehalose, proline, or mannitol. According to one embodiment of the present invention, the stabilizer comprises 220-340 mM or 240-320 mM trehalose, proline, or mannitol, preferably trehalose or proline; more preferably, the stabilizer comprises 260-300 mM, 270-290 mM, or about 280 mM trehalose, proline, or mannitol, preferably trehalose or proline. In a preferred embodiment according to the present invention, the pharmaceutical formulation comprises trehalose, proline, or mannitol as a stabilizer, preferably trehalose or proline as a stabilizer, more preferably trehalose, such as trehalose dihydrate, in combination with a histidine buffer. In one embodiment of the present invention, the pharmaceutical formulation comprises 150-400 mM, preferably 200-360 mM, trehalose, proline, or mannitol as a stabilizer in combination with a histidine buffer. According to one embodiment of the present invention, the pharmaceutical formulation comprises 220-340 mM or 240-320 mM trehalose, proline, or mannitol as a stabilizer, preferably trehalose or proline as a stabilizer, more preferably the formulation comprises 260-300 mM, 270-290 mM, or about 280 mM trehalose, proline, or mannitol as a stabilizer, preferably trehalose or proline.In one embodiment of the present invention, the approximately 280 mM stabilizer can be approximately 275-285 mM trehalose, proline, or mannitol, preferably trehalose or proline.

[0033] In one embodiment according to the present invention, when the formulation comprises a Tris buffer, the stabilizer comprises sodium chloride. In one embodiment of the present invention, the formulation comprises a Tris buffer and sodium chloride as a stabilizer. In one embodiment of the present invention, the formulation comprises a Tris buffer in combination with 100-200 mM, preferably 130-180 mM or 140-160 mM, of sodium chloride as a stabilizer. According to one embodiment of the present invention, the formulation comprises 100-200 mM, preferably 130-180 mM or 140-160 mM, of sodium chloride as a stabilizer in combination with a Tris buffer. According to one embodiment of the present invention, the formulation comprises about 150 mM sodium chloride as a stabilizer in combination with a Tris buffer. In one embodiment of the present invention, the about 150 mM stabilizer can be about 145-155 mM sodium chloride as a stabilizer in combination with a Tris buffer.

[0034] A pharmaceutical formulation according to one embodiment of the present invention may further comprise a surfactant. Preferably, the surfactant comprises polysorbate, more preferably, the surfactant comprises polysorbate 20. In one embodiment of the present invention, the formulation comprises polysorbate, preferably polysorbate 20, as a non-ionic surfactant. In one embodiment of the present invention, the formulation comprises 0.01-0.1% (w / v), preferably 0.01-0.05% (w / v), of a non-ionic surfactant. In one embodiment of the present invention, the formulation comprises 0.01-0.1% (w / v), preferably 0.01-0.05% (w / v), of a polysorbate, preferably polysorbate 20. According to one embodiment of the present invention, the formulation comprises 0.01-0.03% (w / v) of a polysorbate, preferably polysorbate 20. In one embodiment of the present invention, the formulation contains about 0.02% (w / v), e.g., 0.015-0.025% (w / v), of a polysorbate, preferably polysorbate 20. The addition of a non-ionic surfactant such as polysorbate 20 provides increased stability, which can be observed as lower turbidity during storage. Additionally, non-ionic surfactants such as polysorbate 20 are used as surfactants to facilitate the formulation process and further stabilize molecules in liquid formulations.

[0035] A pharmaceutical formulation according to one embodiment of the present invention may further comprise an antioxidant, preferably L-methionine, to further improve the stability of the formulation. The addition of methionine has shown slightly improved stabilization. In one embodiment of the present invention, the formulation comprises 5 to 40 mM, preferably 15 to 25 mM, or 18 to 22 mM, of an antioxidant. In one embodiment of the present invention, the formulation comprises 5 to 40 mM, preferably 15 to 25 mM, or 18 to 22 mM, of L-methionine as an antioxidant. In one embodiment of the present invention, the formulation comprises approximately 20 mM of an antioxidant, preferably L-methionine. In one embodiment of the formulation, approximately 20 mM of an antioxidant, preferably L-methionine, can be 19 to 21 mM or 19.5 to 20.5 mM of an antioxidant, preferably L-methionine.

[0036] According to one embodiment of the present invention, a stable pharmaceutical formulation comprises 1-100 mg / ml of an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a histidine or Tris buffer, a stabilizer, a surfactant, and an antioxidant. In one embodiment according to the invention, a stable pharmaceutical formulation comprises 1-100 mg / ml of an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a histidine buffer as a stabilizer, trehalose or proline, a surfactant, and an antioxidant. In another embodiment according to the invention, a stable pharmaceutical formulation comprises 1-100 mg / ml of an anti-CLEVER-1 antibody or antigen-binding fragment thereof, a Tris buffer, sodium chloride as a stabilizer, a surfactant, and an antioxidant.

[0037] According to one embodiment of the present invention, the formulation comprises: (i) 1 to 100 mg / ml of an anti-CLEVER-1 antibody or an antigen-binding fragment thereof; (ii) 5 to 50 mM histidine buffer; (iii) 150 to 400 mM trehalose, proline, or mannitol as a stabilizer; (iv) 0.01 to 0.1% (w / v) polysorbate 20 as a nonionic surfactant, and (v) containing 5 to 40 mM L-methionine as an antioxidant; The pH of the composition is 5.5 to 6.5, preferably 5.8 to 6.2, The anti-CLEVER-1 antibody or antigen-binding fragment thereof has the following sequence in the complementarity-determining region (CDR) of the heavy chain: CDR1: TSGMGIG (SEQ ID NO: 1), CDR2: HIWWDDDKRYNPALKS (SEQ ID NO: 2), and CDR3: HYGYDPYYAMDY (SEQ ID NO: 3), and The following sequence of the light chain complementarity determining region (CDR): CDR1: TASSSVSSSYLH (SEQ ID NO: 4), CDR2: RTSNLAS (SEQ ID NO: 5), and CDR3: HQYHRSPPT (SEQ ID NO: 6), Preferably, the anti-CLEVER-1 antibody is an anti-CLEVER-1 antibody comprising a heavy chain SEQ ID NO:7 and a light chain SEQ ID NO:8.

[0038] According to one embodiment of the present invention, the formulation comprises: (i) 20 to 40 mg / ml or 20 to 30 mg / ml of an anti-CLEVER-1 antibody or an antigen-binding fragment thereof; (ii) 5 to 20 mM or 5 to 15 mM histidine buffer; (iii) 200 to 360 mM, preferably 240 to 320 mM or 260 to 290 mM, of trehalose, proline, or mannitol as a stabilizer; (iv) 0.01 to 0.1% (w / v), preferably 0.01 to 0.05% (w / v) of polysorbate 20 as a nonionic surfactant, and (v) containing 5 to 40 mM L-methionine as an antioxidant; The pH of the composition is 5.5 to 6.5, preferably 5.8 to 6.2, The anti-CLEVER-1 antibody or antigen-binding fragment thereof has the following sequence in the complementarity-determining region (CDR) of the heavy chain: CDR1: TSGMGIG (SEQ ID NO: 1), CDR2: HIWWDDDKRYNPALKS (SEQ ID NO: 2), and CDR3: HYGYDPYYAMDY (SEQ ID NO: 3), and The following sequence of the light chain complementarity determining region (CDR): CDR1: TASSSVSSSYLH (SEQ ID NO: 4), CDR2: RTSNLAS (SEQ ID NO: 5), and CDR3: HQYHRSPPT (SEQ ID NO: 6), Preferably, the anti-CLEVER-1 antibody is an anti-CLEVER-1 antibody comprising a heavy chain SEQ ID NO:7 and a light chain SEQ ID NO:8.

[0039] According to one embodiment of the present invention, the formulation comprises: (i) 1 to 100 mg / ml of an anti-CLEVER-1 antibody or an antigen-binding fragment thereof; (ii) a 5 to 50 mM Tris buffer, preferably a 5 to 20 mM or 5 to 15 mM Tris buffer; (iii) 100 to 200 mM, preferably 130 to 180 mM, sodium chloride as a stabilizer; (iv) 0.01 to 0.1% (w / v), preferably 0.01 to 0.05% (w / v) of polysorbate 20 as a nonionic surfactant, and (v) containing 5 to 40 mM L-methionine as an antioxidant; The pH of the composition is 7.0 to 7.6, The anti-CLEVER-1 antibody or antigen-binding fragment thereof has the following sequence in the complementarity-determining region (CDR) of the heavy chain: CDR1: TSGMGIG (SEQ ID NO: 1), CDR2: HIWWDDDKRYNPALKS (SEQ ID NO: 2), and CDR3: HYGYDPYYAMDY (SEQ ID NO: 3), and The following sequence of the light chain complementarity determining region (CDR): CDR1: TASSSVSSSYLH (SEQ ID NO: 4), CDR2: RTSNLAS (SEQ ID NO: 5), and CDR3: HQYHRSPPT (SEQ ID NO: 6), Preferably, the anti-CLEVER-1 antibody is an anti-CLEVER-1 antibody comprising a heavy chain SEQ ID NO:7 and a light chain SEQ ID NO:8.

[0040] In one preferred embodiment, the formulation according to the invention comprises: (i) 1 to 100 mg / ml of an anti-CLEVER-1 antibody or an antigen-binding fragment thereof; (ii) 10 mM L-histidine-HCl buffer; (iii) 280 mM trehalose or proline as a stabilizer, preferably 280 mM trehalose or trehalose dihydrate; (iv) 0.02% (w / v) polysorbate 20 as a nonionic surfactant, and (v) 20 mM L-methionine as an antioxidant; The pH of the composition is 5.8 to 6.2; The anti-CLEVER-1 antibody or antigen-binding fragment thereof has the following sequence in the complementarity-determining region (CDR) of the heavy chain: CDR1: TSGMGIG (SEQ ID NO: 1), CDR2: HIWWDDDKRYNPALKS (SEQ ID NO: 2), and CDR3: HYGYDPYYAMDY (SEQ ID NO: 3), and The following sequence of the light chain complementarity determining region (CDR): CDR1: TASSSVSSSYLH (SEQ ID NO: 4), CDR2: RTSNLAS (SEQ ID NO: 5), and CDR3: HQYHRSPPT (SEQ ID NO: 6), Preferably, the anti-CLEVER-1 antibody is an anti-CLEVER-1 antibody comprising a heavy chain SEQ ID NO:7 and a light chain SEQ ID NO:8.

[0041] More preferably, in one embodiment according to the present invention, the pharmaceutical formulation comprises: (i) 20 to 40 mg / ml, 20 to 30 mg / ml, or 25 mg / ml ± 2.5 mg / ml of an anti-CLEVER-1 antibody or antigen-binding fragment thereof; (ii) 10 mM L-histidine-HCl buffer; (iii) 280 mM trehalose or proline as a stabilizer, preferably 280 mM trehalose or trehalose dihydrate; (iv) 0.02% (w / v) polysorbate 20 as a nonionic surfactant, and (v) 20 mM L-methionine as an antioxidant; The pH of the composition is 5.8 to 6.2; The anti-CLEVER-1 antibody or antigen-binding fragment thereof has the following sequence in the complementarity-determining region (CDR) of the heavy chain: CDR1: TSGMGIG (SEQ ID NO: 1), CDR2: HIWWDDDKRYNPALKS (SEQ ID NO: 2), and CDR3: HYGYDPYYAMDY (SEQ ID NO: 3), and The following sequence of the light chain complementarity determining region (CDR): CDR1: TASSSVSSSYLH (SEQ ID NO: 4), CDR2: RTSNLAS (SEQ ID NO: 5), and CDR3: HQYHRSPPT (SEQ ID NO: 6), Preferably, the anti-CLEVER-1 antibody is an anti-CLEVER-1 antibody comprising a heavy chain SEQ ID NO:7 and a light chain SEQ ID NO:8.

[0042] The formulation according to the invention is preferably a liquid formulation.

[0043] According to an exemplary embodiment of the present invention, a liquid antibody formulation can be made by taking an anti-CLEVER-1 antibody in liquid form and buffer-exchanging it into the desired formulation. In this embodiment, there is no lyophilization step. The drug substance in the final buffer is concentrated to the desired concentration. Excipients such as trehalose dihydrate and polysorbate 20 are added to the solution and diluted with an appropriate buffer to the final protein concentration. The final formulated drug substance is filtered using a 0.22 μm filter and filled into the final container (e.g., glass vial).

[0044] According to one embodiment of the present invention, the formulation may also be a lyophilized formulation, i.e., a lyophilizate. The term "lyophilizate" refers to the product of lyophilization. The term "lyophilizing" with respect to the pharmaceutical formulations of the present invention is intended to refer to the freeze-drying of a solution of the formulation. Lyophilized formulations of therapeutic proteins may offer advantages such as better chemical stability. Lyophilized formulations may also be reconstituted at different concentrations depending on clinical factors such as the route of administration or dosage. The term "reconstitution" refers to the dissolution of the lyophilizate to reach an aqueous solution.

[0045] Pharmaceutical formulations according to the present invention have been observed to stabilize anti-CLEVER-1 antibodies or antigen-binding fragments thereof against degradation during storage. According to embodiments of the present invention, formulations comprising anti-CLEVER-1 antibodies or antigen-binding fragments thereof are storage-stable at 2-8°C. Even at concentrations of 100 mg / ml of anti-CLEVER-1 antibodies or antigen-binding fragments thereof, good stability can be achieved at high concentrations. A "stable" pharmaceutical formulation of the present invention is one comprising an anti-CLEVER-1 antibody or antigen-binding fragment thereof that does not show significant changes at refrigerated temperatures of 2-8°C for at least 3 months. Formulations according to the present invention have been observed to be storage-stable for at least 18 or 24 months at temperatures of 2-8°C. Pharmaceutical formulations are considered to be storage-stable at temperatures of 2-8°C for at least 30 months, and even for 36 months.

[0046] An antibody "retains its physical stability" in a pharmaceutical formulation if it does not show a significant increase in aggregation and / or denaturation, and / or a significant increase in color and / or clarity, as measured by SE-HPLC. An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. An antibody "retains its biological activity" in a pharmaceutical formulation if the antibody's biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation was prepared. The biological activity of an antibody can be determined, for example, by an antigen binding assay. Typical acceptance criteria for stability are as follows: Typically, no more than about 10%, preferably no more than about 5%, of the antibody monomer aggregates as measured by SE-HPLC. The pharmaceutical antibody formulation is colorless or clear to slightly opaque by visual analysis. The concentration varies by no more than + / - 10%. Typically, no more than about 10%, preferably no more than about 5%, of degradation or variants is observed. Titers are typically within 50-200% of the reference.

[0047] The pharmaceutical formulation of the present invention may be administered to a patient. The formulation according to one embodiment of the present invention is targeted for intravenous or intratumoral administration. A liquid formulation can be diluted before administration. A lyophilized formulation can be reconstituted at different concentrations before administration. According to a preferred embodiment of the present invention, the pharmaceutical formulation is administered intravenously. It may be administered via intravenous infusion or bolus injection. The term "intravenous" or "IV" administration refers to administration into a blood vessel.

[0048] The formulation according to the present invention comprising an anti-CLEVER-1 antibody or an antibody fragment thereof can be used as a pharmaceutical.

[0049] The preparation according to the present invention comprising an anti-CLEVER-1 antibody or an antibody fragment thereof can be used to remove tumor- or antigen-induced immunosuppression. The pharmaceutical preparation according to the present invention is suitable for use in the treatment or prevention of cancer. In one embodiment according to the present invention, the pharmaceutical preparation can shrink tumors, inhibit tumor growth, or prevent metastasis. The pharmaceutical preparation is applicable to all forms of cancer. Any benign or malignant tumor or metastasis of malignant tumors can be treated. Leukemia, lymphoma, and multiple myeloma can also be treated.

[0050] Formulations according to the present invention comprising anti-CLEVER-1 antibodies or antibody fragments thereof can also be used to treat or prevent chronic infections or acute inflammatory infections that result in immune exhaustion, and modulation of macrophage phenotype is achieved by the anti-CLEVER-1 antibodies.

[0051] The pharmaceutical formulations according to the invention can also be used as adjuvants for vaccines: anti-CLEVER-1 antibodies achieve repolarization of macrophages, thus eliminating or at least reducing immune suppression against vaccine antigens.

[0052] Furthermore, it has been observed that antibodies capable of binding to CLEVER-1 have the ability to inhibit and / or block the uptake of modified low-density lipoproteins, particularly acetylated low-density lipoprotein (acLDL), by CLEVER-1 and the generation of foam cells, i.e., precursors of atherosclerotic plaques, and therefore the pharmaceutical formulations according to the present invention are suitable for use in the treatment of hypercholesterolemia, dyslipidemia, and / or atherosclerotic cardiovascular disease.

[0053] The term "treatment" or "treating" should be understood to include complete cure of a disease, as well as amelioration or relief of the disease. The term "prophylaxis" should be understood to include complete prevention, prophylaxis, and reducing an individual's risk of developing the disease or disorder.

[0054] The dose of the anti-CLEVER-1 antibody or antigen-binding fragment thereof selected should be pharmacologically effective, and thus sufficient to produce the desired therapeutic result, e.g., reducing malignant tumor growth and / or inhibiting metastatic spread and / or blocking negative regulation of T cells in cancer, chronic infections, infectious diseases, or other immune-exhausted states. According to one embodiment of the present invention, the anti-CLEVER-1 antibody is administered in the range of 0.1 to 50 mg / kg, preferably 0.1 to 30 mg / kg or 0.1 to 10 mg / kg, depending on the patient's body weight. In one embodiment according to the present invention, the anti-CLEVER-1 antibody is administered in the range of 0.3 to 10 mg / kg, preferably 0.3 to 3 mg / kg, depending on the patient's body weight. In one embodiment according to the present invention, the method for treating cancer, chronic infection, infectious disease, or other immune-compromised conditions comprises the administration of a pharmaceutical formulation according to the present invention in an amount of preferably 0.1 to 50 mg / kg, preferably 0.1 to 30 mg / kg or 0.1 to 10 mg / kg, depending on the patient's weight.

[0055] The formulations of the invention comprising anti-CLEVER-1 antibodies or antibody fragments thereof can be used alone or in combination with other drugs or pharmaceutical products. In one embodiment according to the invention, the pharmaceutical formulations comprising anti-CLEVER-1 antibodies or antigen-binding fragments thereof are used to treat cancer alone or in combination with other immunotherapeutic agents.

[0056] Experimental part In this experimental part, the stability of pharmaceutical formulations according to the present invention containing anti-CLEVER-1 antibodies is tested and confirmed.

[0057] drug substance FP-1305 is a humanized monoclonal immunoglobulin G4κ anti-CLEVER-1 antibody produced in CHO cells. More specifically, FP-1305 is the humanized monoclonal anti-CLEVER-1 antibody bexmarilimab (International Nonproprietary Name (INN) disclosed in WHO Drug Information, Vol. 33, No. 4, pp. 814-815 (2019) as a proposed INN and in WHO Drug Information, Vol. 34, No. 3 (2020), pp. 699-700 as a recommended INN). The amino acid sequences of the heavy and light chains of FP-1305 comprise SEQ ID NO: 7 and SEQ ID NO: 8 (also presented in Figure 1). FP-1305 comprises the CDR sequences of SEQ ID NO: 1 to SEQ ID NO: 6. The cell line for producing FP-1305 has been deposited at DSMZ - German Collection of Microorganisms and Cell Cultures GmbH under accession number DSM ACC3361.

[0058] Analysis method Analytical methods for evaluating the stability of formulations include dynamic laser light scattering (DLS), nano-differential scanning calorimetry (nanoDSC), composition gradient multi-angle light scattering (CG-MALS), size exclusion chromatography (SE-HPLC), reduced RP-HPLC, and capillary zone electrophoresis (CZE). Standard methods for the above analyses are used.

[0059] DLS is used to determine protein-protein interactions. At higher concentrations of macromolecules, interactions between neighboring particles result in non-ideal diffusion behavior (intermolecular stability is affected). This is due to the second hydrodynamic virial coefficient, k D It can be explained by k D The value represents the tendency for nonspecific molecular association under a given set of solution conditions. onset ), i.e., the onset of the unfolding transition (denaturation onset temperature), can also be determined by DLS.

[0060] NanoDSC measures the denaturation temperature (T) of proteins and other macromolecules in solution with the versatility and precision to perform molecular stability screening. onset ) and thermal denaturation enthalpy.

[0061] In CG-MALS, interactions between protein molecules in solution were characterized by changes in light scattering behavior at different concentrations. This series of light scattering measurements allowed the calculation of the second virial coefficient, A2, a characteristic parameter for measuring molecular interactions. A negative A2 indicates attractive interactions between molecules of dissolved substances, while a positive A2 is characteristic of repulsive interactions between dissolved protein molecules.

[0062] SE-HPLC (size exclusion chromatography) is a standard method for detecting aggregated and fragmented protein species.

[0063] Capillary zone electrophoresis (CZE) is a method for analyzing the charge heterogeneity of proteins in their native state. CZE was performed according to standard methods.

[0064] Reduced RP-HPLC is the standard method for the detection of oxidized antibody species.

[0065] The concentration was determined by UV280 absorbance measurement.

[0066] The absorbance of the undiluted samples was measured at wavelengths of 350 nm and 510 nm to determine the increase in turbidity. The turbidity of the samples was also determined using a turbidimeter according to the European Pharmacopoeia. For visual inspection, the samples were visually inspected under backlight.

[0067] Evaluate buffer and pH conditions of anti-CLEVER-1 formulations The formulations presented in Table 1 were selected to study the general behavior of the drug substance FP-1305 with respect to pH, buffer composition, and ionic strength in terms of the best colloidal and thermodynamic stability of the protein. Samples were prepared by dialyzing FP-1305 (10-15 mg / mL) into the buffer system in question.

[0068] Protein-protein interactions as a measure of colloidal stability in buffer systems were determined by measuring the hydrodynamic radius of FP-1305 at increasing concentrations of FP-1305. The data were used to predict protein aggregation during handling and storage.

[0069] The denaturation temperature (thermodynamic stability) in a buffer system was determined by measuring the hydrodynamic radius of FP-1305 as the temperature increased. As the protein domains began to unfold, the hydrodynamic radius of the protein increased significantly. The onset temperature of unfolding could be used as an indicator of the secondary structure stability of the protein. The data were used to predict protein denaturation during handling and storage.

[0070] The pH of the formulation was adjusted to 5.5–7.4 (an acceptable range for intravenous or bolus injection). The L-histidine buffer selected covers a neutral to slightly acidic pH range. Tris buffer was tested as an alternative buffer component to cover the physiological pH range. The pH was adjusted with sufficient amounts of 6N hydrochloric acid and, if necessary, 10N sodium hydroxide.

[0071] The ionic strength of the formulation with Tris buffer (maximum ionic strength set at isotonicity) was adjusted by adding sodium chloride.

[0072] [Table 1]

[0073] From the results presented in Table 1, it was observed that the colloidal stability of FP-1305 was higher at acidic pH values ​​compared to neutral pH values. However, high ionic strength significantly reduced repulsive interactions at low pH and should be avoided. Clear repulsive protein interactions were observed for sodium chloride-free histidine / HCl buffer at pH 5.5 and 6.0. At pH 7.4, the addition of sodium chloride reduced attractive interactions, thus having a beneficial effect on colloidal stability.

[0074] Thermodynamic stability is somewhat opposite to colloidal stability. The addition of sodium chloride is beneficial to thermodynamic stability in Tris buffer systems. In the case of L-histidine / HCl, the addition of sodium chloride reduces the onset temperature of unfolding. Therefore, formulations with histidine buffers preferably do not contain sodium chloride (NaCl).

[0075] Based on the findings of these studies, the formulation variants presented in Table 2 were selected and tested using CG-MALS, nano-DSC, DLS, and SE-HPLC with the addition of the stabilizers trehalose, mannitol, and L-proline at concentrations leading to isotonicity. Variant number 6 was evaluated as an alternative variant at physiological pH. The results are presented in Table 3.

[0076] [Table 2]

[0077] [Table 3]

[0078] The addition of stabilizers to the L-histidine / HCl buffer system did not significantly affect both colloidal and thermodynamic stability. Intermolecular interactions were near zero (slightly repulsive to slightly attractive) in all cases.

[0079] Concentration provocation test A wide pH range (pH shows the strongest effect on colloidal and thermodynamic stability) was used without the addition of additional stabilizers in concentration-challenged studies. The tested variants are presented in detail in Table 1.

[0080] The concentration of FP-1305 drug substance was increased to 16 mg / mL using a cross-flow device. The FP-1305 solution was centrifuged at 4000 rpm for 5 minutes to remove any insoluble particles. The solution was then filtered using a 0.22 μm membrane filter. Samples for accelerated stability testing were prepared by dialyzing concentrated FP-1305 drug substance (DS) into selected buffer variants in three dialysis steps to achieve quantitative buffer exchange. 50 mL of DS containing approximately 16 mg / mL was transferred to conditioned dialysis tubing (in dialysis buffer). The loaded dialysis tubing was incubated in 1000 mL of target buffer for 2 hours before the first buffer exchange (1000 mL). After an additional 2 hours of dialysis, a second buffer exchange (1000 mL) was performed, and dialysis was completed overnight.

[0081] The solution was then concentrated to approximately 100 mg / mL by ultrafiltration. After the concentration step, the solution was sterile filtered using a 0.22 μm syringe filter under laminar flow. 0.5 mL of the concentrated sample was filled into sterile standard 2R glass vials. The samples were stored at 5 ± 3°C (2-8°C) and 35°C for 7 days. The samples were analyzed by UV at 280 nm, absorbance at 350 nm and 510 nm, and SE-HPLC was performed before and after storage. The samples were also analyzed visually.

[0082] The results of concentration measurements using UV-280 nm are shown in Figure 2. FP-1305 concentrations of over 100 mg / mL could be achieved in all formulation variants. Concentrations remained unchanged during short-term stability testing of highly concentrated samples.

[0083] The results of the adsorption measurements at 350 nm and 510 nm are shown in Figure 3. None of the formulations showed an increase in turbidity during the stability study, either at 5±3°C or at 35°C. Upon visual inspection, all formulations showed a clear, slightly red solution at the start of the stability study. After 7 days of storage at both 5±3°C and 35°C, none of the samples showed visible particles.

[0084] As shown in Figure 4, soluble aggregate formation was detected in all formulation variants at both 5 ± 3 °C and 35 °C by SE-HPLC as described in the analytical methods above. At 35 °C, aggregate evolution was faster than at 5 ± 3 °C. Compared to the Tris / HCl formulation, aggregate formation was lower in the L-histidine / HCl formulation. There was no significant effect of pH on aggregate formation in the L-histidine formulation, with a trend toward lower aggregate formation at more acidic pHs, i.e., pH 5.5 and pH 6.0.

[0085] Forced decomposition test Based on the stability studies described above, the formulations presented in Table 4 were selected for further testing in forced degradation studies to determine the formulation variant that exhibited the best stability against multiple stress conditions (light stress, heat stress with agitation, and freeze / thaw stress). Formulations were prepared with and without polysorbate 20 (PS20). To investigate the stabilizing effect of L-methionine against radical-induced chemical degradation, further light stress studies were conducted using variants 3-1 + PS20, 3-2 + PS20, and 3-3 + PS20, which contained 20 mM L-methionine in the formulation.

[0086] [Table 4]

[0087] The concentration of FP-1305 drug substance was increased to 25 mg / mL using a cross-flow device. The FP-1305 solution was centrifuged at 4000 rpm for 5 minutes to remove any insoluble particles. The solution was then filtered using a 0.22 μm membrane filter. Samples for accelerated stability testing were prepared by dialyzing the concentrated FP-1305 drug substance into the selected buffer variant in three dialysis steps to achieve quantitative buffer exchange. Dialysis of FP-1305 was completed in three dialysis steps to achieve quantitative buffer exchange. 25 mL of solution containing approximately 25 mg / mL FP-1305 was transferred to conditioned dialysis tubing (in dialysis buffer). The filled dialysis tubing was incubated in 1000 mL of target buffer for 2 hours before the first buffer exchange (1000 mL). After an additional 2 hours of dialysis, a second buffer exchange (1000 mL) was performed, and dialysis was completed overnight.

[0088] After the dialysis step, the solution was supplemented with polysorbate 20 or polysorbate 20 and L-methionine to achieve the desired formulation variant. The sample was then sterile filtered using a 0.22 μm syringe filter under laminar flow. 0.8 mL of sample was filled into a sterile standard 6R glass vial. The sample was exposed to the following stress conditions: 1) Forced heat stress with agitation: storage at 35°C under agitation at 200 rpm. 2) Light exposure: 750W / m 2 / 7.5 hours at 25℃. 3) Freeze-thaw stress: Liquid samples were frozen from room temperature to -50°C at a controlled freezing rate and warmed back to room temperature at a controlled heating rate (1°C / min to simulate bulk freezing conditions).

[0089] Two liquid samples of each variant were analyzed visually for precipitation before and after each time point from each stress condition. Samples were analyzed for concentration by UV at 280 nm, turbidity by absorbance at 350 nm and 510 nm, aggregate state by SE-HPLC, chemical degradation by CZE, and reduced RP-HPLC. The results are shown in Figures 5-9.

[0090] Figure 5 shows the results of concentration determination by UV-280nm. The concentration of the samples remained constant for all variants. A solution of L-methionine was also added to the samples (variants 3-1 + PS20 + methionine, 3-2 + PS20 + methionine, and 3-3 + PS20 + methionine) to dilute them. The target concentration of these samples was approximately 24 mg / mL. The concentration was measured by UV-280nm.

[0091] The results of the adsorption measurements at 350 nm and 510 nm are presented in Figure 6. Freeze / thaw stress did not show an increase in the turbidity of the samples. After light stress, the absorbance at 350 nm increased slightly. Some beneficial effects of polysorbate 20 were observed. Methionine had no beneficial effect on turbidity after light stress. After 2 weeks of heat stress, some formulations showed turbidity (variants 3-2 and 3-3).

[0092] After freeze / thaw stress and light stress, all formulation variants with polysorbate 20 yielded visually clear samples. Heat stress induced the formation of gel-like particles. After 2 weeks of heat stress, all variants with polysorbate 20 remained clear, while all variants without polysorbate 20 contained gel-like particles.

[0093] Possible aggregation was tested by SE-HPLC. Figure 7 shows the aggregate content of the formulation variants at the start of the stability study and after each stress test (bars correspond to the variants mentioned in Figure 7 from top to bottom, in order from left to right).

[0094] After freeze / thaw stress, increased aggregation was observed for variants 3-2 and 3-2+PS20 containing mannitol. A slight increase in aggregation was observed for variants 6 and 6+PS20 containing Tris buffer. No additional aggregates were formed in the other variants.

[0095] Light stress induced aggregation in all formulation variants, with the lowest aggregation in variant 3-3, followed by variants 3-1, 3-2, and 6. Some minor beneficial effects of polysorbate 20 and L-methionine were observed. Aggregates increased in all formulations under heat stress / agitation, with the strongest in variant 6. After 2 weeks at 35°C, the aggregate content increased in all formulation variants.

[0096] Capillary zone electrophoresis (CZE) was used to monitor the charge heterogeneity of the antibody, a measure of the chemical stability of the samples. Figure 8 shows the charge heterogeneity of the formulation variants at the start of the stability test and after each stress test (the bars correspond to the variants listed in Figure 8 from top to bottom, from left to right). After stress, a strongly deamidated subvariant and an acidic subvariant with other charge heterogeneities were formed. Freeze / thaw stress only slightly affected the charge heterogeneity of the samples. The highest degradation was observed after light stress. Variant No. 6 showed higher degradation compared to the other variants. Polysorbate 20 and methionine did not have a significant effect on the chemical stability of FP-1305. The formation of charge variants was observed for all formulation variants during heat stress. The highest stability was observed for all histidine / HCl buffer-containing variants. The lowest stability was observed for variant No. 6 (Tris / HCl buffer). Polysorbate 20 had no positive or negative effect on the stability of FP-1305 against chemical degradation.

[0097] Samples were also analyzed by reduced RP-HPLC to detect oxidized degradation products. Figure 9 shows the purity by reduced RP-HPLC of formulation variants at the start of the stability study and after each stress test (bars, ordered from left to right, correspond to the variants mentioned in Figure 9 from top to bottom). No degradation / oxidation was observed after freeze / thaw and heat stress with stirring at 35°C for up to 2 weeks. During light stress, sample purity decreased, with variant 3-1 having the highest purity, followed by variants 3-2, 3-3, and 6. Polysorbate 20 did not affect the purity of FP-1305 by reduced RP-HPLC. L-methionine had a slightly positive effect on the purity of FP-1305 after light stress.

[0098] In conclusion, a histidine / HCl buffer system at pH 6.0 was preferred over a Tris / HCl buffer system due to its higher chemical stability and lower tendency to aggregate. However, although the stability of FP-1305 was superior in the slightly acidic histidine buffer system, the Tris / HCl buffer system appears to offer an alternative to the histidine / HCl buffer system. The addition of polysorbate 20 proved to increase stability, particularly under freeze / thaw stress, as samples without polysorbate 20 exhibited turbidity after freeze / thaw testing. The stabilizers trehalose and proline were preferred over mannitol-containing histidine buffers due to their higher stability during freeze / thaw stress. The addition of methionine showed a slight stabilizing effect on FP-1305 against radical-induced degradation (formation of aggregates and impurities during light stress).

[0099] Accelerated Stability Testing The two formulations presented in Table 5 were placed into an accelerated stability study to test the stability of FP-1305 at 5°C ± 3°C, 25°C, and 35°C.

[0100] [Table 5]

[0101] The concentration of FP-1305 drug substance was increased to 25 mg / mL using a cross-flow device. Samples for accelerated stability testing were prepared by dialyzing concentrated FP-1305 drug substance into selected buffer variants in three dialysis steps to achieve quantitative buffer exchange similar to that disclosed above for the forced degradation test. After the dialysis steps, the solution was supplemented with polysorbate 20 stock solution and L-methionine solution to obtain the desired formulation variant. Samples were then sterile filtered using a 0.22 μm syringe filter under laminar flow. 1.4 mL of sample was filled into sterile standard 6R glass vials.

[0102] Samples were stored upside down at 5°C ± 3°C (2-8°C), 25°C ± 2°C, and 35°C ± 2°C for 12 weeks without humidity control. Two liquid samples were collected per time point (T0, T4 weeks, T8 weeks, T12 weeks) and temperature. All samples were analyzed separately at each time point for concentration by UV at 280 nm, turbidity by nephelometry, aggregate state by SE-HPLC, and chemical degradation by reduced RP-HPLC and CZE. The results are shown in Figures 10-16.

[0103] Figure 10 shows the results of concentration determination by UV-280 nm. The concentration of the samples remained constant during the stability test, regardless of storage temperature and formulation. Also, samples of all formulation variants remained clear during the stability test, regardless of storage temperature, as visually inspected.

[0104] Figure 11 shows the results of non-turbidimetric turbidity measurements. During the stability test, the turbidity of the samples remained constant at 5°C ± 3°C and 25°C. At 35°C, a slight increase in turbidity was observed for both formulation variants. Formulation variant 3-1 + PS20 was slightly less turbid than formulation variant 3-3 + PS20. An increase in turbidity is an indication of the formation of insoluble aggregates. However, the turbidity of the samples remained relatively low even after 12 weeks at 35°C. After 12 weeks, a slight decrease in turbidity was observed at 25°C and 35°C, which may be caused by a possible increase in particle size of the precipitated aggregates.

[0105] Figures 12-14 show the monomer, aggregate, and fragment content of the formulation variants during the stability study. SE-HPLC analysis revealed that the relative monomer content remained nearly constant at 5°C ± 3°C and 25°C. At 35°C, a slight decrease in the relative monomer peak area was observed for both formulation variants. The aggregate content remained constant at storage temperatures of 5°C ± 3°C. In contrast, the aggregate content decreased during the stability study at 25°C and 35°C. This was most likely caused by precipitation of aggregates formed at 25°C and 35°C, in line with an increase in sample turbidity. The fragment content increased at 25°C and 35°C but remained nearly constant in both formulations at 5°C ± 3°C.

[0106] Capillary zone electrophoresis (CZE) is used to monitor the charge heterogeneity of antibodies, a measure of the chemical stability of the samples. Figure 15 shows the charge heterogeneity of the formulation variants during the stability study. The charge heterogeneity of the samples remained fairly constant during the first four weeks of the stability study, regardless of storage temperature. After eight weeks, the main peak area decreased at all three storage temperatures for both formulation variants. Higher degradation was observed at higher temperatures. After 12 weeks, the main peak area for both formulations remained unchanged at storage temperatures of 5°C ± 3°C and 25°C.

[0107] Figure 16 shows the purity by reduced RP-HPLC of formulation variants 3-1+PS20 and 3-3+PS20 during stability testing. The dashed lines in the figure indicate the minimum and maximum purity of the FP-1305 standard measured during stability testing (at each time point, an FP-1305 standard was analyzed at the beginning and end of each sequence). Reduced RP-HPLC primarily detects oxidative degradation products of the antibody. RP-HPLC analysis showed a slight increase in degradation products (oxidized forms) for both formulation variants 3-1+PS20 and 3-3+PS20 during stability testing at 25°C and 35°C. Both formulation variants showed comparable chemical degradation. Chemical degradation was found to be the most important degradation pathway for FP-1305 drug substance.

[0108] Based on the results, both formulations 3-1+PS20 and 3-3+PS20 provide stable formulations of anti-CLEVER-1 antibodies. According to a preferred embodiment of the present invention, the pH of the formulation solution is adjusted to 6.0 and buffered with L-histidine. The pH can be adjusted with a sufficient quantity (qs) of 6N hydrochloric acid, and, if necessary, with 10N sodium hydroxide. L-methionine is added to improve stability and acts as an antioxidant. Trehalose dihydrate or proline is also added to the formulation to improve stability and act as a stabilizer. Nonionic polysorbate 20 is used as a surfactant to further stabilize molecules in the liquid formulation. Formulation 3-1+PS20 had slightly lower turbidity than Formulation 3-3+PS20. Therefore, according to a preferred embodiment of the present invention, the formulation includes trehalose, such as trehalose dihydrate, as a stabilizer. The formulation is stable at least at 2-8°C (5°C ± 3°C).

[0109] Stability test (18 months at 5°C ± 3°C) Formulation 3-1+PS20 (FP-1305 drug substance concentration was 25 mg / mL) has been stored in hydrolysis class I DIN 10R glass vials at 5°C ± 3°C for 18 months. Bromobutyl rubber stoppers with FluroTec coating were selected to minimize interactions between the drug product and the closure system and maintain the integrity of the container closure system.

[0110] After 18 months of storage at 5°C ± 3°C, no significant changes were observed in pH, osmolality, subvisible particles (HIAC), concentration (UV), identity (image capillary isoelectric focusing (icIEF)), or purity (SE-HPLC). Regarding solution appearance, the sample remains clear (equivalent to water), unlike previous time points (slightly opaque solutions). The results are shown in Figure 18.

[0111] Efficacy Testing Formulation 3-1+PS20 (the concentration of FP-1305 drug substance was 25 mg / mL) was stored at 5°C ± 3°C, and after 18 months of storage, a biological activity assay was performed to measure the potency of the anti-CLEVER-1 antibody. A cell-based assay was used to determine biological function. The antibody concentration required to achieve half-maximal efficacy was determined as EC 50 The potency of the test samples was assessed by comparing the curves of the test samples to the standard by EC50 ratio. Potency was expressed as a percentage of relative potency of the standard (FP-1305 was stored at -70°C). Formulations according to the present invention exhibit long-term biological activity (including up to at least about 18 months). The results of the biological activity assay are presented in Figure 17. The relative potency after the storage period was 92.3%.

[0112] Clinical trials The anti-CLEVER-1 antibody FP-1305 is currently being tested for safety and preliminary efficacy in a Phase I / II trial in patients with advanced solid tumors (clinicaltrials.gov NCT03733990: A Study to Evaluate Safety, Tolerability, and Preliminary Efficacy of FP-1305 in Cancer Patients (MATINS)). Accumulated safety information indicates that FP-1305 is well tolerated, a highly positive indicator of product performance, as traditional cytotoxic anticancer therapies are associated with dose-dependent and treatment-defined toxicities. The level of tolerability and unwanted side effects observed in humans is also consistent with preclinical observations of inhibiting CLEVER-1 function in rodents and monkeys. First-in-human data also suggest favorable clinical efficacy, with 8 of 30 treated patients with highly advanced metastatic tumors showing no progression in target lesions, and 3 patients showing clear tumor shrinkage at follow-up (Figure 19). Figure 19 shows baseline and follow-up computed tomography scans of metastatic lesions in patients with the best response. Arrows point to shrinking lung metastases in patients with microsatellite-stable (MSS) metastatic colorectal cancer (CRC), melanoma, and ovarian cancer. The size and time course of target lesions are shown on the right side of each figure.

[0113] In a Phase I / II study, it was also observed that patients treated with the anti-CLEVER-1 antibody FP-1305 experienced increased plasma LDL (P-LDL) levels, as shown in Table 6, indicating that LDL binding and uptake is inhibited or blocked by CLEVER-1 monocytes / macrophages. The anti-CLEVER-1 antibody FP-1305 prevents macrophage LDL cholesterol uptake and foam cell formation because acLDL is not digested. This is seen as an increase in LDL levels in cancer patients treated with FP-1305.

[0114] The first (pre-dose) fasting plasma sample was collected before starting FP-1305. The second (post-dose) fasting plasma sample was collected at the end of the first 3-week treatment cycle of the anti-CLEVER-1 antibody FP-1305. The results are shown in Table 6, and plasma LDL (P-LDL) levels are expressed as mmol / L.

[0115] [Table 6]

Claims

1. A stable pharmaceutical formulation comprising: -1 to 100 mg / ml of an anti-CLEVER-1 antibody or an antigen-binding fragment thereof; - 5-50 mM histidine buffer in combination with 150 mM-400 mM trehalose as a stabilizer, wherein the pH of the pharmaceutical formulation ranges from 5.5 to 6.5; and - containing 0.01-0.1% (w / v) of polysorbate as a non-ionic surfactant, A stable pharmaceutical formulation, wherein the anti-CLEVER-1 antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:7 for the heavy chain and the amino acid sequence of SEQ ID NO:8 for the light chain.

2. 2. The formulation of claim 1, wherein the anti-CLEVER-1 antibody is bexmarilimab.

3. The formulation of claim 1 or 2, wherein the anti-CLEVER-1 antibody is antibody FP-1305 (DSM ACC3361).

4. 4. The formulation of any one of claims 1 to 3, wherein the formulation comprises a histidine buffer and has a pH of 5.5 to 6.2, or 5.8 to 6.

2.

5. The formulation of any one of claims 1 to 4, wherein the histidine buffer comprises L-histidine.

6. 6. The formulation of any one of claims 1 to 5, wherein the formulation comprises 5 to 20 mM or 5 to 15 mM of the histidine buffer.

7. 7. The formulation of any one of claims 1 to 6, wherein the formulation comprises 200 to 360 mM, or 240 to 320 mM, or 270 to 290 mM trehalose as a stabilizing agent.

8. A formulation according to any one of claims 1 to 7, wherein the formulation comprises polysorbate 20 as a non-ionic surfactant.

9. 9. The formulation of any one of claims 1 to 8, wherein the formulation comprises 0.01 to 0.05% (w / v) polysorbate.

10. 10. The formulation of claim 9, wherein the polysorbate is polysorbate 20.

11. The formulation of any one of claims 1 to 10, wherein the formulation further comprises an antioxidant.

12. 12. The formulation of claim 11, wherein the antioxidant comprises L-methionine.

13. 13. The formulation of claim 11 or 12, wherein the formulation comprises 5 to 40 mM, or 15 to 25 mM, of L-methionine.

14. The formulation (i) 1 mg / ml to 100 mg / ml, or 20 to 40 mg / ml, or 20 to 30 mg / ml of an anti-CLEVER-1 antibody or antigen-binding fragment thereof; (ii) 5 to 50 mM, or 5 to 20 mM, or 5 to 15 mM histidine buffer; (iii) 150 to 400 mM, or 200 to 360 mM, or 240 to 320 mM, or 260 to 290 mM trehalose as the stabilizer; (iv) 0.01 to 0.1% (w / v), or 0.01 to 0.05% (w / v) of polysorbate as the nonionic surfactant, and (v) 5 to 40 mM, or 15 to 25 mM, of L-methionine as an antioxidant; 14. The formulation of any one of claims 1 to 13, wherein the pH of the formulation is 5.5 to 6.5, or 5.8 to 6.

2.

15. 15. The formulation of claim 14, wherein the polysorbate is polysorbate 20.

16. The formulation of any one of claims 1 to 15, wherein the formulation is a liquid formulation or in lyophilized form.

17. A formulation according to any one of claims 1 to 16 for use as a medicament.

18. 18. The formulation of any one of claims 1 to 17 for use in the treatment of cancer, chronic infections, acute inflammatory infections, hypercholesterolemia, dyslipidemia, or atherosclerotic cardiovascular disease.

19. 20. The formulation for use according to claim 18, wherein the formulation is administered intravenously.

20. 20. The formulation for use according to claim 18 or 19, wherein the formulation is administered at a dose of 0.1 mg / kg to 50 mg / kg, calculated as the antibody or antigen-binding fragment thereof.

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