Pharmaceutical preparation containing a CD80 extracellular domain-Fc fusion protein

Abstract

The present disclosure provides pharmaceutical compositions comprising CD80 extracellular domain (ECD)-fragment crystallizable (Fc) fusion molecules. The present disclosure also provides methods of treating solid tumors by administering such pharmaceutical compositions.

Description

Technical Field

[0001] 1. Field Provided are pharmaceutical compositions (formulations) comprising a fusion protein. The fusion protein comprises a CD80 (B7-1) extracellular domain (ECD) and an immunoglobulin fragment crystallizable (Fc) domain. Also provided is a method of using such a formulation.

Background Art

[0002] 2. Background The regulation of T cells is related to a combination of multiple signaling pathways: signaling through the T cell receptor (TCR) complex and signaling through co-signaling receptors for both co-stimulation and co-inhibition. CD80 (cluster of differentiation 80, also known as B7, B7.1, B7-1) is a well-characterized co-signaling ligand. It is expressed in professional antigen-presenting cells (APCs), such as dendritic cells and activated macrophages. When an antigen peptide is presented due to major histocompatibility complex (MHC) molecules, the APC presents the antigen peptide to T cells. Upon TCR recognition of the peptide-MHC complex, CD80 functions as a co-stimulatory ligand through its interaction with CD28, a differentiation antigen group 28 expressed on T cells, which is its receptor. In addition to signaling through CD28, CD80 also interacts with cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a co-inhibitory molecule. The interaction between CD80 and CTLA-4 plays a central role in suppressing T cell responses when activated T cell responses are no longer needed. According to some reports, CD80 interacts with PD-L1. However, whether this interaction occurs is not clear, and even if it occurs, its biological significance is not clear. At the same time, co-stimulatory and co-inhibitory signals ensure both tolerance to self-antigens and the ability to initiate an appropriate immune response to non-self antigens.

[0003] The immune system often initially mounts an effective immune response against tumor cells via TCR-dependent and -independent mechanisms, but tumors can evade this immune response. Mechanisms that bring about this effect include upregulation of pathways that enforce peripheral tolerance to self-antigens such as CTLA-4 and PD-L1. Recent immuno-oncology approaches have focused on reprogramming the immune system to mount an effective immune response against tumors that have evaded the initial immune response. These approaches include the use of "checkpoint inhibitors." For example, blocking antibodies against both the programmed cell death protein (PD-1) / PD-L1 axis and the CTLA-4 axis are effective in anti-tumor immunity, such as improving progression-free survival (PFS) and overall survival (OS) in some patients. In some patients with certain types of tumors, the use of blocking antibodies against the PD-1 / PD-L1 axis or the CTLA-4 axis has achieved long-term disease control, but in the majority of patients, there is no response or recurrence after the response.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The CD80 signaling axis may provide additional opportunities for cancer treatment, and for that purpose, CD80 extracellular domain (ECD)-Fc fusion proteins have been developed. Accordingly, there is a long-felt need for a pharmaceutical composition comprising a CD80 ECD-Fc fusion protein formulated for administration in such a treatment.

Means for Solving the Problems

[0005] 3. Overview This specification provides a pharmaceutical composition comprising a CD80 extracellular domain (ECD)-fragment crystallizable (Fc) fusion molecule. In certain embodiments, the pharmaceutical composition comprises (i) a CD80 ECD-Fc fusion molecule, (ii) histidine, and (iii) a sugar selected from the group consisting of sucrose and sorbitol, and the pH of the composition is from about 5 to about 7.5.

[0006] In certain embodiments, the composition contains less than 10% of the fusion molecules of high molecular weight species (HMWS) after 4 weeks at 40°C.

[0007] In certain embodiments, the composition contains about 1% to about 10% of the fusion molecules of HMWS after 4 weeks at 40°C.

[0008] In certain embodiments, the composition contains less than 2.5% of the CD80 ECD-Fc fusion molecules of low molecular weight species (LMWS) after 4 weeks at 40°C.

[0009] In certain embodiments, the composition contains about 0.5% to about 2.5% of the CD80 ECD-Fc fusion molecules of LMWS after 4 weeks at 40°C.

[0010] In certain embodiments, the composition contains about 1% to about 6% of the fusion molecules of HMWS after 4 weeks at 40°C.

[0011] In certain embodiments, the composition contains about 1% to about 4% of the fusion molecules of HMWS after 4 weeks at 40°C.

[0012] In certain embodiments, the composition contains about 0.5% to about 1.6% of the CD80 ECD-Fc fusion molecules of LMWS after 4 weeks at 40°C.

[0013] In certain embodiments, the composition contains about 0.5% to about 1.5% of the CD80 ECD-Fc fusion molecules of LMWS after 4 weeks at 40°C.

[0014] In certain embodiments, the pH of the composition is about 5 to about 7.5. In certain embodiments, the pH of the composition is about 5.5 to about 7.0. In certain embodiments, the pH of the composition is about 6.4 to about 7.0. In certain embodiments, the pH of the composition is about 6.7.

[0015] In certain embodiments, the composition comprises histidine, optionally, the histidine is L-histidine. In certain embodiments, the concentration of histidine (e.g., L-histidine) is from about 15 to about 25 mM. In certain embodiments, the concentration of histidine (e.g., L-histidine) is from about 18 mM to about 22 mM. In certain embodiments, the concentration of histidine (e.g., L-histidine) is about 20 mM.

[0016] In certain embodiments, the composition further comprises a sugar selected from the group consisting of sucrose and sorbitol. In certain embodiments, the sugar is sucrose. In certain embodiments, the sugar is sorbitol.

[0017] In certain embodiments, the concentration of the sugar is from about 225 mM to about 300 mM. In certain embodiments, the concentration of the sugar is from about 250 mM to about 290 mM. In certain embodiments, the concentration of the sugar is about 270 mM.

[0018] In certain embodiments, the concentration of the sugar is about 10 to about 15 times the concentration of histidine, optionally, the concentration of the sugar is about 13.5 times the concentration of the buffer.

[0019] In certain embodiments, the composition further comprises a surfactant.

[0020] In certain embodiments, the surfactant is polysorbate, optionally, the polysorbate is polysorbate 20. In certain embodiments, the concentration of the surfactant is from about 0.025% to about 0.075% weight / volume (w / v). In certain embodiments, the concentration of the surfactant is from about 0.035% to about 0.065% weight / volume (w / v). In certain embodiments, the concentration of the surfactant is about 0.05% weight / volume (w / v).

[0021] In certain embodiments, the concentration of the CD80 ECD-Fc fusion molecule is from about 5 mg / ml to about 15 mg / ml. In certain embodiments, the concentration of the CD80 ECD-Fc fusion molecule is about 10 mg / ml.

[0022] In certain embodiments, the composition is a liquid.

[0023] In certain embodiments, the composition is for intravenous administration.

[0024] In certain embodiments, the composition comprises about 20 mM histidine, 270 mM sucrose, about 10 mg / ml of a CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and has a pH of about 6.7.

[0025] In certain embodiments, the composition comprises a sugar concentration that is about 13.5 times the concentration of histidine, about 10 mg / ml of a CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and has a pH of about 6.7.

[0026] In certain embodiments, the composition comprises about 20 mM histidine (e.g., L-histidine), about 270 mM sorbitol, about 10 mg / ml of a CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and has a pH of about 6.7.

[0027] In certain embodiments, the composition comprises a sorbitol concentration that is about 13.5 times the concentration of histidine, about 10 mg / ml of a CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and has a pH of about 6.7.

[0028] In certain embodiments, the CD80 ECD-Fc fusion molecule comprises a human CD80 ECD and a human IgG1 Fc domain.

[0029] In certain embodiments, the composition comprises a sialylated CD80 ECD-Fc fusion molecule.

[0030] In certain embodiments, the CD80 ECD-Fc fusion molecule comprises at least 15 moles of sialic acid (SA) per mole of the fusion protein.

[0031] In certain embodiments, the CD80 ECD-Fc fusion molecule comprises 15 to 60 moles of SA per mole of the fusion protein. In certain embodiments, the CD80 ECD-Fc fusion molecule comprises 15 to 40 moles of SA per mole of the fusion protein. In certain embodiments, the CD80 ECD-Fc fusion molecule comprises 15 to 30 moles of SA per mole of the fusion protein. In certain embodiments, the CD80 ECD-Fc fusion molecule comprises 20 to 30 moles of SA per mole of the fusion protein.

[0032] In certain embodiments, the CD80 ECD-Fc fusion molecule comprises a human CD80 ECD comprising the amino acid sequence of SEQ ID NO: 1.

[0033] In certain embodiments, the CD80 ECD-Fc fusion molecule comprises a human IgG1 Fc domain comprising the amino acid sequence of SEQ ID NO: 4.

[0034] In certain embodiments, the CD80 ECD-Fc fusion molecule comprises an Fc domain of human IgG1 attached to the carboxy terminus of the ECD of human CD80.

[0035] In certain embodiments, the CD80 ECD-Fc fusion molecule comprises the amino acid sequence of SEQ ID NO: 5.

[0036] In certain embodiments, the composition alone elicits less release of interferon gamma or TNF alpha from T cells in vitro than TGN1412 alone.

[0037] In certain embodiments, the composition alone induces interferon gamma or TNF alpha release at a strength that is at least 1000-fold less than that of TGN1412 alone.

[0038] In certain embodiments, after a single administration of the composition at 0.3 - 0.6 mg / kg, the composition enables at least 90% inhibition of tumor growth in at least one murine syngeneic cancer model over a period of at least 1 week, 10 days, 2 weeks, or 3 weeks.

[0039] In certain embodiments, the murine syngeneic cancer model is a CT26 tumor model.

[0040] In certain embodiments, the pharmaceutical composition consists of (i) a sialylated CD80 ECD-Fc fusion molecule, (ii) about 20 mM of L-histidine, (iii) about 270 mM of sucrose, and (iv) about 0.05% weight / volume of polysorbate 20, and the pH of the composition is about 6.7.

[0041] In certain embodiments, the pharmaceutical composition consists of (i) a sialylated CD80 ECD-Fc fusion molecule, (ii) about 20 mM of L-histidine, (iii) about 270 mM of sorbitol, and (iv) about 0.05% weight / volume of polysorbate 20, and the pH of the composition is about 6.7.

[0042] In certain embodiments, provided herein is a syringe or vial containing the pharmaceutical composition.

[0043] In certain embodiments, provided herein is a method of treating a solid tumor in a subject. In certain embodiments, the method comprises administering to the subject the pharmaceutical composition provided herein. In certain embodiments, the subject is a human.

[0044] In certain embodiments, the solid tumor is an advanced solid tumor.

[0045] In certain embodiments, the solid tumor is not a primary central nervous system tumor.

[0046] In certain embodiments, the solid tumor is colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, or endometrial cancer.

[0047] In certain embodiments, the solid tumor is renal cell carcinoma.

[0048] In certain embodiments, the solid tumor is melanoma.

[0049] In certain embodiments, the patient has not previously received treatment with a PD-1 / PD-L1 antagonist.

[0050] In certain embodiments, the patient has received prior treatment with at least one PD-1 / PD-L1 antagonist selected from a PD-L1 antagonist and a PD-1 antagonist. In certain embodiments, the at least one PD-1 / PD-L1 antagonist is nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab.

[0051] In certain embodiments, the at least one PD-1 / PD-L1 antagonist is administered in an advanced or metastatic state.

[0052] In certain embodiments, the patient has received prior treatment with at least one angiogenesis inhibitor. In certain embodiments, the angiogenesis inhibitor is sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab.

[0053] In certain embodiments, the angiogenesis inhibitor is administered in an advanced or metastatic state.

[0054] In certain embodiments, the patient has a BRAF mutation.

[0055] In certain embodiments, the patient has previously received treatment with at least one BRAF inhibitor.

[0056] In certain embodiments, the BRAF inhibitor is vemurafenib or dabrafenib.

[0057] In certain embodiments, the BRAF inhibitor is administered in an advanced or metastatic state.

[0058] In certain embodiments, the solid tumor recurs or progresses after treatment selected from surgery, chemotherapy, radiotherapy, and combinations thereof.

[0059] In certain embodiments, the pharmaceutical composition is administered intravenously. In certain embodiments, for example, the following items are provided. (Item 1) A pharmaceutical composition comprising (i) a CD80 extracellular domain (ECD)-fragment crystallizable (Fc) fusion molecule, (ii) histidine, and (iii) a sugar selected from the group consisting of sucrose and sorbitol, wherein the pH of the composition is from about 5 to about 7.5, said pharmaceutical composition. (Item 2) A pharmaceutical composition comprising a CD80 ECD-Fc fusion molecule, wherein the composition contains less than 10% of a high molecular weight species (HMWS) of the fusion molecule after 4 weeks at 40°C, said pharmaceutical composition. (Item 3) A pharmaceutical composition comprising a CD80 ECD-Fc fusion molecule, wherein the composition contains from about 1% to about 10% of a HMWS of the fusion molecule after 4 weeks at 40°C, said pharmaceutical composition. (Item 4) A pharmaceutical composition comprising a CD80 ECD-Fc fusion molecule, wherein the composition contains less than 2.5% of a low molecular weight species (LMWS) of the CD80 ECD-Fc fusion molecule after 4 weeks at 40°C, said pharmaceutical composition. (Item 5) A pharmaceutical composition comprising a CD80 ECD-Fc fusion molecule, wherein the composition contains from about 0.5% to about 2.5% of a LMWS of the CD80 ECD-Fc fusion molecule after 4 weeks at 40°C, said pharmaceutical composition. (Item 6) The composition according to any one of Items 1 to 5, wherein the composition contains from about 1% to about 6% of a HMWS of the fusion molecule after 4 weeks at 40°C. (Item 7) The composition according to Item 6, wherein the composition contains from about 1% to about 4% of a HMWS of the fusion molecule after 4 weeks at 40°C. (Item 8) The composition according to any one of Items 1 to 7, wherein the composition contains from about 0.5% to about 1.6% of a LMWS of the CD80 ECD-Fc fusion molecule after 4 weeks at 40°C. (Item 9) The composition according to Item 8, wherein the composition contains from about 0.5% to about 1.5% of a LMWS of the CD80 ECD-Fc fusion molecule after 4 weeks at 40°C. (Item 10) The composition according to any one of Items 2 to 9, wherein the pH of the composition is from about 5 to about 7.5. (Item 11) The composition according to any one of Items 2 to 10, wherein the composition optionally contains histidine, and optionally, the histidine is L-histidine. (Item 12) The composition according to any one of Items 2 to 11, wherein the composition further contains a sugar selected from the group consisting of sucrose and sorbitol. (Item 13) The composition according to any one of items 1, 11, and 13, wherein the concentration of the histidine is from about 15 mM to about 25 mM. (Item 14) The composition according to item 13, wherein the concentration of the histidine is from about 18 mM to about 22 mM. (Item 15) The composition according to item 14, wherein the concentration of the histidine is about 20 mM. (Item 16) The composition according to any one of items 1 and 12 - 15, wherein the concentration of the sugar is from about 225 mM to about 300 mM. (Item 17) The composition according to item 16, wherein the concentration of the sugar is from about 250 mM to about 290 mM. (Item 18) The composition according to item 17, wherein the concentration of the sugar is about 270 mM. (Item 19) The composition according to any one of items 1 and 12 - 18, optionally, wherein the concentration of the sugar is from about 10 to about 15 times the concentration of the histidine, and optionally, wherein the concentration of the sugar is about 13.5 times the concentration of the buffer. (Item 20) The composition according to any one of items 1 - 19, further comprising a surfactant. (Item 21) The composition according to item 20, wherein the surfactant is polysorbate, and optionally, wherein the polysorbate is polysorbate 20. (Item 22) The composition according to item 20 or 21, wherein the concentration of the surfactant is from about 0.025% to about 0.075% weight / volume (w / v). (Item 23) The composition according to item 22, wherein the concentration of the surfactant is from about 0.035% to about 0.065% weight / volume (w / v). (Item 24) The composition according to item 23, wherein the concentration of the surfactant is about 0.05% weight / volume (w / v). (Item 25) The composition according to any one of items 1 - 24, wherein the concentration of the CD80 ECD - Fc fusion molecule is from about 5 mg / ml to about 15 mg / ml. (Item 26) The composition according to item 25, wherein the concentration of the CD80 ECD - Fc fusion molecule is about 10 mg / ml. (Item 27) The composition according to any one of items 1 - 26, wherein the pH of the composition is from about 5.5 to about 7.0. (Item 28) The composition according to any one of items 1 - 27, wherein the pH of the composition is from about 6.4 to about 7.0. (Item 29) The composition according to any one of items 1 - 28, wherein the pH of the composition is about 6.7. (Item 30) The composition according to any one of items 1 - 29, wherein the composition is a liquid. (Item 31) The composition according to any one of items 1 - 30, wherein the composition is for intravenous administration. (Item 32) The composition according to any one of Items 1 to 31, wherein the sugar is sucrose. (Item 33) The composition according to any one of Items 1 to 32, comprising about 20 mM histidine, about 270 mM sucrose, about 10 mg / ml CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and having a pH of about 6.7. (Item 34) The composition according to any one of Items 1 to 32, comprising a sugar concentration that is about 13.5 times the concentration of histidine, about 10 mg / ml CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and having a pH of about 6.7. (Item 35) The composition according to any one of Items 1 to 31, wherein the sugar is sorbitol. (Item 36) The composition according to any one of Items 1 to 31 or 35, comprising about 20 mM histidine, about 270 mM sorbitol, about 10 mg / ml CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and having a pH of about 6.7. (Item 37) The composition according to any one of Items 1 to 31 or 35, comprising a sorbitol concentration that is about 13.5 times the concentration of histidine, about 10 mg / ml CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and having a pH of about 6.7. (Item 38) The composition according to any one of Items 1 to 37, wherein the CD80 ECD-Fc fusion molecule comprises a human CD80 ECD and a human IgG1 Fc domain. (Item 39) The composition according to any one of Items 1 to 38, wherein the composition comprises a sialylated CD80 ECD-Fc fusion molecule. (Item 40) The composition according to Item 39, wherein the CD80 ECD-Fc fusion molecule comprises at least 15 moles of sialic acid (SA) per mole of the fusion protein. (Item 41) The composition according to Item 39, wherein the CD80 ECD-Fc fusion molecule comprises 15 to 60 moles of SA per mole of the fusion protein. (Item 42) The composition according to Item 39, wherein the CD80 ECD-Fc fusion molecule comprises 15 to 40 moles of SA per mole of the fusion protein. (Item 43) The composition according to Item 39, wherein the CD80 ECD-Fc fusion molecule comprises 15 to 30 moles of SA per mole of the fusion protein. (Item 44) The composition according to item 39, wherein the CD80 ECD-Fc fusion molecule contains 20 to 30 moles of SA per mole of the fusion protein. (Item 45) The composition according to any one of items 1 to 44, wherein the CD80 ECD-Fc fusion molecule contains a human CD80 ECD containing the amino acid sequence of SEQ ID NO: 1. (Item 46) The composition according to any one of items 1 to 45, wherein the CD80 ECD-Fc fusion molecule contains a human IgG1 Fc domain containing the amino acid sequence of SEQ ID NO: 4. (Item 47) The composition according to any one of items 1 to 46, wherein the CD80 ECD-Fc fusion molecule contains an Fc domain of human IgG1 bound to the carboxy terminus of the ECD of human CD80. (Item 48) The composition according to any one of items 1 to 47, wherein the CD80 ECD-Fc fusion molecule contains the amino acid sequence of SEQ ID NO: 5. (Item 49) The composition according to any one of items 1 to 48, wherein the composition alone shows less release of interferon gamma or TNF alpha from T cells in vitro than in the case of TGN1412 alone. (Item 50) The composition according to item 49, wherein the composition alone is at least 1000 times less potent in inducing the release of interferon gamma or TNF alpha as compared to the case of TGN1412 alone. (Item 51) The composition according to any one of items 1 to 50, wherein after a single administration of the composition at 0.3 to 0.6 mg / kg, the composition enables inhibition of tumor growth by at least 90% in at least one mouse syngeneic cancer model over a period of at least 1 week, 10 days, 2 weeks, or 3 weeks. (Item 52) The composition according to item 51, wherein the mouse syngeneic cancer model is a CT26 tumor model. (Item 53) A pharmaceutical composition comprising (i) a sialylated CD80 ECD-Fc fusion molecule, (ii) about 20 mM of L-histidine, (iii) about 270 mM of sucrose, and (iv) about 0.05% weight / volume of polysorbate 20, and the pH of the composition is about 6.7. (Item 54) A pharmaceutical composition comprising (i) a sialylated CD80 ECD-Fc fusion molecule, (ii) about 20 mM of L-histidine, (iii) about 270 mM of sorbitol, and (iv) about 0.05% weight / volume of polysorbate 20, and the pH of the composition is about 6.7. (Item 55) A syringe or vial containing the pharmaceutical composition according to any one of Items 1 to 54. (Item 56) A method for treating a solid tumor in a human subject, comprising administering to the subject the pharmaceutical composition according to any one of Items 1 to 55. (Item 57) The method according to Item 56, wherein the solid tumor is an advanced solid tumor. (Item 58) The method according to any one of Items 56 and 57, wherein the solid tumor is not a primary central nervous system tumor. (Item 59) The method according to any one of Items 56 to 58, wherein the solid tumor is colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, or endometrial cancer. (Item 60) The method according to any one of Items 56 to 58, wherein the solid tumor is renal cell carcinoma. (Item 61) The method according to any one of Items 56 to 58, wherein the solid tumor is melanoma. (Item 62) The method according to any one of Items 56 to 61, wherein the patient has not previously received treatment with a PD-1 / PD-L1 antagonist. (Item 63) The method according to any one of Items 56 to 61, wherein the patient has previously received treatment with at least one PD-1 / PD-L1 antagonist selected from a PD-L1 antagonist and a PD-1 antagonist. (Item 64) The method according to Item 63, wherein at least one PD-1 / PD-L1 antagonist is nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. (Item 65) The method according to Item 63 or 64, wherein at least one PD-1 / PD-L1 antagonist is administered in an advanced or metastatic state. (Item 66) The method according to any one of Items 56 to 65, wherein the solid tumor recurs or progresses after undergoing treatment selected from surgery, chemotherapy, radiotherapy, and combinations thereof. (Item 67) The method according to any one of Items 56 to 65, wherein the pharmaceutical composition is administered intravenously.

[0060] 4. Brief Description of the Drawings.

Brief Description of the Drawings

[0061]

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BRIEF DESCRIPTION OF THE INVENTION

[0062] 5. DETAILED DESCRIPTION This specification provides a pharmaceutical composition comprising a CD80 extracellular domain (ECD)-fragment crystallizable (Fc) fusion molecule. The pharmaceutical composition can be stabilized, for example, under long-term storage conditions, through repeated freeze-thaw cycles (e.g., at least 5 cycles), and / or through agitation.

[0063] The pharmaceutical composition provided herein comprises a CD80 ECD-Fc fusion molecule (e.g., at a concentration of about 5 to about 15 mg / ml), histidine (e.g., L-histidine), sugar (e.g., sucrose or sorbitol), and / or a surfactant (such as polysorbate, e.g., polysorbate 20 (PS20), but not limited thereto).

[0064] In certain embodiments, an aqueous liquid pharmaceutical composition is provided herein that comprises a 10 mg / mL CD80 ECD-Fc fusion molecule (e.g., a human CD80 ECD-human IgG1 fusion molecule as shown in SEQ ID NO: 5), 20 mM histidine (e.g., L-histidine), 270 mM sucrose, and 0.05% PS20, and has a pH of 6.7. In another specific embodiment, an aqueous liquid pharmaceutical composition is provided herein that comprises a 10 mg / mL CD80 ECD-Fc fusion molecule (e.g., a human CD80 ECD-human IgG1 fusion molecule as shown in SEQ ID NO: 5), 20 mM histidine (e.g., L-histidine), 270 mM sorbitol, and 0.05% PS20, and has a pH of 6.7.

[0065] The pharmaceutical composition provided herein is useful in the treatment of conditions, such as solid tumors.

[0066] 5.1 TERMINOLOGY As used herein, "fusion molecule" refers to a molecule composed of two or more different molecules that do not exist together in nature and form a new molecule by covalent or non-covalent bonding. For example, a fusion molecule can be composed of a polypeptide and a polymer such as PEG, or of two different polypeptides. "Fusion protein" refers to a fusion molecule composed of two or more polypeptides that do not exist as a single molecule in nature.

[0067] "CD80 extracellular domain" or "CD80 ECD" refers to the extracellular domain polypeptide of human CD80, such as its native variants and genetically engineered variants. CD80 ECD can, for example, comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 1 or 2. "CD80 ECD fusion molecule" refers to a molecule comprising CD80 ECD and a fusion partner. The fusion partner can be covalently bound, for example, to the N-terminus or C-terminus of CD80 ECD, or at an internal position. The CD80 ECD fusion molecule can be a fusion protein comprising CD80 ECD and another polypeptide that does not naturally bind to CD80 ECD, such as an Fc domain. Such a CD80 ECD-Fc fusion molecule can, for example, comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 4 or 5.

[0068] The terms "polypeptide", "peptide", and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such a polymer of amino acid residues may contain natural or unnatural amino acid residues and includes, for example, but is not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. The definition includes both full-length proteins and fragments thereof. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Further, in the present invention, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence as long as the protein maintains the desired activity. These modifications can be intentional via site-directed mutagenesis or can be accidental via mutations in the host producing the protein or errors resulting from PCR amplification.

[0069] As used herein, the term "isolated" refers to a molecule that has been separated from at least a portion of the components with which it is normally associated in nature. For example, a polypeptide is considered "isolated" if it is separated from at least a portion of the components of the cell in which it was produced. After a polypeptide is expressed and secreted from the cell, physically separating the supernatant containing the polypeptide from the cell in which it was produced is considered isolating the polypeptide. Similarly, a polynucleotide is considered "isolated" if it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) that is normally found in nature or if, for example, in the case of an RNA polynucleotide, the polynucleotide is separated from at least a portion of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector within a host cell can be considered "isolated" as long as the polynucleotide is not found in nature in that vector.

[0070] As used herein, the term "host cell" refers to any type of cell, such as primary cells, cells in culture, or cells derived from cell lines. In certain embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example, due to mutations that may occur in subsequent generations or environmental influences, or due to the incorporation of the nucleic acid molecule into the host cell genome.

[0071] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a formulation in a form that permits the biological activity of the active ingredient to be effective and that contains no additional ingredients that would impart unacceptable toxicity to the subject to which the formulation is administered. The formulation can be made sterile.

[0072] The term "pharmaceutical product" refers to the final formulation, such as a liquid formulation, that contains, for example, the active pharmaceutical ingredient and generally, although not necessarily, one or more other ingredients.

[0073] The term "active pharmaceutical ingredient" refers to the active ingredient, for example, the CD80 ECD-Fc fusion molecule, and is intended to provide pharmacological or biological activity, or other direct effects, in the diagnosis, cure, mitigation, treatment, or prevention of disease, but does not include intermediates used in the synthesis of those ingredients.

[0074] As used herein, the term "buffer" refers to a component contained in a solution that can prevent a change in the pH of the solution. Examples of buffers include acetate, citrate, succinate, and histidine.

[0075] The term "stable" formulation refers to a formulation in which its active ingredient (e.g., CD80 ECD-Fc fusion molecule) substantially retains its physical stability and / or chemical stability and / or biological activity during storage. Stability can be measured over a selected period under selected conditions (e.g., temperature). The formulations provided herein are stable at room temperature (about 25°C) for at least 6 months and / or at about 2 - 8°C for at least 1 year. The formulations provided herein are stable after freezing (e.g., down to -70°C) and thawing of the formulation, hereinafter referred to as "freeze / thaw cycle". The formulations provided herein are stable after agitation.

[0076] As used herein, the terms "administer", "administering", "administration" refer to a method (e.g., intravenous administration) that can be used to enable delivery of a drug, e.g., a CD80 ECD-Fc fusion molecule, to a desired site for a biological effect. Administration techniques that can be used with the agents and methods described herein are recognized, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington’s, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0077] As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be an animal. In some embodiments, the subject is a mammal such as a non-human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey, or other primates, etc.). In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a human.

[0078] The term "therapeutically effective amount" refers to the amount of a drug, e.g., a CD80 ECD-Fc fusion molecule, that is effective in treating a disease or disorder in a subject. In the case of solid tumors, a therapeutically effective amount of an agent reduces the number of cancer cells; reduces the size or burden of the tumor; inhibits, to some extent, the invasion of cancer cells into peripheral organs; inhibits, to some extent, the metastasis of the tumor; inhibits, to some extent, the growth of the tumor; alleviates, to some extent, one or more symptoms associated with the cancer; and / or results in a favorable response such as a decrease in progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete remission (CR), partial remission (PR), or, in some cases, stable disease (SD), progressive disease (PD), a shortening of the time to progression (TTP), or any combination thereof. To the extent that a drug can prevent and / or kill the growth of existing cancer cells, the drug may have cytostatic and / or cytotoxic properties.

[0079] The terms "treating", "treatment", "treat", "alleviating", and "alleviate" refer to treatment means that cure, suppress, relieve the symptoms of, and / or arrest the progression of a pathological condition or disorder. Accordingly, those in need of treatment include those who have already been diagnosed as having a disorder or those suspected of having it. In certain embodiments, a patient exhibits a decrease or complete absence of cancer cells, a reduction in tumor size, inhibition or absence of cancer cell infiltration into peripheral organs such as when cancer metastasizes to soft tissue and bone; inhibition or absence of tumor metastasis, inhibition or absence of tumor growth, alleviation of one or more symptoms associated with a particular cancer, reduction in morbidity and mortality; improvement in quality of life, reduction in tumorigenicity, tumorigenic frequency, or tumorigenic ability of a tumor, reduction in the number or frequency of cancer stem cells in a tumor, differentiation of tumorigenic cells into a non-tumorigenic state, improvement in progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete remission (CR), partial remission (PR), stable disease (SD), reduction in progressive disease (PD), shortening of time to progression (TTP), or any combination thereof, then the subject is successfully "treating" cancer according to the methods of the present invention.

[0080] The terms "cancer" and "cancerous" refer to or describe a physiological state of a mammal characterized by disordered cell growth in a population of cells. Examples of cancer include, but are not limited to, colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, and endometrial cancer. Cancer can be a primary tumor or a progressive or metastatic cancer.

[0081] "Refractory" cancer refers to cancer that continues to progress despite anti-tumor treatment such as chemotherapy in a cancer patient.

[0082] "Recurrent" cancer refers to cancer that regrows at either the primary site or a distal site after a response to initial treatment.

[0083] A "recurrent" patient is a patient who exhibits signs or symptoms of cancer after recovery. In certain embodiments, the patient recurs after adjuvant or neoadjuvant therapy.

[0084] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0085] When an embodiment is described herein using the word "comprising", it is understood that other similar embodiments are also provided that are described with "consisting of" and / or "consisting essentially of". In this disclosure, terms such as "comprises", "comprising", "containing", and "having" can be given the meaning ascribed to them in U.S. patent law and can mean "includes" and "including", etc.; similarly, "consisting essentially of" or "consisting essentially" has the meaning ascribed to it in U.S. patent law, and the term is not limiting and allows the presence of matters outside the scope of the recited matter, provided that the essential or novel features of the recited matter are not changed by the presence of matters outside the scope of the recited matter, except for prior art embodiments.

[0086] Unless otherwise specified or clear from the context, the term "or" as used herein is to be understood as inclusive. When the term "and / or" is used in a phrase such as "A and / or B", it is intended to include "A and B", "A or B", and both "A" and "B". Similarly, when the term "and / or" is used in a phrase such as "A, B, and / or C", the following embodiments are intended: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0087] When the terms "about" and "approximately" are used herein to modify a numerical value or numerical range, a difference of up to 10% above and up to 10% below the numerical value or numerical range means that the value or range falls within the scope of the intended purpose.

[0088] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0089] 5.2 Pharmaceutical Composition Containing CD80 ECD-Fc Fusion Molecule Provided herein is a pharmaceutical composition (e.g., an aqueous pharmaceutical composition) containing a CD80 ECD-Fc fusion molecule (e.g., as described in Section 5.3 below).

[0090] In certain embodiments, the pharmaceutical compositions provided herein are stable to multiple freeze-thaw cycles. A freeze-thaw cycle can include freezing the pharmaceutical composition (e.g., at a temperature of about -70°C) and then thawing the pharmaceutical composition (e.g., at room temperature). The pharmaceutical composition is stable after at least 5 freeze-thaw cycles. The freeze-thaw cycles (e.g., at least 5 freeze-thaw cycles) do not cause detectable changes in appearance, soluble aggregates, or invisible particulate matter.

[0091] In certain embodiments, the pharmaceutical compositions provided herein are stable upon agitation. The agitation can include shaking at room temperature for about 3 days (e.g., about 300 revolutions per minute on an orbital shaker). The agitation does not result in detectable changes in appearance, soluble aggregates, charge variant profile, or subvisible particulate matter.

[0092] In certain embodiments, the pharmaceutical compositions provided herein are stable under long-term storage conditions. The long-term storage conditions can include storage at about 5°C (e.g., about 2°C to about 8°C) for about 6 months or about 1 year. The long-term storage conditions can include storage at about 25°C for about 6 months or about 1 year. The long-term storage conditions can include storage at about 40°C for about 3 months, about 6 months, or about 1 year.

[0093] In certain embodiments, the pharmaceutical compositions provided herein are stable to multiple (e.g., at least 5) freeze-thaw cycles, stable upon agitation, and / or stable under long-term storage conditions.

[0094] In certain embodiments, the pharmaceutical compositions provided herein are stable for about 1 year when stored at about -70°C and when stored at about 2°C to about 8°C.

[0095] In certain embodiments, the pharmaceutical composition can contain a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5). In certain embodiments, the concentration of the CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5) in the formulation is about 5 mg / ml to about 15 mg / ml. In certain embodiments, the concentration of the CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5) in the pharmaceutical composition is about 10 mg / ml.

[0096] In certain embodiments, the concentration of the CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5) in the formulation is from 5 mg / ml to 15 mg / ml. In certain embodiments, the concentration of the CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5) in the pharmaceutical composition is 10 mg / ml.

[0097] As described herein, the pharmaceutical composition can contain a buffer. In certain embodiments, the buffer is histidine (e.g., L-histidine). In certain embodiments, the concentration of histidine (e.g., L-histidine) is from about 15 mM to about 25 mM. In certain embodiments, the concentration of histidine (e.g., L-histidine) is from about 18 mM to about 22 mM. In certain embodiments, the concentration of histidine (e.g., L-histidine) is about 20 mM.

[0098] In certain embodiments, the concentration of histidine (e.g., L-histidine) is from 15 mM to 25 mM. In certain embodiments, the concentration of histidine (e.g., L-histidine) is from 18 mM to 22 mM. In certain embodiments, the concentration of histidine (e.g., L-histidine) is 20 mM.

[0099] As described herein, the pharmaceutical composition can contain an excipient, such as a sugar, e.g., sucrose or sorbitol. In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is from about 225 mM to about 300 mM. In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is from about 250 mM to about 290 mM. In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is about 270 mM.

[0100] In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is from 225 mM to 300 mM. In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is from 250 mM to 290 mM. In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is 270 mM.

[0101] As described herein, the pharmaceutical composition can include excipients such as histidine (e.g., L-histidine) and sugar (e.g., sucrose or sorbitol). In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is about 10 to about 15 times the concentration of the histidine (e.g., L-histidine). In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is about 13.5 times the concentration of the histidine (e.g., L-histidine).

[0102] In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is 10 to 15 times the concentration of the histidine (e.g., L-histidine). In some embodiments, the concentration of the sugar (e.g., sucrose or sorbitol) is 13.5 times the concentration of the histidine (e.g., L-histidine).

[0103] As described herein, the pharmaceutical composition can include a surfactant, such as polysorbate. The polysorbate can be, for example, polysorbate 20 (PS20). In some embodiments, the concentration of the surfactant (e.g., PS20) is about 0.025 to 0.075 weight / volume (w / v)%. In some embodiments, the concentration of the surfactant (e.g., PS20) is about 0.035 to about 0.065% w / v. In some embodiments, the concentration of the surfactant (e.g., PS20) is about 0.05% w / v.

[0104] In some embodiments, the concentration of the surfactant (e.g., PS20) is 0.025 to 0.075 weight / volume (w / v)%. In some embodiments, the concentration of the surfactant (e.g., PS20) is 0.035 to 0.065% w / v. In some embodiments, the concentration of the surfactant (e.g., PS20) is 0.05% w / v.

[0105] As described herein, in some embodiments, the pH of the pharmaceutical composition is from about 5 to about 7.5. In some embodiments, the pH of the pharmaceutical composition is from about 5.5 to about 7. In some embodiments, the pH of the pharmaceutical composition is from about 6.4 to about 7. In some embodiments, the pH of the pharmaceutical composition is from about 6.5 to about 7. In some embodiments, the pH of the pharmaceutical composition is about 6.7.

[0106] In some embodiments, the pH of the pharmaceutical composition is from 5 to 7.5. In some embodiments, the pH of the pharmaceutical composition is from 5.5 to 7. In some embodiments, the pH of the pharmaceutical composition is from 6.4 to 7. In some embodiments, the pH of the pharmaceutical composition is from 6.5 to 7. In some embodiments, the pH of the pharmaceutical composition is 6.7.

[0107] As described herein, the pharmaceutical composition can be in liquid form. The pharmaceutical composition (e.g., liquid pharmaceutical composition) can be for parenteral administration, for example, for intravenous administration.

[0108] In certain embodiments, the pharmaceutical composition comprises from about 5 mg / mL to about 15 mg / mL of a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5), from about 15 mM to about 25 mM of histidine (e.g., L-histidine), from about 225 mM to about 300 mM of sucrose or sorbitol, and from about 0.025% to about 0.075% of PS20. In certain embodiments, the pH of the pharmaceutical composition is from about 6.4 to about 7 or from about 6.5 to about 7, for example, about 6.7. In certain embodiments, the pharmaceutical composition is in liquid form.

[0109] In certain embodiments, the pharmaceutical composition comprises 5 mg / mL to 15 mg / mL of a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5), 15 mM to 25 mM of histidine (e.g., L-histidine), 225 mM to 300 mM of sucrose or sorbitol, and 0.025% to 0.075% of PS20. In certain embodiments, the pH of the pharmaceutical composition is from 6.4 to 7 or from 6.5 to 7, for example, 6.7. In certain embodiments, the pharmaceutical composition is in liquid form.

[0110] In certain embodiments, the pharmaceutical composition comprises a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5) at about 5 mg / mL to about 15 mg / mL, histidine (e.g., L-histidine) at about 18 mM to about 22 mM, sucrose or sorbitol at about 250 mM to about 290 mM, and PS20 at about 0.035% to about 0.065%. In certain embodiments, the pH of the pharmaceutical composition is about 6.4 to about 7 or about 6.5 to about 7, for example, about 6.7. In certain embodiments, the pharmaceutical composition is liquid.

[0111] In certain embodiments, the pharmaceutical composition comprises a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5) at 5 mg / mL to 15 mg / mL, histidine (e.g., L-histidine) at 18 mM to 22 mM, sucrose or sorbitol at 250 mM to 290 mM, and PS20 at 0.035% to 0.065%. In certain embodiments, the pH of the pharmaceutical composition is 6.4 to 7 or 6.5 to 7, for example, 6.7. In certain embodiments, the pharmaceutical composition is liquid.

[0112] In certain embodiments, the pharmaceutical composition comprises a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5) at about 10 mg / mL, histidine (e.g., L-histidine) at about 20 mM, sucrose or sorbitol at about 270 mM, and PS20 at about 0.05%. In certain embodiments, the pH of the pharmaceutical composition is about 6.7. In certain embodiments, the pharmaceutical composition is liquid.

[0113] In certain embodiments, the pharmaceutical composition comprises a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5) at 10 mg / mL, histidine (e.g., L-histidine) at 20 mM, sucrose or sorbitol at 270 mM, and PS20 at 0.05%. In certain embodiments, the pH of the pharmaceutical composition is 6.7. In certain embodiments, the pharmaceutical composition is liquid.

[0114] In some embodiments (including, but not limited to, any of the embodiments of the pharmaceutical composition described above), the pharmaceutical composition comprises a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5), and the composition comprises less than 10% of high molecular weight species (HMWS) of the CD80 ECD-Fc fusion molecule and / or less than 2.5% of low molecular weight species (LMWS) of the CD80 ECD-Fc fusion molecule after 4 weeks at 40°C.

[0115] In some embodiments, the pharmaceutical composition comprises a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5), and the composition comprises from about 1% to about 10%, from about 1% to about 6%, or from about 1% to about 4% of HMWS of the CD80 ECD-Fc fusion molecule and / or from about 0.5% to about 2.5%, from about 0.5% to about 1.6%, or from about 0.5% to about 1.5% of LMWS of the CD80 ECD-Fc fusion molecule after 4 weeks at 40°C. In some embodiments, the pharmaceutical composition comprises a CD80 ECD-Fc fusion molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 5), and the composition comprises 1% - 10%, 1% - 6%, or 1% - 4% of HMWS of the CD80 ECD-Fc fusion molecule and / or 0.5% - 2.5%, 0.5% - 1.6%, or 0.5% - 1.5% of LMWS of the CD80 ECD-Fc fusion molecule after 4 weeks at 40°C.

[0116] In some embodiments, the above-described pharmaceutical composition is provided in a syringe or vial. In some embodiments, the syringe or vial contains from about 0.5 to about 5 mls of the pharmaceutical composition provided herein. In some embodiments, the syringe or vial contains from about 1 to about 5 mls of the pharmaceutical composition provided herein.

[0117] In some embodiments, the syringe or vial contains the 2 mls pharmaceutical composition provided herein. Thus, by way of example, the syringe or vial can contain 20 mg of the CD80 ECD-Fc fusion molecule in a 2 ml volume, i.e., about 10 mg / ml of the CD80 ECD-Fc fusion molecule. In some embodiments, the syringe or vial contains the 1 ml pharmaceutical composition provided herein. Thus, by way of example, the syringe or vial can contain 10 mg of the CD80 ECD-Fc fusion molecule in a 1 ml volume, i.e., about 10 mg / ml of the CD80 ECD-Fc fusion molecule.

[0118] 5.3 CD80 ECD-Fc Fusion Molecule Provided herein are pharmaceutical compositions comprising a CD80 ECD-Fc fusion molecule. Exemplary CD80 ECD-Fc fusion molecules are provided, for example, in WO2017 / 079117, which is hereby incorporated by reference in its entirety.

[0119] The CD80 ECD can be, for example, a human CD80 ECD. In certain embodiments, the human CD80 ECD can comprise, consist essentially of, or consist of, the amino acid sequence set forth in SEQ ID NO: 1.

[0120] The Fc domain can be the Fc domain of IgG. The Fc domain can be the Fc domain of a human immunoglobulin. In certain embodiments, the Fc domain is a human IgG Fc domain. In certain embodiments, the Fc domain is a human IgG1 Fc domain. In certain embodiments, the human IgG1 Fc domain can comprise, consist essentially of, or consist of, the amino acid sequence set forth in SEQ ID NO: 4.

[0121] The CD80 ECD and the Fc domain can be directly linked such that the N-terminal amino acids of the Fc domain follow the C-terminal amino acids of the CD80 ECD. In certain embodiments, the CD80 ECD and the Fc domain are translated as a single polypeptide from coding sequences encoding both the CD80 ECD and the Fc domain. In certain embodiments, the CD80 ECD-Fc fusion molecule comprises a human CD80 ECD and a human IgG1 Fc domain. In certain embodiments, the CD80 ECD-Fc fusion molecule can comprise, consist essentially of, or consist of, an amino acid sequence as set forth in SEQ ID NO:5.

[0122] CD80 ECD-Fc fusion molecules can have different levels of specific glycosylation modifications depending on how they are produced. For example, the CD80 ECD-Fc fusion molecules can be sialylated and can have different amounts of sialic acid (SA) residues.

[0123] In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 10 to 60 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 15 to 60 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 10 to 40 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 15 to 30 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 15 to 25 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 20 to 40 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 20 to 30 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 30 to 40 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises 10, 15, 20, 25, 30, 35, or 40 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises at least 15 molecules of SA. In certain embodiments, the CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO: 5) comprises at least 20 molecules of SA.In certain embodiments, a CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO:5) comprises at least 25 molecules of SA. In certain embodiments, a CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO:5) comprises at least 30 molecules of SA. In certain embodiments, a CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO:5) comprises at least 35 molecules of SA. In certain embodiments, a CD80 ECD-Fc fusion molecule (e.g., comprising a human CD80 ECD and a human IgG1 Fc domain, or comprising SEQ ID NO:5) comprises at least 40 molecules of SA.

[0124] 5.4 Therapeutic Uses and Methods In one aspect, the present disclosure provides a method of treating a solid tumor in a subject (e.g., a human subject), the method comprising administering to a subject in need thereof a pharmaceutical composition comprising a CD80 ECD-Fc fusion molecule provided herein.

[0125] In certain embodiments, the present disclosure provides a pharmaceutical composition for treating a solid tumor selected from the group consisting of colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, or endometrial cancer. In certain embodiments, the solid tumor is renal cell carcinoma. In certain embodiments, the solid tumor is melanoma.

[0126] The solid tumor can be, for example, an advanced solid tumor. In certain examples, the solid tumor is not a primary central nervous system tumor.

[0127] A patient to be treated according to the method provided herein may have previously received treatment with at least one PD-1 / PD-L1 antagonist selected from a PD-1 antagonist and a PD-L1 antagonist. The PD-1 / PD-L1 antagonist can be, for example, nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The PD-1 / PD-L1 antagonist can be administered in an advanced or metastatic state. In other examples, a patient to be treated according to the method provided herein has not previously received treatment with a PD-1 / PD-L1 antagonist.

[0128] A patient to be treated according to the method provided herein may have previously received treatment with an angiogenesis inhibitor. The angiogenesis inhibitor can be, for example, sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. The angiogenesis inhibitor can be administered in an advanced or metastatic state.

[0129] A patient to be treated according to the method provided herein, for example, a patient with melanoma, may have a BRAF mutation. The patient may have previously received treatment with a BRAF inhibitor. The BRAF inhibitor can be, for example, vemurafenib and dabrafenib. The BRAF inhibitor can be administered in an advanced or metastatic state.

[0130] A tumor to be treated according to the method provided herein can recur or progress after undergoing treatment selected from surgery, chemotherapy, radiotherapy, and combinations thereof.

[0131] The tumors to be treated according to the methods provided herein can be those that are resistant or non-responsive to PD-1 / PD-L1 antagonists such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The tumors to be treated according to the methods provided herein can be those that are resistant or non-responsive to angiogenesis inhibitors such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. The tumors to be treated according to the methods provided herein can be those that are resistant or non-responsive to BRAF inhibitors such as vemurafenib or dabrafenib.

[0132] The tumors to be treated according to the methods provided herein can be those that are refractory to PD-1 / PD-L1 antagonists such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The tumors to be treated according to the methods provided herein can be those that are refractory to angiogenesis inhibitors such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. The tumors to be treated according to the methods provided herein can be those that are refractory to BRAF inhibitors such as vemurafenib or dabrafenib.

[0133] The tumors to be treated according to the methods provided herein can recur after treatment with PD-1 / PD-L1 antagonists such as nivolumab, pembrolizumab, atezolizumab, durvalumab, or avelumab. The tumors to be treated according to the methods provided herein can recur after treatment with angiogenesis inhibitors such as sunitinib, sorafenib, pazopanib, axitinib, tivozanib, ramucirumab, or bevacizumab. The tumors to be treated according to the methods provided herein can recur after treatment with BRAF inhibitors such as vemurafenib or dabrafenib.

[0134] The pharmaceutical composition described in this specification can be delivered to a patient via the intravenous route. Usually, the patient is a human, but non-human mammals such as transgenic mammals can also be treated.

Examples

[0135] 6. Examples The examples in this section (i.e., Section 6) are for illustrative purposes and not for limiting purposes.

[0136] Example 1: Methods for Use in Formulation Research I. General Formulation Procedures The human CD80 ECD-human IgG1 Fc fusion protein containing the amino acid sequence of SEQ ID NO: 5 (hereinafter referred to as "hCD80-Fc") was expressed in a CHO cell line and purified from the same CHO cell line. Samples of hCD80-Fc were dialyzed against a polysorbate-free drug substance using a 20 kD molecular weight cut-off (MWCO) dialysis membrane and prepared in various formulations. After dialysis, the concentration of hCD80-Fc was measured by UV spectroscopy using an extinction coefficient of 1.35 cm -1 [g / L] -1 The protein concentration of the buffer exchange samples was adjusted to the desired value with the dialysis buffer, and a 10% polysorbate 20 (PS20) stock solution was dispensed into each formulation to a final concentration of 0.05% (w / v) PS20. The formulations were sterile filtered using a 0.22 μm filter unit and filled into appropriate containers / closure systems in a laminar flow hood. Samples were placed under various storage conditions for each study design and removed at predetermined times for stability analysis using various methods.

[0137] II. Analytical Procedures Visual inspection: Visual evaluation was performed under fluorescent illumination against both black and white backgrounds. Samples were examined for color, clarity, and the presence of visible particles.

[0138] Protein concentration: The protein concentration was 1.35 cm -1 [g / L] -1The theoretical absorption coefficient was used to determine the UV absorbance at 280 nm. Samples were diluted within the linear range of absorbance using Dulbecco’s Phosphate Buffer Saline (DPBS) and measured against blank DPBS. Absorbance was measured using an Agilent Cary 8454 UV-Vis Spectrophotometer (Agilent Technologies, CA).

[0139] pH: The pH of the buffer was determined using a Beckman Coulter pHi560 meter (Beckman Coulter, Inc., CA) equipped with a scale.

[0140] Osmotic pressure: The osmotic pressure of the buffer was measured by vapor pressure using a Wescor VAPRO system (Wescor, Inc., UT).

[0141] Differential scanning calorimetry (DSC) analysis: DSC measurements were performed on a MicroCal VP-Capillary DSC platform (GE Healthcare, UK). Protein samples were diluted to a concentration of 1 mg / mL with the respective formulation buffer. The formulation buffer meeting the reference was used as a reference. Samples were scanned from 15 °C to 110 °C at a rate of 1 °C / min. Data were first normalized against the protein concentration, then baseline corrected, and buffer subtraction was performed using Origin 7.0 software (OriginLab, MA). The melting transition was analyzed with the DSC peak fit function launched by the cursor using a non-two-state unfolding model stored in the original software.

[0142] Denaturation Temperature (Tm) by the UNit System: The denaturation temperature (Tm) of a protein provides a measure of the physical stability of the molecule. The denaturation temperature is defined as the temperature at which equal amounts of native and denatured protein exist in equilibrium. The UNit system from Unchained Labs (Pleasanton, CA) uses intrinsic fluorescence spectroscopic changes to determine the temperature at which heat induces protein denaturation. A 1 mg / mL sample was scanned from 20 °C to 90 °C at a rate of 1 °C per minute. Tm was determined using Unchained Labs' UNcle software.

[0143] Imaged Capillary Isoelectric Focusing (iCIEF): Imaged capillary isoelectric focusing (iCIEF) was performed on a Protein Simple iCE3 instrument equipped with a 720NV autosampler (ProteinSimple, San Jose, CA) to analyze charge variants. Data were analyzed using iCIEF CFR software, and the areas of the peaks observed in the profile were integrated to determine the relative amounts of the main, acidic, and basic peaks.

[0144] Size Exclusion High Performance Liquid Chromatography (SE-HPLC): Samples were analyzed on an Agilent 1100 series HPLC equipped with a diode array detector, and absorbance was monitored at 280 nm. Samples were diluted to 1 mg / ml with the mobile phase (100 mM sodium phosphate, 400 mM sodium chloride pH 6.8), and 50 μL was injected onto a pre-equilibrated Sepax Zenix SEC-300 7.8 × 200 mm column (Sepax Technologies, Inc., Delaware). The SEC separation column and guard column were used at 25 °C. A flow rate of 1.0 mL / min was used, and the run time was 12 minutes. Peaks for aggregates, monomers, and fragments were quantified using the instrument software for data analysis.

[0145] Capillary electrophoresis using sodium dodecyl sulfate gel (CE-SDS): Purity was determined under reducing and non-reducing conditions using the CE-SDS method. Samples were analyzed on a Beckman Coulter PA800 plus system (Beckman Coulter, CA) using an uncoated 50 μM I.D. capillary. Absorbance was monitored at 220 nm. The purity of hCD80-Fc under reducing conditions was determined by measuring the peak area of the single-chain monomer peak and comparing it to the total area of all detected peaks. The purity of hCD80-Fc under non-reducing conditions was determined by measuring the peak area of the major intact protein peak and comparing it to the total area of all detected peaks.

[0146] Visual non-detectable particulate matter in HIAC: A HIAC 9703+ particle counter (Hach, Loveland, Colorado) equipped with an HRDL-150 detector and a 1 mL syringe was used. Prior to use, the system was washed with particle-free Milli-Q water (Millipore, MA) to obtain a clean baseline. Four consecutive 0.4 mL aliquots were withdrawn from the sample, and the last three aliquots were counted for particles, averaged, and reported.

[0147] CTLA4-binding ELISA: The relative efficacy of hCD80-Fc was determined based on the specific and quantitative binding of hCD80-Fc to recombinant human cytotoxic T-lymphocyte antigen 4 (CTLA-4) protein against a reference material (RM) using the ELISA method. In this assay, 96-well microplates were coated with recombinant human CTLA-4 protein. After blocking with 1% BSA buffer, hCD80-Fc RM, control samples, and serially diluted test samples were added to the plates and incubated with the coating ligand. Horseradish peroxidase (HRP)-conjugated mouse anti-human Fc secondary antibody, secondary antibody-HRP, and TMB substrate were sequentially added to the plates with the wash solution. The ELISA plates were read, and absorbance signals at 450 nm were measured using a plate reader. The relative potency (RP) was calculated based on the EC 50 values of the RM and test samples and then reported.

[0148] CD28 Cell-Based Bioassay: Method QM5963 is based on Promega's T Cell Activation Assay (IL-2) (Madison, WI), which utilizes Jurkat T cells stably transfected with a luciferase reporter driven by the IL-2 promoter. Activated cells (HEK293-OKT3-CD64) and effector cells (provided in Promega's kit) are thawed and then resuspended in IL-2 luciferase reporter assay buffer, plated, and incubated overnight (18-22 hours) with hCD80-Fc reference material (RM), test sample (TS), and Assay Control Standard (ACS) diluent. During incubation, hCD80-Fc binds, in conjunction with OKT3 / CD3, to CD64 (activated cells) and CD28 (effector cells) localized on the surface, causing activation of the IL-2 Luc pathway and resulting in increased luciferase expression. The degree of the signaling response is proportional to the amount of hCD80-Fc present at each dilution level, which is quantified by adding Bio-Glo™ luciferase assay reagent and counting with a plate reader that shows glow-type luminescence measurements.

[0149] Example 2: Biochemical Analysis of Amino Acid Residues of hCD80-Fc Among protein therapeutics, Fc fusion proteins are generally more difficult to formulate than antibodies (e.g., monoclonal antibodies), which is to say that the latter have a relatively stable Fab portion, while Fc fusion proteins have a protein structure outside the Fc, potentially leading to a high degree of complexity and reduced stability. Compared to monoclonal antibodies, relatively few Fc fusion proteins have received FDA approval. Specifically, by the end of 2017, a total of 11 Fc fusion proteins had been approved, whereas by the same time, a total of approximately 73 monoclonal antibodies had been approved. (See, for example, Jafari et al (2017) Curr. Med. Chem. 24, 1228-1237; and https: / / www.creative-biolabs.com / blog / index.php / new-monoclonal-antibody-drug-approvals-in-2017 / , posted February 8, 2018.)

[0150] hCD80-Fc is an Fc fusion protein comprising the amino acid sequence of SEQ ID NO: 5. Using a knowledge-based formulation development approach, an appropriate composition was identified that confers maximum stability to the protein. To do this, both the properties inherent to the molecule and exogenous formulation components that can affect the stability of the protein were considered.

[0151] The primary amino acid sequence of hCD80-Fc is shown in Table 1. The theoretical pI of the hCD80-Fc protein is 6.0. However, the presence of sialic acid in this highly glycosylated protein results in a decrease in the actual pI. Indeed, when iCIEF analysis of hCD80-Fc was performed, complex results were obtained due to the presence of sialic acid in the protein. As shown in Figure 5A, iCIEF showed a series of peaks representing a wide range of pIs from approximately 4.0 to 6.5 in the acidic region (pI < 4.73), the main region (pI 4.73 - 5.35), and the basic region (pI > 5.35). (Note that there are two pI markers at 3.59 and 7.55.) For comparison, Figure 5B shows the iCIEF profile of a typical therapeutic monoclonal antibody, which shows a main peak of intensity and several different charge variant peaks. Therefore, the change in the charge profile of hCD80-Fc cannot be reasonably discriminated using iCIEF, and this method cannot be used for further formulation selection. The inability to use iCIEF as a tool to determine charge variants makes it even more difficult to develop an appropriate formulation with long-term stability.

[0152] Taking these results into account, the initial screening of formulations with pH in the range of 4 - 7.5 was planned using methods other than iCIEF.

Table 1

[0153] Analysis of the primary sequence of hCD80-Fc revealed that some amino acid residues are subject to biochemical modifications. These include deamidation at asparagine, isomerization at aspartic acid, and oxidation at methionine, cysteine, histidine, tryptophan, phenylalanine, and tyrosine. The potential sites for biochemical degradation in hCD80-Fc are indicated in the sequence in Table 1 in bold and gray squares. The underlined amino acid sequence in Table 1 indicates the Fc (IgG1) region. The potential sites for biochemical degradation are shown in Table 2.

Table 2

[0154] To develop a liquid formulation that imparts good stability to hCD80-Fc, various conditions such as pH, type of buffer, excipients, etc. were evaluated. The stability of the protein was monitored based on the biophysical and biochemical properties of hCD80-Fc in each study. The details of the studies and their results are described herein.

[0155] Example 3: Initial pH Screening Study The pH of the formulation plays an important role in the stability of the protein and affects biochemical degradation pathways such as deamidation, isomerization, oxidation, etc., as well as biophysical degradation such as aggregation and fragmentation due to protein-protein interactions and protein-environment interactions. A pH screening study was conducted to determine the pH range that confers stability to hCD80-Fc and to understand the degradation mechanism of the protein under these conditions. The details of the formulation compositions evaluated in this study are shown in Table 3. The materials used in this study were provided after Protein A purification.

Table 3

[0156] The stability of hCD80-Fc at various pH values was evaluated at temperature conditions of 5°C, 25°C, and 40°C for up to 11 days. During the study period, all samples remained clear and colorless, and no particles were observed. Changes in aggregates and fragments were determined by size exclusion high performance liquid chromatography (SE-HPLC). The SE-HPLC assay separates protein molecules based on size. The monomer peak is identified as a protein of a size that can be considered from its retention time. Aggregate or HMW peak(s) appear before the monomer peak. Fragment or LMW peak appears after the monomer peak.

[0157] At T0, for the studied formulations, aggregates were observed at a concentration of 1 mg / ml of hCD80-Fc at about 8 - 9%. At 40°C, after 11 days, soluble aggregates increased significantly in formulations at pH 4, 5, and 7.5, but in the formulation at pH 6.7, the increase in aggregates was only slight (Figure 1). Compared with formulations at pH 6.7 or 7.5, there were slightly more fragments in formulations at pH 4 or 5 (Figure 2). The results of this study revealed that hCD80-Fc is most stable around pH 5 - 7.5, as shown by SE-HPLC.

[0158] Example 4: Detailed pH Screening Study The results of the preliminary pH screening study indicated that hCD80-Fc was more stable around pH 5 - 7.5. Further pH studies were conducted to determine that maximum stability was obtained in the range of pH 5.5 - 7.0. The formulations of the tested compositions are shown in Table 4. All formulations contained 270 mM sucrose and 0.05% polysorbate 20. The stability of 1 mg / mL hCD80-Fc was examined based on appearance, aggregation, and fragmentation (by SE-HPLC), and charge variants (iCIEF) under stress (40°C) conditions for up to 4 weeks.

Table 4

[0159] To determine the thermal stability of hCD80-Fc under different pH conditions, in another study, a UNit instrument was used to measure the denaturation temperature with intrinsic fluorescence changes. The shift in the tryptophan fluorescence emission wavelength indicates that a denaturation event occurs when the sample is heated. Table 5 shows the denaturation temperatures (Tm1) of hCD80-Fc measured under various pH conditions. Since this study focuses on the overall stability of hCD80-Fc rather than domain-dependent denaturation, only the minimum Tm (Tm1) shown by the UNit system is reported. The results indicate that Tm1 is hardly dependent on pH. Therefore, formulation compositions with pH values ranging from 5.5 to 7.0 provide similar thermal stability. The effect of formulation compositions with pH values ranging from 5.5 to 7.0 on colloidal stability has not been determined.

Table 5

[0160] During the study period, all samples remained clear and colorless, and no particles were observed. The results of SE-HPLC showed the presence of low levels of aggregates in hCD80-Fc at a concentration of 1 mg / ml at T0. At 40°C, 4 weeks of thermal stress increased aggregates in a pH-dependent manner in the order of pH 7.0, <6.5, =5.5, <6.0 (Figure 3). The cause of the large amount of aggregates observed at pH 6.0 has not yet been identified. No significant differences in fragmentation were observed under different pH conditions (Figure 4).

[0161] In summary, the results of this study indicate that formulations with pH values ranging from 6.0 to 7.0 are most likely to control the formation of aggregates and fragments for hCD80-Fc most successfully.

[0162] Example 5: Screening Study of Buffer Species Similar to pH, the type of buffer also affects the stability of proteins to varying degrees. Citrate and histidine are two frequently used buffer types, and their effective pH range is 5.5 - 7.0, which is based on their pKa. To determine the buffer that provides maximum stability for 10 mg / mL hCD80-Fc at pH 5.5 - 7.0, it was evaluated in citrate and histidine buffers from pH 5.5 to pH 7.0 in 0.5 unit increments. In this study, a protein concentration of 10 mg / mL was used to confirm the pH effect observed at a protein concentration of 1 mg / mL. The composition of the formulation is shown in Table 6. These buffers were examined for their effects on protein stability at 25°C for up to 3 months and at 40°C for up to 2 months based on appearance, aggregation, and fragmentation.

Table 6

[0163] During the study period, all samples remained clear and colorless, and no particles were observed. Among the citrate- and histidine-based formulations at pH 5.5 - 7.0, fewer aggregates were observed in the histidine formulations compared to the citrate formulations. In particular, the histidine-based formulations at pH 6.5 and 7.0 are desirable compared to other formulations based on the results of SE-HPLC shown in Figure 6. Similar levels of fragmentation were observed in all formulations evaluated (Figure 7).

[0164] Based on these results, the results of this study can be concluded that hCD80-Fc has better stability in histidine formulations at pH 6.5 - 7.0 than in the other formulations tested.

[0165] Example 6: Excipient Selection Study Formulation excipients such as bulking agents affect the stability of the product. To evaluate the effect of excipients on the stability of hCD80-Fc, 1 mg / mL of hCD80-Fc was formulated using citrate, histidine, and phosphate formulations containing arginine, sucrose, or NaCl at isotonic concentrations. The citrate formulation was formulated at pH 5.5; the histidine formulation was formulated at pH 6.0 or 6.5; and the phosphate formulation was formulated at pH 7.0. The detailed formulation compositions are shown in Table 7. The effect of the excipients on the protein stability was examined based on appearance, aggregation, and fragmentation under storage conditions of 5 °C and 25 °C for 3 months and 40 °C for up to 2 months.

Table 7

[0166] Another study was conducted to compare sucrose and sorbitol with respect to the stability of hCD80-Fc using histidine-based formulations at pH 6.0, 6.5, and 7.0. The composition of the formulations is shown in Table 8. The effect of the excipients on the protein stability was examined based on appearance, aggregation, and fragmentation under storage conditions of 5 °C and 25 °C for 3 months and 40 °C for up to 2 months.

Table 8

[0167] During the study period, all samples remained clear and colorless, and no particles were observed. The SE-HPLC results shown in Figure 8 indicate that formulations containing arginine or NaCl showed more rapid progression of hCD80-Fc aggregate formation compared to the corresponding formulations containing sucrose at pH 5.5, 6.5, and 7.0. Also, in the case of histidine buffer, more aggregates were observed in the sorbitol-containing formulation than in the sucrose-containing formulation at pH 6.0 - 7.0 (Figure 9). All formulations contained the same amount of fragments as determined by SE-HPLC analysis. In the preferred pH range of 6.5 - 7.0 determined by the pH study, sucrose provided the best stability for hCD80-Fc among all the excipients evaluated. Also, sorbitol showed a better stability profile than arginine and NaCl.

[0168] In summary, the results of the studies on the selection of pH, buffer species, and excipients showed that the formulation of hCD80-Fc with 20 mM L-histidine, 270 mM sucrose, and 0.05% PS20 at pH 6.5 - 7.0 was the most stable. Since the side chain pKa of L-histidine is 6.0, it is considered that the buffering capacity is minimized with L-histidine at pH above 7.0. Therefore, a formulation containing 20 mM L-histidine, 270 mM sucrose, and 0.05% PS20 with a pH of 6.7 ± 0.3 was selected as having desirable properties. A second formulation containing sorbitol instead of sucrose was also selected.

[0169] The differential scanning calorimetry (DSC) profile of a formulation containing hCD80-Fc, 20 mM L-histidine, 270 mM sucrose, and 0.05% PS20 with a pH of 6.7 was collected and shown in Figure 10. Two peaks were observed at Tm1 = 62.7 °C and Tm2 = 84.8 °C.

[0170] Example 7: Freeze-thaw stability of hCD8-Fc The freeze / thaw study was conducted on a formulation containing 10 mg / mL protein, 20 mM L-histidine, 270 mM sucrose, and 0.05% PS20 with a pH of 6.7, using the hCD80-Fc bulk drug substance at a scale of 500 mL. The formulation was frozen at -70 °C and thawed at ambient temperature for 5 cycles. No obvious changes in appearance, soluble aggregates, or invisible particulate matter were detected among the formulations (Table 9). [Table 9]

[0171] Example 8: Stirring Stability of hCD80-Fc The hCD80-Fc bulk drug substance was filled into 3 cc glass vials. The sample vials were placed horizontally on an orbital shaker, and the samples were shaken at 300 RPM for 72 hours at room temperature (20 ± 5 °C) to apply stirring stress to hCD80-Fc. hCD80-Fc was obtained as a formulation containing 10 mg / mL protein, 20 mM L-histidine, 270 mM sucrose, and 0.05% PS20 with a pH of 6.7. No obvious changes in appearance, soluble aggregates, charge variants, or invisible particulate matter were detected in the hCD80-Fc formulation samples (Table 10). [Table 10]

[0172] Example 9: Verification of Stability Study In a first formulation containing 10 mg / mL of protein, 20 mM of L-histidine, 270 mM of sucrose, and 0.05% of PS20, with a pH of 6.7, and a second formulation containing 10 mg / mL of protein, 20 mM of L-histidine, 270 mM of sorbitol, and 0.05% of PS20, with a pH of 6.7, a 12-month stability study was initiated to evaluate the stability of hCD80-Fc. The solution of the formulation (1.5 ml) was filled into one 3 cc type glass vial with a 13 mm neck, capped with a 13 mm West 4023 / 50 gray bromobutyl serum stopper, and sealed with an aluminum seal. The compatibility of the container closure system with hCD80-Fc was confirmed by inverting the vial. Storage conditions of 5 °C, 25 °C, and 40 °C were used for the stability study, and stability data were collected over 12 months. The stability data of the first formulation are shown in Tables 11 to 13. The stability data of the preliminary formulation are shown in Tables 14 to 16.

Table 11-1

Table 11-2

Table 12-1

Table 12-2

Table 13

Table 14-1

Table 14-2

Table 15

Table 16

[0173] Stability data of the hCD80-Fc pharmaceutical in the first and second formulations under long-term storage conditions of 2 - 8°C were collected in real time over 12 months. All stability data met the acceptance criteria (Tables 11 and 14). No clear trend in the change of stability was observed for the items tested. The results demonstrate that the hCD80-Fc pharmaceutical is stable in both formulations under long-term storage conditions for at least 12 months.

[0174] Stability data of the hCD80-Fc pharmaceutical in the first and second formulations under accelerated conditions of 25°C were collected over 6 months. Overall, after storage for 6 months under accelerated conditions, the purity determined by reduced and non-reduced CE-SDS decreased slightly, and the efficacy decreased slightly in CTLA4 binding ELISA and CD28 cell-based bioassays. No clear changes were observed in other items related to the product. All stability data were within the acceptance criteria.

[0175] Stability data of the hCD80-Fc pharmaceutical under stress conditions of 40°C were collected over 2 months. Some changes were more prominent in the data collected over time. These trends were similar to those observed under accelerated storage conditions at 25°C. An increase in aggregates and fragments with a decrease in monomers was observed by SE-HPLC. In addition, the purity decreased under this condition, as demonstrated by reduced and non-reduced CE-SDS analysis. A decrease in efficacy was also observed in CD28 cell-based bioassays. These results are consistent with the changes expected for protein therapeutics stored under this condition.

[0176] The research in the above-described examples was conducted to determine the formulation conditions that provide the maximum stability for hCD80-Fc. Therefore, studies on pH screening, buffer species selection, and excipient selection were carried out. hCD80-Fc was most stable in the pH range of 6.5 to 7.0. Also, hCD80-Fc was stable when sucrose was used as an excipient. A first formulation containing 10 mg / mL protein, 20 mM L-histidine, 270 mM sucrose, and 0.05% polysorbate 20 with a pH of 6.7 was selected. A second formulation containing 10 mg / mL protein, 20 mM L-histidine, 270 mM sorbitol, and 0.05% polysorbate 20 with a pH of 6.7 was also selected.

[0177] hCD80-Fc was stable in the first formulation even under freeze-thaw and agitation conditions. The first formulation containing hCD80-Fc is considered to be stable when stored at -70 °C as a formulation raw material or at 2 - 8 °C as a pharmaceutical for at least 12 months.

[0178] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications in addition to those described herein will be apparent to those skilled in the art in light of the above description and the accompanying drawings. The appended claims are intended to cover such variations.

[0179] All documents cited herein (e.g., publications or patents or patent applications) are hereby incorporated by reference in their entirety, and, in this specification, are incorporated by reference in their entirety as if each individual document (e.g., publication or patent or patent application) were specifically and individually recited herein.

[0180] The appended claims cover other embodiments.

[0181] Sequence Listing The following table provides a list of the specific sequences referenced herein.

Table 17-1

Table 17-2

Claims

1. A pharmaceutical composition comprising a CD80 extracellular domain (ECD) fragment crystallizable (Fc) fusion molecule, wherein the pharmaceutical composition comprises about 15 to about 25 mM of histidine, about 225 mM to about 300 mM of sugar, and about 0.025% to about 0.075% weight / volume (w / v) of polysorbate 20, wherein the sugar is sucrose or sorbitol, the pH is about 6.5 to 7.0, the composition contains less than 10% of the aggregated fusion molecule after 4 weeks at 40° C., or the composition contains about 1% to about 10% of the aggregated fusion molecule after 4 weeks at 40° C., said pharmaceutical composition.

2. The composition according to claim 1, wherein the composition contains about 0.5% to about 1.6% of the CD80 ECD-Fc fusion molecule of the fragment after 4 weeks at 40° C.

3. The composition according to claim 1 or 2, wherein the concentration of the histidine is about 20 mM.

4. The composition according to any one of claims 1 to 3, wherein the concentration of the sugar is about 270 mM.

5. The composition according to claim 1 or 2, wherein the concentration of the sugar is about 10 to about 15 times the concentration of the histidine.

6. The composition according to any one of claims 1 to 5, wherein the concentration of the polysorbate 20 is about 0.05% weight / volume (w / v).

7. The composition according to any one of claims 1 to 6, wherein the concentration of the CD80 ECD-Fc fusion molecule is about 5 mg / ml to about 15 mg / ml.

8. The composition according to claim 7, wherein the concentration of the CD80 ECD-Fc fusion molecule is about 10 mg / ml.

9. The composition according to any one of claims 1 to 8, wherein the pH of the composition is about 6.

7.

10. The composition according to any one of claims 1 to 9, wherein the composition is liquid.

11. The composition according to any one of claims 1 to 10, wherein the composition is for intravenous administration.

12. The composition according to any one of claims 1 to 11, comprising about 20 mM of histidine, about 270 mM of sucrose, about 10 mg / ml of the CD80 ECD-Fc fusion molecule, and about 0.05% of polysorbate 20, and having a pH of about 6.

7.

13. The composition according to any one of claims 1 to 11, wherein the sugar concentration is about 13.5 times the histidine concentration, the CD80 ECD-Fc fusion molecule is about 10 mg / ml, and polysorbate 20 is about 0.05%, and the pH is about 6.

7.

14. The composition according to any one of claims 1 to 11, comprising about 20 mM histidine, about 270 mM sorbitol, about 10 mg / ml of the CD80 ECD-Fc fusion molecule, and about 0.05% polysorbate 20, and the pH is about 6.

7.

15. The composition according to any one of claims 1 to 11, wherein the sorbitol concentration is about 13.5 times the histidine concentration, the CD80 ECD-Fc fusion molecule is about 10 mg / ml, and polysorbate 20 is about 0.05%, and the pH is about 6.

7.

16. The composition according to any one of claims 1 to 15, wherein the CD80 ECD-Fc fusion molecule comprises a human CD80 ECD and a human IgG1 Fc domain.

17. The composition according to any one of claims 1 to 16, wherein the composition comprises a sialylated CD80 ECD-Fc fusion molecule.

18. The composition according to claim 17, wherein the CD80 ECD-Fc fusion molecule comprises at least 15 moles of sialic acid (SA) per mole of the fusion protein.

19. The composition according to claim 18, wherein the CD80 ECD-Fc fusion molecule comprises 15 to 60 moles of SA per mole of the fusion protein.

20. The composition according to any one of claims 1 to 19, wherein the CD80 ECD-Fc fusion molecule comprises the amino acid sequence of SEQ ID NO:

5.

21. A syringe or vial containing the composition according to any one of claims 1 to 20.

22. The composition according to any one of claims 1 to 21 for use in the treatment of solid tumors in a human subject.

23. The composition for use according to claim 22, wherein the solid tumor is colorectal cancer, breast cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, squamous cell carcinoma of the head and neck, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatocellular carcinoma, bladder cancer, or endometrial cancer.

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