Pharmaceutical compositions comprising fusion proteins targeting pd-l1 and tgf-beta

Abstract

The present application provides a pharmaceutical composition comprising a fusion protein targeting PD-L1 and TGF-β and uses thereof. Specifically, the present application provides a pharmaceutical composition comprising (a) a fusion protein targeting PD-L1 and TGF-β, (b) a buffer system, (c) a surfactant, and (d) a stabilizer, and uses of the pharmaceutical composition in the preparation of an antitumor drug.

Description

[0001] All documents cited or referenced in this application (including, but not limited to, documents, patents, and patent applications), and all documents cited or referenced in the documents cited in this application, as well as any manufacturer's descriptions, specifications, and product pages of any products mentioned in this application or any documents incorporated herein by reference, are incorporated by reference and may be used in the practice of this invention. More specifically, all references cited in this application are incorporated by reference to the same extent as if each document were specifically and individually indicated to be incorporated herein by reference. Moreover, in any country, any reference to these publications in this application does not constitute an endorsement that such publications are common general knowledge in the art. Technical Field

[0002] This invention belongs to the field of pharmaceutical formulations. Specifically, this invention relates to pharmaceutical compositions comprising a fusion protein targeting PD-L1 and TGF-β and their uses. Background Technology

[0003] Programmed death-ligand 1 (PD-L1) is one of the ligands of programmed death receptor 1 (PD-1). After PD-L1 binds to PD-1 to form a receptor-ligand complex, it emits inhibitory signals, including inducing the production of IL-10 (an inflammatory and immunosuppressive factor), downregulating the anti-apoptotic gene bcl-2 to promote apoptosis of antigen-specific T cells, and inhibiting the proliferation of CD8+ T cells in lymph nodes. In addition to binding to PD-1 with a strong affinity, PD-L1 can also bind to CD80 with a weaker affinity, inhibiting T cell activity. Studies have found high expression of PD-L1 protein in various human tumor tissues, including breast cancer, lung cancer, gastric cancer, colorectal cancer, kidney cancer, and melanoma, enabling tumor cells to evade the immune system. Furthermore, the expression level of PD-L1 is closely related to the clinical condition and prognosis of the subjects. This indicates that PD-L1 is a highly promising target for tumor immunotherapy.

[0004] Transforming growth factor β (TGF-β) is a multifunctional cytokine that plays a crucial regulatory role in cell proliferation and differentiation, migration and adhesion, extracellular matrix production, angiogenesis and connective tissue formation, and immune function. TGF-β exhibits both tumor-suppressive and tumor-promoting effects; however, as tumors progress, it promotes tumor metastasis, immune escape, and induces tumor angiogenesis through the epithelial-mesenchymal transition (EMT), ultimately leading to disease progression. TGF-β forms a complex by recognizing TGF-β receptor II (TGFβRII), followed by the addition of TGFβRI in a dimer form, forming a heterotetrameric receptor complex for signal transduction. Studies have shown that inhibiting TGF-β signaling using the TGFβ receptor can reduce tumor metastasis, making it a key area of ​​focus in current cancer drug development.

[0005] Inhibitors of PD-L1 and other immune checkpoint proteins have low efficacy, providing only long-term effects in a subset of subjects and potentially leading to fatal autoimmune adverse events. Improving their therapeutic efficacy and reducing toxicity remains a key focus of research in immunotherapy. Studies have found that TGF-β is a significant cause of immune checkpoint inhibitor failure. Therefore, developing drugs that inhibit both the PD-L1 / PD-1 pathway and the TGF-β signaling pathway can effectively enhance anti-tumor efficacy, for example, through fusion proteins targeting PD-L1 and TGF-β. However, fusion protein drugs are large in molecular weight, structurally complex, and prone to degradation, polymerization, and / or undesirable chemical modifications, making them unstable. To ensure that fusion proteins are suitable for production and administration to patients, and to maintain their biological activity and stability during storage and subsequent use, there remains a need in the field to develop stable pharmaceutical compositions containing fusion proteins targeting PD-L1 and TGF-β. Summary of the Invention

[0006] The present invention provides a pharmaceutical composition comprising a fusion protein targeting PD-L1 and TGF-β, the pharmaceutical composition being capable of achieving stable effects and suitable for administration in subjects.

[0007] The pharmaceutical composition provided by the present invention comprises: (a) a fusion protein targeting PD-L1 and TGF-β, (b) a buffer system comprising one or more buffer solutions, (c) a surfactant, and (d) a stabilizer.

[0008] In some embodiments, the buffer system in the aforementioned pharmaceutical composition includes one or two of phosphate buffer, histidine buffer, Tris-HCl buffer, or citrate buffer.

[0009] In some embodiments, the buffer system in the aforementioned pharmaceutical composition comprises a first buffer, which is a phosphate buffer or a histidine buffer. In some embodiments, the buffer system in the aforementioned pharmaceutical composition comprises a first buffer, a phosphate buffer, and further comprises a second buffer, which is a histidine buffer, a Tris-HCl buffer, or a citrate buffer. In some embodiments, the buffer system in the aforementioned pharmaceutical composition comprises a first buffer, a histidine buffer, and further comprises a second buffer, which is a phosphate buffer, a Tris-HCl buffer, or a citrate buffer. In some embodiments, the buffer system in the aforementioned pharmaceutical composition comprises a first buffer, a phosphate buffer, and a second buffer, a histidine buffer.

[0010] In some specific embodiments, the phosphate buffer includes sodium phosphate buffer or potassium phosphate buffer, and the citrate buffer includes sodium citrate buffer, potassium citrate buffer or calcium citrate buffer.

[0011] In some embodiments, the buffer system in the aforementioned pharmaceutical composition includes one or two of sodium phosphate buffer, histidine buffer, Tris-HCl buffer, or sodium citrate buffer.

[0012] In some embodiments, the buffer system in the aforementioned pharmaceutical composition comprises a first buffer, which is a sodium phosphate buffer or a histidine buffer. In some embodiments, the buffer system in the aforementioned pharmaceutical composition comprises a first buffer, a sodium phosphate buffer, and further comprises a second buffer, which is a histidine buffer, a Tris-HCl buffer, or a sodium citrate buffer. In some embodiments, the buffer system in the aforementioned pharmaceutical composition comprises a first buffer, a histidine buffer, and further comprises a second buffer, which is a sodium phosphate buffer, a Tris-HCl buffer, or a sodium citrate buffer. In some embodiments, the buffer system in the aforementioned pharmaceutical composition comprises a first buffer, a sodium phosphate buffer, and a second buffer, a histidine buffer.

[0013] In some specific embodiments, the sodium phosphate buffer is composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, the sodium citrate buffer is composed of citric acid and sodium citrate, the Tris-HCl buffer is composed of Tris and hydrochloric acid, and the histidine buffer is composed of histidine, or histidine and histidine hydrochloride.

[0014] In some embodiments, the concentration of the first buffer solution in the aforementioned pharmaceutical composition is from 1 mM to 100 mM, preferably from 2 mM to 80 mM, more preferably from 5 mM to 60 mM, and even more preferably from 10 mM to 40 mM. In some embodiments, the concentration of the first buffer solution in the aforementioned pharmaceutical composition is about 10 mM. In some embodiments, the concentration of the first buffer solution in the aforementioned pharmaceutical composition is about 18 mM. In some embodiments, the concentration of the first buffer solution in the aforementioned pharmaceutical composition is about 20 mM. In some embodiments, the concentration of the first buffer solution in the aforementioned pharmaceutical composition is about 22 mM. In some embodiments, the concentration of the first buffer solution in the aforementioned pharmaceutical composition includes, but is not limited to: about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM.

[0015] In some embodiments, the concentration of the second buffer solution in the aforementioned pharmaceutical composition is from 1 mM to 100 mM, preferably from 2 mM to 80 mM, more preferably from 5 mM to 60 mM, and even more preferably from 10 mM to 40 mM. In some embodiments, the concentration of the second buffer solution in the aforementioned pharmaceutical composition is about 10 mM. In some embodiments, the concentration of the second buffer solution in the aforementioned pharmaceutical composition is about 18 mM. In some embodiments, the concentration of the second buffer solution in the aforementioned pharmaceutical composition is about 20 mM. In some embodiments, the concentration of the second buffer solution in the aforementioned pharmaceutical composition is about 22 mM. In some embodiments, the concentration of the second buffer solution in the aforementioned pharmaceutical composition includes, but is not limited to: about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM.

[0016] In some embodiments, the pH of the buffer system in the aforementioned pharmaceutical composition is 5 to 9, 6 to 9, preferably 6.5 to 8.5, and more preferably 6.5 to 8. In some embodiments, the pH of the buffer system in the aforementioned pharmaceutical composition is about 6.5. In some embodiments, the pH of the buffer system in the aforementioned pharmaceutical composition is about 7. In some embodiments, the pH of the buffer system in the aforementioned pharmaceutical composition is about 7.5. In some embodiments, the pH of the buffer system in the aforementioned pharmaceutical composition is about 8. In some embodiments, the pH of the buffer system in the aforementioned pharmaceutical composition includes, but is not limited to: about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.2, about 8.4, or about 8.5.

[0017] In some embodiments, the concentration of the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition is from 1 mg / mL to 200 mg / mL, preferably from 2 mg / mL to 150 mg / mL, more preferably from 5 mg / mL to 100 mg / mL, and more preferably from 10 mg / mL to about 60 mg / mL. In some embodiments, the concentration of the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition is about 20 mg / mL. In some embodiments, the concentration of the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition is about 25 mg / mL. In some embodiments, the concentration of the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition is about 40 mg / mL. In some embodiments, the concentration of the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition is about 50 mg / mL. In some embodiments, the concentration of the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition includes, but is not limited to: about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 80 mg / mL, or about 100 mg / mL.

[0018] In some embodiments, the surfactant in the aforementioned pharmaceutical composition is selected from polysorbates (such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85), poloxamers (such as poloxamer 181, poloxamer 188, poloxamer 407), polyethylene glycol, or polyhydroxylates. In some embodiments, the surfactant in the aforementioned pharmaceutical composition is polysorbate 80. In some embodiments, the surfactant in the aforementioned pharmaceutical composition is polysorbate 20.

[0019] In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is from 0.01 mg / mL to 1 mg / mL, preferably from 0.04 mg / mL to 0.8 mg / mL, more preferably from 0.06 mg / mL to 0.6 mg / mL, and even more preferably from 0.08 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.1 mg / mL. In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.2 mg / mL. In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition is about 0.4 mg / mL. In some embodiments, the concentration of the surfactant in the aforementioned pharmaceutical composition includes, but is not limited to: about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, or about 0.6 mg / mL.

[0020] In some embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sugars and / or other stabilizers, wherein the sugars comprise conventional compositions (CH2O)n or derivatives thereof, such as sucrose, trehalose, lactose, sorbitol, mannitol, sorbitol, etc.; the other stabilizers comprise sodium chloride, arginine or a pharmaceutically acceptable salt thereof, glycine, or proline, etc. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises trehalose, mannitol, or sucrose. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sucrose. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises arginine or a pharmaceutically acceptable salt thereof, glycine, or proline.

[0021] In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sugar, and / or other stabilizers selected from sodium chloride, arginine or a pharmaceutically acceptable salt thereof, glycine, or proline. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sucrose, and / or other stabilizers selected from sodium chloride, arginine or a pharmaceutically acceptable salt thereof, glycine, or proline. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises trehalose, and / or other stabilizers selected from sodium chloride, arginine or a pharmaceutically acceptable salt thereof, glycine, or proline. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises mannitol, and / or other stabilizers selected from sodium chloride, arginine or a pharmaceutically acceptable salt thereof, glycine, or proline. The arginine or its pharmaceutically acceptable salt includes arginine, arginine hydrochloride, or arginine acetate, etc. In some specific embodiments, the stabilizer in the aforementioned pharmaceutical composition comprises sucrose and arginine hydrochloride, or comprises sucrose and proline.

[0022] In some embodiments, the concentration of the sugar in the aforementioned pharmaceutical composition is from 10 mg / mL to 200 mg / mL, preferably from 20 mg / mL to 150 mg / mL, more preferably from 40 mg / mL to 120 mg / mL, and even more preferably from 50 mg / mL to 100 mg / mL. In some embodiments, the concentration of the sugar in the aforementioned pharmaceutical composition is about 70 mg / mL. In some embodiments, the concentration of the sugar in the aforementioned pharmaceutical composition is about 80 mg / mL. In some embodiments, the concentration of the sugar in the aforementioned pharmaceutical composition is about 85 mg / mL. In some embodiments, the concentration of the sugar in the aforementioned pharmaceutical composition is about 90 mg / mL. In some embodiments, the concentration of sugar in the aforementioned pharmaceutical composition includes, but is not limited to: about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL.

[0023] In some embodiments, the concentration of the other stabilizer in the aforementioned pharmaceutical composition is from 20 mM to 800 mM, preferably from 40 mM to 600 mM, more preferably from 60 mM to 500 mM, and even more preferably from 80 mM to 400 mM. In some embodiments, the concentration of the other stabilizer in the aforementioned pharmaceutical composition is about 100 mM. In some embodiments, the concentration of the other stabilizer in the aforementioned pharmaceutical composition is about 150 mM. In some embodiments, the concentration of the other stabilizer in the aforementioned pharmaceutical composition is about 200 mM. In some embodiments, the concentration of the other stabilizer in the aforementioned pharmaceutical composition is about 250 mM. In some embodiments, the concentrations of other stabilizers in the aforementioned pharmaceutical composition include, but are not limited to: about 20 mM, about 50 mM, about 60 mM, about 80 mM, about 100 mM, about 120 mM, about 140 mM, about 150 mM, about 160 mM, about 180 mM, about 200 mM, about 220 mM, about 240 mM, about 250 mM, about 260 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0024] In some embodiments, the pH of the aforementioned pharmaceutical composition is 5 to 9, 6 to 9, preferably 6.5 to 8.5, more preferably 6.5 to 8. In some embodiments, the pH of the buffer system in the aforementioned pharmaceutical composition is about 6.5. In some embodiments, the pH of the aforementioned pharmaceutical composition is about 7. In some embodiments, the pH of the aforementioned pharmaceutical composition is about 7.5. In some embodiments, the pH of the aforementioned pharmaceutical composition is about 8. In some embodiments, the pH of the aforementioned pharmaceutical composition includes, but is not limited to: about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.2, about 8.4, or about 8.5.

[0025] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 1 mg / mL to about 200 mg / mL; (b) a first buffer at a concentration of about 1 mM to about 100 mM; and / or a second buffer at a concentration of about 1 mM to about 100 mM; (c) a surfactant at a concentration of about 0.01 mg / mL to about 1 mg / mL; and (d) a sugar at a concentration of about 10 mg / mL to about 200 mg / mL; and / or other stabilizers selected from sodium chloride, arginine or pharmaceutically acceptable salts thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0026] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 2 mg / mL to about 150 mg / mL; (b) a first buffer at a concentration of about 2 mM to about 80 mM; and / or a second buffer at a concentration of about 2 mM to about 80 mM; (c) a surfactant at a concentration of about 0.04 mg / mL to about 0.8 mg / mL; and (d) a sugar at a concentration of about 20 mg / mL to about 150 mg / mL; and / or other stabilizers selected from sodium chloride, arginine or pharmaceutically acceptable salts thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0027] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 5 mg / mL to about 100 mg / mL; (b) a first buffer at a concentration of about 5 mM to about 60 mM; and / or a second buffer at a concentration of about 5 mM to about 60 mM; (c) a surfactant at a concentration of about 0.06 mg / mL to about 0.6 mg / mL; and (d) a sugar at a concentration of about 40 mg / mL to about 120 mg / mL; and / or other stabilizers selected from sodium chloride, arginine or pharmaceutically acceptable salts thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0028] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL; (b) a first buffer at a concentration of about 10 mM to about 40 mM; and / or a second buffer at a concentration of about 10 mM to about 40 mM; (c) a surfactant at a concentration of about 0.08 mg / mL to about 0.4 mg / mL; and (d) a sugar at a concentration of about 50 mg / mL to about 100 mg / mL; and / or other stabilizers selected from sodium chloride, arginine or pharmaceutically acceptable salts thereof, glycine or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0029] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 1 mg / mL to about 200 mg / mL; (b) a first buffer of about 1 mM to about 100 mM phosphate buffer; and / or a second buffer of about 1 mM to about 100 mM selected from histidine buffer, Tris-HCl buffer, or citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.01 mg / mL to about 1 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 10 mg / mL to about 200 mg / mL; and / or other stabilizers selected from sodium chloride, arginine, or pharmaceutically acceptable salts thereof, glycine, or proline at a concentration of about 20 mM to about 800 mM; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0030] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 2 mg / mL to about 150 mg / mL; (b) a first buffer of about 2 mM to about 80 mM phosphate buffer; and / or a second buffer of about 2 mM to about 80 mM selected from histidine buffer, Tris-HCl buffer, or citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.04 mg / mL to about 0.8 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 20 mg / mL to about 150 mg / mL; and / or another stabilizer selected from sodium chloride, arginine, or a pharmaceutically acceptable salt thereof, glycine, or proline at a concentration of about 40 mM to about 600 mM; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0031] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 5 mg / mL to about 100 mg / mL; (b) a first buffer of about 5 mM to about 60 mM phosphate buffer; and / or a second buffer of about 5 mM to about 60 mM selected from histidine buffer, Tris-HCl buffer, or citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.06 mg / mL to about 0.6 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 40 mg / mL to about 120 mg / mL; and / or other stabilizers selected from sodium chloride, arginine, or pharmaceutically acceptable salts thereof, glycine, or proline at a concentration of about 60 mM to about 500 mM; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0032] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL; (b) a first buffer of about 10 mM to about 40 mM phosphate buffer; and / or a second buffer of about 10 mM to about 40 mM selected from histidine buffer, Tris-HCl buffer, or citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.08 mg / mL to about 0.4 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 50 mg / mL to about 100 mg / mL; and / or another stabilizer selected from sodium chloride, arginine, or a pharmaceutically acceptable salt thereof, glycine, or proline at a concentration of about 80 mM to about 400 mM; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0033] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 1 mg / mL to about 200 mg / mL; (b) a first buffer, histidine buffer, at a concentration of about 1 mM to about 100 mM; and / or a second buffer, at a concentration of about 1 mM to about 100 mM selected from phosphate buffer, Tris-HCl buffer, or citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.01 mg / mL to about 1 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 10 mg / mL to about 200 mg / mL; and / or another stabilizer, at a concentration of about 20 mM to about 800 mM selected from sodium chloride, arginine, or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0034] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 2 mg / mL to about 150 mg / mL; (b) a first buffer, histidine buffer, at a concentration of about 2 mM to about 80 mM; and / or a second buffer, at a concentration of about 2 mM to about 80 mM, selected from phosphate buffer, Tris-HCl buffer, or citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.04 mg / mL to about 0.8 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 20 mg / mL to about 150 mg / mL; and / or another stabilizer, at a concentration of about 40 mM to about 600 mM, selected from sodium chloride, arginine, or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0035] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 5 mg / mL to about 100 mg / mL; (b) a first buffer, histidine buffer, at a concentration of about 5 mM to about 60 mM; and / or a second buffer, at a concentration of about 5 mM to about 60 mM, selected from phosphate buffer, Tris-HCl buffer, or citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.06 mg / mL to about 0.6 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 40 mg / mL to about 120 mg / mL; and / or another stabilizer, at a concentration of about 60 mM to about 500 mM, selected from sodium chloride, arginine, or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0036] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL; (b) a first buffer, histidine buffer, at a concentration of about 10 mM to about 40 mM; and / or a second buffer, at a concentration of about 10 mM to about 40 mM selected from phosphate buffer, Tris-HCl buffer, or citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.08 mg / mL to about 0.4 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 50 mg / mL to about 100 mg / mL; and / or another stabilizer, at a concentration of about 80 mM to about 400 mM selected from sodium chloride, arginine, or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0037] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 1 mg / mL to about 200 mg / mL; (b) a first buffer, sodium phosphate buffer, at a concentration of about 1 mM to about 100 mM; and / or a second buffer, at a concentration of about 1 mM to about 100 mM, selected from histidine buffer, Tris-HCl buffer, or sodium citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.01 mg / mL to about 1 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 10 mg / mL to about 200 mg / mL; and / or another stabilizer, at a concentration of about 20 mM to about 800 mM, selected from arginine or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0038] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 2 mg / mL to about 150 mg / mL; (b) a first buffer, sodium phosphate buffer, at a concentration of about 2 mM to about 80 mM; and / or a second buffer, selected from histidine buffer, Tris-HCl buffer, or sodium citrate buffer, at a concentration of about 2 mM to about 80 mM; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.04 mg / mL to about 0.8 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 20 mg / mL to about 150 mg / mL; and / or another stabilizer selected from arginine or a pharmaceutically acceptable salt thereof, glycine, or proline, at a concentration of about 40 mM to about 600 mM; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0039] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 5 mg / mL to about 100 mg / mL; (b) a first buffer, sodium phosphate buffer, at a concentration of about 5 mM to about 60 mM; and / or a second buffer, at a concentration of about 5 mM to about 60 mM, selected from histidine buffer, Tris-HCl buffer, or sodium citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.06 mg / mL to about 0.6 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 40 mg / mL to about 120 mg / mL; and / or another stabilizer, at a concentration of about 60 mM to about 500 mM, selected from arginine or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0040] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL; (b) a first buffer, sodium phosphate buffer, at a concentration of about 10 mM to about 40 mM; and / or a second buffer, selected from histidine buffer, Tris-HCl buffer, or sodium citrate buffer, at a concentration of about 10 mM to about 40 mM; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.08 mg / mL to about 0.4 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 50 mg / mL to about 100 mg / mL; and / or another stabilizer selected from arginine or a pharmaceutically acceptable salt thereof, glycine, or proline, at a concentration of about 80 mM to about 400 mM; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0041] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 1 mg / mL to about 200 mg / mL; (b) a first buffer, histidine buffer, at a concentration of about 1 mM to about 100 mM; and / or a second buffer, at a concentration of about 1 mM to about 100 mM selected from sodium phosphate buffer, Tris-HCl buffer, or sodium citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.01 mg / mL to about 1 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 10 mg / mL to about 200 mg / mL; and / or another stabilizer, at a concentration of about 20 mM to about 800 mM selected from arginine or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0042] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 2 mg / mL to about 150 mg / mL; (b) a first buffer, histidine buffer, at a concentration of about 2 mM to about 80 mM; and / or a second buffer, at a concentration of about 2 mM to about 80 mM, selected from sodium phosphate buffer, Tris-HCl buffer, or sodium citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.04 mg / mL to about 0.8 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 20 mg / mL to about 150 mg / mL; and / or another stabilizer, at a concentration of about 40 mM to about 600 mM, selected from arginine or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0043] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 5 mg / mL to about 100 mg / mL; (b) a first buffer, histidine buffer, at a concentration of about 5 mM to about 60 mM; and / or a second buffer, at a concentration of about 5 mM to about 60 mM, selected from sodium phosphate buffer, Tris-HCl buffer, or sodium citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.06 mg / mL to about 0.6 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 40 mg / mL to about 120 mg / mL; and / or another stabilizer, at a concentration of about 60 mM to about 500 mM, selected from arginine or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0044] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL; (b) a first buffer, histidine buffer, at a concentration of about 10 mM to about 40 mM; and / or a second buffer, at a concentration of about 10 mM to about 40 mM selected from sodium phosphate buffer, Tris-HCl buffer, or sodium citrate buffer; (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.08 mg / mL to about 0.4 mg / mL; and (d) trehalose, mannitol, or sucrose at a concentration of about 50 mg / mL to about 100 mg / mL; and / or another stabilizer, at a concentration of about 80 mM to about 400 mM selected from arginine or a pharmaceutically acceptable salt thereof, glycine, or proline; wherein the pH of the pharmaceutical composition is about 5 to about 9, about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 6.5 to about 8.

[0045] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 1 mg / mL to about 200 mg / mL; (b) a first buffer, sodium phosphate buffer, at a concentration of about 1 mM to about 100 mM; and / or a second buffer, histidine buffer, at a concentration of about 1 mM to about 100 mM; (c) polysorbate 80 at a concentration of about 0.01 mg / mL to about 1 mg / mL; and (d) sucrose at a concentration of about 10 mg / mL to about 200 mg / mL; and a stabilizer selected from arginine or a pharmaceutically acceptable salt thereof or proline at a concentration of about 20 mM to about 800 mM; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0046] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 2 mg / mL to about 150 mg / mL; (b) a first buffer, sodium phosphate buffer, at a concentration of about 2 mM to about 80 mM; and / or a second buffer, histidine buffer, at a concentration of about 2 mM to about 80 mM; (c) polysorbate 80 at a concentration of about 0.04 mg / mL to about 0.8 mg / mL; and (d) sucrose at a concentration of about 20 mg / mL to about 150 mg / mL; and a stabilizer selected from arginine or a pharmaceutically acceptable salt thereof or proline at a concentration of about 40 mM to about 600 mM; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0047] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 5 mg / mL to about 100 mg / mL; (b) a first buffer, sodium phosphate buffer, at a concentration of about 5 mM to about 60 mM; and / or a second buffer, histidine buffer, at a concentration of about 5 mM to about 60 mM; (c) polysorbate 80 at a concentration of about 0.06 mg / mL to about 0.6 mg / mL; and (d) sucrose at a concentration of about 40 mg / mL to about 120 mg / mL; and a stabilizer selected from arginine or a pharmaceutically acceptable salt thereof or proline at a concentration of about 60 mM to about 500 mM; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0048] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL, (b) a first buffer, sodium phosphate buffer, at a concentration of about 10 mM to about 40 mM, and / or a second buffer, histidine buffer, at a concentration of about 10 mM to about 40 mM, (c) polysorbate 80 at a concentration of about 0.08 mg / mL to about 0.4 mg / mL, and (d) sucrose at a concentration of about 50 mg / mL to about 100 mg / mL, and a stabilizer selected from arginine or a pharmaceutically acceptable salt thereof or proline at a concentration of about 80 mM to about 400 mM; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0049] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL, (b) a first buffer, sodium phosphate buffer, at a concentration of about 10 mM to about 40 mM, and / or a second buffer, histidine buffer, at a concentration of about 10 mM to about 40 mM, (c) polysorbate 20 or polysorbate 80 at a concentration of about 0.1 mg / mL to about 0.4 mg / mL, and (d) a phytohexidine buffer, at a concentration of about 50 mg / mL to about 100 mg / mL. The pharmaceutical composition contains trehalose, mannitol, or sucrose, and about 80 mM to about 400 mM of other stabilizers selected from arginine or its pharmaceutically acceptable salts or proline; wherein the pH of the pharmaceutical composition is 6.5 to 8.5, preferably 6.5 to 8, for example, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.2, about 8.4, or about 8.5.

[0050] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL, (b) a first buffer, sodium phosphate buffer, at a concentration of about 10 mM to about 40 mM, and / or a second buffer, histidine buffer, at a concentration of about 10 mM to about 40 mM, (c) polysorbate 80 at a concentration of about 0.1 mg / mL to about 0.4 mg / mL, and (d) sucrose at a concentration of about 50 mg / mL to about 100 mg / mL; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0051] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL, (b) a first buffer of about 20 mM sodium phosphate buffer, and / or a second buffer of about 20 mM histidine buffer, (c) about 0.2 mg / mL polysorbate 80, and (d) about 50 mg / mL to about 100 mg / mL sucrose, and about 80 mM to about 400 mM of other stabilizers selected from arginine, arginine hydrochloride, or proline; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0052] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 10 mg / mL to about 60 mg / mL, (b) a first buffer sodium phosphate buffer at a concentration of about 20 mM, and / or a second buffer histidine buffer at a concentration of about 20 mM, (c) polysorbate 80 at a concentration of about 0.2 mg / mL, and (d) sucrose at a concentration of about 50 mg / mL to about 100 mg / mL; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0053] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of about 20 mg / mL to about 50 mg / mL, (b) a first buffer, sodium phosphate buffer, at a concentration of about 20 mM, and / or a second buffer, histidine buffer, at a concentration of about 20 mM, (c) polysorbate 80 at a concentration of about 0.2 mg / mL, and (d) sucrose at a concentration of about 40 mg / mL to about 120 mg / mL or about 50 mg / mL to about 100 mg / mL, and a stabilizer selected from arginine, arginine hydrochloride, or proline at a concentration of about 80 mM to about 400 mM; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0054] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at about 20 mg / mL to about 50 mg / mL, (b) a first buffer sodium phosphate buffer at about 20 mM, and / or a second buffer histidine buffer at about 20 mM, (c) polysorbate 80 at about 0.2 mg / mL, and (d) sucrose at about 50 mg / mL to about 100 mg / mL; wherein the pH of the pharmaceutical composition is about 6.5 to about 8.5, preferably about 6.5 to about 8.

[0055] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 50 mg / mL of a fusion protein targeting PD-L1 and TGF-β, (b) about 20 mM of a first buffer sodium phosphate buffer, and / or about 20 mM of a second buffer histidine buffer, (c) about 0.2 mg / mL of polysorbate 80, and (d) about 80 mg / mL of sucrose, and about 100 mM of arginine hydrochloride; wherein the pH of the pharmaceutical composition is about 7.5.

[0056] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 50 mg / mL of a fusion protein targeting PD-L1 and TGF-β, (b) about 20 mM of a first buffer sodium phosphate buffer, and / or about 20 mM of a second buffer histidine buffer, (c) about 0.2 mg / mL of polysorbate 80, and (d) about 80 mg / mL of sucrose, and about 100 mM of arginine hydrochloride; wherein the pH of the pharmaceutical composition is about 6.5.

[0057] In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 50 mg / mL of a fusion protein targeting PD-L1 and TGF-β, (b) about 20 mM of a first buffer sodium phosphate buffer, and / or about 20 mM of a second buffer histidine buffer, (c) about 0.2 mg / mL of polysorbate 80, and (d) about 80 mg / mL of sucrose, and about 200 mM of proline; wherein the pH of the pharmaceutical composition is about 7.5. In some embodiments, the aforementioned pharmaceutical composition comprises: (a) about 50 mg / mL of a fusion protein targeting PD-L1 and TGF-β, (b) about 20 mM of a first buffer sodium phosphate buffer, and / or about 20 mM of a second buffer histidine buffer, (c) about 0.2 mg / mL of polysorbate 80, and (d) about 80 mg / mL of sucrose; wherein the pH of the pharmaceutical composition is about 7.5.

[0058] In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises:

[0059] (a) An antibody or antigen-binding fragment thereof that binds to human programmed death-ligand 1 (PD-L1) (e.g., at least one heavy chain variable domain and at least one light chain variable domain); and

[0060] (b) Human TGFβ binding domain.

[0061] In some embodiments, the human TGFβ binding domain is human TGFβRII or a TGFβ binding fragment thereof, such as a polypeptide or peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:36, or any fragment described herein.

[0062] In some embodiments, the heavy chain CDR1 of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:15, the heavy chain CDR2 contains the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:16, and the heavy chain CDR3 contains the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:17. The light chain CDR1 of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:18, the light chain CDR2 contains the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:19, and the light chain CDR3 contains the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:20.

[0063] In some embodiments, the antibody or its antigen-binding fragment comprises: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; or HCDR1 shown in SEQ ID NO:15, HCDR2 shown in SEQ ID NO:16, HCDR3 shown in SEQ ID NO:17, LCDR1 shown in SEQ ID NO:18, LCDR2 shown in SEQ ID NO:19, and LCDR3 shown in SEQ ID NO:20.

[0064] In some embodiments, the heavy chain variable domain of the antibody or its antigen-binding fragment has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:21, and the light chain variable domain of the antibody or its antigen-binding fragment has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:22. In some embodiments, the heavy chain variable domain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:21, and the light chain variable domain of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:22. In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain variable domain shown in SEQ ID NO:7 and the light chain variable domain shown in SEQ ID NO:8. In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain variable domain shown in SEQ ID NO:21 and the light chain variable domain shown in SEQ ID NO:22.

[0065]

[0066] In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition may further include a linker peptide that connects an antibody or its antigen-binding fragment to a human TGFβ-binding domain, for example, a linker peptide that connects the C-terminus of an antibody or its antigen-binding fragment to the N-terminus of a human TGFβ-binding domain. The linker peptide may be a flexible or rigid linker peptide, and optionally, it may be composed of a combination of glycine and serine, for example, (G4S). x G represents the linker peptide, where x is optionally an integer from 3 to 6, preferably 4 to 5, and most preferably 4. A linker peptide-free linking method can also be used.

[0067] In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition includes at least one light chain variable domain and at least one heavy chain variable domain of the antibody, wherein the light chain variable domain and the heavy chain variable domain combine to form an antigen-binding site that binds to human PD-L1.

[0068] In optional embodiments, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition may further comprise a constant region of an immunoglobulin, or a fragment, analog, variant, or derivative of said constant region. In some embodiments, said constant region is derived from human immunoglobulin heavy chains, such as IgG1, IgG2, IgG3, and IgG4, or heavy chains of other classes of immunoglobulins, preferably from the heavy chain of IgG1. In some embodiments, said constant region may comprise any modifications described herein, such as insertion, deletion, substitution, or chemical modification of amino acids. In some embodiments, said constant region comprises mutations that alter effector function, for example, mutating a lysine residue at the C-terminus of the antibody constant region to a hydrophobic amino acid, such as alanine or leucine, reducing proteolytic cleavage and increasing serum half-life. In some embodiments, any amino acid residue of said constant region may be substituted with any allotype amino acid residue of IgG1, preferably with amino acid residues of G1m(3) and / or nG1m(1).

[0069] In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises an antibody or antigen-binding fragment thereof that binds to human PD-L1, and a human TGFβ-binding domain. The antibody or antigen-binding fragment thereof may optionally comprise a modified constant region, such as any modification described herein, including a K-to-A mutation at the C-terminus of the constant region, or an allotype amino acid substitution; the human TGFβ-binding domain comprises human TGFβRII or a fragment or variant thereof capable of binding TGFβ, or an extracellular domain of human TGFβRII.

[0070] In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises (a) a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain variable domain amino acid sequence shown in SEQ ID NO:7 and a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable domain amino acid sequence shown in SEQ ID NO:8, or ...97%, 98%, 99%, or 100% identity with the light chain variable domain amino acid sequence shown in SEQ ID NO:8, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain variable domain amino acid sequence shown in SEQ ID NO:8. The heavy chain variable domain amino acid sequence shown in NO:21 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of the light chain variable domain shown in SEQ ID NO:22, and (b) human TGFβ binding domain. In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises (a) the heavy chain variable domain shown in SEQ ID NO:7 and the light chain variable domain shown in SEQ ID NO:8, or the heavy chain variable domain shown in SEQ ID NO:21 and the light chain variable domain shown in SEQ ID NO:22, and (b) a human TGFβ-binding domain. In some specific embodiments, the human TGFβ-binding domain is human TGFβRII or a TGFβ-binding fragment or variant thereof, or a polypeptide or peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:36.

[0071] EVQLVESGGGLVKPGGSLRLSCAASGFIFRSYGMSWVRQAPGKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYDCARGYDSGFAYWGQGTLVTVSS(SEQ ID NO:7);

[0072] DIVLTQSPASLAVSPGQRATITCRASQSVSTSSSSFMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSWEIPYTFGQGTKLEIK(SEQ ID NO:8);

[0073] QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGKALEWLGVIWRGVTTDYNAAFMSRLTITKDNSKNQVVLTMNNMDPVDTATYYCARLGFYAMDYWGQGTLVTVSS(SEQ ID NO:21);

[0074] DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYAANRYTGVPDRFSGSGYGTDFTFTISSLQPEDIATYFCQQDYTSPYTFGQGTKLEIK(SEQ ID NO:22);

[0075] IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ IDNO:36)。

[0076] In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (a) a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain amino acid sequence shown in SEQ ID NO:9 and a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain amino acid sequence shown in SEQ ID NO:10, or ...97%, 98%, 99%, or 100% identity with the light chain amino acid sequence shown in SEQ ID NO:10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain amino acid sequence shown in SEQ ID NO:10. The heavy chain amino acid sequence shown in NO:23 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain amino acid sequence shown in SEQ ID NO:24, and (b) a human TGFβ binding domain.In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (a) a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain amino acid sequence shown in SEQ ID NO:29 and a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain amino acid sequence shown in SEQ ID NO:10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain amino acid sequence shown in SEQ ID NO:10, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 98%, 99%, or 100% identity with the light chain amino acid sequence shown in SEQ ID NO:29. The heavy chain amino acid sequence shown in NO:32 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain amino acid sequence shown in SEQ ID NO:24, and (b) a human TGFβ binding domain. In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (a) the heavy chain shown in SEQ ID NO:9 and the light chain shown in SEQ ID NO:10, or the heavy chain shown in SEQ ID NO:23 and the light chain shown in SEQ ID NO:24, and (b) a human TGFβ-binding domain. In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (a) the heavy chain shown in SEQ ID NO:29 and the light chain shown in SEQ ID NO:10, or the heavy chain shown in SEQ ID NO:32 and the light chain shown in SEQ ID NO:24, and (b) a human TGFβ-binding domain. In some specific embodiments, the human TGFβ binding domain is human TGFβRII or its TGFβ binding fragment or variant, or a polypeptide or peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:36.

[0077] In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (1) a first polypeptide having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:30, or SEQ ID NO:33; and (2) a second polypeptide having an amino acid sequence having an identity with the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:25, SEQ ID NO:30, or SEQ ID NO:33; The amino acid sequence shown in NO:27 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (1) a first polypeptide having the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:30; and (2) a second polypeptide having the amino acid sequence shown in SEQ ID NO:13. In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (1) a first polypeptide having the amino acid sequence shown in SEQ ID NO:25 or SEQ ID NO:33; and (2) a second polypeptide having the amino acid sequence shown in SEQ ID NO:27.In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (1) two identical first polypeptides having an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:25, or an amino acid sequence thereof that is identical to the amino acid sequence shown in SEQ ID NO:30 or SEQ ID NO:25. (1) The amino acid sequence shown in NO:33 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:27. In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (1) two identical first polypeptides having the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:30; and (2) two identical second polypeptides having the amino acid sequence shown in SEQ ID NO:13. In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition comprises: (1) two identical first polypeptides having the amino acid sequence shown in SEQ ID NO:25 or SEQ ID NO:33; and (2) two identical second polypeptides having the amino acid sequence shown in SEQ ID NO:27.

[0078] The amino acid sequence of the first polypeptide, from N-terminus to C-terminus, consists of: the variable domain of the antibody heavy chain that recognizes the PD-L1 antigen epitope or antigen, the antibody constant region, the linker peptide, and the sequence of the TGFβ binding fragment of human TGFβRII.

[0079] In an optional embodiment, the fusion protein targeting PD-L1 and TGF-β in the aforementioned pharmaceutical composition is the fusion protein targeting PD-L1 and TGF-β as described in PCT / CN2020 / 111983.

[0080] The present invention also provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of contacting the aforementioned fusion protein targeting PD-L1 and TGF-β with a buffer system, for example, by displacing the fusion protein targeting PD-L1 and TGF-β into the buffer system. In one embodiment, the buffer system comprises a first buffer, phosphate buffer, and further comprises a second buffer, the second buffer being a histidine buffer, a citrate buffer, or a Tris-HCl buffer. In another embodiment, the buffer system comprises a first buffer, histidine buffer, and further comprises a second buffer, the second buffer being a phosphate buffer, a citrate buffer, or a Tris-HCl buffer. The concentration of the first buffer is 1 mM to 100 mM, preferably 2 mM to 80 mM, preferably 5 mM to 60 mM, more preferably 10 mM to 40 mM. The concentration of the second buffer is 1 mM to 100 mM, preferably 2 mM to 80 mM, preferably 5 mM to 60 mM, more preferably 10 mM to 40 mM. The pH of the buffer system is 5 to 9, 6 to 9, preferably 6.5 to 8.5, and more preferably 6.5 to 8. The method for preparing the aforementioned pharmaceutical composition further includes adding a stabilizer and a surfactant, in no particular order, wherein the stabilizer includes trehalose, mannitol, sucrose, sodium chloride, arginine or a pharmaceutically acceptable salt thereof, glycine and / or proline, etc., and the surfactant includes polysorbate 20 or polysorbate 80, etc.

[0081] This invention also provides a method for preparing a lyophilized formulation comprising a fusion protein targeting PD-L1 and TGF-β, comprising the step of lyophilizing the aforementioned pharmaceutical composition. In some embodiments, the lyophilization is performed according to methods known in the art, including but not limited to steps including pre-freezing, primary drying, and secondary drying. Those skilled in the art will understand that any method for removing water from the pharmaceutical composition of this invention is applicable to this invention.

[0082] The present invention also provides a lyophilized formulation comprising a fusion protein targeting PD-L1 and TGF-β, which is prepared by the aforementioned method for preparing lyophilized formulations.

[0083] The present invention also provides a lyophilized formulation comprising a fusion protein targeting PD-L1 and TGF-β, wherein the lyophilized formulation can be reconstituted to form the aforementioned pharmaceutical composition.

[0084] The present invention also provides an article comprising a container containing the aforementioned pharmaceutical composition or the aforementioned lyophilized preparation.

[0085] The pharmaceutical compositions or lyophilized formulations of the present invention can be administered according to known methods, such as by infusion or injection over a period of time in a suitable manner, for example, by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intra-articular routes, by local application, inhalation, or by continuous or delayed release.

[0086] The present invention also provides the use of the pharmaceutical compositions, lyophilized formulations or articles described herein in the preparation of medicaments for treating cancer, inhibiting tumor growth or enhancing antitumor responses. In some implementations, the cancers or tumors include, but are not limited to: squamous cell carcinoma of the lung, adenocarcinoma of the lung, mucinous adenoma, low-grade glioma of the brain, glioblastoma multiforme, mesothelioma, melanoma, sarcoma, soft tissue sarcoma, osteosarcoma, thyroid cancer, kidney cancer, liver cancer, acute myeloid leukemia, esophageal adenocarcinoma, esophageal squamous cell carcinoma, lymphoma, non-small cell lung cancer, breast cancer, pancreatic ductal carcinoma, pancreatic cancer, prostate cancer, colorectal cancer, colon cancer, gastric adenocarcinoma, gastric cancer, gastrointestinal stromal tumor, bile duct cancer, cholangiocarcinoma, gallbladder cancer, penile cancer, anal cancer, vaginal cancer, cervical cancer, endometrioid carcinoma of the uterine corpus, endometrial cancer, bladder cancer, human papillomavirus infection, head and neck cancer, skin cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasopharyngeal cancer, oropharyngeal cancer, pharyngeal cancer, paranasal sinus and nasal cavity cancer, and / or salivary gland cancer.

[0087] This application also provides a method for treating cancer, inhibiting tumor growth, or enhancing anti-tumor responses, comprising administering the pharmaceutical composition, lyophilized formulation, or article of the present invention. In some implementations, the tumors or cancers mentioned include, but are not limited to: squamous cell carcinoma of the lung, adenocarcinoma of the lung, mucinous adenoma, low-grade glioma of the brain, glioblastoma multiforme, mesothelioma, melanoma, sarcoma, soft tissue sarcoma, osteosarcoma, thyroid cancer, kidney cancer, liver cancer, acute myeloid leukemia, esophageal adenocarcinoma, esophageal squamous cell carcinoma, lymphoma, non-small cell lung cancer, breast cancer, pancreatic ductal carcinoma, pancreatic cancer, prostate cancer, colorectal cancer, colon cancer, gastric adenocarcinoma, gastric cancer, gastrointestinal stromal tumor, bile duct cancer, cholangiocarcinoma, gallbladder cancer, penile cancer, anal cancer, vaginal cancer, cervical cancer, endometrioid carcinoma of the uterine corpus, endometrial cancer, bladder cancer, human papillomavirus infection, head and neck cancer, skin cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasopharyngeal cancer, oropharyngeal cancer, pharyngeal cancer, paranasal sinus and nasal cavity cancer, and / or salivary gland cancer. Attached Figure Description

[0088] Figure 1 A schematic diagram of the structure of the anti-PD-L1 antibody-TGFβRII fusion protein, showing that the C-terminus of the anti-PD-L1 antibody heavy chain is linked to the N-terminus of the extracellular domain of the TGFβ receptor II via a (G4S)4G linker peptide.

[0089] Figure 2AReporter gene assay for detecting the PD-L1 end biological activity of hu5G11-hIgG1-TGFβRII; Figure 2B The reporter gene assay was used to detect the biological activity of the PD-L1 terminus of M7824.

[0090] Figure 3 The in vitro binding activity of hu5G11-hIgG1-TGFβRII to PD-L1 was detected by ELISA.

[0091] Figure 4 The in vitro binding activity of hu5G11-hIgG1-TGFβRII to TGF-β1 was detected by ELISA. Detailed Implementation

[0092] The following description of the present invention is only for illustrating various embodiments of the invention. Unless otherwise specified, the technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. Where a term in this application has multiple definitions, unless otherwise specified, the definition used in this section shall prevail.

[0093] Phosphate buffer is a buffer solution containing phosphate ions. Examples of phosphate buffers include sodium phosphate buffer, potassium phosphate buffer, etc., with sodium phosphate buffer being the preferred phosphate buffer. For example, sodium phosphate buffer is prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0094] "Citrate buffer" is a buffer solution containing citrate ions. Examples of citrate buffers include sodium citrate buffer, potassium citrate buffer, calcium citrate buffer, etc., with sodium citrate buffer being the preferred citrate buffer. For example, sodium citrate buffer is prepared from citric acid and sodium citrate.

[0095] "Tris-HCl buffer" is prepared by mixing Tris (tromethamine) and hydrochloric acid.

[0096] "Histidine buffer" is a buffer solution containing histidine ions. Examples of histidine buffers include histidine buffer, histidine-acetic acid buffer, histidine-hydrochloric acid buffer, histidine-histidine hydrochloride buffer, etc. For example, the histidine buffer suitable for use in this invention is prepared from histidine (L-Histidine) and does not contain acetic acid or hydrochloric acid. The histidine buffer suitable for use in this invention can also be composed of histidine and histidine hydrochloride.

[0097] "Buffer solution" or "buffer system" refers to a pharmaceutically acceptable method for maintaining the pH of a pharmaceutical composition to a desired pH range. Buffer systems suitable for use in this invention include at least one buffer solution, such as one or more combinations of phosphate buffer, citrate buffer, Tris-HCl buffer, and histidine buffer. In some embodiments, the buffer system suitable for use in this invention comprises both phosphate buffer and histidine buffer.

[0098] The term "pharmaceutical composition" is intended to encompass products containing a specific active ingredient (e.g., a fusion protein) in optionally specific amounts, and any product directly or indirectly generated by combining a specific active ingredient in optionally specific amounts. The purpose of a pharmaceutical composition is to make the fusion protein suitable for production and administration to patients, and to maintain its biological activity and / or stability during storage and subsequent use. In some embodiments, the pharmaceutical composition is a water-soluble injection, including but not limited to un-lyophilized water-soluble formulations or water-soluble formulations reconstituted from lyophilized powders. In other embodiments, the pharmaceutical composition is a lyophilized formulation. In this invention, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0099] A “stable” or “stabilized” pharmaceutical composition is one in which the active ingredient (e.g., a fusion protein) substantially retains its physical and / or chemical stability and / or biological activity when stored therein. Various analytical techniques exist in the art for measuring the stability of active ingredients, as reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at selected temperatures and other storage conditions over selected time periods. For example, if the active ingredient does not show significant increase in aggregation, precipitation, and / or denaturation after visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and differential scanning calorimetry (DSC), then the active ingredient “retains its physical stability” in the pharmaceutical composition. Preferably, when using the pharmaceutical compositions of the present invention, 5% or less, 4% or less, preferably 3% or less of the active ingredient forms aggregates (also known as high molecular weight impurities) when measured by SEC-UPLC or any other suitable method for measuring aggregate formation. If the active ingredient (e.g., a fusion protein) does not show significant chemical changes, then the active ingredient “retains its chemical stability” in the pharmaceutical composition. Chemical stability can be assessed by detecting and quantifying the form of chemical changes. Processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing electrophoresis, peptide mapping, isoaspartate measurement, etc.), and isomerization (evaluated by measuring isoaspartate content, peptide mapping, etc.). If the biological activity of the active ingredient (e.g., a fusion protein) is within a predetermined range of the biological activity exhibited when the pharmaceutical composition is prepared, then the active ingredient “retains its biological activity” in the pharmaceutical composition for a given time, as determined, for example, by an antigen binding assay.

[0100] "Polymer impurities" or "aggregates" refer to the general term for impurities with a molecular weight greater than that of the target active ingredient (such as a fusion protein).

[0101] The articles “a,” “an,” and “the” are used here to refer to one or more of the grammatical objects of the article (i.e., at least one). For example, “a pharmaceutical composition” refers to one or more pharmaceutical compositions.

[0102] "About" or "approximately" means a value within the acceptable error range of a specific value determined by a person skilled in the art, the value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in the art, "about" or "approximately" may mean within or above 1 standard deviation. Or "about" or "approximately" may mean the value plus or minus a range of 15%, 10%, 5%, or 1%. Furthermore, particularly for biological systems or processes, the term may mean at most one order of magnitude or at most five times the value. Unless otherwise stated herein, "about XX" or "approximately XX" or "substantially including 'XX'" means a value within the acceptable error range of the specific value "XX" (including the value "XX" itself, and values ​​within the acceptable error range determined by a person skilled in the art).

[0103] As stated herein, any percentage range, ratio range, or integer range should be understood to include the value of any integer within the enumerated range, and, where appropriate, its fractions (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0104] Throughout this application, unless the context otherwise requires, the words “comprising,” “including,” and “containing” will be understood to mean including the stated steps or elements or a group of steps or elements, but not excluding any other steps or elements or a group of steps or elements. “Comprising…” means including and limited to what follows the phrase “comprising…”. Therefore, the phrase “comprising…” indicates that the listed elements are necessary or required, and no other elements may be present. “Substantially comprising…” means including any elements listed following this phrase, and is limited to other elements that do not impede or contribute to the activity or function of the listed elements as detailed herein. Therefore, the phrase “substantially comprising…” indicates that the listed elements are necessary or required, but other elements are optional and may be present or absent depending on whether they affect the activity or function of the listed elements.

[0105] “TGFβRII” or “TGFβ receptor II” refers to a polypeptide or protein having the same type B sequence as wild-type human TGFβ receptor 2, such as the polypeptide shown in SEQ ID NO:35, or a polypeptide having a sequence substantially identical to the amino acid sequence shown in SEQ ID NO:35.

[0106] The term "TGFβ-binding fragment" or "TGFβ-binding fragment" in TGFβRII refers to a fragment of TGFβRII that has TGFβ-binding activity, comprising at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, 99%, or 100% of the TGFβRII sequence. This fragment is typically a soluble fragment, such as the extracellular domain of human TGFβRII or a variant thereof.

[0107] As used herein, the term "antibody" refers to a binding protein having at least one antigen-binding domain. The antibodies and fragments thereof in this application may be whole antibodies or any fragment thereof. Therefore, the antibodies and fragments thereof in this application include monoclonal antibodies or fragments thereof and antibody variants or fragments thereof, as well as immunoconjugates. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab)'2 fragments, Fv fragments, Fd fragments, Fd' fragments, isolated CDR regions, single-chain Fv molecules (scFv), and other antibody fragments known in the art, as well as antibodies modified in any way known in the art (e.g., glycosylation, chemical modification, etc.). Antibodies and fragments thereof may also include recombinant peptides, fusion proteins, and bispecific antibodies. The anti-PD-L1 antibodies and fragments thereof disclosed herein may be IgG1, IgG2, IgG3, or IgG4 isotypes. The term "isotype" refers to the type of antibody encoded by the heavy chain constant region gene.

[0108] "Antigen-binding fragment" refers to a fragment that retains the antigen-binding function of a full-length antibody, including Fab, Fab', F(ab')2, scFv, Fv, Fd, Fd', isolated CDR regions and single-domain VHH fragments, and other antibody fragments known in the art, or fragments that have been modified in any way known in the art.

[0109] As used herein, the term "EC50" refers to the effective concentration, the 50% maximum response of the antibody. As used herein, the term "IC50" refers to the inhibitory concentration, the 50% maximum response of the antibody. Both EC50 and IC50 can be measured by ELISA or FACS analysis or any other method known in the art.

[0110] "Identity" refers to the similarity between two reference sequences. The identity percentage refers to the percentage obtained by comparing sequences or specified regions of sequences using sequence comparison algorithms well known to those skilled in the art, such as the default settings of the BLASTN / BLASTP algorithm available on the National Center for Biotechnology Institute website.

[0111] "Substantially identical" means that sequences have at least about 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of identity.

[0112] "Subject" refers to mammals, including humans and non-human animals, with humans being the preferred subject.

[0113] "Treatment" includes therapeutic treatment as well as preventive treatment or preventive measures, which reduce or alleviate the development of at least one symptom of the disease, disorder, or condition (e.g., cancer or tumor) by administering a therapeutic agent to the subject.

[0114] "Cancer" refers to a collection of cells that proliferate in an abnormal manner.

[0115] Example

[0116] For clarity, this application is further illustrated with examples, but these examples are not intended to limit the scope of this application. The fusion protein hu5G11-hIgG1-TGFβRII in the examples is hu5G11-hIgG1-TGFβRII as described in PCT / CN2020 / 111983.

[0117] Example 1: Construction and expression of transient transgenic expression plasmid of fusion protein

[0118] Construct a fusion protein hu5G11-hIgG1-TGFβRII targeting PD-L1 and TGF-β, such as... Figure 1 As shown. The light chain of the molecule has the amino acid sequence of SEQ ID NO:27; the heavy chain of the molecule (SEQ ID NO:33) is a fusion protein comprising the heavy chain of the anti-human PD-L1 antibody (SEQ ID NO:32), the extracellular domain amino acid sequence of human TGFβRII (SEQ ID NO:36), and a (G4S)4G linker peptide (SEQ ID NO:37) linking the former two.

[0119] The amino acid sequence of the hu5G11-hIgG1-TGFβRII light chain (SEQ ID NO:27):

[0120] DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYAANRYTGVPDRFSGSGYGTDFTFTISSLQPEDIATYFCQQDYTSPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0121] Amino acid sequence of hu5G11-hIgG1-TGFβRII heavy chain (SEQ ID NO:33):

[0122] QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGKALEWLGVIWRGVTTDYNAAFMSRLTITKDNSKNQVVLTMNNMDPVDTATYYCARLGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0123] Amino acid sequence of PD-L1 antibody heavy chain (SEQ ID NO:32):

[0124] QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGKALEWLGVIWRGVTTTDYNAAFMSRLTITKDNSKNQVVLTMNNMDPVDTATYYCARLGFYAMDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA

[0125] The amino acid sequence of the extracellular domain of human TGFβRII (SEQ ID NO:36):

[0126] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0127] The amino acid sequence of the linker peptide (SEQ ID NO:37):

[0128] GGGGSGGGGSGGGGSGGGGSG

[0129] The heavy chain nucleic acid sequence (SEQ ID NO:34) and light chain nucleic acid sequence (SEQ ID NO:28) of hu5G11-hIgG1-TGFβRII were synthesized using the whole genome and ligated into a eukaryotic expression vector, preferably pcDNA3.1(+), to construct the target expression plasmid. ExpiFectamine was used. TM CHO transfection technology introduces expression plasmids of heavy and light chains into ExpiCHO-S TMIntracellularly, the target protein is transiently expressed, and the obtained cell culture supernatant is used for protein purification.

[0130] Example 2: Isolation, purification, and identification of the fusion protein

[0131] The cell culture supernatant obtained in Example 1 was loaded onto a protein A affinity chromatography column, such as GE's MabSelect. The column was equilibrated with a phosphate-containing equilibration buffer (e.g., 10 mM phosphate buffer, pH = 6.0), followed by elution with a sodium chloride-containing eluent (e.g., 25 mM phosphate buffer containing 0.5 M sodium chloride, pH = 7.0–7.4). Finally, the target protein product bound to the column was eluted with an elution buffer to obtain hu5G11-hIgG1-TGFβRII. The elution can be achieved using conventional methods, such as high-salt buffers or pH adjustments, for example, using 1 M arginine hydrochloride (pH = 3–4) or 50 mM citrate buffer (pH = 3–4). The phosphate buffer was prepared using disodium hydrogen phosphate and sodium dihydrogen phosphate, and the citrate buffer was prepared using citric acid and trisodium citrate. The elution buffer was quantified using UV analysis.

[0132] Example 3: In vitro binding affinity detection of fusion proteins

[0133] The affinity of hu5G11-hIgG1-TGFβRII, M7824, for PD-L1 and TGFβ was analyzed using a Biacore T200 analyzer. M7824 is the anti-PD-L1 / TGFβ trap described in CN106103488, and its heavy and light chain sequences are shown in SEQ ID NOs:42 and 43 of this application, respectively. After synthesizing the DNA sequences encoding the heavy and light chains, they were cloned into expression vectors, and the light and heavy chain expression vectors were co-transfected into ExpiCHO-S TM In this process, the cells were incubated in an 8% CO2, 37°C incubator, and then the culture supernatant was purified using protein A packing material according to the method described in Example 2 to obtain M7824.

[0134] 1) Detection of the affinity between the fusion protein and PD-L1: Anti-hIgG1(Fc)Antibody was coupled to the surface of a CM5 chip via amino-coupling, with a coupling amount of approximately 8000–9000 RU. The CM5 chip was used to capture hu5G11-hIgG1-TGFβRII and M7824, respectively. Then, PD-L1 protein (Sinobiological, 10084-H08H) was diluted to 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM using running buffer. The interaction signals between different concentrations of PD-L1 protein and the blank control (running buffer) with hu5G11-hIgG1-TGFβRII and M7824 were measured to obtain binding and dissociation curves. The mixture was then regenerated with 3 mol / L MgCl2 buffer until the baseline stabilized.

[0135] 2) Detection of the affinity between the fusion protein and TGFβ: Anti-hIgG1(Fc)Antibody was coupled to the surface of a CM5 chip via amino-coupling, with a coupling amount of approximately 8000–9000 RU. The CM5 chip was used to capture hu5G11-hIgG1-TGFβRII and M7824, respectively. Then, the TGFβ protein (Sinobiological, 10804-H08H) was diluted to 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM using running buffer. The interaction signals between different concentrations of TGFβ protein and the blank control (running buffer) with hu5G11-hIgG1-TGFβRII and M7824 were measured to obtain binding and dissociation curves. The mixture was then regenerated with 3 mol / L MgCl2 buffer until the baseline stabilized.

[0136] BiaControl Software 2.0 acquires data signals in real time, and BiaEvaluation Software 2.0 performs data analysis. After data subtraction (i.e., the blank control signal is subtracted from the sample signal in each cycle), the data is fitted with the Langmuir 1:1 model to calculate the binding rate constant Ka, dissociation rate constant Kd, and equilibrium constant KD.

[0137] Table 1. Affinity of each test sample to PD-L1 and TGFβ

[0138]

[0139] As can be seen from the table above, compared with the control M7824, hu5G11-hIgG1-TGFβRII exhibits smaller KD and Kd values, meaning that hu5G11-hIgG1-TGFβRII has higher affinity for both PD-L1 and TGFβ.

[0140] Example 4: Detection of PD-L1 terminal biological activity of hu5G11-hIgG1-TGFβRII using reporter gene assay

[0141] CHO-PDL1-CD3L cells (China National Institutes for Food and Drug Control) were used, and the viable cell density was adjusted to 4–5 × 10⁻⁶ cells / mL using CHO-PDL1-CD3L complete culture medium. 5 Jurkat-PD-1-NFAT cells (China National Institutes for Food and Drug Control) in the logarithmic growth phase were added to 100 μL per well of a 96-well plate of whole white blood cells and incubated at 37°C in a 5% CO2 cell culture incubator. After incubation for 16–20 h, Jurkat-PD-1-NFAT cells (China National Institutes for Food and Drug Control) in the logarithmic growth phase were harvested and the viable cell density was adjusted to 1.25–2 × 10⁻⁶ cells / mL using analytical medium. 6 cells / mL. Remove the above 96-well whole white blood cell plate, discard the supernatant, and add 50 μL / well of Jurkat-PD-1-NFAT cell suspension. Pre-dilute hu5G11-hIgG1-TGFβRII to a concentration of 56 nmol / L, then perform 2-fold serial dilutions for a total of 10 dilution gradients (i.e., dilute to approximately 56 nmol / L, approximately 28 nmol / L, approximately 14 nmol / L, approximately 7 nmol / L, approximately 3.5 nmol / L, approximately 1.75 nmol / L, approximately 0.875 nmol / L, approximately 0.4375 nmol / L, approximately 0.21875 nmol / L, and approximately 0.109375 nmol / L); pre-dilute PD-L1 antibody to a concentration of 68 nmol / L, then perform 2-fold dilutions for a total of 10 dilution gradients; pre-dilute M7824 to a concentration of 56 nmol / L, then perform 2-fold dilutions for a total of 10 dilution gradients. 50 μL of serially diluted hu5G11-hIgG1-TGFβRII, PD-L1 antibody, and M7824 were added to each well of the cell culture plate described above, and incubated at 37°C in a 5% CO2 cell culture incubator for 6 h. 100 μL of Bio-Glo Luciferase reagent (Promega, G7940) was added to each well of the cell culture plate, and incubated at room temperature in the dark for 2–3 min. Relative optical units (RLU) were read using a Thermo, Varioskan Flash microplate reader. The PD-L1 end biological activity results of hu5G11-hIgG1-TGFβRII are shown below. Figure 2A As shown in Table 2. The results in Table 2 indicate that hu5G11-hIgG1-TGFβRII has higher PD-L1 end biological activity.

[0142] Biological activity (%) of test sample = (EC50 value of reference sample / EC50 value of test sample) × 100%

[0143] Table 2. Reporter gene assay for PD-L1 terminal biological activity in each test sample.

[0144] sample biological activity EC50 (nmol / L) PD-L1 antibody 100％ 4.603 hu5G11-hIgG1-TGFβRII 75.4％ 6.106 sample biological activity EC50 (nmol / L) PD-L1 antibody 100％ 4.151 M7824 72.5％ 5.724

[0145] Note: The PD-L1 antibody (reference sample) is the humanized 5G11-IgG1 antibody in CN107001463, and the heavy chain and light chain sequences are shown in SEQ ID NOs:23 and 24 of this application, respectively; M7824 is the anti-PD-L1 / TGFβ trap in CN106103488, and the heavy chain and light chain sequences are shown in SEQ ID NOs:42 and 43 of this application, respectively. The preparation method is described in Example 3.

[0146] Example 5: Enzyme-linked immunosorbent assay (ELISA) for detecting the in vitro binding activity of hu5G11-hIgG1-TGFβRII with PD-L1 and TGF-β, and the in vitro binding assay of the fusion protein with PD-L1.

[0147] 1) Coating: Dilute PD-L1 recombinant protein (Sinobiological, 10084-H08H) to 2 μg / mL with PBS, and coat 100 μL / well in a 96-well plate. Incubate overnight at 4°C; 2) Wash the 96-well plate once with PBS containing 0.05% Tween 20, add 250 μL of blocking buffer (PBS containing 3% BSA) to each well, and block for 1–2 hours; 3) Coat with diluent (containing 1% BSA and 0.05% Tween 20). Pre-dilute hu5G11-hIgG1-TGFβRII to approximately 4000 ng / mL using PBS solution containing 20 g of Tween, then perform 4-fold serial dilutions, resulting in 7 dilution gradients (i.e., approximately 4000 ng / mL, approximately 1000 ng / mL, approximately 250 ng / mL, approximately 62.5 ng / mL, approximately 15.625 ng / mL, approximately 3.90625 ng / mL, and approximately 0.9765625 ng / mL); wash the 96-well plate three times with PBS solution containing 0.05% Tween, then add the serially diluted hu5G11-hIgG1-TGFβRII and blank control, 100 μL / well, and incubate at 25°C for 1–2 hours; 4) add 0.05% Tween... Wash the 96-well plate three times with PBS solution containing 20% ​​Tween 20, add 100 μL of HRP-labeled goat anti-human secondary antibody (PE, NEF802001EA) diluted 1:3500 to each well, and incubate at 25°C for 1 hour; 5) Wash the 96-well plate three times with PBS solution containing 0.05% Tween 20, add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) to each well, and incubate at 25°C in the dark for 5 minutes; finally, add 1M H2SO4 to terminate the reaction; 6) Detect the absorbance at 450 nm using a microplate reader (Thermo Scientific, Varioskan Flash), and calculate the EC50 value of hu5G11-hIgG1-TGFβRII with the average absorbance value as the ordinate and the logarithm of the hu5G11-hIgG1-TGFβRII concentration as the abscissa.

[0148] In vitro binding assay of fusion protein to TGF-β

[0149] 1) Coating: Dilute TGF-β1 recombinant protein (Sinobiological, 10804-H08H) with PBS to 2 μg / mL, coat 100 μL / well in a 96-well plate, and incubate overnight at 4°C; other steps are as described above.

[0150] The results of hu5G11-hIgG1-TGFβRII binding to PD-L1 in vitro are as follows: Figure 3 As shown in Table 3, the results of in vitro binding of TGF-β1 are as follows: Figure 4As shown in Table 3, the ELISA results indicate that it retains its binding activity with PD-L1 and TGF-β.

[0151] Table 3. EC50 values ​​of hu5G11-hIgG1-TGFβRII binding to PD-L1 and TGFβ in ELISA assays.

[0152] PD-L1 terminal TGF-β end sample <![CDATA[EC 50 (ng / mL)]]> <![CDATA[EC 50 (ng / mL)]]> PD-L1 / TGFβRII 34.31 67.03

[0153] Example 6 Pharmacokinetic Analysis

[0154] 1) Single-dose pharmacokinetic study: This study used cynomolgus monkeys as animal models and randomly divided them into 3 groups of 6 monkeys in each group, half male and half female. Each group was given a single intravenous infusion of 1, 10, and 60 mg / kg of hu5G11-hIgG1-TGFβRII. Whole blood was collected intravenously at 0 h before administration and at 1 min, 3 h, 8 h, 24 h, 48 h, 72 h, 120 h, 168 h, 216 h, 264 h, 336 h, 504 h, and 672 h after administration. The supernatant was separated and collected, and the blood drug concentration in the supernatant was detected by ELISA. The results are shown in Table 4.

[0155] 2) Repeated-dose pharmacokinetic study: This study used cynomolgus monkeys as animal models, divided into 3 groups of 10 monkeys in each group, half male and half female. They were given intravenous infusions of 20, 60, and 200 mg / kg of hu5G11-hIgG1-TGFβRII once a week for 4 consecutive weeks (5 times in total). Blood was collected intravenously at 0 h before the first dose and at 1 min, 3 h, 8 h, 24 h, 48 h, 72 h, 120 h, and 168 h after the start of the first dose; at 0 h before the third and fourth doses and at 1 min after the start of the first dose; and at 0 h before the fifth dose and at 1 min, 3 h, 8 h, 24 h, 48 h, 72 h, 120 h, 168 h, 216 h, 264 h, 336 h, 504 h, and 672 h after the start of the fifth dose. The supernatant was separated and collected, and the blood drug concentration in the supernatant was detected by ELISA. The results are shown in Table 5. The results in Table 5 show that no significant accumulation of hu5G11-hIgG1-TGFβRII was observed in the serum.

[0156] The ELISA procedure is as follows:

[0157] 1) Coating: Human PD-L1 / B7-H1 / CD274 Protein, Fc ta (Sinobiological, LC11NO24O2) was diluted to 1 μg / mL with PBS, and 100 μL / well was coated into a 96-well plate and incubated overnight at 4°C; 2) The 96-well plate was washed three times with PBS containing 0.05% Tween 20, and 300 μL of blocking buffer (PBS containing 0.05% Tween 20 and 1% BSA) was added to each well, and the plate was blocked at 25°C for 2–3 hours; 3) The 96-well plate was washed three times with PBS containing 0.05% Tween 20, and 100 μL of blank control sample, standard curve sample, and test sample were added to each well, respectively, and the plate was incubated at 25°C for 1–1.5 hours; 4) PBS containing 0.05% Tween 20 was added to each well. 5) Wash the 96-well plate three times with PBS solution containing 0.05% Tween 20. Add 100 μL of Human TGF-βRII Biotinylated Antibody (R&D, XL0519051) diluted with blocking buffer to a final concentration of 0.05 μg / mL to each well and incubate at 25°C for 1–1.5 hours; 6) Wash the 96-well plate three times with PBS solution containing 0.05% Tween 20. Add 100 μL of Streptavidin-HRP diluted 1:200 with blocking buffer to each well and incubate at 25°C for 30 minutes; 7) Wash the 96-well plate three times with PBS solution containing 0.05% Tween 20. Add 100 μL of TMB substrate chromogenic solution to each well and incubate at 25°C in the dark for 5–10 minutes; finally, add 100 μL of 0.5 M H2SO4 to each well to terminate the reaction; 8) Detect the absorbance value at 450 nm using a microplate reader.

[0158] Table 4. Pharmacokinetic parameters of a single dose of hu5G11-hIgG1-TGFβRII

[0159]

[0160] Table 5. Pharmacokinetic parameters of repeated doses of hu5G11-hIgG1-TGFβRII

[0161]

[0162] Example 7: Inhibitory effect of fusion protein on mouse MC38 xenografts

[0163] The DNA sequence of IgG1 was synthesized and cloned into the pcDNA3.1 expression vector. The light and heavy chain expression vectors were co-transfected into the ExpiCHO-S vector using the ExpiCHO transfection kit (Thermo Fisher, A29133). TMCells were cultured in an 8% CO2, 37°C incubator. The culture supernatant was purified using protein A packing material according to the method described in Example 2 to obtain IgG1. The heavy and light chain amino acid sequences of IgG1 are shown in SEQ ID NO:38 and SEQ ID NO:39 of this application.

[0164] The DNA sequence of IgG1-TGFβRII was synthesized and cloned into the pcDNA3.1 expression vector. The light and heavy chain expression vectors were co-transfected into the ExpiCHO-S vector using the ExpiCHO transfection kit (Thermo Fisher, A29133). TM Cells were cultured in an 8% CO2, 37°C incubator. The culture supernatant was purified using protein A packing material according to the method described in Example 2 to obtain IgG1-TGFβRII. The heavy and light chain amino acid sequences of IgG1-TGFβRII are shown in SEQ ID NO:40 and SEQ ID NO:41 of this application.

[0165] PD-L1 humanized mice were subcutaneously inoculated with 3×10 5 A mouse colon cancer model was established using MC38 / hPD-L1 cells per mouse. Tumors were allowed to grow to 50–70 mm. 3 Based on tumor volume, animals were grouped into 10 groups and intravenously injected with (IV) hu5G11-hIgG1-TGFβRII, IgG1, and IgG1-TGFβRII every 2 days for a total of 6 times, with an injection volume of 0.1 mL / 10 g body weight. Tumor diameter was measured twice weekly using calipers. The T / C% or tumor inhibition rate (TGI%) was calculated using the following formula to assess the effect of the drugs on tumor growth. The experiment ended, the experimental endpoint was reached, or the tumor volume reached 1500 mm². 3 Animals were euthanized with CO2 anesthesia, then dissected to remove the tumor and photographed. The tumor volume (V) was calculated using the formula: V = 1 / 2 × a × b 2 , where a and b represent length and width respectively; T / C (%) = (T-T0) / (C-C0) × 100, where T and C are the tumor volumes of the treatment group and the isotype control group at the end of the experiment; T0 and C0 are the tumor volumes of the treatment group and the isotype control group at the beginning of the experiment; tumor inhibition rate (TGI) (%) = 100 - T / C.

[0166] The results are shown in Table 6. hu5G11-hIgG1-TGFβRII (3.7 and 12.3 mg / kg) significantly inhibited the growth of MC38 / hPD-L1 subcutaneous xenografts, with inhibition rates of 56% and 69%, respectively, showing a dose-dependent effect. IgG1-TGFβRII (2.3 mg / kg) had an inhibition rate of only 4% on MC38 / hPD-L1 subcutaneous xenografts, showing no significant therapeutic effect. Notably, the dosages of IgG1-TGFβRII (2.3 mg / kg) and hu5G11-hIgG1-TGFβRII (3.7 mg / kg) were in the same order of magnitude, indicating no substantial difference. This confirms that hu5G11-hIgG1-TGFβRII exhibits a significantly higher inhibition rate compared to fusion proteins with other components (i.e., fusion proteins containing IgG1 and TGFβRII). Tumor-bearing mice tolerated all the drugs well, with no significant weight loss or other symptoms observed.

[0167] Table 6. Efficacy of each tested sample against subcutaneous xenografts of colon cancer cells MC38 / hPD-L1.

[0168]

[0169] Note: Randomized groups were assigned, with the first administration date being D0; the administration frequency and number of doses were once every 2 days for a total of 6 doses, administered via intravenous injection (IV). P<0.05, P<0.01, and P<0.001 were compared with the IgG1 group (isotype control group) using a t-test.

[0170] Example 8: Electrochemiluminescence method for detecting hu5G11-hIgG1-TGFβRII-stimulated cytokine secretion

[0171] 1) Liquid phase method: Adjust the PBMC cell concentration to approximately 1–2 × 10⁻⁶ cells using RPMI 1640 complete medium. 6Cells / mL, 100 μl / well added to a 96-well cell culture plate. IgG1 (SEQ ID NO:38 and SEQ ID NO:39, preparation method see Example 7), LPS (SIGMA, L4391-1MG), and hu5G11-hIgG1-TGFβRII were diluted with RPMI 1640 complete medium to prepare 1000 μg / mL IgG1; 10 μg / mL LPS; and 100 μg / mL, 300 μg / mL, and 1000 μg / mL hu5G11-hIgG1-TGFβRII solutions. RPMI 1640 complete medium was used as a negative control. 100 μL of each of the prepared solutions was added to a 96-well cell culture plate, mixed thoroughly, and cultured at 37°C in a 5% CO2 cell culture incubator. Cell supernatants were collected from 96-well plates at 24 and 48 hours, and the levels of cytokines IL-2, IL-4, IL-6, TNF-α, and IFN-γ were detected using the V-PLEX Proinflammatory Panel 1 (human) kit (MSD, K15049D-2). The results are shown in Table 7.

[0172] 2) Solid-phase method: IgG1 (SEQ ID NO:38 and SEQ ID NO:39, preparation method as described in Example 7), LPS (SIGMA, L4391-1MG), and hu5G11-hIgG1-TGFβRII were diluted with PBS to prepare 500 μg / mL IgG1, 5 μg / mL LPS, and 50 μg / mL, 150 μg / mL, and 500 μg / mL hu5G11-hIgG1-TGFβRII solutions; PBS was used as a negative control. The prepared solutions were added at 200 μL / well to the corresponding wells of a 96-well high-adsorption plate, and coated at 37°C for 2 h. The cell culture plates were then removed, the solutions were discarded, and the plates were washed three times with PBS. The PBMC cell concentration was adjusted to approximately 1–2 × 10⁻⁶ cells / well using RPMI 1640 complete medium. 6 Cells / mL were added at a rate of 200 μL / well to 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. Cell supernatant was collected from the 96-well plates at 24 and 48 hours, and the levels of cytokines IL-2, IL-4, IL-6, TNF-α, and IFN-γ were detected using the V-PLEX Proinflammatory Panel 1 (human) kit (MSD, K15049D-2). The results are shown in Table 8.

[0173] Table 7 Results of liquid chromatography detection of cytokine secretion in various test samples

[0174]

[0175] Table 8 Results of solid-phase method for detecting cytokine secretion in various test samples

[0176]

[0177] As can be seen from the results in the table above, hu5G11-hIgG1-TGFβRII exhibits a low likelihood of causing a cytokine storm. Therefore, when administered to subjects, there is virtually no risk of overactivating the immune system and triggering systemic inflammation.

[0178] Example 9: Toxicity test of the fusion protein on cynomolgus monkeys

[0179] Rectal temperature was measured using a Temp-14 thermistor thermometer; respiratory rate and lead II electrocardiogram were measured using a large animal non-invasive physiological signal telemetry system; blood pressure (systolic, diastolic, and mean arterial pressure) was measured using a non-invasive sphygmomanometer. The electrocardiogram waveforms were qualitatively analyzed, and stable, continuous electrocardiograms within a 30-second time interval at each time point were analyzed using ecgAUTO software. Indicators included heart rate, RR interval, P wave duration, PR interval, QRS duration, QT interval, and corrected QT interval.

[0180] Single-dose toxicity: Six cynomolgus monkeys (three in each group, both male and female) were used in this experiment. Two groups were established, and each group received a single intravenous injection of 300 mg / kg hu5G11-hIgG1-TGFβRII, respectively. The animals were observed for 14 days. During the experiment, general observations and monitoring of body weight, food intake, body temperature, lead II electrocardiogram, blood pressure, hematological parameters, blood biochemistry, and urine were performed. Gross necropsy was conducted at the end of the experiment. No animals died or showed signs of near-death during the experiment, and no abnormalities were observed in any of the indicators.

[0181] Repeated-dose toxicity: Forty cynomolgus monkeys were used in this study and divided into four groups of ten each (half male and half female). These groups were the blank control group and the hu5G11-hIgG1-TGFβRII groups (20, 60, and 200 mg / kg). Administration was once weekly for four weeks (five weeks in total), followed by a six-week recovery period after drug withdrawal. The toxicokinetic parameters for the repeated intravenous administration toxicity test are shown in Table 5 of Example 6.

[0182] At the end of administration, mild anemia-like changes, mainly characterized by decreased RBC, HGB, and HCT, and increased RET and RET% were observed in the 60 and 200 mg / kg groups. The 200 mg / kg group also showed a mild increase in the proportion of erythroid lineage and late erythroblasts in bone marrow smears. Histopathological examination of the bone marrow and spleen revealed no related changes. By the end of the recovery period, one female monkey in the 200 mg / kg group still showed no significant recovery in HGB and HCT, and erythrocyte morphology showed decreased hemoglobin content. The other cynomolgus monkeys showed varying degrees of recovery in the aforementioned indicators.

[0183] At the end of administration, mild mononuclear cell infiltration was observed in the thyroid glands of the 20, 60, and 200 mg / kg groups; mild to mild mononuclear cell infiltration was observed in the kidneys of the 60 and 200 mg / kg groups; and mild mononuclear cell infiltration was observed in the meninges of the 20 mg / kg and 200 mg / kg groups. At the end of the recovery period, except for the 200 mg / kg group where no significant recovery of meningeal lesions was observed, all other organ lesions in the cynomolgus monkeys showed some degree of recovery.

[0184] In addition, no significant abnormalities were observed in the general observation, weight, food intake, body temperature, lead II electrocardiogram, respiratory rate and blood pressure, blood biochemistry, urine, ophthalmological examination, bone marrow smear, immunoglobulins, complement, circulating immune complexes, lymphocyte subsets and cytokines of the cynomolgus monkeys in each group.

[0185] Therefore, hu5G11-hIgG1-TGFβRII exhibits low acute and long-term toxicity, and thus the fusion protein can be expected to demonstrate good safety in clinical applications.

[0186] Examples of pharmaceutical compositions

[0187] Size exclusion chromatography (SEC-UPLC): Used to determine the purity of antibodies in a sample, employing Thermo Vanquish F high-performance liquid chromatography, ACQUITY UPLC Protein BEH SEC Column ( 1.7μm, 4.6mm × 300mm) chromatographic column, ACQUITY UPLC Protein BEH SEC Guard Column ( A 1.7 μm, 4.6 mm × 30 mm column was used as the pre-column, and elution was performed using a mobile phase of 50 mmol / L phosphate buffer-200 mmol / L sodium chloride solution (pH 7.0). The detection wavelength was 280 nm. The percentage content of high molecular weight impurities and immunoglobulin monomers was calculated using the area normalization method.

[0188] DLS particle size determination method: The particle size of the sample was determined using a nanoparticle size analyzer (Malvern, ZEN3600). The procedure was as follows: temperature 25℃, equilibration time 120s.

[0189] The detection methods for Tonset and Tm were as follows: The inflection point temperature (Tonset) and melting temperature (Tm) of the sample were analyzed using a protein stability analyzer (NanoTemper, Prometheus NT.48). 10 μL of sample was placed in the sample chamber for detection. The procedure was as follows: the scan start temperature was 25℃, the scan end temperature was 95℃, and the heating rate was 0.3℃ / min.

[0190] The aggregation temperature (Tagg) of the sample was determined using a multi-well plate direct-reading dynamic and static light scattering instrument (Wyatt, Dynaproplate Reader Ⅲ). After centrifugation, 30 μL of the supernatant was added to a 384-well sample tray, the plate was sealed with a sealing film, and the 384-well sample tray was centrifuged to remove air bubbles before detection. The procedure was as follows: the temperature program was from 25℃ to 85℃, and the scan time was 5 seconds.

[0191] The diffusion coefficient (kD) of the samples was determined using a Wyatt Dynaproplate ReaderⅢ direct-reading dynamic and static light scattering instrument. The samples were pre-diluted to 10 mg / ml, and then serially diluted to 8 mg / ml, 6 mg / ml, 4 mg / ml, and 2 mg / ml. After filtration and centrifugation, 30 μL of the supernatant sample was added to a 384-well sample dish, sealed with a sealing membrane, and then the 384-well sample dish was centrifuged to remove air bubbles before detection. The procedure was as follows: temperature set at 25℃, 5 data acquisitions per sample, and acquisition time of 5 seconds.

[0192] The second virial coefficient (A2) of the samples was detected using a Wyatt Dynaproplate Reader Ⅲ multi-well plate direct-reading dynamic and static light scattering instrument. The samples were pre-diluted to 10 mg / ml, and then serially diluted to 8 mg / ml, 6 mg / ml, 4 mg / ml, and 2 mg / ml. After filtration and centrifugation, 30 μL of the supernatant sample was added to a 384-well sample tray, and the plate was sealed with a sealing membrane. The 384-well sample tray was then centrifuged to remove air bubbles before detection. The procedure was as follows: temperature set at 25℃, 5 data acquisitions per sample, and acquisition time of 5 seconds.

[0193] Biological activity detection method: See Example 5.

[0194] CE-SDS reduction electrophoresis: A Beckman Coulter Pa 800Plus biopharmaceutical analysis system was used, employing an uncoated fused silica capillary with an inner diameter of 50 μm, a total length of 31 cm, and an effective length of 21 cm, and a PDA detector. Detection was performed at a wavelength of 220 nm. The percentage of the corrected peak area of ​​the degradation products was calculated using the area normalization method.

[0195] Preparation of buffer systems: Buffer systems can be prepared using conventional methods in the art. For example, the buffer system in F1: 20 mM sodium phosphate buffer (pH 7.5) is prepared from approximately 5.5441 g / L Na2HPO4·12H2O and approximately 0.6237 g / L NaH2PO4·H2O; the buffer system in F4: 20 mM sodium phosphate buffer + 20 mM histidine buffer (pH 7.5) is prepared from approximately 5 g / L Na2HPO4·12H2O, approximately 0.833 g / L NaH2PO4·H2O, and approximately 3.1 g / L histidine; the buffer system in F12: 20 mM sodium phosphate buffer + 20 mM histidine buffer (pH 6.5) is prepared from 2.11 g / L Na2HPO4·12H2O and 1.95 g / L histidine. It was prepared by mixing NaH2PO4·H2O with 2.03 g / L histidine and 1.44 g / L histidine hydrochloride monohydrate.

[0196] Example 9: Screening of Buffer Systems

[0197] The hu5G11-hIgG1-TGFβRII was replaced into the buffer system selected in Table 9 by ultrafiltration centrifugation. After replacement, the mixture was concentrated, and excipients were added according to the drug compositions selected in Table 9. After thorough mixing, the mixture was filtered and sterilized. The results of SEC-UPLC, DLS particle size, and biological activity of different drug compositions are shown in Table 10.

[0198] Table 9. Screened Drug Compositions

[0199]

[0200] Note: " / " indicates that it has not been added.

[0201] Table 10 shows the results of SEC-UPLC, DLS particle size, and biological activity of the screened drug compositions.

[0202]

[0203] Note: " / " indicates unmeasured, and D indicates day.

[0204] Example 10: Screening of Stabilizers and pH

[0205] The hu5G11-hIgG1-TGFβRII was replaced into the buffer system selected in Table 11 by ultrafiltration centrifugation. After replacement, the mixture was concentrated, and excipients were added to the drug compositions selected in Table 11. After thorough mixing, the mixture was filtered for sterilization. The results of the detection of Tonset, Tm, Tagg, kD, and A2 of different drug compositions are shown in Table 12, and the results of the stability study of different drug compositions at 40℃ and 2–8℃ are shown in Table 13.

[0206] Table 11 Screened Drug Compositions

[0207]

[0208] Note: " / " indicates that it has not been added.

[0209] Table 12 shows the results of Tm, Tagg, kD, and A2 tests for the screened drug compositions.

[0210]

[0211] Note: " / " indicates that it was not measured.

[0212] Table 13 shows the stability test results of the screened drug compositions at 40°C and 2–8°C.

[0213]

[0214] Note: D represents day, W represents week, and M represents month.

[0215] Example 11 Screening of Stabilizers and pH

[0216] The hu5G11-hIgG1-TGFβRII was replaced into the buffer system selected in Table 14 by ultrafiltration and centrifugation. After replacement, the mixture was concentrated, and excipients were added to the drug compositions selected in Table 14. After thorough mixing, the mixture was filtered and sterilized. The stability of different drug compositions at 40℃ and 2–8℃ are shown in Table 15.

[0217] Table 14 Screened Drug Compositions

[0218]

[0219] Note: " / " indicates that it has not been added.

[0220] Table 15 shows the stability test results of the screened drug compositions at 40°C and 2–8°C.

[0221]

[0222] Note: D represents day, M represents month, and " / " indicates not measured.

[0223] Those skilled in the art should further understand that the present invention can be implemented in other specific forms without departing from its spirit and core attributes. In the foregoing description, only exemplary embodiments have been disclosed, and it should be understood that other variations are also included within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims regarding the scope and content of the invention. sequence list <110> Chia Tai Tianqing Pharmaceutical Group Co., Ltd. <120> Pharmaceutical compositions containing fusion proteins targeting PD-L1 and TGF-β <150> CN202110227775.2 <151> 2021-03-01 <160> 43 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Mus sp. <400> 1 Ser Tyr Gly Met Ser 1 5 <210> 2 <211> 16 <212> PRT <213> Mus sp. <400> 2 Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys Gly 1 5 10 15 <210> 3 <211> 8 <212> PRT <213> Mus sp. <400> 3 Gly Tyr Asp Ser Gly Phe Ala Tyr 1 5 <210> 4 <211> 14 <212> PRT <213> Mus sp. <400> 4 Ala Ser Gln Ser Val Ser Thr Ser Ser Ser Ser Ser Phe Met His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mus sp. <400> 5 Tyr Ala Ser Asn Leu Glu Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Mus sp. <400> 6 Gln His Ser Trp Glu Ile Pro Tyr Thr 1 5 <210> 7 <211> 116 <212> PRT <213> Synthetic sequence <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Asp Cys Ala 85 90 95 Arg Gly Tyr Asp Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 111 <212> PRT <213> Synthetic sequence <400> 8 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Ser Ser Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 446 <212> PRT <213> Synthetic sequence <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Asp Cys Ala 85 90 95 Arg Gly Tyr Asp Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 10 <211> 218 <212> PRT <213> Synthetic sequence <400> 10 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Ser Ser Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 11 <211> 603 <212> PRT <213> Synthetic sequence <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Asp Cys Ala 85 90 95 Arg Gly Tyr Asp Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met 465 470 475 480 Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys 485 490 495 Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met 500 505 510 Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys 515 520 525 Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val 530 535 540 Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala 545,550,555,560 Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr 565,570,575 Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile 580,585,590 Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595,600 <210> 12 <211> 1809 <212> DNA <213> Synthetic sequence <400> 12 gaggtgcagc tggtcgagtc aggagggggg ctggtcaagc caggagggtc actgcgactg 60 agctgcgcag cttccgggtt catctttagg tcttatggca tgagttgggt gcgccaggca 120 ccagggaaag gactggagtg ggtcgcttca atcagctccg gaggcagcac ttactatcct 180 gactccgtga agggccggtt caccattctt agagataacg ccaaaatag tctgtacctg 240 cagatgaact ctctgcgagc agaagacaca gccgtctacg attgtgctag aggatatgac 300 agcggctttg catactgggg ccaggggacc ctggtgacag tctcgagcgc ctccactaag 360 ggcccatccg tgttccctct ggcaccctcc 420 ctgggctgcc tcgtgaagga ctacttccca gaacckgtga ccgtcagctg gaatagcggc gctctgacca gcggagtcca cactttcccc gcagtgctgc agtccagcgg cctgtacagc 540 ctgagcagcg tggtcactgt gccaagcagc agcctgggca ctcagaccta catctgcaac 660. gtcaaccaca agcccagcaa cacaaaggtg gacaagaagg tcgagcccaa gtcctgcgat aagacccaca cctgccctcc atgtcccgcc cccgagctgc tgggaggacc cagcgtcttc 720 ctgtttcccc ccaagccaaa ggacaccctg atgatcagca ggacccccga agtgacctgc 780 gtcgtggtgg ccgtgagcca cgaagatccc gaggtgaagt tcaactggta cgtggacggc 840 gtggaagtgc acaacgccaa gacaaaaccc agggaggagc agtatacag cacctacagg gtcgtgagcg tcctgaccgt gctgcaccaa gactggctga acggcaagga gtataagtgc 960 aaggtgagca acaaggcact gcccgccccc atcgagaga ccatttccaa ggccaagggg caacctaggg agccacaggt ctacactctg ccccctagca gggacgagct gaccaagaac caggtctccc tgacttgcct ggtgaagggg ttttaccca gcgacatcgc cgtcgagtgg 1140 gagagcaatg gccagcccga aaacaacac agaccacac cccctgtgct ggacagcgac 1200 ggcagcttct ttctgtatag caaacgaca gtgataaga gcagatggca gcaggggcaac 1260 gtgttctcct gctccgtgat gcacgaggcc ctgcacaatc actacaccca gaagtccctg 1320 agcctgtccc ccggaaagg aggagga tctggaggag gcggcagcgg cggagga 1380 agtggaggag gcggatccgg catccctccc cacgtgcaga agagcgtgaa taacgacatg 1440 atcgtgaccg acaataacgg cgctgtgaag ttccctcagc tgtgcaagtt ctgcgatgtg 1500 cggttctcca cctgcgacaa tcagagagc tgcatgagca actgcagcat cacctccatc 1560 tgcgagaagc ctcaggaggt gtgtgtggcc gtgtggcgga agaatgacga gatatcacc 1620 ctggagaccg tgtgccacga cccaagctg ccttatcacg atttcatcct ggaggacgcc 1680 gctagcccca agtgcatcat gaaggagaag aagaagcccg gcgagacctt cttcatgtgt 1740 agctgtagca gcgatgagtg caacgataat atcatcttta gcgaggagta taacaccagc 1800 aatcccgat 1809 <210> 13 <211> 218 <212> PRT <213> Synthetic sequence <400> 13 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Ser Ser Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Serving Lys Asp Serving Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Valid Thr Lys Ser PhE As An Arg Gly Glu Cys 210 215 <210> 14 <211> 654 <212> DNA <213> Synthetic sequence <400> 14 gatatgtgc tgactcagag ccccgcttca ctggcagtgt ctccaggggca gcggggcacc 60 atcacatgca gagcctcaca gagcgtctcc accagctcct ctagtttcat gcactggtac 120 cagcagaagc ccggacagcc ccctaagctg ctgatcaaat atgctagcaa cctggagtcc 180 ggcgtgccag ccaggttctc tggcagtggg tcaggaccg actttactct gaccattaat 240 cccgtcgaag ccaacgatac agctattac tattgtcagc attcctggga gatcccttac acattttggcc aggggactaa gctggagatc aagcgtacgg tggccgcacc aagcgtcttc 360 atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgt cacagagcag cacagcaagg acagcaccta cagcctcagc 540 agcaccctga cgctgagcaa agcacctc gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca aagagcttta acagaggcga gtgc 654 <210> 15 <211> 5 <212> PRT <213> Mus sp. <400> 15 Thr Tyr Gly Val His 1 5 <210> 16 <211> 16 <212> PRT <213> Mus sp. <400> 16 Click Download to save Val Ile Trp Arg Gly Val Thr Thr Asp Tyr mp3 youtube com 1 5 10 15 <210> 17 <211> 8 <212> PRT <213> Mus sp. <400> 17 Leu Gly Phe Tyr Ala Met Asp Tyr 1 5 <210> 18 <211> 11 <212> PRT <213> Mus sp. <400> 18 Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala 1 5 10 <210> 19 <211> 7 <212> PRT <213> Mus sp. <400> 19 Tyr Ala Ala Asn Arg Tyr Thr 1 5 <210> 20 <211> 9 <212> PRT <213> Mus sp. <400> 20 Gln Gln Asp Tyr Thr Ser Pro Tyr Thr 1 5 <210> 21 <211> 116 <212> PRT <213> Synthetic sequence <400> 21 Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Arg Gly Val Thr Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Val Leu 65 70 75 80 Thr Met Asn Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Leu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 22 <211> 107 <212> PRT <213> Synthetic sequence <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ala Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Thr Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 446 <212> PRT <213> Synthetic sequence <400> 23 Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Arg Gly Val Thr Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Val Leu 65 70 75 80 Thr Met Asn Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Leu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 24 <211> 214 <212> PRT <213> Synthetic sequence <400> 24 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ala Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Thr Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 25 <211> 603 <212> PRT <213> Synthetic sequence <400> 25 Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Arg Gly Val Thr Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Val Leu 65 70 75 80 Thr Met Asn Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Leu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met 465 470 475 480 Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys 485,490,495 Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met 500 505 510 Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys 515,520,525 Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val 530 535 540 Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala 545 550 555 560 Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Pro Gly Glu Thr 565,570,575 Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asn Asn Ile Ile 580,585,590 Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595,600 <210> 26 <211> 1809 <212> DNA <213> Synthetic sequence <400> 26 cagatcacac tgaagaag cggccctacc ctggtcaagc caaccagac cctgacactg 60 acttgcaccg tgtctgggtt ctctctgagt acatacggag tccactggat caggcagccc 120 cctggcaaag ctctggagtg gctgggagtg atttggcggg gcgtcaccac agactataac 180 gccgctttta tgtcaagact gacaatcact aaggataaca gcaaaaatca ggtggtcctg 240 accatgaaca atatggaccc cgtggatacc gcaacatact attgtgcccg gctggggttc 300 tacgccatgg actattgggg ccaggggact ctggtgaccg tctcgagcgc ctccactaag 360 ggaccatccg tgttccctct ggcaccctcc agcaagagca caagcggagg caccgccgca 420 ctgggctgcc tcgtgaagga ctacttccca gaacccgtga ccgtcagctg gaatagcggc 480 gctctgacca gcggagtcca cactttcccc gcagtgctgc agtccagcgg cctgtacagc 540 ctgagcagcg tggtcactgt gccaagcagc agcctgggca ctcagaccta catctgcaac 600 gtcaaccaca agcccagcaa cacaaaggtg gacaagaagg tcgagcccaa gtcctgcgat 660 aagacccaca cctgccctcc atgtcccgcc cccgagctgc tgggaggacc cagcgtcttc 720 ctgtttcccc ccaagccaaa ggacaccctg atgatcagca ggacccccga agtgacctgc 780 gtcgtggtgg ccgtgagcca cgaagatccc gaggtgaagt tcaactggta cgtggacggc 840 gtggaagtgc acaacgccaa gacaaaaccc agggaggagc agtatacag cacctacagg gtcgtgagcg tcctgaccgt gctgcaccaa gactggctga acggcaagga gtataagtgc 960 aaggtgagca acaaggcact gcccgccccc atcgagaga ccatttccaa ggccaagggg caacctaggg agccacaggt ctacactctg ccccctagca gggacgagct gaccaagaac caggtctccc tgacttgcct ggtgaagggg ttttatccca gcgacatcgc cgtcgagtgg 1140 gagagcaatg gccagcccga aaacaactac aagaccacac cccctgtgct ggacagcgac ggcagcttct ttctgtatag caaactgaca gtggataaga gcagatggca gcagggcaac gtgttctcct gctccgtgat gcacgaggcc ctgcacaatc actacaccca gaagtccctg 1320 agcctgtccc ccggaaaagg aggagagga tctggaggag gcggcagcgg cggaggagga 1380 agtgaggag gcggatccgg catccctccc cacgtgcaga agagcgtgaa taacgacatg atcgtgaccg acaataacgg cgctgtgaag ttccctcagc tgtgcaagtt ctgcgatgtg cggttctcca cctgcgacaa tcagaagagc tgcatgagca actgcagcat cacctccatc tgcgagaagc ctcaggaggt gtgtgtggcc gtgtggcgga agaatgacga gaatatcacc 1620 ctggagaccg tgtgccacga ccccaagctg ccttatcacg atttcatcct ggaggacgcc 1680 gctagcccca agtgcatcat gaaggagaag aagaagcccg gcgagacctt cttcatgtgt 1740 agctgtagca gcgatgagtg caacgataat atcatcttta gcgaggagta taacaccagc 1800 aatcccgat 1809 <210> 27 <211> 214 <212> PRT <213> Synthetic sequence <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Ala Ala Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Asp Tyr Thr Ser Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 28 <211> 642 <212> DNA <213> Synthetic sequence <400> 28 gataccaga tgactcagtc tccaagcagc ctgtctgcat ctgtggggga cagggtcacc 60 atcacatgca aagcatctca gagtgtgtca aacgatgtcg cctggtacca gcagaagccc 120 ggaaaagctc ctaagctgct gatttactat gccgctaatc ggtacactgg cgtgccagac 180 agattcagcg gatccgata tggaccgat ttcacttta ccatcagctc cctgcagcca 240 gaggacattg ccacatattt ctgtcagcag gattacacaa gcccctatac tttggccag 300 gggaccaac tggaaatca gcgtacggtg gccgcaccaa gcgtcttcat cttcccgcca 360 tctgatgagc agttgaatc tggactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaagtaca gtggaaggtg gataacgccc tccaatcgggg taactcccag 480 gagagtgtca cagagcagga cagcaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaag cagactacga gaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaa gagctttaac agaggcgagt gc 642 <210> 29 <211> 446 <212> PRT <213> Synthetic sequence <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Asp Cys Ala 85 90 95 Arg Gly Tyr Asp Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala 435 440 445 <210> 30 <211> 603 <212> PRT <213> Synthetic sequence <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Asp Cys Ala 85 90 95 Arg Gly Tyr Asp Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met 465 470 475 480 Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys 485 490 495 Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met 500 505 510 Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys 515 520 525 Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val 530 535 540 Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala 545 550 555 560 Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr 565 570 575 Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile 580 585 590 Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 <210> 31 <211> 1809 <212> DNA <213> Synthetic sequence <400> 31 gaggtgcagc tggtcgagtc aggagggggg ctggtcaagc caggagggtc actgcgactg 60 agctgcgcag cttccgggtt catctttagg tcttatggca tgagttgggt gcgccaggca 120 180. ccagggaag ggtggagtg ggtcgcttca atcagctccg gaggcagcac ttactatcct 240. gactccgtga agggccggtt caccatttct agagataacg ccaaaaatag tctgtacctg slightly ctctgcgagc slightly gccgtctacg attgtgctag slightly agcggctttg catactgggg ccaggggacc ctggtgacag tctcgagcgc ctccactaag 360 ggcccatccg tgttccctct ggcaccctcc 420 ctgggctgcc tcgtgaagga ctacttccca gaacckgtga ccgtcagctg gaatagcggc gctctgacca gcggagtcca cactttcccc gcagtgctgc agtccagcgg cctgtacagc 540 ctgagcagcg tggtcactgt gccaagcagc agcctgggca ctcagaccta catctgcaac 660. gtcaaccaca agcccagcaa cacaaaggtg gacaagaagg tcgagcccaa gtcctgcgat aagacccaca cctgccctcc atgtcccgcc cccgagctgc tgggaggacc cagcgtcttc 720 ctgtttcccc ccaagccaaa ggacaccctg atgatcagca ggacccccga agtgacctgc 780 gtcgtggtgg ccgtgagcca cgaagatccc gaggtgaagt tcaactggta cgtggacggc 840 gtggaagtgc acaacgccaa gacaaaaccc agggaggagc agtatacag cacctacagg gtcgtgagcg tcctgaccgt gctgcaccaa gactggctga acggcaagga gtataagtgc 960 aaggtgagca acaaggcact gcccgccccc atcgagaga ccatttccaa ggccaagggg caacctaggg agccacaggt ctacactctg ccccctagca gggacgagct gaccaagaac caggtctccc tgacttgcct ggtgaagggg ttttatccca gcgacatcgc cgtcgagtgg 1140 gagagcaatg gccagcccga aaacaactac aagaccacac cccctgtgct ggacagcgac ggcagcttct ttctgtatag caaactgaca gtggataaga gcagatggca gcagggcaac gtgttctcct gctccgtgat gcacgaggcc ctgcacaatc actacaccca gaagtccctg 1320 agcctgtccc ccggagccgg aggagagga tctggaggag gcggcagcgg cggaggagga 1380 agtgaggag gcggatccgg catccctccc cacgtgcaga agagcgtgaa taacgacatg atcgtgaccg acaataacgg cgctgtgaag ttccctcagc tgtgcaagtt ctgcgatgtg cggttctcca cctgcgacaa tcagaagagc tgcatgagca actgcagcat cacctccatc 1560 tgcgagaagc ctcaggaggt gtgtgtggcc gtgtggcgga agaatgacga gaatatcacc 1620 ctggagaccg tgtgccacga ccccaagctg ccttatcacg atttcatcct ggaggacgcc 1680 gctagcccca agtgcatcat gaaggagaag aagaagcccg gcgagacctt cttcatgtgt 1740 agctgtagca gcgatgagtg caacgataat atcatcttta gcgaggagta taacaccagc 1800 aatcccgat 1809 <210> 32 <211> 446 <212> PRT <213> Synthetic sequence <400> 32 Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Arg Gly Val Thr Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Val Leu 65 70 75 80 Thr Met Asn Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Leu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala 435 440 445 <210> 33 <211> 603 <212> PRT <213> Synthetic sequence <400> 33 Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Arg Gly Val Thr Thr Asp Tyr Asn Ala Ala Phe Met 50 55 60 Ser Arg Leu Thr Ile Thr Lys Asp Asn Ser Lys Asn Gln Val Val Leu 65 70 75 80 Thr Met Asn Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Leu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met 465 470 475 480 Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys 485 490 495 Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met 500 505 510 Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys 515 520 525 Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val 530 535 540 Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala 545,550,555,560 Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr 565,570,575 Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile 580,585,590 Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595,600 <210> 34 <211> 1809 <212> DNA <213> Synthetic sequence <400> 34 cagatcacac tgaaagaaag cggccctacc ctggtcaagc caactcagac cctgacactg 60 acttgcaccg tgtctgggtt ctctctgagt acatacggag tccactggat caggcagccc 120 cctggcaaag ctctggagtg gctgggagtg atttggcggg gcgtcaccac agactataac 180 gccgctttta tgtcaagact gacaatcact aaggataaca gcaaaaatca ggtggtcctg 240 accatgaaca atatggaccc cgtggatacc gcaacatact attgtgcccg gctggggttc 300 tacgccatgg actattgggg ccaggggact ctggtgaccg tctcgagcgc ctccactaag 360 ggaccatccg tgttccctct ggcaccctcc caagcggagg caccgccgca 420 ctgggctgcc tcgtgaagga ctacttccca gaacckgtga ccgtcagctg gaatagcggc gctctgacca gcggagtcca cactttcccc gcagtgctgc agtccagcgg cctgtacagc 540 ctgagcagcg tggtcactgt gccaagcagc agcctgggca ctcagaccta catctgcaac 660. gtcaaccaca agcccagcaa cacaaaggtg gacaagaagg tcgagcccaa gtcctgcgat aagacccaca cctgccctcc atgtcccgcc cccgagctgc tgggaggacc cagcgtcttc 720 ctgtttcccc ccaagccaaa ggacaccctg atgatcagca ggacccccga agtgacctgc 780 gtcgtggtgg ccgtgagcca cgaagatccc gaggtgaagt tcaactggta cgtggacggc 840 gtggaagtgc acaacgccaa gacaaaaccc agggaggagc agtatacag cacctacagg gtcgtgagcg tcctgaccgt gctgcaccaa gactggctga acggcaagga gtataagtgc 960 aaggtgagca acaaggcact gcccgccccc atcgagaga ccatttccaa ggccaagggg caacctaggg agccacaggt ctacactctg ccccctagca gggacgagct gaccaagaac caggtctccc tgacttgcct ggtgaagggg ttttaccca gcgacatcgc cgtcgagtgg 1140 gagagcaatg gccagcccga aaacaacac agaccacac cccctgtgct ggacagcgac 1200 ggcagcttct ttctgtatag caaacgaca gtgataaga gcagatggca gcaggggcaac 1260 gtgttctcct gctccgtgat gcacgaggcc ctgcacaatc actacaccca gaagtccctg 1320 agcctgtccc cggagccgg aggagga tctggaggag gcggcagcgg cggagga 1380 agtggaggag gcggatccgg catccctccc cacgtgcaga agagcgtgaa taacgacatg 1440 atcgtgaccg acaataacgg cgctgtgaag ttccctcagc tgtgcaagtt ctgcgatgtg 1500 cggttctcca cctgcgacaa tcagagagc tgcatgagca actgcagcat cacctccatc 1560 tgcgagaagc ctcaggaggt gtgtgtggcc gtgtggcgga agaatgacga gatatcacc 1620 ctggagaccg tgtgccacga cccaagctg ccttatcacg atttcatcct ggaggacgcc 1680 gctagcccca agtgcatcat gaaggagaag aagaagcccg gcgagacctt cttcatgtgt 1740 agctgtagca gcgatgagtg caacgataat atcatcttta gcgaggagta taacaccagc 1800 aatcccgat 1809 <210> 35 <211> 567 <212> PRT <213> Synthetic sequence <400> 35 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115 120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu Met Glu Phe Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220 Ser Asp Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225 230 235 240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala 245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu Ile Thr Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu Thr Arg His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys 355 360 365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn 370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu 385 390 395 400 Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp Asp Leu Ala Asn Ser 405 410 415 Gly Gln Val Gly Thr Ala Arg Tyr Met Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu Glu Asn Val Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met Ala Leu Val Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460 Gly Glu Val Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465 470 475 480 His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly 485 490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser Glu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn Thr Thr Lys 565 <210> 36 <211> 136 <212> PRT <213> Synthetic sequence <400> 36 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 37 <211> 21 <212> PRT <213> Synthetic sequence <400> 37 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly 20 <210> 38 <211> 446 <212> PRT <213> Synthetic sequence <400> 38 Glu Val Gln Leu Glu Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Ser Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Asp Ser Thr Tyr Tyr Asn Glu Lys Val 50 55 60 Lys Gly Lys Val Thr Phe Thr Ala Asp Ala Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Phe Tyr Val Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 39 <211> 214 <212> PRT <213> Synthetic sequence <400> 39 Asp Ile Glu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Thr Ser Gln Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Val Tyr Phe Cys Gln Gln Ser Gly Ser Trp Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 40 <211> 603 <212> PRT <213> Synthetic sequence <400> 40 Glu Val Gln Leu Glu Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Ser Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Asp Ser Thr Tyr Tyr Asn Glu Lys Val 50 55 60 Lys Gly Lys Val Thr Phe Thr Ala Asp Ala Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Gly Phe Tyr Val Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 145 150 155 160 Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175 Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190 Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200 205 Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 260 265 270 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met 465 470 475 480 Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys 485 490 495 Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met 500 505 510 Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys 515 520 525 Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val 530 535 540 Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala 545 550 555 560 Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr 565 570 575 Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile 580 585 590 Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 <210> 41 <211> 214 <212> PRT <213> Synthetic sequence <400> 41 Asp Ile Glu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Thr Ser Gln Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Val Tyr Phe Cys Gln Gln Ser Gly Ser Trp Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 42 <211> 607 <212> PRT <213> Artificial Sequence <400> 42 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val 465 470 475 480 Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro 485 490 495 Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln 500 505 510 Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 515 520 525 Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 530 535 540 Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 545 550 555 560 Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 565 570 575 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn 580 585 590 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp 595 600 605 <210> 43 <211> 216 <212> PRT <213> Artificial Sequence <400> 43 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215

Claims

1. A pharmaceutical composition comprising: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of 10 mg / mL to 60 mg / mL; (b) a sodium phosphate buffer at a concentration of 10 mM to 40 mM and a histidine buffer at a concentration of 10 mM to 40 mM; (c) polysorbate 80 or polysorbate 20 at a concentration of 0.08 mg / mL to 0.4 mg / mL; and (d) sucrose at a concentration of 50 mg / mL to 100 mg / mL; wherein the pH of the pharmaceutical composition is 6 to 7.5; wherein, The fusion protein targeting PD-L1 and TGF-β includes: (a) an antibody or antigen-binding fragment thereof that binds to human programmed death ligand 1 (PD-L1); and (b) human TGFβ RII or a TGFβ-binding fragment thereof; The heavy chain CDR1 sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO:15, the heavy chain CDR2 sequence is shown in SEQ ID NO:16, and the heavy chain CDR3 sequence is shown in SEQ ID NO:17; the light chain CDR1 sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO:18, the light chain CDR2 sequence is shown in SEQ ID NO:19, and the light chain CDR3 sequence is shown in SEQ ID NO:20; and the sequence of the human TGFβ RII or its TGFβ binding fragment is shown in SEQ ID NO:

36.

2. The pharmaceutical composition according to claim 1, wherein, The heavy chain variable domain of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:21, and the light chain variable domain of the antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO:

22.

3. The pharmaceutical composition according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: a heavy chain amino acid sequence as shown in SEQ ID NO:32, and a light chain amino acid sequence as shown in SEQ ID NO:

24.

4. The pharmaceutical composition according to claim 1, wherein, The fusion protein targeting PD-L1 and TGF-β further includes a linker peptide that links the antibody or its antigen-binding fragment to the human TGFβ RII or its TGFβ-binding fragment, wherein the linker peptide is (G4S). x G, where x is an integer between 3 and 6.

5. The pharmaceutical composition according to claim 4, wherein, The value of x is 4.

6. The pharmaceutical composition according to claim 1, wherein, The fusion protein targeting PD-L1 and TGF-β comprises: (1) a first polypeptide whose amino acid sequence is at least 80% identical to the amino acid sequence shown in SEQ ID NO:33; and (2) a second polypeptide whose amino acid sequence is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

27.

7. The pharmaceutical composition according to claim 1, wherein, The fusion protein targeting PD-L1 and TGF-β comprises: (1) a first polypeptide having the amino acid sequence shown in SEQ ID NO:33; and (2) a second polypeptide having the amino acid sequence shown in SEQ ID NO:

27.

8. The pharmaceutical composition according to any one of claims 1-7, wherein, The concentration of the fusion protein targeting PD-L1 and TGF-β is from 40 mg / mL to 60 mg / mL.

9. The pharmaceutical composition according to claim 8, wherein, The concentration of the fusion protein targeting PD-L1 and TGF-β is 50 mg / mL.

10. The pharmaceutical composition according to any one of claims 1-7, wherein, The concentration of the sodium phosphate buffer solution is 10 mM to 30 mM.

11. The pharmaceutical composition according to claim 10, wherein, The concentration of the sodium phosphate buffer solution is 20 mM.

12. The pharmaceutical composition according to any one of claims 1-7, wherein, The concentration of the histidine buffer is 10 mM to 30 mM.

13. The pharmaceutical composition according to claim 12, wherein, The concentration of the histidine buffer is 20 mM.

14. The pharmaceutical composition according to any one of claims 1-7, wherein, The concentration of the polysorbate 80 or polysorbate 20 is from 0.1 mg / mL to 0.3 mg / mL.

15. The pharmaceutical composition according to claim 14, wherein, The concentration of polysorbate 80 or polysorbate 20 is 0.2 mg / mL.

16. The pharmaceutical composition according to any one of claims 1-7, wherein, The concentration of the sucrose is from 70 mg / mL to 90 mg / mL.

17. The pharmaceutical composition according to claim 16, wherein, The concentration of sucrose is 80 mg / mL.

18. The pharmaceutical composition according to any one of claims 1-7, wherein, The pH is 6 to 7.

19. The pharmaceutical composition according to claim 18, wherein, The pH value is 6.

5.

20. The pharmaceutical composition according to any one of claims 1-7, wherein, The pharmaceutical composition also contains 80 mM to 400 mM of arginine hydrochloride.

21. The pharmaceutical composition according to claim 20, wherein, The concentration of arginine hydrochloride is 80 mM to 120 mM.

22. The pharmaceutical composition according to claim 21, wherein, The concentration of arginine hydrochloride is 100 mM.

23. The pharmaceutical composition according to any one of claims 1-7, wherein, The pharmaceutical composition comprises: (a) a fusion protein targeting PD-L1 and TGF-β at a concentration of 10 mg / mL to 60 mg / mL, (b) a sodium phosphate buffer at a concentration of 10 mM to 40 mM, and a histidine buffer at a concentration of 10 mM to 40 mM, (c) polysorbate 80 at a concentration of 0.08 mg / mL to 0.4 mg / mL, and (d) sucrose at a concentration of 50 mg / mL to 100 mg / mL, and arginine or a pharmaceutically acceptable salt thereof at a concentration of 80 mM to 400 mM; wherein the pH of the pharmaceutical composition is 6 to 7.

5.

24. The pharmaceutical composition according to any one of claims 1-7, wherein, The pharmaceutical composition comprises: (a) 50 mg / mL of a fusion protein targeting PD-L1 and TGF-β, (b) 20 mM sodium phosphate buffer and 20 mM histidine buffer, (c) 0.2 mg / mL polysorbate 80, and (d) 80 mg / mL sucrose and 100 mM arginine hydrochloride; wherein the pH of the pharmaceutical composition is 6.

5.

25. A lyophilized formulation comprising a fusion protein targeting PD-L1 and TGF-β, said lyophilized formulation being obtained by freeze-drying the pharmaceutical composition of any one of claims 1-24.

26. A lyophilized formulation comprising a fusion protein targeting PD-L1 and TGF-β, wherein the lyophilized formulation, upon reconstitution, can form the pharmaceutical composition according to any one of claims 1-24.

27. Use of the pharmaceutical composition of any one of claims 1-24 or the lyophilized formulation of claim 25 or 26 in the preparation of a medicament for treating cancer, inhibiting tumor growth, or enhancing antitumor responses; wherein, The cancers or tumors mentioned are selected from: squamous cell carcinoma of the lung, adenocarcinoma of the lung, mesothelioma, melanoma, sarcoma, thyroid cancer, kidney cancer, liver cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, non-small cell lung cancer, breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, colon cancer, gastric cancer, gastrointestinal stromal tumor, bile duct cancer, cholangiocarcinoma, gallbladder cancer, penile cancer, anal cancer, vaginal cancer, cervical cancer, endometrial cancer, bladder cancer, head and neck cancer, skin cancer, hypopharyngeal cancer, laryngeal cancer, oral cancer, nasopharyngeal cancer, oropharyngeal cancer, pharyngeal cancer, paranasal sinus and nasal cavity cancer, and salivary gland cancer.

28. The use according to claim 27, wherein, The sarcoma is either a soft tissue sarcoma or an osteosarcoma.

29. The use according to claim 27, wherein, The pancreatic cancer mentioned is pancreatic ductal carcinoma.

30. The use according to claim 27, wherein, The stomach cancer mentioned is gastric adenocarcinoma.

31. The use according to claim 27, wherein, The endometrial cancer mentioned is endometrioid carcinoma of the uterine body.

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