Formulations of dual vegf / il-6 inhibitors, anti-vegf antibodies, and conjugates thereof

Abstract

This disclosure provides formulations comprising an IL-6-binding antagonistic antibody, a fusion protein thereof with a VEGF trap, and conjugates thereof, as well as methods for their preparation and use. The anti-IL-6 antibody, fusion protein, conjugate thereof, and formulation thereof can be used alone or in combination with other therapeutic agents to treat diseases. Furthermore, formulations comprising an anti-VEGF antibody and its conjugate are also provided.

Description

[0001] Incorporation of priority claims All applications claiming foreign or domestic priority as identified in the application data sheet are subject to 37 CFR. 1.57 This application is incorporated herein by reference. This application claims priority to U.S. Provisional Application No. 63 / 606284, filed December 5, 2023, and No. 63 / 656917, filed June 6, 2024. The contents of the foregoing applications are incorporated herein by reference.

[0002] sequence list This application is accompanied by an electronic sequence list. The sequence list is provided as a file named KDIAK222WOseqlist.xml, created on December 3, 2024, and is 381,304 bytes in size. The information in this electronic sequence list is incorporated herein by reference in its entirety.

[0003] Declaration of biological sample preservation In some embodiments, anti-IL-6 antibodies, fusion proteins, and conjugates thereof are provided, which were deposited on March 13, 2019, at the American Center for Type Culture Collection (ATCC) under the Budapest Treaty, with accession numbers PTA-125807 and PTA-125808.

[0004] The aforementioned deposit was made in accordance with the International Treaty on the Recognition of Microbial Deposits for Use in Patent Proceedings and its related provisions (the Budapest Treaty). This ensures the preservation of the deposit for 30 years from the date of deposit. ATCC will provide the deposit in accordance with the terms of the Budapest Treaty and the agreement between the applicant and ATCC, which ensures the permanent and unrestricted availability to the public upon the grant of the relevant U.S. patent or the publication of any U.S. or foreign patent application (whichever comes first), and ensures its availability in accordance with 35 USC. 122 and the rules established by the Director of the U.S. Patent and Trademark Office accordingly (including 37 CFR) 1.14), to provide the deposit to persons deemed by the Director to be entitled to it. The provision of the deposited biological material shall not be construed as licensing others to practice the invention in violation of any right granted under any government patent law. Technical Field

[0005] This article provides fusion constructs containing IL-6 and / or VEGF, or formulations containing anti-VEGF-A antibodies and their conjugates. Background Technology

[0006] Vascular endothelial growth factor A (VEGF-A) is a signaling protein that mediates pro-angiogenic functions such as endothelial cell survival, proliferation, migration, and intercellular permeability. Studies have shown that its activity promotes the progression of retinal diseases, such as choroidal neovascularization (CNV) in age-related macular degeneration (AMD) and diabetic macular edema (DME). Inhibition of VEGF-A signaling has been proven to be an effective means of preventing the progression of neovascular retinal diseases (Ferrara et al., Retina, 2006). Currently, various therapeutic molecules have been developed to inhibit VEGF function. Among them, anti-VEGF monoclonal antibodies such as ranibizumab and bevacizumab have been proven to be safe and effective drugs for treating pathological angiogenesis. Recently, recombinant VEGFR fusion proteins such as aflibercept (Eylea) and conbercept (China) have been shown to be more effective and have a longer duration of action than their antibody competitors as VEGF "traps".

[0007] Inflammation is involved in the pathogenesis of retinal diseases, and anti-inflammatory therapies such as steroids have shown efficacy in treating uveitis and diabetic macular edema (DME). Detailed studies on ocular inflammation and infection have shown that the pro-inflammatory cytokine interleukin-6 (IL-6) is significantly elevated in the ocular fluid of patients with refractory / chronic uveitis, and that inhibiting IL-6 in animal models can suppress uveitis flare-ups. High levels of IL-6 have also been found in the ocular fluid of patients with DME and retinal vein occlusion. Furthermore, chronic inflammatory cells are visible on the surface of Bruch's membrane in the eyes of patients with neovascular AMD, and serum IL-6 levels have been reported to be elevated in AMD patients. Interestingly, IL-6 has also been observed to stimulate defective angiogenesis. In addition to autoimmune diseases such as rheumatoid arthritis, anti-IL-6 therapy has proven effective in treating uveitis and uveitis-related macular edema. Summary of the Invention

[0008] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer solution; and a surfactant, and optionally, a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0009] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer comprising sodium acetate, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0010] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer, wherein the buffer comprises a mixture of sodium acetate and acetic acid; a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80; and optionally a penetration enhancer. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the fusion protein comprises: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the rest of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%, wherein if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0011] In some embodiments, a pharmaceutical formulation is provided, the formulation comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer; a surfactant; and optionally a penetration enhancer. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the unconjugated antibody is present in the formulation in an amount of approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0012] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer containing sodium acetate; and a surfactant comprising polysorbate 20 or polysorbate 80, and optionally a penetration enhancer. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the unconjugated fusion protein is present in a concentration of approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein in the formulation. In some embodiments, the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114, wherein the fusion protein has a light chain with at least 80% sequence identity with SEQ ID NO: 169, and wherein the fusion protein has a heavy chain with at least 80% sequence identity with SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0013] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising sodium acetate and a penetration enhancer; and a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114, wherein the fusion protein has a light chain with at least 80% sequence identity with SEQ ID NO: 169, and wherein the fusion protein has a heavy chain with at least 80% sequence identity with SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17A) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0014] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer solution containing histidine; a surfactant; and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0015] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer comprising histidine acetate, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0016] In some embodiments, a pharmaceutical formulation is provided, the formulation comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylated choline polymer or a zwitterionic monomer; a buffer, the buffer comprising a mixture of histidine and acetic acid; a penetration enhancer; and a surfactant, wherein the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the fusion protein comprises: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO:174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO:114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0017] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate; a penetration enhancer; and a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80, and the penetration enhancer. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0018] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate, an osmotic agent; and a surfactant. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114, wherein the fusion protein has a light chain with at least 80% sequence identity with SEQ ID NO: 169, and wherein the fusion protein has a heavy chain with at least 80% sequence identity with SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0019] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate and a penetration enhancer; and a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114, wherein the fusion protein has a light chain with at least 80% sequence identity with SEQ ID NO: 169, and wherein the fusion protein has a heavy chain with at least 80% sequence identity with SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17A) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0020] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer solution comprising sodium acetate; an penetration enhancer; and a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170.

[0021] In some embodiments, a pharmaceutical formulation is provided, comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising sodium acetate and a penetration enhancer; and a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170. In some embodiments, the fusion protein comprises the following structures: Formula (17Br) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the rest of the polymer, where X is Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%.

[0022] In some embodiments, a pharmaceutical formulation is provided, comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate, an osmotic agent; and a surfactant. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap. In some embodiments, the fusion protein comprises SEQ ID NO: 169 and 170, wherein the fusion protein comprises the following structures: Formula (17Br) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the rest of the polymer, where X is Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%.

[0023] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer solution ( wherein the buffer solution contains histidine), an emulsifier, and a penetration enhancer. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap.

[0024] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer comprising sodium acetate, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0025] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer ( wherein the buffer contains sodium acetate), an infiltration enhancer; and a surfactant. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170 (with or without the C-terminal lysine of SEQ ID NO: 170).

[0026] This article also provides a pharmaceutical formulation comprising about 40 mM to about 60 mM sodium acetate, about 0.01% to about 0.04% polysorbate 20, and a mixture of OG1950 and OG1953 at about 40 mg / mL to about 60 mg / mL (total protein concentration), the mixture containing about 15% to about 25% OG1950 and about 75% to about 85% OG1953 by molar weight, and a pH of about 4.5 to about 5.5.

[0027] This article provides a pharmaceutical formulation comprising, consisting of, or consisting primarily of: about 50 mM sodium acetate, about 0.025% polysorbate 20, and a mixture of OG1950 and OG1953 at about 50 mg / mL (total protein concentration), the mixture containing about 20% OG1950 and about 80% OG1953 by molar volume, and having a pH of about 5.

[0028] This article provides a pharmaceutical formulation comprising: a fusion protein conjugate comprising a first fusion protein conjugated to a phosphocholine-containing polymer; an unconjugated fusion protein comprising a second fusion protein unconjugated to a phosphocholine-containing polymer; a buffer containing histidine; and a surfactant, wherein the first fusion protein and the second fusion protein each comprise: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap, wherein the unconjugated fusion protein in the formulation comprises 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the unconjugated fusion protein.

[0029] Furthermore, a pharmaceutical formulation is provided, comprising: a first fusion protein comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO:170, with or without a C-terminal lysine; and a light chain comprising the amino acid sequence shown in SEQ ID NO:169; and a phosphorocholine-containing polymer conjugated to said heavy chain, wherein said fusion protein conjugate comprises the following structure: Equation (17A) Wherein: a portion of each heavy chain of the first fusion protein is represented by the letter H, and each light chain of the first fusion protein is represented by the letter L; the polymer is bonded to the heavy chain of the first fusion protein via a thiol group of C443 (EU number), and this bond is shown on one of the heavy chains; PC is... The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) -H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 2500 ± 15%; an uncoupled fusion protein comprising a second fusion protein uncoupled to a phosphocholine-containing polymer, the uncoupled fusion protein comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 170 (with or without a C-terminal lysine), or a variant thereof having L449 according to SEQ ID NO: 170; and comprising SEQ ID NO: The formulation comprises: a light chain of the amino acid sequence shown in 169, wherein the total concentration of the first fusion protein and the second fusion protein is 40 mg / mL to 60 mg / mL, wherein the content of the uncoupled fusion protein in the formulation is approximately 30% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, and the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the uncoupled fusion protein; 1 mM to 60 mM of histidine acetate; and a surfactant selected from polysorbate 20, polysorbate 80, and poloxamer 188, wherein the content of the surfactant in the formulation is 0.01% (w / w) to 0.1% (w / w), and wherein the pH of the pharmaceutical formulation is 4.9 to 6.2.

[0030] A pharmaceutical formulation is also provided, the pharmaceutical formulation comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is approximately 50 mg / mL; a phosphoric acid choline polymer, wherein the fusion protein is coupled to or uncoupled from the polymer; approximately 15 mM of histidine acetate; and approximately 0.025% (w / w) of a surfactant, wherein the surfactant comprises polysorbate 20, polysorbate 80, and / or poloxamer 188, wherein the pH of the pharmaceutical formulation is approximately 5.6, wherein the uncoupled fusion protein in the formulation is approximately 30% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the uncoupled fusion protein, and wherein the fusion protein comprises: a light chain comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 169; and a heavy chain comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 170, wherein the fusion protein conjugate comprises the following structure: Equation (17A) Wherein: a portion of each heavy chain of the fusion protein is represented by the letter H, and each light chain of the fusion protein is represented by the letter L; the polymer is bonded to the heavy chain via a thiol group of C443 (EU number), and this bond is shown on one of the heavy chains; PC is... The curve represents the connection point with the rest of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) -H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 2500 ± 15%.

[0031] This document also provides a pharmaceutical formulation comprising: a fusion protein comprising: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap; and a buffer, wherein the pH of the pharmaceutical formulation is in the range of 4.5 to 6.8, and optionally, wherein the formulation comprises a surfactant.

[0032] This article provides a pharmaceutical formulation comprising: a fusion protein comprising: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap; 10 mM to 50 mM sodium acetate or histidine acetate; 0 to 0.1% (w / w) of polysorbate 20; and 0 to 8% (w / v) of sucrose or trehalose, wherein the pH of the pharmaceutical formulation is in the range of 4.5 to 6.8, and the content of the fusion protein is 20 mg / mL to 200 mg / mL.

[0033] This article further provides a pharmaceutical formulation comprising: a fusion protein comprising: a light chain comprising SEQ ID NO: 169, or a sequence having at least 80% identity with it; and a heavy chain comprising SEQ ID NO: 170 (with or without a C-terminal lysine), or a sequence having at least 80% identity with it; about 15 mM of sodium acetate; about 0.025% (w / w) of polysorbate 20; and about 5.2% (w / v) of sucrose; wherein the pH of the pharmaceutical formulation is about 5.7, and the content of the fusion protein is about 100 mg / mL.

[0034] This article also provides a pharmaceutical formulation comprising: a fusion protein comprising: a light chain comprising SEQ ID NO: 169, or a sequence having at least 80% identity with it; and a heavy chain comprising SEQ ID NO: 170 (with or without a C-terminal lysine), or a sequence having at least 80% identity with it; about 15 mM of histidine acetate; about 0.025% (w / w) of polysorbate 20; and about 5.2% (w / v) of sucrose; wherein the pharmaceutical formulation has a pH of about 6.3 and the content of the fusion protein is about 100 mg / mL. Attached Figure Description

[0035] Figure 1 The sequence of IL-6 is shown.

[0036] Figure 2A Compound L is shown.

[0037] Figure 2B Compound K is shown.

[0038] Figure 2C The process of synthesizing OG1802 from R3707 is shown.

[0039] Figure 2D OG1786 is shown.

[0040] Figure 2E The process of synthesizing OG1546 from OG1550 is shown.

[0041] Figure 2F The process of synthesizing OG1784 from OG1546 and OG1563 is shown.

[0042] Figure 2G The process of synthesizing OG1405 from OG1784 is shown.

[0043] 0053] Figure 2H The process of synthesizing OG1785 from OG1405 is shown.

[0044] Figure 2I The process of synthesizing OG1786 from OG1785 is shown.

[0045] Figure 2J OG1801 is shown.

[0046] Figure 2K OG1802 is shown.

[0047] Figure 2L Compound E is shown.

[0048] Figure 3 A flowchart of antibody screening and optimization is shown.

[0049] Figure 4 The ELISA data shown indicate that the anti-IL-6 monoclonal antibody can bind to IL-6 but not to the IL-6 / IL-6R complex, and that the anti-IL-6 monoclonal antibody can inhibit the formation of the IL-6 / IL-6R complex.

[0050] Figure 5 Some embodiments of the heavy and light chain variable regions of the IL-6 antibody (IL-6-Ab) are shown. Embodiments of the CDR are shown in the boxed areas. These sequences can also be used in IL-6 antibody-VEGF trap fusion constructs.

[0051] Figure 6 Some embodiments of the IL-6-VEGF trap fusion protein are shown. The VEGF trap domain is located at the N-terminus immediately before the variable domain (left side), or between the Fab region and the hinge region of the antibody (right side).

[0052] Figure 7 Sensor diagrams and tables are shown, demonstrating that the binding affinity of the dual inhibitor molecules VEGFR-anti-IL-6 and anti-IL-6-VEGFR to VEGF-A is similar to that of the anti-VEGF antibodies OG1950 and Eylea. Therefore, the location of the VEGF trap does not alter its affinity for the target.

[0053] Figure 8Sensor maps of VEGF-A and IL-6 binding independently or in combination to the dual inhibitors are shown. The IL-6 sensor map of the mixed targets was compared with the theoretical curve (the sum of individual IL-6 and VEGF-A sensor maps). The results show that the theoretical curve coincides with the experimental curve, qualitatively indicating that both targets can bind to the dual inhibitor molecule without affecting each other's binding.

[0054] Figure 9 The OD450 nm values ​​for various ELISA IL-6 assays are shown. In the bridging ELISA (top), both dual inhibitors bridged btVEGF to IL-6, indicating that both conformations can bind to both targets. The EC50 for the VEGF trap-anti-IL-6 was 0.079 nM, and the EC50 for the anti-IL-6-VEGF trap was 0.026 nM. Eylea and anti-IL-6 were used as negative controls. Figure 9 The results of the IL-6 / IL-6R complex ELISA are also shown (middle). The dual inhibitors and anti-IL-6 showed similar levels of inhibition of IL-6 / IL-6R complex formation. IC50 values: anti-IL-6 = 0.36 nM, VEGF trap-anti-IL-6 = 0.47 nM, anti-IL-6-VEGF trap = 0.32 nM. Eylea was used as a negative control. Figure 9 The results of the VEGF / VEGFR competitive ELISA are also shown (bottom). The dual inhibitors, Eylea, and OG1950 showed varying degrees of inhibition against VEGF / VEGFR binding. Eylea was comparable to the anti-IL-6-VEGF trap construct (4.24 nM vs. 4.53 nM), while the VEGF trap-anti-IL-6 construct was approximately twice as good (1.74 nM), and OG1950 showed the greatest inhibitory effect (1.55 nM) compared to the other inhibitors.

[0055] Figure 10 Some embodiments of methods for preparing antibody conjugates are illustrated (which can also be applied to antibody-capture bodies or capture body-antibody conjugates). Although antibodies are depicted in the figures, those skilled in the art will understand in the present context that... Figure 10 The antibody description in the text can be replaced with the capture body fusion (e.g.) Figure 6 (As shown). For simplicity, the generic antibody depicted here represents both antibody options and fusion arrangement options in the context of fusion capture (unless it has been depicted as a fusion).

[0056] Figures 11A to 11B The SDS-PAGE bands of SeeBlue®Plus2 standard, anti-IL-6, anti-IL-6-VEGFR, and VEGFR-anti-IL-6 are shown. Figure 11CThe VEGFR-anti-IL-6 conjugate construct is shown, which is a fusion of anti-VEGF (VEGFR1 / 2) and anti-IL-6 coupled with a phosphorylcholine polymer.

[0057] Figure 12 shows the results of the transfer to the PVDF membrane and the N-terminal Edman sequencing results, which show that the cleavage products have the same N-terminal sequence (LTHRQT), indicating that the cleavage site is located in the VEGF trapping region.

[0058] Figures 13A to 13B The SDS-PAGE strips of SeeBlue®Plus2 standard and 19 VEGF capture constructs are shown.

[0059] Figures 13C to 13E The sequence tables for VEGF_capture_variant_1 and VEGF_capture_variant_3 are shown.

[0060] Figures 13F to 13G The SDS-PAGE bands of the SeeBlue®Plus2 standard and four VEGF trap variants are shown, including the VEGFR variant 3 with double point mutations of T94I and H95I.

[0061] Figures 14A to 14F The results of the Biacore assay and the measured affinity for VEGF-A are shown.

[0062] Figure 15 A cell-based assay for VEGF-stimulated VEGFR reporter gene is shown.

[0063] Figure 16A An assay is shown to inhibit VEGF / IL-6-mediated lumen formation in human umbilical vein endothelial cells (“HUVEC”).

[0064] Figures 16B to 16C The statistical data of lumen formation determination under different parameters are shown.

[0065] Figure 17 The results of HUVEC proliferation assays are shown.

[0066] Figure 18 An implementation of the anti-IL-6 heavy chain variable region sequence is shown. The CDR is underlined.

[0067] Figure 19 Various implementations of VEGF trapping sequences are shown. Differences between sequences are highlighted in bold and underlined.

[0068] Figure 20Some implementations of the linker (GS) sequence are shown. It can exist in the form of a double-repeating Gly-Gly-Gly-Gly-Ser linker (GS).

[0069] Figures 21A to 21B Some implementations of the heavy chain sequence of the anti-IL-6 molecule are shown. CDRs are underlined.

[0070] Figures 22A to 22B Some implementations of the light chain sequence of the anti-IL-6 molecule are shown. CDRs are underlined.

[0071] Figures 23A to 23B Some implementations of the heavy chain sequence of the anti-IL-6 molecule are shown. CDRs are underlined.

[0072] Figures 24A to 24B It shows Figures 21A to 23B Some implementations of CDR combination in the text.

[0073] Figure 25 Some implementations of the VEGFR-Fc sequence variants are shown. Differences between sequences are highlighted in bold and underlined.

[0074] Figures 26A to 26C The affinity binding data are shown.

[0075] Figure 27 Sequences of some embodiments of the VEGFR-anti-IL-6 sequence are shown. CDRs (as defined by Kabat) are underlined. Gray sections represent VEGFR constructs. Bold text indicates linker sections. Mutations L234A, L235A, G237A, and L443C (EU numbers) are double-underlined. Each of these sections is interchangeable with other corresponding sections provided herein (e.g., alternative linkers or CDRs, etc.).

[0076] Figure 28The SDS-PAGE electrophoresis image of the VEGFR-anti-IL-6 reduction (cysteine ​​decapping) reaction products is shown. Lanes are as follows: 1. VEGFR-anti-IL-6; 2. VEGFR-anti-IL-6-complete reduction (TCEP); 3. Novex Sharp pre-stained protein standard; 4. VEGFR-anti-IL-6 + 30x TCEP, starting point; 5. VEGFR-anti-IL-6 + 30x TCEP, after 30 minutes; 6. VEGFR-anti-IL-6 + 30x TCEP, after 60 minutes; 7. VEGFR-anti-IL-6 treated with TCEP, buffer replacement; 8. VEGFR-anti-IL-6 + 15x dHAA, starting point; 9. VEGFR-anti-IL-6 + 15x dHAA, after 30 minutes; 10. VEGFR-anti-IL-6 + 15x dHAA, after 30 minutes. dHAA, after 60 minutes; 11. VEGFR-anti-IL-6 decapping; 12. VEGFR-anti-IL-6 decapping, complete reduction (TCEP); Gel: NuPAGE Bis-Tris 4% to 12% Protein content: 4 μg / lane; 30x TCEP = 30 times molar excess TCEP; 15x dHAA = 15 times molar excess dHAA Figure 29 The SDS-PAGE electrophoresis image of the VEGFR-anti-IL-6-OG1802 conjugate by CEX chromatography is shown. Non-reducing gel: NuPAGE Bis-Tris 4% to 12%. Buffer A: 20 mM sodium acetate, pH 5.5. Buffer B: 20 ​​mM sodium acetate, pH 5.5, 500 mM NaCl. Lanes are as follows: 1. VEGFR-anti-IL-6; 2. VEGFR-anti-IL-6-OG1802 (loading); 3. NovexSharp pre-stained protein standard; 4. Flow-through buffer; 5. Elution buffer; 6. 30% Buffer B - aliquot 1; 7. 30% Buffer B - aliquot 2; 8. 30% Buffer B - aliquot 3; 9. 30% Buffer B - aliquot; 10. 40% Buffer B - aliquot 1; 11. 40% Buffer B - aliquot 2; 12. 40% Buffer B - aliquot 3; 13. 40% Buffer B - aliquot 4; 14. 60% Buffer B - aliquot 1; 15. 60% Buffer B - aliquot 2; 16. 60% Buffer B - aliquot 3; 17. 100% Buffer B - elution. Lanes 6-13 show protein conjugates that were too large to enter the gel. Lanes 14-17 show mixtures of proteins that may contain aggregated, conjugated, and unconjugated protein material. Figure 30SDS-PAGE electrophoresis images of the protein-polymer conjugate and the unconjugated protein are shown. Gel analysis revealed that when the bioconjugate (lane 3) was compared to the VEGFR-anti-IL-6 reference standard (lane 2), the band intensities of the heavy and light chains were 57% and 96%, respectively, indicating the presence of one OG1802 polymer per VEGFR-anti-IL-6 molecule. Reduction gels were prepared with NuPAGE Bis-Tris 4% to 12%.

[0077] Figure 31 The SEC-MALS chromatogram of VEGFR-anti-IL-6-OG1802 is shown. The molecular weight of the VEGFR-anti-IL-6 conjugate was determined by integrated size exclusion chromatography (Shodex-SB806M-HQ) and light scattering (MALS). Top: The chromatogram shows a single elution peak. The absence of additional peaks and shoulders indicates the absence of aggregates and degradation products after conjugation and subsequent CEX separation. Bottom: Protein conjugate analysis of the selected peaks shows an experimentally determined average molecular weight (Mw) of 983 kDa for the VEGFR-anti-IL-6-OG1802 bioconjugate. This value is derived from the conjugation of one VEGFR-anti-IL-6 molecule (Mw approximately 189 kDa) with one OG1802 polymer (Mw approximately 794 kDa).

[0078] Figure 32 The results of VEGFR-anti-IL-6 CEX chromatography are shown. Gel: Novex 8% to 16% Tris-Glycine (reduction conditions), M = SeeBlue® Plus 2 standard. Lanes D12 to F11 correspond to samples aliquoted at different buffer B concentrations as shown in the chromatogram. The intact (I) and cleaved (C) heavy chains are indicated in the figure. In the Poros pXS column, buffer A was 20 mM sodium phosphate, pH 6, and buffer B was 20 mM sodium phosphate, pH 6, 1M NaCl.

[0079] Figure 33 The results of VEGFR-anti-IL-6 HIC chromatography are shown. Gel: Novex 8% to 16% Tris-Glycine / reduction conditions, M = SeeBlue® Plus 2 standard. L = VEGFR-anti-IL-6 intact and lysed mixture (sample loading). The remaining lanes correspond to samples aliquoted at different buffer B concentrations as shown in the chromatogram. The intact (I) and lysed (C) heavy chains are labeled in the figure. In the Hi Trap Butly HP column, buffer A was: 20 mM sodium phosphate, pH 6, 1 M ammonium sulfate. Buffer B was: 20 mM sodium phosphate, pH 6.

[0080] Figure 34 The results of the VEGF / VEGFR competitive ELISA are shown.

[0081] Figure 35 The results of the IL-6 / IL-6R complex ELISA are shown.

[0082] Figure 36 The results of VEGF-stimulated VEGFR reporter gene assays based on cells are shown.

[0083] Figure 37 The formation of tubular structures in HUVECs stimulated by lipopolysaccharide is shown.

[0084] Figure 38 The formation of tubular structures in HUVECs stimulated by lipopolysaccharide is shown.

[0085] Figure 39 The formation of tubular structures in HUVECs stimulated by lipopolysaccharide is shown.

[0086] Figure 40A It demonstrates the inhibitory effect on VEGF / IL-6-mediated HUVEC proliferation.

[0087] Figure 40B The study demonstrated the inhibitory effect of increasing cell number per well on HUVEC proliferation.

[0088] Figures 41A to 41C SDS-PAGE images of antibodies and antibody conjugates after reduction and re-oxidation with different reagents are shown. Lane 41A is as follows: Lane 4: Antibody (IL-6 antibody-VEGF trap) starting material, Lane 5: 3x TCEP, Lane 6: 6x TCEP, Lane 7: 10x TCEP, Lane 8: 30x TCEP. Lane 42B is as follows: Lane 2: Antibody (IL-6 antibody-VEGF trap) starting material, Lane 3: 3x TCEP (re-oxidized over time), Lane 4: 6x TCEP (re-oxidized over time), Lane 5: 3x TCEP after re-oxidation, Lane 6: 6x TCEP after re-oxidation, Lane 7: 10x TCEP after re-oxidation, Lane 8: 30x TCEP after re-oxidation, Lane 9: 3x TCEP after conjugation, Lane 10: 6x TCEP after conjugation, Lane 11: 10x TCEP after conjugation, Lane 12: 30x TCEP after conjugation. Figure 41CThe lanes are as follows: Lane 7: Antibody (IL-6 antibody-VEGF trap) starting material, Lane 8: 30x TCEP after re-oxidation, Lane 9: 30x TCEP after conjugation. IEF PAGE gel (right). Lane 7: Label, Lane 8: Antibody (IL-6 antibody-VEGF trap) starting material, Lane 9: 30x TCEP after re-oxidation, Lane 10: 30x TCEP after conjugation.

[0089] Figure 42 A set of SEC-HPLC chromatograms with different excess TCEP is shown. Top panel: SEC-HPLC chromatograms of the antibody (IL-6 antibody-VEGF trap) after reduction with different excess TCEP, followed by conjugation with the biopolymer OG1802 to produce the fusion protein. 3x TCEP (blue), 6x TCEP (magenta), 10x TCEP (brown), and 30x TCEP (black) after conjugation. Bottom panel: A graph showing the conversion rate of the fusion protein versus the fold excess of TCEP.

[0090] Figure 43 The SDS-PAGE image is shown. The lanes are as follows: Lane 1: Antibody (IL-6 antibody-VEGF trap) starting material, Lane 2: 30x TCEP reduction, Lane 3: 10x DHAA re-oxidation, Lane 4: Label, Lane 5: 15x dHAA re-oxidation, Lane 6: 20x DHAA re-oxidation, Lane 7: 30x DHAA re-oxidation.

[0091] Figure 44 and 45 Flowcharts for the production of fusion antibodies (IL-6 antibody-VEGF trap) and combined dual inhibitor molecules (VEGFR-anti-IL-6-OG1802) are shown respectively.

[0092] Figure 46 The design scheme is shown, which uses SEC-HPLC, VEGF potency, viscosity, and osmolarity to screen pH, sucrose percentage, NaCl concentration, sodium acetate buffer strength, and protein concentration of OG2072 formulation. The sample buffer is sodium acetate with 0.025% polysorbate 20 added, and the samples are incubated at 37°C.

[0093] Figures 47A to 47B It shows Figure 46 The SEC-HPLC evaluation results of the OG2072 main peak in the sample formulation are shown. The sample formulation was analyzed on days 0, 7, 14, and 28.

[0094] Figures 48A to 48B It shows Figure 46The VEGF potency evaluation results of the sample formulation are shown. The sample formulation was analyzed on days 0, 7, 14, and 28.

[0095] Figures 49A to 49B It shows Figure 46 The viscosity evaluation results of the sample formulation are shown. The sample formulation was analyzed on day 0.

[0096] Figures 50A to 50B It shows Figure 46 The osmotic pressure assessment results of the sample formulation are shown. The sample formulation was analyzed on day 0.

[0097] Figure 51 It shows Figures 47A to 50B The results are summarized, where the effects of formulation parameters on SEC-HPLC, potency, viscosity, or osmotic pressure are represented by '+', '-', or 0. The more significant the effect, the more times the symbol is repeated, i.e., '+++'.

[0098] Figure 52 The parameters for the OG2072 (uncoupled) and OG2074 (coupled) formulations are shown, indicating the concentration of sodium acetate or histidine acetate, sucrose percentage, polysorbate 20% percentage, pH value, protein concentration, and the ratio of conjugate to uncoupled product.

[0099] Figure 53 The formulation information for six formulations containing OG2074 (conjugated) and OG2072 (unconjugated) antibodies is shown, indicating the ratio of conjugated to unconjugated antibodies, protein concentration, pH value, and the presence or absence of sucrose.

[0100] Figure 54 It is a table showing the sample settings for the long-term stability program of various formulations of OG1953 conjugates, with free protein content ranging from 7.5% to 20% and total combined protein concentration ranging from 50 mg / mL to 65 mg / mL.

[0101] Figure 55 and 56 It is a series of tables and charts showing ELISA assay results for measuring the potency of various OG1953 conjugate formulations containing 7.5% to 20% free protein and a total combined protein concentration of 50 mg / mL to 65 mg / mL after storage.

[0102] Figure 57 It consists of a table and a chart showing the formulation protein concentrations as measured by the SoloVPE OD280nm method.

[0103] Figure 58 It is a data table that shows a summary of the results of the formulation stability test.

[0104] Figure 59A This is the SEC-HPLC chromatogram of a series of formulations #4 (20% OG1950, 80% OG1953, 50 mM sodium acetate, 0.025% Tween 20, pH 5.0) after being stored at the temperature highlighted for 6 months. Figure 59B A table and several charts are provided to show formulation size exclusion chromatography analysis of OG1953 conjugate aggregation and degradation levels.

[0105] Figure 60A A schematic diagram of a tandem HPLC method is shown, which combines CEX-HPLC and SEC-HPLC chromatography columns in series.

[0106] Figure 60B It consists of a series of maps, a table, and multiple charts, showing the tandem method analysis of the OG1950 free protein and its aggregate forms (P1 and P2).

[0107] Figure 60C and 60D It consists of a series of graphs and a table showing the tandem method analysis of the formulation.

[0108] Figures 61A to 61C It is a series of charts showing the levels of impurities (e.g., aggregation and / or degradation levels) in OG1953 conjugate formulations over time.

[0109] Figure 62A and 62B It is a series of charts and tables showing the potency comparisons of various OG1953 formulations using ELISA or cell-based assays.

[0110] Figures 63A to 63C These are a series of schematic diagrams illustrating the components of non-limiting embodiments of the formulations disclosed herein.

[0111] Figure 64 The release data for batches 1-3 of KSI-501DS is shown.

[0112] Figure 65 The comparison of injection power between (Group A) OG1953 (100%) conjugate and KSI-301 mixture and (Group B) OG2074 (100%) conjugate and KSI-501_batch 2 mixture is shown when using 27G or 29G injection needles.

[0113] Figure 66 The viscosity of (Group A) OG1953 (100%) coupling compound and KSI-301 mixture at ambient temperature is shown; the viscosity of (Group B) OG2074 (100%) coupling compound and different batches of KSI-501 mixture is also shown.

[0114] Figures 67A to 67D show the tandem separation method over a continuous 80-minute period using a photodiode array (PDA) with a detection range of 200 nm to 350 nm. Figure 67A shows a two-dimensional contour plot of elution time versus wavelength; Figure 67B shows the extraction wavelength distribution at 280 nm, and the peak identification of various eluted components collected for subsequent SDS-PAGE analysis and silver staining characterization. The results show... Figure 67C (Non-reducing gum) and Figure 67D In (reducing gel).

[0115] Figure 68 Some implementations of the OG1950 heavy chain amino acid sequence are shown.

[0116] Figure 69 Some implementations of the OG1950 light chain amino acid sequence are shown.

[0117] Figures 70A to 70B Some implementation methods of experimental design (DoE) analysis for studying the effect of formulation parameters on the stability of OG2074 by CEX-HPLC are shown.

[0118] Figures 71A to 71B Some implementation methods of DoE analysis for studying the effect of formulation parameters on the stability of OG2072 by SEC-HPLC are shown.

[0119] Figures 72A to 72B Some implementation methods of DoE analysis for studying the effect of formulation parameters on the stability of OG2072 using a tandem HPLC model are shown.

[0120] Figures 73A to 73B Some implementation methods of DoE analysis for studying the effect of formulation parameters on impurity stability using a tandem HPLC model are shown.

[0121] Figures 74A to 74B The efficacy of VEGF was demonstrated. Figure 74A ) and IL-6 efficacy ( Figure 74B Some implementation methods for evaluation.

[0122] Figures 75A to 75B Some implementations of DoE analysis are shown, with exemplary CEX-HPLC and SEC-HPLC chromatograms demonstrating stability at 37°C.

[0123] Figure 76 Some implementations of DOE analysis are shown, displaying tandem HPLC chromatograms of stability on day 14.

[0124] Figure 77ASome implementations of DoE analysis are shown, in which DoE samples on day 0, day 14, and day 56 were analyzed by tandem HPLC. Figure 77B A summary table of some implementation methods of model analysis from DoE analysis is shown. Figure 77C Some implementations are shown, including the pH value after vacuum-assisted concentration, the actual protein concentration, and the actual buffer strength of the components from tandem HPLC analysis.

[0125] Figure 78 A summary table is shown, outlining some implementation methods for one-way analysis (OFAT) of the effects of formulation parameters on the stability of KSI-501.

[0126] Figures 79A to 79B Some implementation methods of OFAT analysis are shown, demonstrating the use of CEX-HPLC ( Figure 79A ) and SEC-HPLC ( Figure 79B The stability improvement was measured.

[0127] Figures 80A to 80B Some implementation methods for pH fine-tuning experiments with and without sucrose are shown. Figure 80A Some implementations of the sample pH value and sucrose concentration summary table are shown. Figure 80B Some embodiments of sample turbidity at pH values ​​of 5.6, 5.9, 6.2, and 6.5 are shown.

[0128] Figure 81 Some implementations are shown, illustrating stability data for KSI-501 GMP batches at a 3-month time point using two different formulations.

[0129] Figure 82A Some embodiments of the KSI-501 formulation containing 50 mM sodium acetate, pH 5.0, 4% sucrose, 0.025% polysorbate 20, 30% OG2072, and 70% OG2074 (at a concentration of 50.0 mg / mL) are shown. Figure 82B Some embodiments of the KSI-501 formulation containing 15 mM histidine acetate, pH 5.6, 0.025% polysorbate 20, 30% OG2072, and 70% OG2074 (at a concentration of 50 mg / mL) are shown.

[0130] Figures 83A to 83B The SEC-HPLC evaluation results of the OG2072 main peak in the OG2072 sample formulation in the sodium acetate buffer system are shown.

[0131] Figures 84A to 84B The results of the VEGF efficacy evaluation of the OG2072 sample formulation in the sodium acetate buffer system are shown.

[0132] Figure 85 It shows Figures 83A to 84B The results are summarized, where the effects of formulation parameters on SEC-HPLC, potency, viscosity, or osmotic pressure are indicated by '+', '-', or 0, with more significant effects indicated by repeated symbols, i.e., '+++'.

[0133] Figure 86 The parameters for each of the 24 experimental formulations are shown.

[0134] Figures 87A to 87C The results of SEC-HPLC evaluation of the OG2072 main peak in the OG2072 sample formulation in the histidine hydrochloride buffer system are shown. Figure 87C The stability of OG2072 was demonstrated after incubation at 37°C for 28 days.

[0135] Figures 88A to 88C The results of VEGF efficacy evaluation of the OG2072 sample formulation in the histidine hydrochloride buffer system are shown. Figure 87C The efficacy of VEGF was demonstrated after incubation at 37°C for 28 days.

[0136] Figure 89 The viscosity evaluation results of the OG2072 sample formulation in the histidine hydrochloride buffer system are shown.

[0137] Figure 90 The osmotic pressure assessment results of the OG2072 sample formulation in the histidine hydrochloride buffer system are shown.

[0138] Figure 91 It shows Figures 87A to 90 The results are summarized, where the effects of formulation parameters on SEC-HPLC, potency, viscosity, or osmotic pressure are indicated by '+', '-', or 0, with more significant effects indicated by repeated symbols, i.e., '+++'.

[0139] Figures 92A to 92B An overlay of SEC-HPLC chromatograms is provided, and some implementation methods of one-factor analysis (OFAT) for studying the effects of formulation parameters on the stability of KSI-101 are summarized.

[0140] Figures 93A to 93B The corrected VEGF potency and OG2072 SEC-HPLC peak area after 28 days of incubation at 37°C are shown.

[0141] Figures 94A to 94B The osmotic pressure and viscosity of the KSI-101 formulation in the sodium acetate buffer system and the histidine hydrochloride buffer system are shown.

[0142] Figure 95A flowchart of the production of the fusion protein (IL-6 antibody-VEGF trap) is shown. Detailed Implementation

[0143] To directly alleviate the concurrent inflammation and defective angiogenesis driving the pathogenesis of neovascular retinopathy, this paper presents molecular formulations designed to simultaneously block the function of the pro-inflammatory cytokine IL-6 and the pro-angiogenic signaling protein VEGF, as well as methods for preparing these formulations. These molecules consist of an anti-IL-6 monoclonal antibody fused to the VEGF-binding domains of two VEGF receptors (VEGFRs), (1) and (2). The anti-IL-6 moiety specifically binds to IL-6 and inhibits its interaction with the IL-6 receptor (IL-6R). The VEGF trapping moiety comprises a fusion of two VEGF-binding domains (VEGFR1 domain 2 and VEGFR2 domain 3), which acts as a VEGF trapping agent, preventing VEGF from binding to the VEGF receptor. Furthermore, in some embodiments, each of these dual inhibitor molecules is equipped with an unpaired cysteine ​​residue at its C-terminus, which can be conjugated to a phosphorocholine-based biopolymer with an extended half-life. In some embodiments, as provided herein, various fusion protein conjugate formulations, such as KSI-501, have been tested. When testing monovalent buffers (such as sodium acetate or imidazole), turbidity formation occurs at lower pH values. Histidine acetate only shows turbidity formation at higher pH values. Therefore, it is speculated that the monovalent ions of the buffer act as counterions, binding to the biopolymer of the conjugate to neutralize the charge. Without being bound by theory, trivalent histidine binds to the biopolymer but retains two additional charges, which may help repel molecules from interacting with it, thereby reducing turbidity.

[0144] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer solution; and a surfactant, and optionally, a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0145] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer solution comprising sodium acetate, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0146] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphocholine-containing polymer or a zwitterionic monomer; a buffer solution comprising a mixture of sodium acetate and acetic acid; a penetration enhancer; and a surfactant comprising polysorbate 20 or polysorbate 80 (or poloxamer 188). In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the fusion protein comprises: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments: each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), and this bond is shown on one of the heavy chains; PC is The curve represents the connection point with the rest of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%, wherein if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0147] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer; a surfactant; and optionally a penetration enhancer. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the unconjugated antibody (or fusion protein) is present in the formulation in an amount of approximately 10% to 60% of the total molar amount of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein), wherein the total molar amount is the sum of the molar amounts of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein). In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain).

[0148] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; and a buffer comprising sodium acetate, a penetration enhancer, and a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80 (or poloxamer 188). In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the unconjugated antibody (or fusion protein) is present in the formulation at a concentration of approximately 10% to 60% of the total molar amount of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein). In some embodiments, the total molar amount is the sum of the molar amount of the antibody (or fusion protein) conjugate and the molar amount of the unconjugated antibody (or fusion protein). In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114, wherein the fusion protein has a light chain with at least 80% sequence identity with SEQ ID NO: 169, and wherein the fusion protein has a heavy chain (with or without a C-terminal lysine) with at least 80% sequence identity with SEQ ID NO: 170. In some embodiments, the fusion protein conjugate comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0149] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer containing sodium acetate and an penetration enhancer; and a surfactant comprising polysorbate 20 or polysorbate 80 (or poloxamer 188). In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated antibody (or fusion protein) is present in the formulation at approximately 30% of the total molar amount of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein), wherein the total molar amount is the sum of the molar amounts of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein). In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114, wherein the fusion protein has a light chain with at least 80% sequence identity with SEQ ID NO: 169, and wherein the fusion protein has a heavy chain with at least 80% sequence identity with SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17A) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain).

[0150] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer solution containing histidine; a surfactant; and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0151] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer comprising histidine acetate, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0152] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylated choline polymer or a zwitterionic monomer; a buffer, the buffer comprising a mixture of histidine and acetic acid; an osmotic agent; and a surfactant, wherein the surfactant is polysorbate 20 or polysorbate 80 (or poloxamer 188). In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the fusion protein comprises: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain).

[0153] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer containing histidine acetate; a penetration enhancer; and a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80 (or poloxamer 188), and optionally includes a penetration enhancer. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated antibody (or fusion protein) in the formulation comprises approximately 10% to 60% of the total molar amount of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein), wherein the total molar amount is the sum of the molar amounts of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein). In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain).

[0154] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer containing histidine acetate, an osmotic agent; and a surfactant. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated antibody (or fusion protein) in the formulation comprises approximately 10% to 60% of the total molar amount of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein), wherein the total molar amount is the sum of the molar amounts of the antibody (or fusion protein) conjugate and the unconjugated antibody (or fusion protein). In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114, wherein the fusion protein has a light chain with at least 80% sequence identity with SEQ ID NO: 169, and wherein the fusion protein has a heavy chain with at least 80% sequence identity with SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain).

[0155] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate and an osmotic agent; and a surfactant comprising polysorbate 20 or polysorbate 80 (or poloxamer 188). In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein in the formulation, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114, wherein the fusion protein has a light chain with at least 80% sequence identity with SEQ ID NO: 169, and wherein the fusion protein has a heavy chain with at least 80% sequence identity with SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17A) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain).

[0156] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer solution comprising sodium acetate and a penetration enhancer; and a surfactant comprising polysorbate 20 or polysorbate 80 (or poloxamer 188). In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170. In any formulation or composition herein, in some embodiments, the surfactant may be poloxamer 188 instead of polysorbate 20 or polysorbate 80.

[0157] In some embodiments, a pharmaceutical formulation is provided, comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylated choline polymer or a zwitterionic monomer; a buffer solution comprising sodium acetate and an osmotic agent; and a surfactant, wherein the surfactant comprises polysorbate 20 or polysorbate 80 (or poloxamer 188). In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein in the formulation, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170. In some embodiments, the fusion protein comprises the following structures: Formula (17Br) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is The curve represents the connection point with the rest of the polymer, where X is Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%.

[0158] In some embodiments, a pharmaceutical formulation is provided, comprising: a pharmaceutically effective amount of a fusion protein; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate, an osmotic agent; and a surfactant. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein in the formulation, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap. In some embodiments, the fusion protein comprises SEQ ID NO: 169 and 170, wherein the fusion protein comprises the following structures: Formula (17Br) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is The curve represents the connection point with the rest of the polymer, where X is Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%.

[0159] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer solution containing histidine; and a surfactant and a penetration enhancer. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap.

[0160] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer comprising sodium acetate, a surfactant, and a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap.

[0161] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer (containing sodium acetate and an infiltration enhancer), and a surfactant. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170 (with or without the C-terminal lysine of SEQ ID NO: 170).

[0162] In some embodiments, this document provides a pharmaceutical formulation comprising a pharmaceutically effective amount of a fusion protein, a buffer solution, and an emulsifier. In some embodiments, the fusion protein is... Figure 27 The protein shown, and / or containing Figure 27 The fusion protein may contain one, two, three, four, or more components, and / or include SEQ ID NO: 169 and / or 170 (but may be any fusion protein provided herein). In some embodiments, the fusion protein has a sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to SEQ ID NO: 169 and / or 170, and / or includes three heavy chains and / or three light chains CDRs therein. In some embodiments, the fusion protein is conjugated to a polymer, optionally as shown in Formula 17, 17A, or 17Br. Note that Formulas 17 and 17A show the same active ingredient bound to the antibody. In some embodiments, in Formula 17Br, X is Br.

[0163] In some embodiments, the formulation comprises a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is between about 30 mg / mL and about 85 mg / mL; a polymer, wherein the fusion protein is coupled to the polymer, wherein the polymer comprises a phosphorylated choline polymer or a zwitterionic monomer; a buffer, wherein the buffer comprises a mixture of sodium acetate and acetic acid, wherein the buffer is between about 0.1 mM and about 25 mM, wherein the pH of the buffer is about 5.0; and a surfactant, wherein the surfactant comprises about 0.01% (w / w) to about 0.05% (w / w) of polysorbate 20 or polysorbate 80. In some embodiments, the fusion protein comprises: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap, wherein the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114 (or wherein the fusion protein has Figure 27 (The sequence in the image). In some embodiments, the fusion protein comprises the following structure: Equation (17) Wherein: each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), and this bond is shown on one of the heavy chains; PC is The curve represents the connection point with the rest of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n6, n7, n8, and n9 is 2500 ± 15%, wherein if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0164] In some embodiments, the fusion protein comprises the following structure: Equation (17A) Wherein: each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via the thiol group of C443 (EU number), and this bond is shown on one of the heavy chains; PC is The curve represents the connection point with the rest of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500 ± 15%, wherein if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain.

[0165] Unless otherwise stated, the various embodiments provided herein will employ conventional techniques within the scope of the art, including molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology. These techniques are well explained in the literature, for example: *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989), Cold Spring Harbor; *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Methods in Molecular Biology*, Humana Publishing; *Cell Biology: Laboratory Notes* (edited by J.E. Cellis, 1998), Academic Press; *Animal Cell Culture* (edited by R.I. Freshney, 1987); *Introduction to Cell and Tissue Culture* (JP. Mather and PE. Roberts, 1998), Plenum Publishing; *Cell and Tissue Culture: Laboratory Procedures* (edited by A. Doyle, J.B. Griffiths, and D.G. Newell, 1993–1998), J. Wiley and Sons; *Enzymological Methods* (Academic Press); *Handbook of Experimental Immunology* (edited by DM. Weir and CC. Blackwell); *Mammalian Cell Gene Transfer Vectors* (edited by J.M. Miller and PE. Calos, 1987); *A Concise Protocol for Molecular Biology* (FM... Ausubel et al. (eds., 1987); *PCR: Polymerase Chain Reaction* (eds., Mullis et al., 1994); *Immunology: A Concise Protocol* (eds., JE Coligan et al., 1991); *Short Protocols for Molecular Biology* (Wiley and Sons, 1999); *Immunobiology* (CA Janeway and P. Travers, 1997); *Antibodies* (P. Finch, 1997); *Antibodies: Practical Approaches* (eds., D. Catty, IRL Publishing, 1988-1989); *Monoclonal Antibodies: Practical Approaches* (eds., P. Shepherd and C. Dean, Oxford University Press, 2000); *Antibodies in Use: A Laboratory Handbook* (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); *Antibodies* (eds., M. Zanetti and JD Capra, Harwood Academic Press, 1995).

[0166] Unless otherwise stated, the following terms shall be understood to have the following meanings: “isolated molecule” refers to a molecule (e.g., polypeptide, polynucleotide, or antibody, etc.) that, due to its origin or derivative, possesses the following characteristics: (1) it is not bound to the naturally associated components that accompany it in its natural state; (2) it is substantially free of other molecules from the same source (e.g., species, cells expressing the molecule, libraries, etc.); (3) it is expressed by cells of a different species; or (4) it is not present in nature. Therefore, a chemically synthesized molecule or expressed in a cell system different from its natural origin will be “isolated” from its naturally associated components. Isolation can also render a molecule substantially free of its naturally associated components by purification techniques known in the art. The purity or homogeneity of a molecule can be determined by a variety of methods known in the art. For example, the purity of a polypeptide sample can be visualized using polyacrylamide gel electrophoresis and staining of the gel, a technique known in the art. For certain purposes, higher resolution can be provided by using HPLC or other purification methods known in the art.

[0167] As used herein, unless otherwise specified, "IL-6" or "IL6" refers to human IL-6. Other forms of IL-6 are also contemplated in some embodiments and will be designated by specific references to other organisms, such as dogs, cats, horses, and cattle. An exemplary human IL-6 can be found in UniProt registry number P05231.

[0168] The anti-IL-6 antibodies or other biological agents described herein are typically provided in isolated form. This means that the antibodies are generally at least 50% w / w pure, free from interfering proteins and other contaminants generated during production or purification, but this does not preclude the possibility of combining the antibodies with pharmaceutically acceptable excipients intended to facilitate their use. Sometimes, the antibodies are at least 60%, 70%, 80%, 90%, 95%, or 99% pure by weight, free from interfering proteins and contaminants generated during production or purification. Typically, the antibody (or antibody-drug conjugate) is the major macromolecule remaining after purification.

[0169] An "antibody" is an immunoglobulin molecule capable of specifically binding to a target (such as carbohydrates, polynucleotides, lipids, peptides, etc.) through at least one antigen recognition site located within the variable region of an immunoglobulin molecule. As used herein, the term encompasses not only intact monoclonal or polyclonal antibodies, but also, unless otherwise stated, any antigen-binding moiety competing for specific binding with an intact antibody, fusion proteins containing an antigen-binding moiety, and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site. Antigen-binding moieties include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camel antibodies), fragments containing complementarity-determining regions, single-chain variable fragment antibodies (scFv), large antibodies, micro antibodies, intracellular antibodies, dimeric antibodies, trimeric antibodies, tetrameric antibodies, v-NAR, and bispecific scFv, as well as peptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen-binding ability. Antibodies include any class of antibodies, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies need not belong to any particular class. Immunoglobulins can be classified into different categories based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0170] The “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, whether present alone or in combination. As is known in the art, the variable regions of both the heavy and light chains each consist of four frame regions (FRs), which are linked by three complementarity-determining regions (CDRs, also known as hypervariable regions) and contribute to the formation of the antibody’s antigen-binding site. If a variant of the target variable region is desired, especially when amino acid residues outside the CDR regions (i.e., within the frame regions) are substituted, suitable amino acid substitutions can be determined by comparing the target variable region with the variable regions of other antibodies containing CDR1 and CDR2 sequences of the same typical class as the target variable region, preferably conserved amino acid substitutions (Chothia and Lesk, J MolBiol 196(4): 901-917, 1987).

[0171] In some embodiments, the precise definition of the CDR and the identification of the residues constituting the antibody binding site can be achieved by resolving the structure of the antibody and / or the antibody-ligand complex. In some embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or approximately determine the CDR region. Examples of such methods include, but are not limited to, Kabat definition, Chothia definition, IMGT method (Lefranc et al., 2003) Dev Comp Immunol. 27:55-77), computational programs such as Paratome (Kunik et al., 2012, Nucl Acids Res. W521-4), AbM definition, and conformational definition.

[0172] Kabat definitions are the standard for numbering antibody residues and are commonly used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. Chothia definitions are similar to Kabat definitions, but Chothia definitions take into account the location of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342: 877-83. AbM definitions use an integrated computer program suite produced by the Oxford Molecular Group to model antibody structures. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM..." TM"A Computer Program for Modeling Variable Regions of Antibodies", Oxford, UK; Oxford Molecular, Ltd. AbM definitions utilize a combination of knowledge databases and ab initio computational methods to model the tertiary structure of antibodies from the primary sequence, as described in, for example, Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Contact definitions are based on the analysis of existing complex crystal structures. See, for example, MacCallumetal., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to here as the "conformational definition" of CDRs, the positions of CDRs can be identified as residues that contribute enthalpy to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry. 283:1156-1166. Other CDR boundary definitions may not strictly follow one of the methods described above, but will still overlap with at least a portion of the Kabat CDR, even though they may be shortened or lengthened, based on predictions or experimental results indicating that a particular residue or group of residues has no significant effect on antigen binding. As used herein, a CDR can refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein can utilize a CDR defined according to any of these methods. For any given implementation containing more than one CDR, the CDR can be defined according to any one of the Kabat, Chothia, extended definitions, IMGT, Paratome, AbM, and / or conformational definitions, or any combination of the above definitions.

[0173] As is known in the art, the “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, whether they exist alone or in combination.

[0174] As used herein, "monoclonal antibody" refers to an antibody derived from a group of essentially homogeneous antibodies, meaning that the individual antibodies constituting the group are identical, but may contain a small number of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. The modifier "monoclonal" indicates the characteristic of the antibody being derived from a essentially homogeneous group of antibodies and should not be interpreted as requiring production by any particular method. For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler and Milstein in 1975 (Nature 256:495), or by recombinant DNA methods (as described in U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage libraries generated using techniques such as those described by McCafferty et al. (1990, Nature 348:552-554). The term "humanized" antibody as used herein refers to a form of non-human (e.g., mouse) antibody, namely a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antibody-antigen binding sequence) containing a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (receptor antibody) in which the residues of the receptor CDR are replaced by CDR residues of a non-human species (donor antibody, such as mouse, rat, or rabbit) with the desired specificity, affinity, and capability. The humanized antibody may contain residues that are not present in the receptor antibody, nor in the introduced CDR or frame sequence; these residues are included to further improve and optimize antibody performance.

[0175] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of human-produced antibodies, and / or prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0176] The term "chimeric antibody" is intended to refer to an antibody whose variable region sequence originates from one species and whose constant region sequence originates from another species, such as an antibody whose variable region sequence originates from a mouse antibody and whose constant region sequence originates from a human antibody. The term "epitope" refers to a portion of a molecule that can be recognized and bound by an antibody at one or more antigen-binding regions. Epitopes typically consist of surface groups of a molecule (such as amino acids or sugar side chains) and have specific three-dimensional structural features and specific charge characteristics. In some embodiments, the epitope may be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all interaction sites between the protein and the interacting molecule (such as an antibody) occur linearly along the amino acid sequence of the protein. A "nonlinear epitope" or "conformational epitope" comprises a discontinuous polypeptide (or amino acid) within an antigen protein that specifically binds to the antibody. As used herein, the term "antigen epitope" is defined as a portion of an antigen that an antibody can determine by any method known in the art (e.g., by routine immunoassay) to specifically bind to. Once a desired epitope on an antigen is identified, it is possible to generate an antibody against that epitope, for example using the techniques described in this specification. Alternatively, during the discovery process, the generation and characterization of antibodies can elucidate information about the desired epitope. Based on this information, antibodies that bind to the same epitope can then be competitively screened. One approach to achieving this is to conduct competitive and cross-competitive studies to identify antibodies that compete or cross-compete with each other when binding to IL-6, for example, antibodies competing to bind to antigens.

[0177] As used herein, the term "competition," in the context of antibodies, refers to the binding of a first antibody or its antigen-binding moiety to an epitope in a manner sufficiently similar to that of a second antibody or its antigen-binding moiety binding epitope, such that, in the presence of the second antibody, the binding of the first antibody to its homologous epitope is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. Conversely, a detectable reduction in the binding of the second antibody to its epitope may occur in the presence of the first antibody, but this is not necessary. That is, the first antibody may inhibit the binding of the second antibody to its epitope without the second antibody needing to inhibit the binding of the first antibody to its corresponding epitope. However, if each antibody detectably inhibits the binding of another antibody to its homologous epitope or ligand (whether to the same, greater, or lesser extent), these antibodies are said to "cross-compete" with each other in binding to their respective epitopes. This document provides for competitive antibodies and cross-competitive antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or a portion thereof), those skilled in the art, based on the teachings provided herein, will understand that such competitive and / or cross-competitive antibodies are encompassed and can be used in the methods disclosed herein.

[0178] As used herein, when the equilibrium dissociation constant of the antibody with IL-6 is equal to or less than 20 nM, preferably less than about 6 nM, more preferably less than about 1 nM, and most preferably less than about 0.75 nM, the antibody "interacts" with IL-6. In some embodiments, the affinity of the antibody is between 400 pM and 800 pM, for example, 450 pM to 700 pM, or 500 pM to 600 pM.

[0179] IL-6 antagonist antibodies encompass antibodies that block, antagonize, inhibit, or reduce (to any extent, including significantly reduce) the biological activity of IL-6, such as binding to IL-6R, binding of the IL-6 / IL-6R complex to gp130, phosphorylation and activation of Stat3, cell proliferation, and stimulation of IL-6-mediated inflammatory or pro-angiogenic pathways. For the purposes of this disclosure, it should be clearly understood that the term "IL-6 antagonist antibody" encompasses all previously identified terms, names, and functional states and characteristics where IL-6 itself, IL-6 biological activity, or the consequences of such biological activity are substantially eliminated, reduced, or neutralized to any meaningful extent. In some embodiments, the IL-6 antagonist antibody binds to IL-6. Examples of IL-6 antagonist antibodies are provided herein.

[0180] "Preferential binding" or "specific binding" (used interchangeably herein) of an antibody to a particular epitope are well-known terms in the art, as are methods for determining such specificity or preferential binding. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or binds to a particular cell or substance more frequently, and / or more quickly, and / or for a longer duration, and / or with a stronger affinity. An antibody is "specifically bound" or "preferentially bound" to a target if its affinity, and / or binding rate, and / or duration of binding to the target is superior to its binding to other substances. For example, an antibody that specifically or preferentially binds to an IL-6 epitope means that its affinity, and / or binding rate, and / or duration of binding to that epitope is superior to its binding to other IL-6 epitopes or non-IL-6 epitopes. Reading this definition, it should also be understood that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Binding is usually (but not always) referred to as preferential binding.

[0181] As used herein, “substantially pure” means a substance with a purity of at least 50% (i.e., free of contaminants), preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99%.

[0182] "Host cell" includes a single cell or cell culture that can serve as, or has served as, a recipient for a vector to integrate a polynucleotide insert. Host cells also include the offspring of a single host cell, which may not be identical to the original parent cell in morphology or genomic DNA complementarity due to natural, accidental, or intentional mutations. Host cells also include cells transfected in vivo with the polynucleotides provided herein.

[0183] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at Cys226 or Pro230 to its C-terminus. The residues in the Fc region are numbered using Kabat's EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The Fc region of an immunoglobulin typically contains two constant domains, CH2 and CH3. As is known in the art, the Fc region can exist in dimer or monomeric form.

[0184] As used in the art, “Fc receptor” and “FcR” describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are naturally occurring human FcRs. Furthermore, preferred FcRs are receptors that bind to IgG antibodies (γ receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. For a review of FcRs, see Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and deHaas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).

[0185] A “functional Fc region” possesses at least one effector function of a native Fc region. Exemplary “effector functions” include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors). Such effector functions typically require the Fc region to bind to a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art for evaluating such antibody effector functions.

[0186] The “natural sequence Fc region” contains an amino acid sequence identical to that of the Fc region found in nature. The “variant Fc region” contains an amino acid sequence that differs from the natural sequence Fc region due to at least one amino acid modification, but still retains at least one effector function of the natural sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to the natural sequence Fc region or the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions in the natural sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region described herein preferably has at least about 80% amino acid sequence identity with the natural sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% amino acid sequence identity, and more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity.

[0187] As used in this article, “treatment” is a method of achieving a beneficial or desired clinical outcome.

[0188] As used herein, “IL-6 and / or VEGF-related diseases” include, for example, ocular diseases and systemic diseases. Ocular diseases include inflammatory eye diseases such as scleritis, non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, diabetic macular edema, prevention of diabetic macular edema, prevention of proliferative diabetic retinopathy, wet age-related macular degeneration, prevention of wet age-related macular degeneration, dry age-related macular degeneration, venous, arterial or other occlusions of ocular and / or retinal vessels with or without retinal edema, anterior and posterior uveitis, uveitis-related macular edema, and intraocular tumors. IL-6-related diseases also include diseases resulting from elevated IL-6 activity levels due to the interaction of IL-6 with IL-6R or soluble IL-6R (sIL-6R). In some embodiments, any one or more fusion proteins and / or any one or more conjugates provided herein may be used to treat or prevent any one or more IL-6 and / or VEGF-related diseases. In some embodiments, the disease includes systemic diseases affecting the eye (such as Graves' disease or neuromyelitis optica), or systemic diseases not affecting the eye (such as multiple sclerosis or rheumatoid arthritis). In some embodiments, the disease includes cytokine release syndrome following CAR-T or similar immuno-oncology therapy.

[0189] Furthermore, anti-IL-6 molecules can eliminate the IL-6 expression induction observed after anti-PD-1 / PD-L1 molecule therapy (Tsukamoto et al., Cancer Res; 2018 78(17); 5011-22). Studies have also shown that blocking VEGF signaling can improve the efficacy of anti-PD-L1 therapy (Allen et al., Sci Transl Med 2017 April 12: 9(385)). Therefore, these dual inhibitors can be used in combination with PD-1 / PDL-1 modulators and / or other immune checkpoint inhibitors for synergistic cancer treatment. Other diseases may include cerebral edema in glioblastoma, where anti-IL-6 therapy may show additional benefits over anti-VEGF therapy. Other diseases include those with solid tumors.

[0190] As used herein, “improvement” refers to a reduction or improvement in one or more symptoms compared to the absence of IL-6 antibody, IL-6 antibody-VEGF trap fusion protein, IL-6 antibody conjugate, and / or IL-6 antibody-VEGF trap fusion protein conjugate. “Improvement” also includes a reduction or shortening of the duration of symptoms.

[0191] As used herein, “VEGF trap” or similar terms refer to the VEGF-binding domain (VEGFR1 domain 2, VEGFR2 domain 3). This fragment enables the protein to function as a VEGF trap, preventing VEGF from binding to VEGF receptors expressed by the cell. An example of this sequence can be found in Table 10. In some embodiments, the VEGF trap comprises only VEGFR1 domain 2 and VEGFR2 domain 3. Various embodiments of trap proteins are known in the art, for example, see U.S. Publication No. 20150376271, the entire contents of which, relating to various embodiments of VEGF traps (i.e., VEGFR proteins or fragments thereof) and their fusion proteins, are incorporated herein by reference. In some embodiments, the term “VEGF trap” or similar terms refer to the full-length extracellular region or any portion thereof, or a combination of portions derived from different VEGF receptors, capable of antagonizing signal transduction between at least one VEGF and VEGFR.

[0192] As used herein, "IL-6 antibody-VEGF trap fusion protein," "IL-6 antibody-VEGF trap," "antibody IL-6-VEGF trap," "anti-IL-6-VEGF trap," "VEGFR-anti-IL-6," "VEGFR-anti-IL-6," "VEGF trap-anti-IL-6 antibody fusion protein (TAF)," "VEGF trap-IL-6," "VEGFR IL-6," "IL-6-VEGFR," or similar terms or their inverses (e.g., "VEGF trap-IL-6 antibody," "VEGF trap-IL-6 antibody fusion protein," etc.) all refer to the fusion between an IL-6 antibody and a VEGF trap. The implementation method is as follows: Figure 6As shown. When used generally, the order of the two terms may be interchanged. When used specifically, the order of the two terms indicates the relative positions of the components in the construct. “Antibody-capture body,” “IL-6 antibody-VEGF capture body,” “antibody IL-6 VEGF capture body,” or “antibody IL-6-capture body,” or “anti-IL-6 VEGF capture body,” “capture body-antibody,” anti-IL-6-VEGFR, anti-IL-6-VEGFR, or other similar terms or their inverses (e.g., “VEGF capture body-IL-6 antibody,” “VEGF capture body-IL-6 antibody fusion protein,” etc.) indicate the arrangement in which an antibody is fused to the relevant domain of a VEGF-binding protein to provide a VEGF capture body. As mentioned above, this portion of the VEGF-binding protein is the part that prevents VEGF from binding to the VEGF receptor. As described herein, the arrangement (order) of the capture body and antibody portions can vary. Therefore, unless explicitly stated otherwise or indicated by the context, the phrases in this document relating to antibody-capture bodies (or IL-6 / VEGF capture bodies, etc.) fusion proteins represent all disclosed embodiments regarding antibody and capture body localization. Therefore, unless otherwise explained, the phrase "antibody-capture body" (or "IL-6 / VEGF capture body," etc.) indicates... Figure 6 The left-side implementation method in Figure 6 The implementation method on the right side and Figure 6 Two implementation methods are described. Therefore, for convenience, the general language is considered to disclose all three options. If the orientation is explicitly specified, for example, it can be indicated by stating that "arrangement" can be one of the following: capture body-antibody, capture body IL-6 antibody, VEGF capture body antibody IL-6, VEGF capture body antibody IL-6. Similarly, it should be understood that the context of certain embodiments of the invention relates to a specific orientation or arrangement of molecules, which is indicated by the context of the embodiment. For all the discussions of fusion proteins provided herein, both arrangements (as alternatives and combinations) are explicitly covered. Furthermore, due to the order, it should be understood that when used in the context of fusion proteins, the phrase IL-6 antibody includes both the option that the antibody is sequential ( Figure 6 (Left side), also includes the option of the trap being located "inside" the antibody ( Figure 6 (Right side). Similarly, unless otherwise stated, when used in the context of fusion proteins (or other similar terms), the term "Ab" or "antibody" covers all three options. Figure 6 Left side Figure 6(Right side, and two options). In some implementations, the VEGF trap is fused to IL-6 in one of the following ways: at the N-terminus of the heavy chain containing IL-6 VH; or between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain. When referring to antibodies or fragments thereof in nomenclature, there is no distinction between the names Ab, antibody, “anti”, or other similar terms. There is no distinction between the names “IL-6,” “IL6,” or simply “IL-6.” As used herein, the terms “VEGF,” “VEGFR,” “VEGF trap,” “VEGFR trap,” etc., are used interchangeably when referring to fusion constructs with IL-6. These terms may have different meanings when used separately from the IL-6 fusion construct, depending on the context of the relevant term.

[0193] As used herein, the term "biopolymer" indicates that a polymer has been linked to a target protein. This term can also be used to describe a "coupled" form of protein. This applies to all proteins described herein. Therefore, all IL-6 antibodies and IL-6 antibody-VEGF traps provided herein encompass IL-6 antibody biopolymers and IL-6 antibody-VEGF trap biopolymers. Additionally, VEGF trap biopolymers are also provided.

[0194] As used in this article, "antagonistic antibody" refers to an antibody that blocks one or more functions or activities of the molecules it binds to.

[0195] As used herein, an “effective dose” or “effective amount” of a drug, compound, or pharmaceutical composition means an amount sufficient to achieve any one or more beneficial or intended results. More specifically, an effective amount may prevent, alleviate, or improve disease symptoms, and / or prolong the survival of a subject. For prophylactic use, beneficial or intended results include eliminating or reducing risk, reducing severity, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during disease development. For therapeutic use, beneficial or intended results include clinical outcomes such as reducing one or more symptoms of the disease (e.g., but not limited to AMD, including dry AMD and wet AMD), reducing the dosage of other drugs required to treat the disease, enhancing the efficacy of another drug, and / or delaying the progression of AMD in a patient. An effective dose may be administered by a single or multiple doses. For the purposes of this invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. As understood in the clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved when used in combination with other drugs, compounds, or pharmaceutical compositions. Therefore, "effective dose" can be considered in the context of administering one or more therapeutic agents. If a single agent, when used in combination with one or more other agents, can achieve or does achieve the desired result, then the single agent can be considered to be administered in an effective dose.

[0196] An effective regimen of anti-IL-6 antibody administration refers to the use of specific doses, routes of administration, and frequencies to delay disease onset, reduce severity, inhibit further deterioration, and / or improve at least one sign or symptom of the disease. If a patient already has the disease, this regimen may be considered a therapeutically effective regimen. If a patient has a higher risk of disease than the general population but has not yet developed symptoms, this regimen may be considered a preventatively effective regimen. In some cases, therapeutic or preventative efficacy can be observed in individual patients relative to historical controls or prior experience with the same patient. In other cases, therapeutic or preventative efficacy can be demonstrated in preclinical or clinical trials in a treated patient population relative to an untreated control population.

[0197] Effective administration of anti-IL-6-VEGF traps refers to the use of specific doses, routes of administration, and frequencies to delay disease onset, reduce severity, inhibit further deterioration, and / or improve at least one sign or symptom of the disease. If a patient already has the disease, this regimen may be considered a therapeutically effective regimen. If a patient has a higher risk of disease relative to the general population but has not yet developed symptoms, this regimen may be considered a preventatively effective regimen. In some cases, therapeutic or preventative efficacy may be observed in individual patients relative to historical controls or prior experience with the same patient. In other cases, therapeutic or preventative efficacy may be demonstrated in preclinical or clinical trials in a treated patient population relative to a control population of untreated patients.

[0198] The "biological half-life" of a substance is a pharmacokinetic parameter that refers to the time required for an organism to eliminate half of a substance after it has entered the body.

[0199] The terms “prevention” or “avoidance” mean (a) preventing the onset of a disease, (b) delaying the onset of a disease or its symptoms, or (c) slowing the progression of an existing condition. Unless otherwise stated, “prevention” does not require the absolute prohibition of the occurrence of the event.

[0200] "Individual" or "subject" refers to a mammal or bird, more preferably a human. Mammals also include, but are not limited to, farm animals (e.g., cattle, pigs, horses, chickens, etc.), livestock, pets, primates, horses, dogs, cats, mice, and rats.

[0201] As used herein, “vector” means a construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors bound to cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0202] As used herein, an "expression control sequence" refers to a nucleic acid sequence that directs nucleic acid transcription. The expression control sequence can be a promoter (e.g., a constitutive or inducible promoter) or an enhancer. The expression control sequence is operatively linked to the nucleic acid sequence to be transcribed.

[0203] As used herein, “pharmaceuticalally acceptable carrier” or “pharmaceuticalally acceptable excipient” includes any substance that, when combined with an active ingredient, allows that ingredient to remain biologically active and unresponsive to the subject’s immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions (e.g., oil / water emulsions), various wetting agents, detergents (e.g., polysorbate 20 for preventing aggregation), and sugars (e.g., sucrose as a cryoprotectant). Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline (PBS) or physiological saline (0.9%). Compositions containing such carriers are formulated using well-known, conventional methods (see, for example, Remington Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington: Pharmaceutical Sciences and Practice, 20th edition, Mack Publishing, 2000).

[0204] As used in this article, the term "k" on "" refers to the rate constant of antibody (or bioconjugate) binding to antigen. Specifically, the rate constant (k on and k off The equilibrium dissociation constant was determined using full-length antibody and / or Fab antibody fragments (i.e., monovalent) and IL-6.

[0205] As used in this article, the term "k" off "" refers to the rate constant of antibody (or bioconjugate) dissociation from antibody / antigen complex.

[0206] As used in this article, the term "K" D "" refers to the equilibrium dissociation constant of antibody-antigen (or bioconjugate-antigen) interaction.

[0207] The use of "about" in this document to refer to a value or parameter includes (and describes) the implementation of that value or parameter itself. For example, a description of "about X" includes a description of "X". Numerical ranges contain the numbers that define the range. "About" indicates a range of 10% above and below a specified value. For example, "about 15 mM" represents a range of 13.5 mM to 16.5 mM, and "about 0.025%" represents a range of 0.0225% to 0.0275%.

[0208] The term "patient" includes humans and other subjects (including mammals) who receive preventive or therapeutic treatment.

[0209] To classify amino acid substitutions as conserved or non-conserved, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions involve exchanging one member of one class for a member of another class.

[0210] Percentage sequence identity is determined by aligning antibody sequences as closely as possible according to the Kabat numbering convention for variable regions or the EU numbering for constant regions. After alignment, if the test antibody region (e.g., the entire mature variable region of the heavy or light chain) is compared with the same region of the reference antibody, the percentage sequence identity between the test antibody region and the reference antibody region is calculated as follows: the number of positions occupied by the same amino acids in both regions, divided by the total number of aligned positions in both regions (excluding gaps), multiplied by 100 to convert to a percentage. Sequence identity for other sequences can be determined by using algorithms such as BESTFIT, FASTA, and TFASTA in Wisconsin Genetics software package version 7.0 (Genetics Computer Group, 575 Science Dr., Madison, WI) with the default gap parameter, or by manual inspection and selection of the best alignment (i.e., the alignment that produces the highest percentage of sequence similarity within the comparison window). Percentage sequence identity is determined by comparing two optimally aligned sequences within a comparison window, identifying the number of positions where the same residues appear in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage sequence identity.

[0211] The term "antibody-dependent cell-mediated cytotoxicity," or ADCC, is a mechanism that induces cell death, dependent on the interaction between antibody-coated target cells (i.e., cells bound to antibodies) and lytically active immune cells (also known as effector cells). Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. ADCC is triggered by the interaction between the Fc region of the antibody bound to the cell and Fcγ receptors (specifically FcγRI and FcγRIII) on immune effector cells such as neutrophils, macrophages, and natural killer cells. Target cells are cleared through phagocytosis or lysis, depending on the type of effector cell mediating the cell. The death of antibody-coated target cells is a consequence of effector cell activity.

[0212] A humanized antibody is a genetically engineered antibody in which a core sequence (CDR) derived from a non-human "donor" antibody is grafted into a human "recipient" antibody sequence (see, for example, Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205, 6,881,557; Foote, US 6,881,557). The recipient antibody sequence can be, for example, a mature human antibody sequence, a combination of such sequences, a common sequence of human antibody sequences, or a germline region sequence. Therefore, a humanized antibody is an antibody whose CDR is wholly or substantially derived from a donor antibody, and whose variable region framework sequence and constant region (if present) are wholly or substantially derived from a human antibody sequence. Similarly, the humanized heavy chain has at least one, two, and usually all three complete or substantially derived CDRs from the donor antibody heavy chain, and a heavy chain variable region framework sequence and a heavy chain constant region (if present) substantially derived from the human heavy chain variable region framework and constant region sequence. Likewise, the humanized light chain has at least one, two, and usually all three complete or substantially derived CDRs from the donor antibody light chain, and a light chain variable region framework sequence and a light chain constant region (if present) substantially derived from the human light chain variable region framework and constant region sequence. Except for nanobodies and dAbs, humanized antibodies comprise humanized heavy chains and humanized light chains. The CDRs in a humanized antibody are substantially derived from the corresponding CDRs in a non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical among their respective CDRs. When, according to Kabat's definition, at least 85%, 90%, 95%, or 100% of the corresponding residues in the variable region frame sequence or constant region sequence of an antibody chain are identical, the variable region frame sequence or constant region sequence of the antibody chain is substantially derived from the human variable region frame sequence or the human constant region, respectively.

[0213] Although humanized antibodies typically contain all six CDRs from mouse antibodies (preferably as defined by Kabat), they can also be made from fewer than all CDRs (e.g., at least 3, 4, or 5 CDRs from mouse antibodies) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36: 1079-1091, 1999; Tamura et al., Journal of Immunology, 164: 1432-1441, 2000).

[0214] Chimeric antibodies are antibodies that bind the mature variable regions of the light and heavy chains of non-human antibodies (such as mouse antibodies) to the constant regions of the human light and heavy chains. These antibodies largely or completely retain the binding specificity of mouse antibodies, and approximately two-thirds of their sequence is human.

[0215] A veneered antibody is a humanized antibody that retains some (usually all) of the core derivation region (CDR) and some of the non-human variable region framework residues from a non-human antibody, but replaces other variable region framework residues that could potentially lead to B-cell or T-cell epitopes (e.g., exposed residues) with residues from the corresponding positions in the human antibody sequence. As a result, the CDR in the antibody is entirely or substantially derived from the non-human antibody, and the aforementioned replacements make the variable region framework of the non-human antibody more humanized. Human antibodies can be isolated from the human body or obtained by expressing human immunoglobulin genes (e.g., in transgenic mice, in vitro, or via phage display). Methods for producing human antibodies include the three-source hybridoma method used by Oestberg et al. (Hybridoma 2:361-367 (1983)); Oestberg, US 4,634,664; and Engleman et al., US 4,634,666, using transgenic mice containing human immunoglobulin genes (see, for example, Lonberg et al., W093 / 12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14,826 (1996), Kucherlapati, WO 91 / 10741 (1991)) and phage display (see, for example, Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743, and US 5,565,332).

[0216] A polymer is a molecule composed of many repeating subunits. These subunits, sometimes also called monomers, can be the same or different. Polymers can be natural or synthetic. DNA, proteins, and complex carbohydrates are examples of natural polymers. Polystyrene and polyacrylamide are examples of synthetic polymers. A polymer composed of repeating units of a single monomer is called a homopolymer. A polymer composed of two or more monomers is called a copolymer, sometimes also called a heteropolymer. Copolymers in which certain types of monomers aggregate together are sometimes called block copolymers. Polymers can be linear or branched. When a polymer is branched, polymer chains with a common starting point are sometimes called polymer arms.

[0217] An "initiator" is a compound that can serve as a substrate on which one or more polymerizations can be carried out using the monomers or comonomers described herein. This polymerization can be a conventional free radical polymerization, or preferably a controlled / "living" free radical polymerization, such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, or nitroxide radical-mediated polymerization (NMP). The polymerization can be a "pseudo" controlled polymerization, such as degenerate transfer polymerization. Initiators suitable for ATRP contain one or more unstable bonds that can homolytically cleave to form an initiator fragment I (i.e., a free radical capable of initiating free radical polymerization) and a free radical scavenger I' (which reacts with the free radicals of the growing polymer chain to reversibly terminate the polymerization). The free radical scavenger I' is typically halogenated, but can also be an organic moiety, such as a nitrile. It is also provided herein that the initiator may contain one or more 2-bromoisobutyrate groups as sites for polymerization via ATRP.

[0218] A “chemical linker” is a chemical part that links two groups together, such as a half-life extension part and a protein. The linker can be cleavable or non-cleavable. Cleavable linkers can be hydrolyzable, enzymatically cleavable, pH-sensitive, light-labile, or disulfide-bonded linkers, etc. Other linkers include homobifunctional and heterobifunctional linkers. A “linking group” is a functional group capable of forming a covalently linked group consisting of one or more bonds with a bioactive agent. Non-limiting examples include those shown in Table 1 of WO2013059137 (incorporated by reference).

[0219] The term "reactive group" refers to a group that can react with another chemical group under suitable reaction conditions to form a covalent bond, typically representing a linking point for another substance. A reactive group is a moiety, such as maleimide or succinimide ester, capable of chemically reacting with functional groups on different moieties to form a covalent bond. Reactive groups typically include nucleophiles, electrophiles, and photoactivators.

[0220] As used in this article, "phosphorylcholine" (also referred to as "PC") refers to: ,in Indicates the junction. Phosphorylcholine is a zwitterionic group, including its salts (such as the inner salt) as well as protonated and deprotonated forms.

[0221] As used in this article, "phosphorylcholine-based polymer" refers to a polymer containing phosphorylcholine. "Zwitterionic polymer" refers to a polymer containing zwitterions.

[0222] Poly(acryloyloxyethyl phosphorylcholine) polymers refer to polymers containing 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate as monomers.

[0223] Poly(methacryloyloxyethyl phosphorylcholine) polymers refer to polymers containing 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate as monomers.

[0224] The term “molecular weight” as used herein, in the context of polymers, can be expressed as number-average molecular weight, weight-average molecular weight, or peak molecular weight. Unless otherwise stated, all references to molecular weight herein refer to peak molecular weight. These molecular weight determinations, namely number-average (Mn), weight-average (Mw), and peak (Mp), can be measured by size exclusion chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values ​​can also be used, such as using end-group analysis or measuring colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) to determine the number-average molecular weight, or using light scattering, ultracentrifugation, or viscometry to determine the weight-average molecular weight. This document also provides that molecular weight can be measured by SEC-MALS (size exclusion chromatography-multi-angle light scattering). The polymer reagents presented herein are typically polydisperse (i.e., the number-average molecular weight and weight-average molecular weight of the polymer are not equal). The polydispersity index (PDI) provides a measure of the dispersion of the polymer in a mixture. The PDI is given by the formula Mw / Mn. In this respect, the PDI for homogeneous proteins is 1.0 (Mn and Mw are the same). Typically, the PDI for polymers will be higher than 1.0. The polymers provided herein preferably have a relatively low polydispersity index (PDI) value, for example, less than about 1.5, which can be determined by, for example, SEC-MALS. In other embodiments, the polydispersity index (PDI) is more preferably in the range of about 1.4 to about 1.2, still more preferably less than about 1.15, even more preferably less than about 1.10, even more preferably less than about 1.05, and most preferably less than about 1.03.

[0225] As used in this article, “protected,” “protected form,” “protecting group,” and “protective group” refer to the presence of a group (i.e., a protecting group) that, under specific reaction conditions, prevents or blocks the reaction of a specific chemically reactive functional group in the molecule. The choice of protecting group depends on the type of chemically reactive group being protected, the reaction conditions used, and the presence of other reactive groups or protecting groups in the molecule (if any). Suitable protecting groups include those described by Greene et al. (“Protective Groups in Organic Synthesis,” 3rd ed., John Wiley and Sons, Inc., New York, 1999).

[0226] As used herein, "alkyl" refers to a straight-chain or branched, saturated, aliphatic group having a specified number of carbon atoms. For example, C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, and decyl. Alkyl groups may contain any number of carbon atoms, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6 carbon atoms.

[0227] The term “lower” in relation to organic groups or compounds, as mentioned above and below, defines a compound or group that may be branched or straight-chained and has a maximum of 7, preferably a maximum of 4, and (if straight-chained) 1 or 2 carbon atoms.

[0228] As used herein, "alkylene" refers to an alkyl group as defined above, which is attached to at least two other groups, namely divalent hydrocarbon groups. The two parts attached to the alkylene group can be attached to the same or different atoms of the alkylene group. For example, a straight-chain alkylene group can be -(CH2). n The divalent group, where n is 1, 2, 3, 4, 5, or 6. Alkylenes include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, secondary butylene, pentylene, and hexylene.

[0229] The substituents of alkyl, alkenyl, alkylene, heteroalkyl, heteroalkylene, heteroalkenyl, ynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups may be one or more groups selected from, but not limited to: -OR', =O, =NR', =N-OR', -NR'R”, -SR', halogen, -SiR'R”R”', -OC(O)R', -C(O)R', -CO2R', -CONR'R”, -OC(O)NR'R R', R”, R”’, R”’, -NR”C(O)2R’, -NR-C(NR’R”R’”)=NR”, -NR-C(NR’R”)=NR’”, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -NRSO2R’, -CN, and -NO2, in quantities ranging from 1 to (2m’+1), where m’ is the total number of carbon atoms in the group. R’, R”, R”’, and R” each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or aralkyl. When R' and R” are attached to the same nitrogen atom, they can combine with that nitrogen atom to form a 5, 6, or 7-membered ring. For example, -NR'R” is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.

[0230] As used herein, “alkoxy” refers to an alkyl group attached to an oxygen atom, forming a -OR group, where R is an alkyl group. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groups can be further substituted by various substituents described herein. For example, alkoxy groups can be substituted with halogens to form “haloalkoxy” groups.

[0231] As used herein, “carboxyalkyl” means an alkyl group substituted with a carboxyl group (as defined herein). The term “carboxycycloalkyl” means a cycloalkyl group substituted with a carboxyl group (as defined herein). The term “alkoxyalkyl” means an alkyl group substituted with an alkoxy group (as defined herein). The term “carboxyl” as used herein refers to carboxylic acids and their esters.

[0232] As used herein, "halogenated alkyl" refers to an alkyl group as defined above, wherein some or all of its hydrogen atoms are replaced by halogen atoms. Halogen (halogenated) preferably represents chlorine or fluorine, but can also be bromine or iodine. For example, haloalkyl groups include trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluorophenyl, etc. The term "perfluoro" defines all compounds or groups in which all available hydrogen atoms are replaced by fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy. Haloalkyl groups can also be called halogen-substituted alkyl groups, such as fluorine-substituted alkyl groups.

[0233] In the context provided here, the term "cytokine" refers to a group of protein signaling molecules that may participate in intercellular communication during immune and inflammatory responses. Cytokines are typically small, water-soluble glycoproteins with a mass of approximately 8 to 35 kDa.

[0234] As used herein, “cycloalkyl” refers to a saturated monocyclic or polycyclic aliphatic ring system containing about 3 to 12, 3 to 10, 3 to 7, or 3 to 6 carbon atoms. When a cycloalkyl group consists of two or more rings, these rings can be linked together in a fused or spirocyclic structure. When a cycloalkyl group consists of three or more rings, these rings can also be linked to form a bridged ring structure. Monocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, bicyclic [1.1.1]pentane, bicyclic [2.1.1]heptane, norbornene, decahydronaphthalene, and adamantane. For example, C 3-8 Cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0235] The term "within the ring" as used in this article refers to the atoms or groups of atoms that constitute part of the ring structure.

[0236] The term "outside the ring" as used in this article refers to atoms or groups of atoms that are connected to the ring structure but do not constitute the ring structure.

[0237] As used herein, “cyclic alkyl ethers” refers to 4- or 5-membered cyclic alkyl groups having 3 or 4 carbon atoms in the ring and 1 oxygen or sulfur atom in the ring (e.g., oxetane, thiobutane, tetrahydrofuran, tetrahydrothiophene); or 6- to 7-membered cyclic alkyl groups having 1 or 2 oxygen or sulfur atoms in the ring (e.g., tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiocyclohexane, 1,4-dithiocyclohexane, 1,4-oxetane).

[0238] As used herein, "alkenyl" refers to a straight-chain or branched hydrocarbon group having at least one double bond and containing 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hextrienyl. Alkenyl groups may also have 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, and 5 to 6 carbon atoms.

[0239] As used herein, "eneyl group" refers to an alkenyl group, i.e., a divalent hydrocarbon group, as defined above, that is, a group attached to at least two other groups. The two parts attached to the alkenyl group can be attached to the same or different atoms of the alkenyl group. Alkenyl groups include, but are not limited to, ethylene, propylene, isopropylene, butene, isobutene, sec-butene, pentene, and hexene.

[0240] As used herein, "alkynyl" refers to a straight-chain or branched hydrocarbon group having at least one triple bond and containing 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butyrynyl, 1-pentynyl, 2-pentynyl, isopentenynyl, 1,3-pentyrynyl, 1,4-pentyrynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hextriynyl. Alynyl groups may also have 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, and 5 to 6 carbon atoms.

[0241] As used in this article, "acetylenic group" refers to an acetylenic group, i.e., a divalent hydrocarbon group, as defined above, that is, a group attached to at least two other groups. The two parts attached to the acetylenic group can be attached to the same or different atoms of the acetylenic group. Acynylenic groups include, but are not limited to, acetylenic, propylenic, butylenic, sec-butylenic, pentylenic, and hexylenic.

[0242] As used herein, "cycloalkyl group" refers to a cycloalkyl group, i.e., a divalent hydrocarbon group, as defined above, that is, a group attached to at least two other groups. The two parts attached to the cycloalkyl group can be attached to the same or different atoms of the cycloalkyl group. Cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutene, cyclopentene, cyclohexene, and cyclooctene.

[0243] As used herein, “heterocyclic alkyl” refers to a ring system having 3 to 20 ring members and containing 1 to 5 heteroatoms (such as N, O, and S). Other heteroatoms may also be used, including but not limited to B, Al, Si, and P. Heteroatoms may also be oxidized, for example, but not limited to -S(O)- and -S(O)2-. For example, heterocycles include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholinyl, pyrrolyl, pyrrololinyl, imidazoalkyl, imidazolinyl, pyrazolyl, pyrazololinyl, piperazinyl, piperidinyl, dihydroindolyl, quininecycloyl, and 1,4-dioxa-8-azaspiro[4.5]dec-8-yl.

[0244] As used herein, "heterocyclic alkylene" refers to a heterocyclic alkyl group as defined above that is attached to at least two other groups. The two parts attached to the heterocyclic alkylene group may be attached to the same or different atoms of the heterocyclic alkylene group.

[0245] As used herein, “aryl” refers to a combination of monocyclic or polycyclic aromatic rings (e.g., fused bicyclic, tricyclic, or more) containing 6 to 16 carbon atoms. For example, an aryl group can be phenyl, benzyl, or naphthyl, preferably phenyl. An aryl group can be monosubstituted, disubstituted, or trisubstituted by one, two, or three groups selected from alkyl, alkoxy, aryl, hydroxyl, halogen, cyano, amino, aminoalkyl, trifluoromethyl, alkylenedioxy, and oxy-C2-C3-alkylene; all of these groups may optionally be further substituted, for example as defined above; or 1- or 2-naphthyl; or 1- or 2-phenanthrene. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of a phenyl group, such as methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of a phenyl group, such as oxyvinyl or oxypropenyl. An example of oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0246] Preferred aryl groups are naphthyl, phenyl, or phenyl monosubstituted or disubstituted with alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl, or phenyl monosubstituted or disubstituted with alkoxy, halogen or trifluoromethyl, especially phenyl.

[0247] Examples of substituted phenyl groups of R include, for example, 4-chlorophenyl-1-yl, 3,4-dichlorophenyl-1-yl, 4-methoxyphenyl-1-yl, 4-methylphenyl-1-yl, 4-aminomethylphenyl-1-yl, 4-methoxyethylaminomethylphenyl-1-yl, 4-hydroxyethylaminomethylphenyl-1-yl, 4-hydroxyethyl-(methyl)-aminomethylphenyl-1-yl, 3-aminomethylphenyl-1-yl, 4-N-acetylaminomethylphenyl-1-yl, 4-aminophenyl-1-yl, 3-aminophenyl-1-yl, 2-aminophenyl-1-yl, 4-phenylphenyl-1-yl, 4 -(imidazol-1-yl)-phenyl, 4-(imidazol-1-ylmethyl)-phenyl-1-yl, 4-(morpholin-1-yl)-phenyl-1-yl, 4-(morpholin-1-ylmethyl)-phenyl-1-yl, 4-(2-methoxyethylaminomethyl)-phenyl-1-yl, and 4-(pyrrolidine-1-ylmethyl)-phenyl-1-yl, 4-(thienyl)-phenyl-1-yl, 4-(3-thienyl)-phenyl-1-yl, 4-(4-methylpiperazin-1-yl)-phenyl-1-yl, and optionally 4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl with substitutions on the heterocycle.

[0248] As used herein, "arylene" refers to an aryl group as defined above that is attached to at least two other groups. The two parts attached to the arylene are attached to different atoms of the arylene. Arylene groups include, but are not limited to, phenylene.

[0249] As used herein, "aryleneoxy group" refers to an arylene group as defined above, wherein one of the portions attached to the arylene group is connected via an oxygen atom. Aryleneoxy groups include, but are not limited to, phenyleneoxy groups.

[0250] Similarly, the substituents of aryl and heteroaryl groups are diverse, selected from: -halogen, -OR', -OC(O)R', -NR'R”, -SR', -R', -CN, -NO2, -CO2R', -CONR'R”, -C(O)R', -OC(O)NR'R”, -NR”C(O)R', -NR”C(O)2R', -NR'-C(O)NR”R”', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=N R', -S(O)R', -S(O)2R', -S(O)2NR'R”, -N3, -CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, ranging from zero to the total number of open valence bonds on the aromatic ring system; wherein R', R”, and R”' are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4))alkyl, and (unsubstituted aryl)oxy-(C1-C4))alkyl.

[0251] The two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CH2). q The -U- group is substituted, where T and U are independently -NH-, -O-, -CH2-, or a single bond, and q is an integer from 0 to 2. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by the formula -A-(CH2). r The -B- group is substituted, where A and B are independently -CH2-, -O-, -NH-, -S(O)-, -S(O)2-, -S(O)2NR'-, or single bonds, and r is an integer from 1 to 3. One single bond in the resulting new ring may optionally be substituted with a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be substituted with the formula -(CH2). s -X-(CH2) t -The substituents are substituted, wherein s and t are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' in -NR'- and -S(O)2NR'- is selected from hydrogen or unsubstituted (C1-C6) alkyl groups.

[0252] As used herein, "heteroaryl" refers to a monocyclic, fused bicyclic, or tricyclic aromatic ring combination containing 5 to 16 ring atoms, wherein 1 to 4 ring atoms are respective N, O, or S heteroatoms. For example, heteroaryl includes pyridinyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothiopheneyl, benzofuranyl, furanyl, pyrroleyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thiopheneyl, or any other group substituted (especially monosubstituted or disubstituted) with, for example, an alkyl, nitro, or halogen group. Pyridinyl represents 2-, 3-, or 4-pyridinyl, preferably 2- or 3-pyridinyl. Thiopheneyl represents 2- or 3-thiopheneyl. Quinolinyl preferably represents 2-, 3-, or 4-quinolinyl. The isoquinolinyl group preferably represents 1-, 3-, or 4-isoquinolinyl. The benzopyranyl and benzothiopyranyl groups preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. The thiazolyl group preferably represents 2- or 4-thiazolyl, with 4-thiazolyl being the most preferred. The triazolyl group is preferably 1-, 2-, or 5-(1,2,4-triazolyl). The tetrazolyl group is preferably 5-tetrazolyl.

[0253] Preferably, the heteroaryl group is pyridyl, indolyl, quinolinyl, pyrroleyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thiophenyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothiaphenyl, oxazolyl, inzolyl, or any substitution of the above groups, especially monosubstituted or disubstituted.

[0254] The term "heteroalkyl" refers to an alkyl group having one to three heteroatoms (such as N, O, and S). Other heteroatoms may also be used, including but not limited to B, Al, Si, and P. Heteroatoms may also be oxidized, for example, but not limited to -S(O)- and -S(O)2-. For example, heteroalkyl groups may include ethers, thioethers, alkylamines, and alkyl thiols.

[0255] The term "heteroalkylene" refers to a heteroalkyl group as defined above that is attached to at least two other groups. The two parts attached to the heteroalkylene group may be attached to the same or different atoms of the heteroalkylene group.

[0256] As used herein, an "electrophile" refers to an ion, atom, or group of atoms (which may be ionic) that has an electrophilic center (i.e., an electron-seeking center) and is capable of reacting with a nucleophile. An electrophile (or electrophilic reagent) is a reagent that forms a bond with its reaction partner (nucleophile) by accepting two bonding electrons from the partner.

[0257] As used herein, a "nucleophile" refers to an ion, atom, or group of atoms (which may be ionic) that has a nucleophilic center (i.e., a center that seeks an electrophilic center or is capable of reacting with an electrophile). A nucleophile (or nucleophilic reagent) is a reagent that forms a bond with its reaction partner (electropophile) by donating two bonding electrons. A "nucleophilic group" refers to a nucleophile that has reacted with a reactive group. Non-limiting examples include amino, hydroxyl, alkoxy, haloalkoxy, etc.

[0258] As used herein, "maleimide group" refers to a pyrrole-2,5-dione-1-yl group having the following structure: Upon reaction with a thiol group (such as a thioalkyl group), it forms a -S-maleimide group with this structure. Where, "•" represents the junction of the maleimide group, "" indicates the connection point between the sulfur atom of the thiol and the rest of the original mercapto-containing group.

[0259] For the purposes of this disclosure, "natural amino acids" found in proteins and peptides are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and / or L-valine. "Non-natural amino acids" found in proteins refer to any amino acid other than the natural amino acids listed above. Non-natural amino acids include, but are not limited to, D-isomers of natural amino acids, and mixtures of D- and L-isomers of natural amino acids. Other amino acids, such as 4-hydroxyproline, desmoline, isodesmoline, 5-hydroxylysine, ε-N-methyllysine, and 3-methylhistidine, although present in natural proteins, are considered, for the purposes of this disclosure, to be non-natural amino acids found in proteins because they are typically introduced through means other than ribosomal mRNA translation.

[0260] As used herein, in relation to the geometry, architecture, or overall structure of a polymer, "linear" refers to a polymer having a single polymer arm.

[0261] As used herein, in relation to the geometry, architecture, or overall structure of a polymer, "branching" refers to a polymer having two or more polymer "arms" extending from a core structure contained within an initiator. This initiator can be used in atom transfer radical polymerization (ATRP) reactions. Branched polymers can have 2 polymer chains (arms), 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms, 9 polymer arms, or more. Each polymer arm extends from a polymer initiation site. Each polymer initiation site can serve as a site for growing polymer chains by adding monomers. For example, but not limited to, when using ATRP, the polymer initiation site on the initiator is typically an organohalide undergoing a reversible redox process catalyzed by a transition metal compound (such as cuprous halide). Preferably, the halide is bromine.

[0262] As used herein, "pharmaceuticalally acceptable excipient" means an excipient that can be included in the compositions provided herein, does not cause significant adverse toxicological effects on patients, and is approved by the FDA or is approved for therapeutic use (particularly for human treatment). Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline, lactated Ringer's solution, conventional sucrose, conventional glucose, etc.

[0263] As used in this article, “OG1786” is a 9-arm initiator for polymer synthesis, with the structure shown below. Figure 2DAs shown in the figure, the salt form of OG1786 with trifluoroacetic acid is illustrated. OG1786 can also be used in other salt forms or as a free base as described herein.

[0264] As used herein, “OG1801” is a polymer of approximately (+ / -25%) 800 kDa (based on Mn or Mp), synthesized via ATRP using OG1786 as an initiator and via monomer HEMA-PC. The structure of OG1801 is as follows: Figure 2J As shown.

[0265] As used in this article, "OG1802" is OG1801 with the addition of maleimide functional groups, and its structure is as follows: Figure 2K As shown, each of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is an integer (positive integer) (from 0 to approximately 3000), such that the total molecular weight (Mw) of the polymer is 800,000 ± 20% Daltons. When the term OG1802 is used to modify protein terms (such as VEGF trap or anti-IL-6 antibody), it indicates that the protein is a conjugated protein.

[0266] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. In case of any conflict, this specification (including the definitions) shall prevail. Throughout the specification and claims, the word “comprising” or variations thereof (such as “including” or “containing”) shall be understood to cover the stated integers or groups of integers, but not exclude any other integers or groups of integers. Unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Any examples following “for example” or “such as” are not exhaustive or limiting.

[0267] An entity that is “a” or “an” means one or more of that entity; for example, a compound means one or more compounds or at least one compound. Therefore, the terms “a (a)” (or “an (an)”), “one or more”, and “at least one” are used interchangeably herein.

[0268] The term "approximately" as used in this article refers to the possible deviations that may occur when measuring with different instruments, samples, and sample preparations.

[0269] The terms "uncoupled protein" and "free protein" are used interchangeably in this article and refer to proteins that are not coupled to polymers (e.g., not coupled to phosphocholine-containing polymers).

[0270] As used herein, “molar quantity” refers to the quantity of moles of a molecule. In some embodiments, molar quantity is molar concentration (e.g., M, mM, μM, nM, etc.). In some embodiments, molar quantity is expressed in molar units (e.g., mole, millimole, micromole, etc.).

[0271] The CDR positions used herein are described in the order in which they appear in the variable structural domains. For example, the heavy chain position may be described as S35H or G66D. The Fc positions used herein follow the EU numbering when described or indicated by reference. For example, an antibody mutation may be described as L234A or L235A, which conforms to the EU numbering. Positions may also be defined according to a specific SEQ ID or a designated position within a sequence provided herein.

[0272] Multi-angle light scattering (MALS) is a technique for analyzing macromolecules, in which a laser beam is irradiated onto the molecule, and the oscillating electric field of the light induces an oscillating dipole within it. This oscillating dipole re-radiates light, which can be measured using a MALS detector such as the Wyatt miniDawn TREOS. The intensity of the radiated light depends on the size of the induced dipole in the macromolecule, which is directly proportional to the polarizability of the macromolecule; the larger the induced dipole, the greater the intensity of the scattered light. Therefore, to analyze the scattering of such macromolecules in solution, it is necessary to know their polarizability relative to the surrounding medium, such as the solvent. This can be measured using a Wyatt Optilab T-rEX differential refractometer. dn / DC ( = Δ n / Δ c The value is used to determine the refractive index of the solution. n With molecular concentration Δ c Change Δ n The two molar weight parameters used in MALS determination are number-average molecular weight (Mn) and weight-average molecular weight (Mw), where the polydispersity index (PDI) is equal to Mw divided by Mn. SEC also allows the determination of another average molecular weight, the peak molecular weight Mp, which is defined as the molecular weight of the highest peak in SEC.

[0273] Polydispersity index (PDI) is used as a measure of the width of the molecular weight distribution of polymers and bioconjugates, derived from the coupling of discrete proteins with polydisperse biopolymers (e.g., OG1802). For protein samples, the polydispersity is close to 1.0 because it is a translational product, and each protein molecule in solution is expected to have nearly identical length and molar mass. In contrast, due to the polydispersity of biopolymers (i.e., polymer chains of varying lengths synthesized during polymerization), determining the PDI of a sample as one of its quality properties of narrow molecular weight distribution is crucial.

[0274] Size exclusion chromatography (SEC) is a chromatographic technique that separates molecules in solution based on their size. Typically, an aqueous solution is used to transport the sample through a chromatography column packed with resins of varying pore sizes. The resin should remain inert as the analytes pass through the column, allowing them to separate based on their unique size and the pore characteristics of the selected column.

[0275] Coupling SEC with MALS, or SEC / MALS, provides a precise distribution of molar mass and size (root mean square radius) without relying on a set of SEC calibration standards. This configuration offers numerous advantages compared to traditional column calibration methods. Since the light scattering and concentration of each eluted fraction are measured, the determination of molar mass and size is independent of the elution site. This is particularly important for non-spherical macromolecules such as biopolymers (OG1802) or bioconjugates; such substances typically do not elute in the manner described by a set of column calibration standards.

[0276] In some implementations, SEC / MALS analysis includes a Waters HPLC system equipped with an Alliance 2695 solvent delivery module and a Waters 2996 photodiode array detector, along with a Shodex SEC-HPLC column (7.8 x 300 mm). This system is connected online to a Wyatt miniDawn TREOS and a Wyatt Optilab T-rEX differential refractometer. Waters' Empower software is used to control the Waters HPLC system, while Wyatt's ASTRAV 6.1.7.16 software is used to acquire MALS data from the Wyatt miniDawn TREOS, dn / dc data from the T-rEX detector, and mass recovery data using the A280 absorbance signal from the Waters 2996 photodiode array detector. SEC can be performed at 1 ml / min in 1 x PBS at pH 7.4. After sample injection, MALS and RI signals can be analyzed using ASTRA software to determine absolute molar mass (Mp, Mw, Mn) and polydispersity index (PDI). In addition, the calculations also involved the input dn / dc values ​​for the polymer and protein, which were 0.142 and 0.183, respectively. For the bioconjugate, the dn / dc value was calculated to be approximately 0.148 based on the weighted molecular weight (MW) of the polymer and protein, as shown in the following formula: The coupling element dn / dc = 0.142x [MW] polymer / (MW polymer + MW protein )] + 0.183x [MW protein quality / (MW polymer + MW protein )] Among them, the MW of OG1802 measured by SEC-MALS polymer Approximately 800 kDa, with an MW of IL-6 resistance measured by SEC-MALS. protein The total molecular weight of the bioconjugate is approximately 145 kDa, and the expected total molecular weight, as determined by SEC-MALS, is approximately 1000 kDa. It is an anti-IL-6 VEGF trap or a VEGF trap-anti-IL-6 MW. protein It is approximately 192 kDa, and the expected total molecular weight of bioconjugates is 1000 kDa to 1100 kDa.

[0277] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of the fusion protein, a buffer, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein is conjugated to a polymer. In some embodiments, the fusion protein comprises an antagonistic antibody or fragment thereof that specifically binds to IL-6 and is conjugated to a polymer. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or fragment thereof comprising: a) a heavy chain amino acid variable region comprising a heavy chain having at least one sequence from SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and b) a light chain amino acid variable region comprising a light chain having at least one sequence from SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) having three complementarity-determining regions: VH(CDR1), VH CDR2, and VH CDR3 having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) having VL CDR1, VL CDR2, and VL CDR3 having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof that binds to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: a) the N-terminus of the heavy chain containing IL-6 VH; or b) between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain. In some embodiments, the buffer comprises sodium acetate. In some embodiments, the surfactant comprises polysorbate 20. In some embodiments, polysorbate 20 comprises about 0.01% (w / w) to about 0.05% (w / w) of the formulation. In some embodiments, the surfactant comprises polysorbate 80.In some embodiments, polysorbate 80 comprises about 0.01% (w / w) to about 0.05% (w / w) of the formulation. In some embodiments, polysorbate 20 comprises about 0.025% (w / w) of the formulation. In some embodiments, polysorbate 80 comprises about 0.025% (w / w) of the formulation. In some embodiments, the sodium acetate concentration is between about 0.1 mM and about 75 mM. In some embodiments, the sodium acetate concentration is about 50 mM. In some embodiments, the formulation is between about pH 4.5 and about pH 6.5. In some embodiments, the formulation is about pH 5.0. In some embodiments, the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer. In some embodiments, the polydispersity index (PDI) is between about 0.5 and about 2. In some embodiments, the polydispersity index (PDI) is 1. In some embodiments, the penetration enhancer is between about 2% and 10% (weight / volume). In some embodiments, the penetrant is any one of sucrose, trehalose, glucose, fructose, maltose, lactose, mannitol, and sorbitol. In some embodiments, the penetrant is sucrose or trehalose. In some embodiments, the penetrant contains about 4% (by weight / volume) sucrose. In some embodiments, the penetrant contains about 4% (by weight / volume) trehalose. In some embodiments, the penetrant contains about 5.2% (by weight / volume) sucrose. In some embodiments, the penetrant contains about 5.2% (by weight / volume) trehalose. In some embodiments, the penetrant contains about 6% (by weight / volume) sucrose. In some embodiments, the penetrant contains about 6% (by weight / volume) trehalose. In some embodiments, the concentration of the fusion protein is between about 20 mg / mL and 75 mg / mL. In some embodiments, the concentration of the fusion protein is about 50 mg / mL. In some embodiments, the formulation is not a formulation of any of embodiments 1-98.

[0278] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer comprising sodium acetate, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein is conjugated to a polymer. In some embodiments, the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer. In some embodiments, the fusion protein comprises a polymer-conjugated IL-6-specific antagonistic antibody or a fragment thereof. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a) a heavy chain amino acid variable region comprising a heavy chain having at least one sequence from SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and b) a light chain amino acid variable region comprising a light chain having at least one sequence from SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) comprising three complementarity-determining regions: VH(CDR1), VH CDR2, and VH CDR3 having amino acid sequences selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof that binds to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: a) the N-terminus of the heavy chain containing IL-6 VH; or b) between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain. In some embodiments, the VEGF trap consists of the sequence of SEQ ID NO: 114.In some embodiments, the surfactant comprises about 0.01% to about 0.05% (by weight) of polysorbate 20. In some embodiments, the surfactant comprises about 0.025% (by weight) of polysorbate 20. In some embodiments, the surfactant comprises about 0.01% to about 0.05% (by weight) of polysorbate 80. In some embodiments, the surfactant comprises about 0.025% (by weight) of polysorbate 80. In some embodiments, the pH of the pharmaceutical preparation is between about 4.5 and about 6.5. In some embodiments, the pH of the pharmaceutical preparation is about 5.0. In some embodiments, the pharmaceutical preparation is stable for at least 52 weeks. In some embodiments, the pharmaceutical preparation is stable for at least 52 weeks at 4 degrees Celsius. In some embodiments, the pharmaceutical preparation is stable for at least 8 weeks at 37 degrees Celsius. In some embodiments, the pharmaceutical preparation is stable under acidic conditions. In some embodiments, the pharmaceutical preparation is formulated for intravitreal injection. In some embodiments, the sodium acetate concentration in the buffer solution is about 0.1 mM to about 75 mM. In some embodiments, the concentration of the fusion protein is between about 20 mg / mL and 75 mg / mL. In some embodiments, the concentration of the fusion protein is about 50 mg / mL. In some embodiments, the infiltration agent comprises about 4% (by weight / volume) sucrose or trehalose. In some embodiments, the pharmaceutical formulation is not the pharmaceutical formulation described in any one of schemes 1-98.

[0279] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is between about 30 mg / mL and about 85 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylated choline polymer or a zwitterionic monomer; a buffer comprising a mixture of sodium acetate and acetic acid, wherein the concentration of the buffer is between about 0.1 mM and about 75 mM, wherein the pH of the buffer is about 5.0; a penetration enhancer, wherein the penetration enhancer comprises about 4% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises about 0.01% to about 0.05% (w / w) polysorbate 20 or polysorbate 80, wherein the pharmaceutical formulation comprises an unconjugated fusion protein and a fusion protein conjugate; and wherein the fusion protein comprises: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap, wherein the VEGF trap is conjugated to the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174. The sequence 114 has at least 80% identity, and the fusion protein contains the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%, wherein if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the fusion protein contains an antagonistic IL-6 antibody or a fragment thereof that specifically binds to IL-6 conjugated to the polymer. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a) a heavy chain amino acid variable region comprising a heavy chain having at least one sequence from SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and b) a light chain amino acid variable region comprising a light chain having at least one sequence from SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) comprising three complementarity-determining regions: VH(CDR1), VH CDR2, and VH CDR3 having amino acid sequences selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof that binds to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: a) the N-terminus of the heavy chain containing IL-6 VH; or b) between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain.In some embodiments, a mutation is included at position 94 or 95 of the VEGF trap sequence, wherein if the mutation occurs at position 94, the mutation is T94I, and if the mutation occurs at position 95, the mutation is H95I. In some embodiments, the fusion protein includes a VEGFR-anti-IL-6 dual inhibitor, wherein the VEGFR-anti-IL-6 dual inhibitor comprises a fusion of an anti-IL-6 antibody or a fragment thereof with a trap antibody against an anti-VEGF trap (VEGFR1 / 2), wherein the dual inhibitor includes at least one point mutation within the VEGFR sequence to reduce VEGFR protein cleavage, and wherein the VEGFR-anti-IL-6 dual inhibitor includes a constant heavy chain, a constant light chain, an antigen-binding fragment, a crystallizable fragment (Fc), a vascular endothelial growth factor receptor (VEGFR), a variable heavy chain, and a variable light chain region. In some embodiments, the anti-IL-6 heavy chain amino acid variable region sequence is selected from SEQ ID NO: 7-13, 89, 90, and / or 256-262, wherein the VEGF trap sequence is selected from at least one of SEQ ID NO: 145, 15, 16, or 17, wherein the linker sequence is SEQ ID NO: 18, or wherein the light chain sequence of the anti-IL-6 molecule comprises at least one, two, or three light chain CDRs from at least one of SEQ ID NO: 76-84. In some embodiments, it comprises a VEGFR-Fc sequence from at least one of SEQ ID NO: 85-88. In some embodiments, the uncoupled fusion protein in the formulation comprises 10% to 60% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the uncoupled fusion protein. In some embodiments, the uncoupled fusion protein in the formulation comprises approximately 30% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the uncoupled fusion protein. In some embodiments, the formulation is not the formulation of any of embodiments 1-98. In some embodiments, X is Br.

[0280] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is approximately 50.0 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylated choline polymer or a zwitterionic monomer; a buffer solution comprising sodium acetate, wherein the sodium acetate is approximately 50 mM; and a surfactant comprising approximately 0.025% (w / w) of polysorbate 20 or polysorbate 80; and a penetration enhancer comprising approximately 4% (w / v) of sucrose or trehalose. The formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein comprises approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein in the formulation, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein, and wherein the fusion protein comprises a protein containing a phosphorylated choline polymer or a zwitterionic monomer. The fusion protein comprises CDRH3 with at least 80% identity to the amino acid sequence 174 (QAWGYYALDI); and a VEGF trap, wherein the VEGF trap has at least 80% identity to the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the fusion protein contains an antagonistic antibody or a fragment thereof that specifically binds to IL-6 conjugated to the polymer. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a) a heavy chain amino acid variable region comprising a heavy chain having at least one sequence from SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and b) a light chain amino acid variable region comprising a light chain having at least one sequence from SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) comprising three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising VLCDR1, VL CDR2, and VL CDR3 having amino acid sequences selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof that binds to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: a) the N-terminus of the heavy chain containing IL-6 VH; or b) between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain.In some embodiments, a mutation is included at position 94 or 95 of the VEGF trap sequence, wherein if the mutation occurs at position 94, the mutation is T94I, and if the mutation occurs at position 95, the mutation is H95I. In some embodiments, the fusion protein includes a VEGFR-anti-IL-6 dual inhibitor, wherein the VEGFR-anti-IL-6 dual inhibitor comprises an anti-IL-6 antibody or a fragment thereof fused with a trap antibody of anti-VEGF trap (VEGFR1 / 2), wherein the dual inhibitor includes at least one point mutation within the VEGFR sequence to reduce VEGFR protein cleavage, and wherein the VEGFR-anti-IL-6 dual inhibitor includes a constant heavy chain, a constant light chain, an antigen-binding fragment, a crystallizable fragment (Fc), a vascular endothelial growth factor receptor (VEGFR), a variable heavy chain region, and a variable light chain region. In some embodiments, the anti-IL-6 heavy chain variable region sequence is selected from options of SEQ ID NO: 7-13, 89, 90, and / or 256-262, wherein the VEGF trap sequence is selected from at least one of SEQ ID NO: 145, 15, 16, or 17, wherein the linker sequence is SEQ ID NO: 18, or wherein the light chain sequence of the anti-IL-6 molecule comprises at least one, two, or three light chain CDRs from at least one of SEQ ID NO: 76-84. In some embodiments, it comprises a VEGFR-Fc sequence from at least one of SEQ ID NO: 85-88. In some embodiments, the formulation is configured for intravitreal administration. In some embodiments, the formulation is not a formulation of any of schemes 1-98. In some embodiments, X is Br.

[0281] In some embodiments, a method for preparing a formulation is provided, the method comprising: culturing a recombinant cell line producing a fusion protein under conditions for producing a fusion protein; recovering the fusion protein; conjugating the fusion protein to a polymer to form a fusion protein conjugate, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; separating the fusion protein conjugate from the polymer and the fusion protein; and transferring a pharmaceutically effective amount of the fusion protein conjugate to a formulation comprising: a buffer containing sodium acetate, wherein the sodium acetate is approximately 50 mM; a surfactant comprising approximately 0.025% (w / w) of polysorbate 20 or polysorbate 80; and a penetration enhancer comprising approximately 4% (w / v) of sucrose or trehalose. In some embodiments, the fusion protein conjugate comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, transferring a pharmaceutically effective amount of the fusion protein conjugate includes ultrafiltration or dialysis. In some embodiments, transferring a pharmaceutically effective amount of the fusion protein conjugate includes dialysis. In some embodiments, the composition comprises an uncoupled fusion protein and a fusion protein conjugate. In some embodiments, the uncoupled fusion protein in the formulation comprises approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the uncoupled fusion protein. In some embodiments, the formulation is not a formulation of any of claims 1-98. In some embodiments, X is Br.

[0282] In some embodiments, a method for preparing a formulation is provided, the method comprising: culturing a cell line for recombinant production of a fusion protein under conditions in which the fusion protein is produced; recovering the fusion protein; and preparing the fusion protein by the following steps: removing thiolates from cysteine ​​residues, wherein the removal of thiolates includes reduction using TCEP and filtering off the resulting TCEP and unbound thiolates; re-oxidizing the fusion protein with DHAA, wherein excess DHAA is removed by filtration; conjugating the fusion protein with a polymer to form a fusion protein conjugate, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; separating the fusion protein conjugate from the polymer and the fusion protein by chromatography; and dialyzing 20 mg / mL of the fusion protein conjugate with a buffer to obtain a post-dialysis buffer. In some embodiments, the buffer comprises: sodium acetate, wherein the sodium acetate concentration is approximately 20 mM; an osmotic agent, wherein the osmotic agent comprises approximately 1.5% sucrose or trehalose; and a surfactant, wherein the surfactant comprises approximately 0.01% (by weight) polysorbate 20 or polysorbate 80. In some embodiments, the fusion protein conjugate comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the reduction using TCEP comprises a 30-fold molar excess of TCEP (tris(2-carboxyethyl)phosphine) relative to the concentration of the fusion protein. In some embodiments, the re-oxidation-reduction of the fusion protein using DHAA (dehydroascorbic acid) involves a 15-fold molar excess of DHAA relative to the concentration of the fusion protein. In some embodiments, the filtration of the resulting TCEP and unbound thiols, as well as the removal of excess DHAA, is performed by tangential flow filtration (TFF). In some embodiments, dialysis is performed by buffer replacement using TFF. In some embodiments, the post-dialysis buffer is concentrated into a concentrated dialysis buffer containing approximately 50.0 mg / mL of the fusion protein conjugate, 50 mM sodium acetate, approximately 0.025% (w / w) of polysorbate 20 or polysorbate 80, and approximately 4.0% (w / v) of an osmotic agent, wherein the osmotic agent is sucrose or trehalose. In some embodiments, the buffer contains approximately 50 mM sodium acetate, approximately 0.025% (w / w) of polysorbate 20, and a pH of approximately 5. In some embodiments, the formulation is not a formulation of any of embodiments 1-98. In some implementations, X is Br.

[0283] In some embodiments, a method for preparing a formulation is provided, the method comprising: culturing a cell line for recombinant production of a fusion protein under conditions that produce the fusion protein; recovering the fusion protein; and preparing the fusion protein by the following steps: removing thiol groups from cysteine ​​residues, wherein removing thiol groups includes reduction with TCEP and filtering off the resulting TCEP and unbound thiol groups; re-oxidizing the fusion protein with DHAA and removing excess DHAA by filtration; conjugating the fusion protein with a polymer to form a fusion protein conjugate, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; separating the fusion protein conjugate from the polymer and the fusion protein by chromatography; and dialyzing a pharmaceutically effective amount of the fusion protein conjugate with a buffer solution. In some embodiments, the buffer comprises: sodium acetate, wherein the sodium acetate concentration is approximately 20 mM; a penetration enhancer, wherein the penetration enhancer comprises approximately 1.5% sucrose or trehalose; and a surfactant, wherein the surfactant comprises approximately 0.01% (w / w) polysorbate 20 or polysorbate 80; and the resulting fusion protein is concentrated, wherein the sodium acetate is concentrated to approximately 50 mM, the surfactant is concentrated to approximately 0.025% (w / w) polysorbate 20 or polysorbate 80, and the penetration enhancer is concentrated to approximately 4% (w / v) sucrose or trehalose. In some embodiments, the fusion protein conjugate comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the rest of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the sodium acetate concentration is between about 0.1 mM and about 75 mM. In some embodiments, the sodium acetate concentration is between about 0.1 mM and about 50 mM. In some embodiments, the anti-IL-6 heavy chain variable region sequence is selected from options SEQ ID NO: 7-13, 89, 90, and / or 256-262, wherein the VEGF trap sequence is selected from at least one of SEQ ID NO: 114, 145, 15, 16, or 17, or wherein the light chain sequence of the anti-IL-6 molecule comprises 1, 2, or 3 light chain CDRs from at least one of SEQ ID NO: 76-84. In some embodiments, a pharmaceutically effective amount refers to an amount sufficient to achieve any one or more beneficial or desired results. In some embodiments, a pharmaceutically effective amount is a concentration greater than 10 mg / mL. In some embodiments, a pharmaceutically effective amount is a concentration greater than 30 mg / mL. In some embodiments, the formulation is not a formulation of any of schemes 1-98. In some embodiments, X is Br.

[0284] In some embodiments, a pharmaceutical formulation comprises: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is about 50 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer containing sodium acetate, wherein the sodium acetate is about 50 mM; and a penetration enhancer, wherein the penetration enhancer is about 4% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises about 0.025% (w / w) of polysorbate 20 or polysorbate 80, wherein the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, and wherein the unconjugated fusion protein in the formulation comprises about 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114, wherein the fusion protein has a light chain having at least 80% identity with the sequence of SEQ ID NO: 169, and wherein the fusion protein has a heavy chain having at least 80% identity with the sequence of SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains, PC being... The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain). In some embodiments, the formulation is not a formulation of any of claims 1-98. In some embodiments, X is Br.

[0285] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is about 50 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer containing sodium acetate, wherein the sodium acetate is about 50 mM; a penetration enhancer, wherein the penetration enhancer comprises about 4% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises about 0.025% (w / w) polysorbate 20 or polysorbate 80, wherein the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, and wherein the unconjugated fusion protein in the formulation comprises about 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114, wherein the fusion protein has a light chain having at least 80% identity with the sequence of SEQ ID NO: 169, and wherein the fusion protein has a heavy chain having at least 80% identity with the sequence of SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17A) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain). In some embodiments, the fusion protein comprises the amino acid sequences of SEQ ID NO: 169 and 170 and is conjugated to a polymer, wherein the polymer is the polymer shown in the structure of Formula 17, Formula 17A, or 17Br. In some embodiments, the formulation is not a formulation of any of schemes 1-98. In some embodiments, X is Br.

[0286] In some embodiments, the formulation comprises: a pharmaceutically effective amount of the fusion protein, wherein the concentration of the fusion protein is 50 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, and the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer containing sodium acetate, wherein the sodium acetate is 50 mM; a penetration enhancer, wherein the penetration enhancer comprises 4% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises 0.025% (w / w) polysorbate 20 or polysorbate 80. The formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the unconjugated fusion protein in the formulation comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170. In some embodiments, the fusion protein comprises the following structures: Equation (17A) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, the formulation is not a formulation of any of claims 1-98. In some embodiments, X is Br.

[0287] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer solution (containing histidine), a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein is conjugated to a polymer. In some embodiments, the fusion protein comprises an antagonistic antibody or a fragment thereof that specifically binds to IL-6 conjugated to the polymer; in some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain amino acid variable region comprising a heavy chain having at least one sequence of SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and a light chain amino acid variable region comprising a light chain having at least one sequence of SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) containing three complementarity-determining regions: VH(CDR1), VH CDR2, and VH CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof binding to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: the N-terminus of the heavy chain containing IL-6 VH; or between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain. In some embodiments, the buffer comprises histidine acetate. In some embodiments, the surfactant comprises polysorbate 20.In some embodiments, the content of polysorbate 20 is from about 0.01% (w / w) to about 0.1% (w / w) of the formulation. In some embodiments, the surfactant comprises polysorbate 80. In some embodiments, the content of polysorbate 80 is from about 0.01% (w / w) to about 0.1% (w / w) of the formulation. In some embodiments, polysorbate 20 is about 0.025% (w / w) of the formulation. In some embodiments, polysorbate 80 is about 0.025% (w / w) of the formulation. In some embodiments, the histidine acetate concentration is from about 10 mM to about 50 mM. In some embodiments, the histidine acetate concentration is about 25 mM. In some embodiments, the pH of the formulation is from about 4.5 to about 6.5. In some embodiments, the pH of the formulation is about 5.5. In some embodiments, the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer. In some embodiments, the polydispersity index (PDI) is from about 0.5 to about 2. In some embodiments, the polydispersity index (PDI) is 1. In some embodiments, the penetration enhancer is about 2% to 10% (weight / volume) of the formulation. In some embodiments, the penetration enhancer is sucrose or trehalose. In some embodiments, sucrose is about 6% (weight / volume) of the formulation. In some embodiments, trehalose is about 6% (weight / volume) of the formulation. In some embodiments, the concentration of the fusion protein is about 20 mg / mL to 75 mg / mL. In some embodiments, the concentration of the fusion protein is about 50 mg / mL. In some embodiments, the formulation is not a formulation of any of embodiments 1-98.

[0288] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein; a buffer comprising histidine acetate, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein is conjugated to a polymer. In some embodiments, the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer. In some embodiments, the fusion protein comprises an antagonistic antibody or a fragment thereof that specifically binds to IL-6 conjugated to the polymer. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a) a heavy chain amino acid variable region comprising a heavy chain having at least one sequence from SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and b) a light chain amino acid variable region comprising a light chain having at least one sequence from SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) comprising three complementarity-determining regions: VH(CDR1), VH CDR2, and VH CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof binding to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: the N-terminus of the heavy chain containing IL-6 VH; or between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain. In some embodiments, the VEGF trap consists of the sequence of SEQ ID NO: 114.In some embodiments, the surfactant comprises about 0.01% to about 0.1% (by weight) of polysorbate 20. In some embodiments, the surfactant comprises about 0.025% (by weight) of polysorbate 20. In some embodiments, the surfactant comprises about 0.01% to about 0.05% (by weight) of polysorbate 80. In some embodiments, the surfactant comprises about 0.025% (by weight) of polysorbate 80. In some embodiments, the pH of the formulation is about 4.5 to about 6.5. In some embodiments, the pH of the formulation is about 5.5. In some embodiments, the formulation is stable for at least 52 weeks. In some embodiments, the formulation is stable at 4°C for at least 52 weeks. In some embodiments, the formulation is stable at 37°C for at least 8 weeks. In some embodiments, the formulation is stable under acidic conditions. In some embodiments, the formulation is formulated for intravitreal injection. In some embodiments, the histidine acetate concentration in the buffer solution is about 0.1 mM to about 50 mM. In some embodiments, the concentration of the fusion protein is between about 20 mg / mL and 100 mg / mL. In some embodiments, the concentration of the fusion protein is about 50 mg / mL. In some embodiments, the infiltration agent comprises about 2% to 10% (by weight / volume) sucrose or trehalose. In some embodiments, the formulation is not a formulation of any of Schemes 1-98.

[0289] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is between about 20 mg / mL and about 100 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer comprising a mixture of histidine and acetic acid, wherein the buffer is between about 10 mM and about 50 mM, wherein the pH of the buffer is about 5.5; a penetration enhancer, wherein the penetration enhancer comprises about 6% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises about 0.01% to about 0.1% (w / w) polysorbate 20 or polysorbate 80. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate. In some embodiments, the fusion protein comprises: CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group of C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the fusion protein contains an antagonistic antibody or a fragment thereof that specifically binds to IL-6 conjugated to the polymer. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain amino acid variable region comprising a heavy chain having at least one sequence of SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and a light chain amino acid variable region comprising a light chain having at least one sequence of SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) comprising three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof that binds to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: the N-terminus of the heavy chain containing IL-6 VH; or between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain.In some embodiments, a mutation is included at position 94 or 95 of the VEGF trap body sequence, wherein if the mutation occurs at position 94, the mutation is T94I, and if the mutation occurs at position 95, the mutation is H95I. In some embodiments, the fusion protein includes a VEGFR-anti-IL-6 dual inhibitor, wherein the VEGFR-anti-IL-6 dual inhibitor comprises a fusion of an anti-IL-6 antibody or a fragment thereof with a trap body antibody against the anti-VEGF trap body (VEGFR1 / 2), wherein the dual inhibitor includes at least one point mutation within the VEGFR sequence to reduce VEGFR protein cleavage, and wherein the VEGFR-anti-IL-6 dual inhibitor includes a constant heavy chain, a constant light chain, an antigen-binding fragment, a crystallizable fragment (Fc), a vascular endothelial growth factor receptor (VEGFR), a variable heavy chain region, and a variable light chain region. In some embodiments, the anti-IL-6 heavy chain variable region sequence is selected from SEQ ID NO: 7-13, 89, 90, and / or 256-262, wherein the VEGF trap sequence is selected from at least one of SEQ ID NO: 145, 15, 16, or 17, wherein the linker sequence is SEQ ID NO: 18, or wherein the light chain sequence of the anti-IL-6 molecule comprises at least one, two, or three light chain CDRs from at least one of SEQ ID NO: 76-84. In some embodiments, it comprises a VEGFR-Fc sequence from at least one of SEQ ID NO: 85-88. In some embodiments, the uncoupled fusion protein in the pharmaceutical formulation comprises 10% to 60% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the uncoupled fusion protein. In some embodiments, the uncoupled fusion protein in the pharmaceutical formulation comprises approximately 30% of the total molar amount of the antibody-conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amounts of the antibody-conjugate and the uncoupled fusion protein. In some embodiments, the pharmaceutical formulation is not the pharmaceutical formulation of any of embodiments 1-98. In some embodiments, X is Br.

[0290] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is approximately 50.0 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate, wherein the histidine acetate is approximately 25 mM; a surfactant comprising approximately 0.025% (w / w) of polysorbate 20 or polysorbate 80; and optionally a penetration enhancer comprising approximately 6% (w / v) of sucrose or trehalose. In some embodiments, the pharmaceutical formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein comprises approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein in the pharmaceutical formulation, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the fusion protein contains an antagonistic antibody or a fragment thereof that specifically binds to IL-6 conjugated to the polymer. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain amino acid variable region comprising a heavy chain containing at least one sequence of SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and a light chain amino acid variable region comprising a light chain containing at least one sequence of SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) comprising three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising VL CDR1, VLCDR2, and VL CDR3 having amino acid sequences selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof that binds to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: the N-terminus of the heavy chain containing IL-6 VH; or between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain.In some embodiments, a mutation is included at position 94 or 95 of the VEGF trap sequence, wherein if the mutation occurs at position 94, the mutation is T94I, and if the mutation occurs at position 95, the mutation is H95I. In some embodiments, the fusion protein includes a VEGFR-anti-IL-6 dual inhibitor, wherein the VEGFR-anti-IL-6 dual inhibitor comprises an anti-IL-6 antibody or a fragment thereof fused with a trap antibody of an anti-VEGF trap (VEGFR1 / 2), wherein the dual inhibitor includes at least one point mutation within the VEGFR sequence to reduce VEGFR protein cleavage, and wherein the VEGFR-anti-IL-6 dual inhibitor includes a constant heavy chain, a constant light chain, an antigen-binding fragment, a crystallizable fragment (Fc), a vascular endothelial growth factor receptor (VEGFR), a variable heavy chain region, and a variable light chain region. In some embodiments, the anti-IL-6 heavy chain variable region sequence is selected from SEQ ID NO: 7-13, 89, 90, and / or 256-262, wherein the VEGF trap sequence is selected from at least one of SEQ ID NO: 145, 15, 16, or 17, wherein the linker sequence is SEQ ID NO: 18, or wherein the light chain sequence of the anti-IL-6 molecule comprises at least one, two, or three light chain CDRs from at least one of SEQ ID NO: 76-84. In some embodiments, the VEGFR-Fc sequence is from at least one of SEQ ID NO: 85-88. In some embodiments, the formulation is configured for intravitreal administration. In some embodiments, the formulation is configured for intravitreal administration. In some embodiments, the formulation is not a formulation of any of schemes 1-98. In some embodiments, X is Br.

[0291] In some embodiments, a method for preparing a formulation is provided, the method comprising: culturing a recombinant cell line producing the fusion protein under conditions for producing the fusion protein; recovering the fusion protein; coupling the fusion protein to a polymer to form a fusion protein conjugate, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; separating the fusion protein conjugate from the polymer and the fusion protein; and transferring a pharmaceutically effective amount of the fusion protein conjugate to a formulation comprising: a buffer containing histidine acetate, wherein the histidine acetate is approximately 25 mM; a surfactant comprising approximately 0.025% (w / w) of polysorbate 20 or polysorbate 80; and a penetration enhancer comprising approximately 6% (w / v) of sucrose or trehalose. In some embodiments, the fusion protein conjugate comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, transferring a pharmaceutically effective amount of the fusion protein conjugate includes ultrafiltration or diafiltration. In some embodiments, transferring a pharmaceutically effective amount of the fusion protein conjugate includes dialysis. In some embodiments, the composition comprises an uncoupled fusion protein and a fusion protein conjugate. In some embodiments, the uncoupled fusion protein in the formulation comprises approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the uncoupled fusion protein. In some embodiments, the formulation is not a formulation of any of claims 1-98. In some embodiments, X is Br.

[0292] In some embodiments, a method for preparing a formulation includes: culturing a cell line that produces a recombinant fusion protein under conditions conducive to fusion protein production; recovering the fusion protein; and preparing the fusion protein by the following steps: removing thiol groups from cysteine ​​residues, wherein the removal of thiol groups includes reduction with TCEP, wherein the resulting TCEP and unbound thiol groups are filtered off; re-oxidizing the fusion protein with DHAA, wherein excess DHAA is removed by filtration; conjugating the fusion protein with a polymer to form a fusion protein conjugate, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; separating the fusion protein conjugate from the polymer and the fusion protein by chromatography; and dialyzing 20 mg / mL of the fusion protein conjugate with a buffer to obtain a post-dialysis buffer solution, wherein the buffer solution comprises histidine acetate, wherein the concentration of histidine acetate is approximately 10 mg / mL. mM; a penetration enhancer, wherein the penetration enhancer comprises about 2.4% (by weight / volume) sucrose or trehalose; and a surfactant, wherein the surfactant comprises about 0.01% (by weight / volume) polysorbate 20 or polysorbate 80. In some embodiments, the fusion protein conjugate comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the reduction with TCEP involves a molar excess of TCEP (tris(2-carboxyethyl)phosphine) of 30 times relative to the concentration of the fusion protein. In some embodiments, the re-oxidation-reduction of the fusion protein with DHAA (dehydroascorbic acid) involves a molar excess of DHAA at 15 times the concentration of the fusion protein. In some embodiments, the filtration of the resulting TCEP and unbound thiol groups, as well as the removal of excess DHAA, is performed by tangential flow filtration (TFF). In some embodiments, dialysis is performed by buffer replacement using TFF. In some embodiments, the post-dialysis buffer is concentrated into a concentrated dialysis buffer containing approximately 50.0 mg / mL of the fusion protein conjugate, 25 mM histidine acetate, approximately 0.025% (w / w) of polysorbate 20 or polysorbate 80, and approximately 6.0% (w / v) of an osmotic agent, wherein the osmotic agent is sucrose or trehalose. In some embodiments, the buffer contains approximately 25 mM histidine acetate, approximately 0.025% (w / w) of PS20, and a pH of approximately 5.5. In some embodiments, the formulation is not the formulation of any of embodiments 1-98. In some implementations, X is Br.

[0293] A method for preparing a formulation, the method comprising: culturing a cell line that produces a recombinant fusion protein under conditions in which the fusion protein is produced; recovering the fusion protein; and preparing the fusion protein by the following steps: removing thiolates from cysteine ​​residues, wherein the removal of thiolates includes reduction with TCEP and filtering off the resulting TCEP and unbound thiolates; re-oxidizing the fusion protein with DHAA, wherein excess DHAA is removed by filtration; and conjugating the fusion protein with a polymer to form a fusion protein conjugate, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; separating the fusion protein conjugate from the polymer and the fusion protein by chromatography; and dialyzing a pharmaceutically effective amount of the fusion protein conjugate with a buffer containing histidine acetate, wherein the concentration of histidine acetate is approximately 10. mM; a penetration enhancer comprising about 2.4% (by weight / volume) sucrose or trehalose; and a surfactant comprising about 0.01% (by weight) polysorbate 20 or polysorbate 80; and concentrating the prepared fusion protein, wherein histidine acetate is concentrated to about 25 mM, wherein the surfactant is concentrated to about 0.025% (by weight) polysorbate 20 or polysorbate 80, and wherein the penetration enhancer is concentrated to about 6% (by weight / volume) sucrose or trehalose. In some embodiments, the fusion protein conjugate comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the coupling contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the anti-IL-6 heavy chain variable region sequence is selected from options of SEQ ID NO: 7-13, 89, 90, and / or 256-262, wherein the VEGF trap sequence is selected from at least one of SEQ ID NO: 114, 145, 15, 16, or 17, or wherein the light chain sequence of the anti-IL-6 molecule comprises at least one, two, or three light chain CDRs from at least one of SEQ ID NO: 76-84. In some embodiments, the pharmaceutically effective amount is an amount sufficient to achieve any one or more beneficial or desired results. In some embodiments, the pharmaceutically effective amount is a concentration greater than 10 mg / mL. In some embodiments, the pharmaceutically effective amount is a concentration greater than 30 mg / mL. In some embodiments, the formulation is not a formulation of any of schemes 1-98. In some embodiments, X is Br.

[0294] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is approximately 50 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, and the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate, wherein the histidine acetate is approximately 25 mM; a penetration enhancer, wherein the penetration enhancer is approximately 6% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises approximately 0.025% (w / w) polysorbate 20 or polysorbate 80. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114, wherein the fusion protein has a light chain having at least 80% identity with the sequence of SEQ ID NO: 169, and wherein the fusion protein has a heavy chain having at least 80% identity with the sequence of SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17) In some embodiments, each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region of the heavy chain and the Fab region (downstream of the CH1 domain). In some embodiments, the formulation is not a formulation of any of claims 1-98. In some embodiments, X is Br.

[0295] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is approximately 50 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate, wherein the histidine acetate is approximately 25 mM; a penetration enhancer, wherein the penetration enhancer is approximately 6% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises approximately 0.025% (w / w) polysorbate 20 or polysorbate 80. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises approximately 10% to 60% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114, wherein the fusion protein has a light chain having at least 80% identity with the sequence of SEQ ID NO: 169, and wherein the fusion protein has a heavy chain having at least 80% identity with the sequence of SEQ ID NO: 170. In some embodiments, the fusion protein comprises the following structure: Equation (17A) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curves represent the connection points with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H, c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, if the conjugate contains a VEGF trap, the VEGF trap is fused to: the N-terminus of the heavy chain; or between the hinge region and the Fab region (downstream of the CH1 domain) of the heavy chain. In some embodiments, the fusion protein comprises the amino acid sequences of SEQ ID NO: 169 and 170 and is conjugated to a polymer, wherein the polymer is the polymer described in the structure of Formula 17, Formula 17A, or Formula 17Br. In some embodiments, the formulation is not a formulation of any of schemes 1-98. In some embodiments, X is Br.

[0296] In some embodiments, the pharmaceutical formulation comprises: a pharmaceutically effective amount of the fusion protein, wherein the concentration of the fusion protein is 50 mg / mL; a polymer, wherein the fusion protein is coupled to the polymer, and the polymer comprises a phosphorylated choline polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate, wherein the histidine acetate is 25 mM; a penetration enhancer, wherein the penetration enhancer is about 6% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises 0.025% (w / w) polysorbate 20 or polysorbate 80. In some embodiments, the pharmaceutical formulation comprises an uncoupled fusion protein and a fusion protein conjugate, wherein the uncoupled fusion protein in the pharmaceutical formulation comprises about 30% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the uncoupled fusion protein. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170, and wherein the fusion protein comprises the following structures: Equation (17A) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%. In some embodiments, the pharmaceutical formulation is not a pharmaceutical formulation of any of claims 1-98. In some embodiments, X is Br.

[0297] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is 50 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, and the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer containing sodium acetate, wherein the sodium acetate is 50 mM; a penetration enhancer, wherein the penetration enhancer is 4% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises 0.025% (w / w) polysorbate 20 or polysorbate 80, wherein the pH is 5.0. In some embodiments, the pharmaceutical formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the pharmaceutical formulation comprises approximately 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170. In some embodiments, the fusion protein comprises the following structure: Formula (17Br) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the rest of the polymer, where X is Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%.

[0298] In some embodiments, a pharmaceutical formulation is provided, comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is 50 mg / mL; a polymer, wherein the fusion protein is conjugated to the polymer, wherein the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer; a buffer solution comprising histidine acetate, wherein the histidine acetate is 25 mM; a penetration enhancer, wherein the penetration enhancer is about 6% (w / v) sucrose or trehalose; and a surfactant, wherein the surfactant comprises 0.025% (w / w) polysorbate 20 or polysorbate 80, wherein the pH is 5.5. In some embodiments, the formulation comprises an unconjugated fusion protein and a fusion protein conjugate, wherein the unconjugated fusion protein in the formulation comprises about 30% of the total molar amount of the fusion protein conjugate and the unconjugated fusion protein, wherein the total molar amount is the sum of the molar amounts of the fusion protein conjugate and the unconjugated fusion protein. In some embodiments, the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170. In some embodiments, the fusion protein comprises the following structures: Formula (17Br) In some embodiments, a portion of each heavy chain of the anti-IL-6 antibody is represented by the letter H, and each light chain of the anti-IL-6 antibody is represented by the letter L; the polymer is bonded to the antibody via a thiol group at C443 (EU number), which is shown on one of the heavy chains; PC is... The curve represents the connection point with the rest of the polymer, where X is Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 3500 ± about 10% to about 20%.

[0299] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, a buffer (containing histidine), a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody and a VEGF trap. In some embodiments, the VEGF trap has at least 80% sequence identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein is conjugated to a polymer. In some embodiments, the fusion protein comprises a polymer-conjugated IL-6-specific antagonistic antibody or a fragment thereof. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain amino acid variable region comprising a heavy chain having at least one sequence from SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and a light chain amino acid variable region comprising a light chain having at least one sequence from SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) containing three complementarity-determining regions: VH(CDR1), VH CDR2, and VH CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO:256; and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO:91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof that binds to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: the N-terminus of the heavy chain containing IL-6 VH; or between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain. In some embodiments, the buffer comprises histidine acetate. In some embodiments, the emulsifier comprises polysorbate 20. In some embodiments, polysorbate 20 comprises about 0.01% (w / w) to about 0.1% (w / w) of the formulation. In some embodiments, the emulsifier comprises polysorbate 80.In some embodiments, polysorbate 80 comprises about 0.01% (w / w) to about 0.1% (w / w) of the formulation. In some embodiments, polysorbate 20 comprises about 0.025% (w / w) of the formulation. In some embodiments, polysorbate 80 comprises about 0.025% (w / w) of the formulation. In some embodiments, the histidine acetate concentration is between about 10 mM and about 50 mM. In some embodiments, the histidine acetate concentration is about 25 mM. In some embodiments, the formulation concentration is between about pH 4.5 and about pH 6.5. In some embodiments, the formulation concentration is about pH 5.5. In some embodiments, the polymer comprises a phosphorylcholine-containing polymer or a zwitterionic monomer. In some embodiments, the polydispersity index (PDI) is between about 0.5 and about 2. In some embodiments, the polydispersity index (PDI) is 1. In some embodiments, the penetration enhancer comprises about 2% to 10% (weight / volume) of the formulation. In some embodiments, the penetration enhancer is sucrose or trehalose. In some embodiments, sucrose comprises about 6.0% (by weight / volume) of the formulation. In some embodiments, trehalose comprises about 6.0% (by weight / volume) of the formulation. In some embodiments, the formulation is not the formulation described in any one of schemes 1-98.

[0300] In some embodiments, the formulation comprises: a pharmaceutically effective amount of the fusion protein; a buffer comprising sodium acetate, a surfactant, and optionally a penetration enhancer. In some embodiments, the fusion protein comprises CDRH3 having at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and a VEGF trap. In some embodiments, the VEGF trap has at least 80% identity with the sequence of SEQ ID NO: 114. In some embodiments, the fusion protein comprises an antagonistic antibody or a fragment thereof that specifically binds to polymer-conjugated IL-6. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof comprising: a) a heavy chain amino acid variable region comprising a heavy chain having at least one sequence of SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and b) a light chain amino acid variable region comprising a light chain having at least one sequence of SEQ ID NO: 91-93, 28-30. In some embodiments, the fusion protein comprises an antagonistic IL-6 antibody or a fragment thereof, comprising: a heavy chain variable region (VH) containing three complementarity-determining regions: VH(CDR1), VH CDR2, and VH CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3, having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 91-93. In some embodiments, the fusion protein further comprises an antagonistic antibody or fragment thereof that binds to IL-6, the antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; at least one of the following mutations (EU numbers): L234A, L235A, and G237A; and at least one of the following mutations (EU numbers): Q347C or L443C. In some embodiments, the VEGF trap is located at one of the following: a) the N-terminus of the heavy chain containing IL-6 VH; or b) between the hinge region of the heavy chain containing IL-6 VH and downstream of the CH1 domain. In some embodiments, the VEGF trap consists of the sequence of SEQ ID NO: 114. In some embodiments, the surfactant comprises about 0.01% to about 0.05% (by weight) of polysorbate 20. In some embodiments, the surfactant comprises about 0.025% (by weight) of polysorbate 20.In some embodiments, the surfactant comprises about 0.01% to about 0.05% (by weight) of polysorbate 80. In some embodiments, the surfactant comprises about 0.025% (by weight) of polysorbate 80. In some embodiments, the pH of the formulation is between about 4.5 and about 6.5. In some embodiments, the pH of the formulation is about 5.5. In some embodiments, the formulation is stable for at least 52 weeks. In some embodiments, the formulation is stable at 4°C for at least 52 weeks. In some embodiments, the formulation is stable at 37°C for at least 8 weeks. In some embodiments, the formulation is stable under acidic conditions. In some embodiments, the formulation is formulated for intravitreal injection. In some embodiments, the sodium acetate concentration in the buffer is about 0.1 mM to about 50 mM. In some embodiments, the sodium acetate concentration in the buffer is about 15 mM sodium acetate. In some embodiments, the concentration of the fusion protein is between about 20 mg / mL and 150 mg / mL. In some embodiments, the concentration of the fusion protein is about 100 mg / mL. In some embodiments, the penetration enhancer comprises about 2% to 10% (by weight / volume) of the formulation. In some embodiments, the penetration enhancer is sucrose or trehalose. In some embodiments, sucrose comprises about 5.2% (by weight / volume) of the formulation. In some embodiments, trehalose comprises about 5.2% (by weight / volume) of the formulation. In some embodiments, the formulation is not a formulation of any of claims 1-98.

[0301] In some embodiments, a pharmaceutical formulation is provided comprising: a pharmaceutically effective amount of a fusion protein, wherein the concentration of the fusion protein is 100 mg / mL; a buffer containing sodium acetate, wherein the concentration of sodium acetate is 15 mM; and a penetration enhancer, wherein the penetration enhancer is 5.2% (w / v) sucrose or trehalose; a surfactant, wherein the surfactant comprises 0.025% (w / w) polysorbate 20 or polysorbate 80, wherein the pH is 5.5; and wherein the fusion protein comprises an anti-IL-6 antibody and a VEGF trap, wherein the fusion protein comprises SEQ ID NO: 169 and 170 (with or without the C-terminal lysine of SEQ ID NO: 170).

[0302] In some embodiments, the formulation excludes any or all of the formulations listed in schemes 1-98.

[0303] In some embodiments, the formulation is the one disclosed herein, but excludes the specific formulation disclosed in any of Examples 1-33.

[0304] In some embodiments, the formulation may contain histidine. In some embodiments, the formulation may contain histidine acetate. In some embodiments, the formulation may contain... Figure 52 The characteristics shown are as follows. In some embodiments, the formulation may use histidine acetate instead of sodium acetate. In some embodiments, the formulation may contain histidine acetate in the range of 10 nm to 50 nm. In some embodiments, the formulation may contain sucrose or trehalose in the range of 2% to 10% (w / v). In some embodiments, the formulation may contain polysorbate 20 in the range of 0.01% to 0.1% (w / w). In some embodiments, the formulation may contain a protein concentration in the range of 20 mg / mL to 100 mg / mL. In some embodiments, the formulation may contain a pH value in the range of 5.0 to 6.5. In some embodiments, the formulation may contain a percentage of uncoupled fusion protein in the range of 10% to 50%. In some embodiments, the formulation may contain 25 mM histidine acetate. In some embodiments, the formulation may contain 6% (w / v) sucrose or trehalose. In some embodiments, the formulation may contain 0.025% (w / w) polysorbate 20. In some embodiments, the formulation may contain a protein concentration of 50 mg / mL. In some embodiments, the formulation may contain a pH of 5.5. In some embodiments, the formulation may contain 30% of the uncoupled fusion protein. In some embodiments, the polymer may be any polymer disclosed herein. In some embodiments, the fusion protein may be any fusion protein disclosed herein.

[0305] In some embodiments, the formulation may contain a pH value in the range of 4.5 to 5.5. In some embodiments, the formulation may contain an osmotic pressure replenisher concentration in the range of 0 to 6%. In some embodiments, the formulation may contain a sodium chloride concentration in the range of 0 to 40 mM. In some embodiments, the formulation may contain a sodium acetate concentration in the range of 10 mM to 50 mM. In some embodiments, the formulation may contain a protein concentration in the range of 50 g / L to 100 g / L. In some embodiments, the formulation may contain a buffer further supplemented with 0.025% polysorbate 20.

[0306] In some embodiments, the formulation comprises 15 mM sodium acetate, pH 5.5; 5.2% permeation replenisher; and 100 g / LOG2072 monoclonal antibody. In some embodiments, parameters of some exemplary formulations of OG2072 and combinations of OG2072 and OG2074 are shown in [the table / image / description]. Figure 52 In some embodiments, the preferred formulation may be... Figure 52The formulation on the right. OG2072 represents a fusion construct comprising an anti-IL-6 antibody fused to a VEGF trap and comprising the heavy chain (with or without a C-terminal lysine) of SEQ ID NO: 170 and the light chain of SEQ ID NO: 169.

[0307] This document describes exemplary methods and materials, although similar or equivalent methods and materials may also be used. These materials, methods, and embodiments are illustrative only and are not intended to be limiting.

[0308] IL-6 antagonist antibodies, IL-6 antibody-VEGF traps, and / or their conjugates This document provides anti-IL-6 antibodies that block, inhibit, or reduce (including significantly reduce) the biological activity of IL-6, including downstream events mediated by IL-6. In some embodiments, the IL-6 antagonist antibody will have one or more CDR sequences provided herein.

[0309] In some implementations, the isolated antagonist antibody specifically binds to IL-6.

[0310] In some embodiments, the antibody preferably reacts with IL-6 in a manner that inhibits IL-6 signaling. In some embodiments, the IL-6 antagonist antibody specifically binds to primate IL-6.

[0311] This document also provides antibodies encompassing the following: monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single-chain (ScFv), mutants thereof, fusion proteins containing antibody moieties (e.g., domain antibodies), humanized antibodies, and any other modified conformations of immunoglobulin molecules containing desired specific antigen recognition sites, including glycosylated variants, amino acid sequence variants, and covalently modified antibodies. These antibodies may be murine, rat, human, or any other source (including chimeric or humanized antibodies). In some embodiments, the IL-6 antagonist antibody is a monoclonal antibody. In some embodiments, the antibody is a human antibody or a humanized antibody.

[0312] In some embodiments, the antibody comprises as shown in Tables 1, 2, 6, 7, 8, and / or 9. Figure 5The heavy chain amino acid variable region is shown. In some embodiments, the isolated antagonist antibody comprises a heavy chain variable region (VH) having VH complementarity-determining regions 1 (CDR1), 2 (CDR2), and 3 (CDR3) having the amino acid sequences shown in Tables 1, 2, 6, 7, 8, and / or 9; and a light chain variable region (VL) having VL CDR1, 2 (CDR2), and 3 (CDR3) having the amino acid sequences shown in the table.

[0313] In some embodiments, a separate antagonist anti-IL-6 antibody is provided. This antibody contains a heavy chain constant region comprising one or more mutations designed to reduce effector function. In some embodiments, the one or more mutations reduce antibody effector function associated with the complement cascade, for example, reducing complement cascade activation. In some embodiments, the reduction in effector function is at least about 50%.

[0314] In some embodiments, an isolated IL-6-specific antagonist antibody is provided, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the antibody comprises mutants L234A, L235A, and G237A (based on EU numbers). In some embodiments, the isolated antagonist antibody comprises mutants L234A, L235A, and G237A. In some embodiments, the binding of the isolated antagonist antibody with mutations to the FCγ receptor or C1q is minimized. In some embodiments, the binding of the isolated antagonist antibody with mutants L234A, L235A, and G237A to the FCγ receptor or C1q is minimized. In some embodiments, an isolated antagonist anti-IL-6 antibody is provided, wherein the mutation is located at one or more of the following amino acid positions (EU numbers): E233, L234, L235, G236, G237, A327, A330, and P331. In some embodiments, an isolated antagonist anti-IL-6 antibody is provided, wherein the mutation is selected from E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S.

[0315] In some embodiments, a separated antagonist anti-IL-6 antibody is provided. The heavy chain constant region also contains cysteine ​​residues introduced via recombinant DNA technology. In some embodiments, the cysteine ​​residue is selected from Q347C and L443C (EU number). In some embodiments, the cysteine ​​residue is L443C (EU number).

[0316] In some embodiments, the antibody contains all three of the following mutations (EU numbers): L234A, L235A, and G237A, and the antibody also contains L443C (EU number). In some embodiments, the antibody is human IgG1, and the heavy chain constant region of the antibody contains one or more mutations that reduce immune-mediated effector function.

[0317] In some embodiments, a separate antagonist antibody is provided that binds to an epitope on human IL-6 that is identical to or overlaps with an epitope recognized by an antibody comprising any one or more of the amino acid sequences shown in Tables 1, 2, and / or 6-9. In some embodiments, an IL-6 antibody (as shown in Formula 17, 17A, or 17Br) is provided having a cysteine ​​residue and being linked to a polymer via that cysteine ​​residue.

[0318] In some embodiments, a separate antagonist antibody that binds to IL-6 is provided. In some embodiments, the separate antagonist antibody that binds to IL-6 comprises a heavy chain containing the amino acid sequences (with or without C-terminal lysine) shown in Tables 1, 2, 6, 7, 8, and / or 9, and a light chain containing the amino acid sequences shown in Tables 1, 2, 6, 7, 8, and / or 9.

[0319] In some embodiments, a separate antagonist antibody binding to IL-6 is provided. The antibody comprises VH, which includes the amino acid sequences shown in Tables 1, 2, 6, 7, 8, and / or 9, or sequences at least 90% identical thereto, and has amino acid substitutions at residues within non-complementarity-determining regions. In some embodiments, the antibody comprises one or more of the following: Figure 5 HCDR1 in Figure 5 HCDR2 in Figure 5 HCDR3 in Figure 5 LCDR1 in Figure 5 LCDR2 in Figure 5 LCDR3 in the array, such as 1, 2, 3, 4, 5 or all 6 CDRs.

[0320] In some embodiments, an antibody that binds to IL-6 is provided, wherein the antibody comprises: CDRH1, which is CDRH1 in Tables 1, 2, 6, 7, 8, and / or 9; CDRH2, which is CDRH2 in Tables 1, 2, 6, 7, 8, and / or 9; CDRH3, which is CDRH3 in Tables 1, 2, 6, 7, 8, and / or 9; CDRL1, which is CDRL1 in Tables 1, 2, 6, 7, 8, and / or 9; CDRL2, which is CDRL2 in Tables 1, 2, 6, 7, 8, and / or 9; CDRL3, which is CDRL3 in Tables 1, 2, 6, 7, 8, and / or 9; at least one of the following mutations: L234A, L235A, and G237A (based on EU number), and at least one of the following mutations: Q347C or L443C (based on EU number).

[0321] In some embodiments, an isolated antagonist anti-IL-6 antibody is provided. The heavy chain variable region of this antibody includes three complementarity-determining regions (CDRs) containing the amino acid sequences shown in Table 1. In some embodiments, an isolated antagonist anti-IL-6 antibody is provided, wherein the light chain variable region of this antibody includes three complementarity-determining regions (CDRs) containing the amino acid sequences shown in Table 2. In some embodiments, the antibody contains... Figure 5 One or more sequences identified in the block, such as one or more CDRs (including 2, 3, 4, 5, or 6 CDRs within the frame) and / or the entire heavy chain and light chain variable region.

[0322] In some embodiments, a separate antagonist anti-IL-6 antibody includes a heavy chain variable region (VH) containing the three complementarity-determining regions (CDRs) shown in Table 1, and the light chain variable region (VL) of the antibody contains the three complementarity-determining regions (CDRs) shown in Table 2.

[0323] In some embodiments, an isolated antagonistic anti-IL-6 antibody is provided. Its VH contains the amino acid sequences shown in Table 1, and the light chain variable region of the antibody contains three CDRs containing the amino acid sequences shown in Table 2.

[0324] In some embodiments, an isolated antagonistic anti-IL-6 antibody is provided, wherein the antibody comprises a VL containing the amino acid sequence shown in Table 2, or a variant thereof having an amino acid substitution at a non-CDR amino acid. In some embodiments, an isolated antagonistic anti-IL-6 antibody is provided, wherein the antibody comprises a VH containing the amino acid sequence shown in Table 1, or a variant thereof having several amino acid substitutions at non-CDR amino acids.

[0325] In some embodiments, a separate antagonistic antibody is provided, wherein the antibody comprises a heavy chain having a C-terminal lysine (containing the amino acid sequence shown in Table 1) and a light chain containing the amino acid sequence shown in Table 2. In some embodiments, a separate antagonistic antibody is provided, wherein the antibody comprises a heavy chain without a C-terminal lysine (containing the amino acid sequence shown in Table 1) and a light chain containing the amino acid sequence shown in Table 2.

[0326] IL-6 antagonistic antibodies can be prepared by any method known in the art. General techniques for producing human and murine antibodies are known in the art and / or described herein.

[0327] IL-6 antagonistic antibodies can be identified or characterized using methods known in the art, by which these methods detect and / or measure a decrease, improvement, or neutralization of IL-6 biological activity. In some embodiments, IL-6 antagonistic antibodies are identified by incubating a candidate antibody with IL-6 and monitoring its binding to IL-6R, or monitoring the binding of IL-6 / IL-6R to gp130, and / or monitoring a concomitant decrease or neutralization of IL-6 biological activity. Binding assays can be performed, for example, using purified IL-6 peptides, or using naturally expressed (e.g., various bacterial strains) or transfected cells expressing IL-6 peptides. In one embodiment, the binding assay is a competitive binding assay, in which the ability of a candidate antibody to compete with a known IL-6 antagonistic antibody for IL-6 binding is evaluated. This assay can be performed in various forms, including ELISA.

[0328] Following initial identification, the activity of candidate IL-6 antagonist antibodies can be further confirmed and optimized using bioassays to test the target biological activity. In some embodiments, in vitro cell assays are used to further characterize the candidate IL-6 antagonist antibodies.

[0329] IL-6 antagonist antibodies can be characterized using methods well-known in the art. One method, for example, is to identify the epitopes they bind to, known as “epitope mapping.” Many methods for mapping and characterizing epitope positions on proteins are known in the art, including resolving the crystal structure of antibody-antigen complexes, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described in Chapter 11 of *Using Antibodies: A Laboratory Handbook* by Harlow and Lane (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999). In another example, epitope mapping can be used to determine the sequence to which IL-6 antagonist antibodies bind. IL-6 antagonist antibody epitope mapping is available from various commercial sources, such as Pepscan Systems (Edelhertweg 15, 8219 PHLelystad, The Netherlands). Epitopes can be linear, i.e., contained within a single amino acid sequence, or conformational, formed by the three-dimensional interactions of amino acids, and not necessarily contained within a single sequence. Peptides of varying lengths (e.g., at least 4 to 6 amino acids long) can be isolated or synthesized (e.g., recombined) and used for binding assays with IL-6 antagonist antibodies. In another example, epitopes binding to IL-6 antagonist antibodies can be identified through systematic screening using overlapping peptides derived from the IL-6 sequence and measuring the binding of the IL-6 antagonist antibody. Based on gene fragment expression assays, open reading frames encoding IL-6 are randomly or fragmented via specific gene constructs, and the reactivity of the expressed IL-6 fragment with the test antibody is measured. For example, gene fragments can be generated by PCR and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled IL-6 fragment is then measured by immunoprecipitation and gel electrophoresis. Some epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) or yeast (yeast display). Alternatively, the binding of a defined library of overlapping peptide fragments to a test antibody can be tested in a simple binding assay. In another example, antigen mutagenesis, domain exchange assays, and alanine scan mutagenesis can be performed to identify the residues required, sufficient, and / or necessary for epitope binding. For instance, alanine scan mutagenesis can be performed using mutant IL-6, in which various residues of the IL-6 peptide have been substituted with alanine. By evaluating the binding of the antibody to mutant IL-6, the importance of specific IL-6 residues for antibody binding can be assessed.

[0330] In some embodiments, an isolated antagonistic anti-IL-6 antibody is provided, wherein the antibody binds to human IL-6 with an affinity of about 0.01 pM to about 10 nM. In some embodiments, an isolated antagonistic anti-IL-6 antibody is provided, wherein the antibody binds to human IL-6 with an affinity of about 0.1 pM to about 2 nM. In some embodiments, an isolated antagonistic anti-IL-6 antibody is provided, wherein the antibody binds to human IL-6 with an affinity of about 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, and 1000 pM.

[0331] The binding affinity (Kb) of IL-6 antagonist antibodies to IL-6 D The binding affinity can be from about 0.001 nM to about 200 nM. In some embodiments, the binding affinity is any one of about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 2 pM, or about 1 pM. In some embodiments, the binding affinity is less than any of the following: about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 0.5 pM, about 0.1 pM, about 0.05 pM, about 0.01 pM, about 0.005 pM, or about 0.001 pM.

[0332] In some embodiments, an isolated antagonistic anti-IL-6 antibody is provided, wherein the antibody binds to human IL-6 at 37 degrees Celsius with a koff of at least 5.0E-03. In some embodiments, the koff is 5E-04. In some embodiments, an isolated antagonistic anti-IL-6 antibody is provided, wherein the antibody binds to human IL-6 at 37 degrees Celsius with a koff better than 5.0E-04.

[0333] In some embodiments, binding affinity may be defined based on one or more of the association constant (k_a), dissociation constant (k_d), and analyte concentration (KD) at which half-maximum binding capacity is achieved. In some embodiments, k_a may range from about 0.50E+05 to about 5.00E+08. In some embodiments, k_a... d The range can be from approximately 0.50E-06 to approximately 5.00E-03. In some embodiments, K D The range is approximately 0.50E-12 to approximately 0.50E-07.

[0334] In some embodiments, a pharmaceutical composition comprising any of the antibodies disclosed herein is provided. In some embodiments, a pharmaceutical composition comprising any of the conjugates disclosed herein is provided. In some embodiments, a pharmaceutical composition comprising any of the antibodies and any of the conjugates disclosed herein is provided. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical composition is a liquid. In some embodiments, the endotoxin level of the pharmaceutical composition is less than about 0.2 EU / ml. In some embodiments, the pharmaceutical composition is a liquid and the endotoxin level is less than about 0.2 EU / ml. In some embodiments, the pharmaceutical composition is a liquid and the endotoxin level is less than about 2.0 EU / ml, 1 EU / ml, 0.5 EU / ml, or 0.2 EU / ml. In some embodiments, for example in intravitreal injection, the endotoxin limit is 0.01 to 0.02 EU / injection / eye.

[0335] Some embodiments provide any of the following, or compositions (including pharmaceutical compositions) comprising antibodies or variants thereof having portions of the light chain and heavy chain sequences shown in Tables 1 and 2. In Tables 1 and 2, the underlined sequences are some embodiments of the CDR sequences provided herein.

[0336] Table 1 Table 1. Anti-IL-6 heavy chain variable region sequence. CDRs are underlined.

[0337] Table 2. Variable region sequence of the anti-IL-6 light chain. CDRs are underlined.

[0338] In some embodiments, the antibody does not have one or more (or any) CDRs listed in Tables 3, 4, and / or 5.

[0339] Table 3 Table 4 Table 5 In some embodiments, the compositions disclosed herein comprise an antibody having a partial or complete light chain sequence and a partial or complete heavy chain sequence, or variants thereof, of any of the options provided in Tables 1, 2, 6, 7, 8, and / or 9. In some embodiments, the antibody (or its binding fragment) may comprise any one or more CDRs provided in Tables 1, 2, 6, 7, 8, and / or 9. In some embodiments, the antibody (or its binding fragment) may comprise any three or more CDRs provided in Tables 1, 2, 6, 7, 8, and / or 9. In some embodiments, the antibody (or its binding fragment) may comprise all six CDRs provided in Tables 1, 2, 6, 7, 8, and / or 9. In some embodiments, the heavy chain and / or light chain may be any one or more other antibody constructs provided herein, such as those comprising… Figure 5 , 18 1, 2, and / or 21-24 and those provided in Tables 1, 3, 4, 5, 6, 7, 8, and / or 9.

[0340] In some embodiments, the compositions disclosed herein comprise an antibody having a partial or complete light chain CDR sequence and a partial or complete heavy chain CDR sequence of any of the options provided in Tables 1, 2, 6, 7, 8, and / or 9.

[0341] In some embodiments, a CDR portion of the IL-6 antagonist antibody is also provided. The determination of the CDR region is within the ordinary skill of those skilled in the art. It should be understood that in some embodiments, the CDR may be a combination of IMGT and Paratome CDRs (also referred to as a "combined CDR" or "extended CDR"). The determination of the CDR is within the ordinary skill of those skilled in the art. In some embodiments, the CDR is an IMGT CDR. In other embodiments, the CDR is a Paratome CDR. In other embodiments, the CDR is an extended, AbM, conformational, Kabat, or Chothia CDR. In embodiments having more than one CDR, the CDR may be any one of IMGT, Paratome, extended, Kabat, Chothia, AbM, conformational CDRs, or a combination thereof. In some embodiments, other CDR definitions may also be used. In some embodiments, only the common residues in definitions 2, 3, 4, 5, 6, or 7 above are used (thus obtaining a shorter sequence). In some embodiments, any residues within any of definitions 2, 3, 4, 5, 6, or 7 above may be used (thus obtaining a longer sequence).

[0342] In some embodiments, the IL-6 antagonist antibody comprises three CDRs for any heavy chain variable region shown in Tables 1, 2, 6, 7, 8, and / or 9. In some embodiments, the antibody comprises three CDRs for any light chain variable region shown in Tables 1, 2, 6, 7, 8, and / or 9. In some embodiments, the antibody comprises three CDRs for any heavy chain variable region shown in Table 1 and three CDRs for any light chain variable region shown in Table 2. In some embodiments, the CDRs are one or more CDRs specified in Tables 6 and / or 7, or 8 and / or 9 below: Table 6. Anti-IL-6 heavy chain CDR sequences Table 7. Anti-IL-6 heavy chain CDR sequences. Kabat Table 8. Anti-IL-6 light chain CDR sequence.

[0343] Table 9. Anti-IL-6 light chain CDR sequence.

[0344] In some embodiments, the antibody for binding IL-6 may be an antibody comprising one or more sequences from Tables 1, 2, 6, 7, 8, and / or 9. In some embodiments, the antibody for binding IL-6 may be an antibody comprising three or more sequences from Tables 1, 2, 6, 7, 8, and / or 9. In some embodiments, the antibody for binding IL-6 may be an antibody that competes for binding with an antibody comprising six specified CDRs from any one of Tables 1, 2, 6, 7, 8, and / or 9.

[0345] In some embodiments, the antibody may be linked to or fused to a VEGF trap sequence. In some embodiments, the trap sequence may be as shown in Table 10. In some embodiments, the sequence has at least 80% identity with the sequences shown in Table 10, for example, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences shown in Table 10. In some embodiments, any VEGF trap molecule may be used herein in U.S. Publication No. 20150376271. In some embodiments, the VEGF trap sequence is fused to IL-6 in one of the following ways: at the N-terminus of the heavy chain containing IL-6 VH ( Figure 6 (left), or between the hinge region of the heavy chain containing IL-6 VH and the downstream of the CH1 domain ( Figure 6(Right). Unless otherwise specified, the embodiments described herein, when discussing any antibody-capture fusion, encompass both of the above-described options and combinations thereof. In some embodiments, the term "capture body" refers to a full-length extracellular region or any portion thereof, or a combination of portions from different VEGF receptors, capable of antagonizing signal transduction between at least one VEGF and VEGFR. Preferably, the extracellular capture body fragment comprises at least one domain from any one of VEGFR-1, -2, or -3, more preferably comprising at least two consecutive domains, such as D2 and D3. Optionally, the extracellular domain comprises at least one domain from at least two different VEGFRs. Preferred extracellular domains comprise or consist primarily of D2 of VEGFR-1 and D3 of VEGFR-2.

[0346] Table 10. Fusion sequence of VEGFR1 domain 2 and VEGFR2 domain 3 In some embodiments, the IL-6 antibody VEGF trap construct may have any of the sequences provided in Table 11. In some embodiments, the construct may be at least identical to the sequences in Table 11, for example, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher. In some embodiments, the fusion protein may meet the above percentages, except where the antibody IL-6 domain does not contain one or more CDRs from Tables 3, 4, and / or 5. In some embodiments, the fusion protein contains... Figure 5 The sequence may include one or more sequences, such as one or more CDRs (including CDRs 2, 3, 4, 5, or 6 within the frame) and / or the entire heavy and light chain variable regions, as well as VEGF trap sequences (e.g., Table 10). In some embodiments, these sequences may be directly fused to each other. In some embodiments, one or more flexible linker sequences or segments may be used. The linker sequence may be located between the antibody sequence and the VEGF trap sequence. These sequences may be 5 to 30 amino acids in length. In some embodiments, the linker sequence may contain G and S in a ratio of approximately 4:1. In some embodiments, the linker contains the sequence: GGGGSGGGGS (SEQ ID NO: 115). In some embodiments, any flexible linker may be used. In some embodiments, the Fc portion of the IL-6 antibody is IgG1.

[0347] Table 11 Heavy and light chain sequences of the dual inhibitor molecule. CDRs are underlined in the heavy and light chains, VEGF trap sequences are bolded in black, and glycine-serine linkers are shown in italics.

[0348] To express the anti-IL-6 antibody and / or IL-6 VEGF trap provided herein, a DNA fragment encoding the VH and VL regions can first be obtained. Various modifications (e.g., mutations, deletions, and / or additions) can also be introduced into the DNA sequence using standard methods known to those skilled in the art. For example, standard methods can be used for mutagenesis, such as PCR-mediated mutagenesis, in which mutated nucleotides are incorporated into PCR primers such that the PCR product contains the desired mutation, or site-directed mutagenesis can be performed.

[0349] This document also provides for modifications to the illustrated variable regions. For example, antibodies containing functionally equivalent variable regions and CDRs without significantly affecting their properties are also provided, as well as variants with enhanced or reduced activity and / or affinity. For example, the amino acid sequence can be mutated to obtain an antibody with the desired IL-6 binding affinity. Peptide modification is a routine practice in the art and will not be detailed here. Examples of modified peptides include: peptides with conserved substitutions of amino acid residues; deletions or additions of one or more amino acids that do not significantly and harmfully alter functional activity or mature (enhance) the peptide's affinity for its ligands; or the use of chemical analogs.

[0350] Amino acid sequence insertions include N-terminal and / or C-terminal fusions of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionine residue or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include N-terminal or C-terminal fusion enzymes of antibodies or peptides that increase the half-life of antibodies in circulation.

[0351] Substitution variants are antibody molecules in which at least one amino acid residue has been removed and a different residue has been inserted. The most concerning sites for substitution mutagenesis include hypervariable regions, but changes to the framework region are also considered. Conservative substitutions are shown under the heading “Conservative Substitutions” in Table 12. If such substitutions result in a change in biological activity, more substantial changes, referred to as “Exemplary Substitutions” in Table 12, may be introduced, or as described below with reference to the amino acid categories, and the product may be screened.

[0352] Table 12 - Amino Acid Substitutions Substantial modification of antibody biological properties can be achieved by selecting substitutions that significantly affect (a) the structure of the substituted polypeptide backbone (e.g., as a β-sheet or helical conformation), (b) the charge or hydrophobicity of the target site molecule, or (c) the side chain volume. Naturally occurring residues are grouped into the following groups based on their common side chain characteristics: (1) Nonpolar: Leucine, Met, Ala, Val, Leu, Ile; (2) No charge polarity: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Alkaline (positively charged): Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatics: Trp, Tyr, Phe, His.

[0353] Non-conservative mutations occur by exchanging one member of these categories with another.

[0354] For example, one possible substitution is to change one or more cysteine ​​residues in the antibody that may be chemically reactive, replacing them with another residue, such as, but not limited to, alanine or serine. For example, atypical cysteine ​​substitutions can be made. This substitution can be made in the CDR or framework region of a variable domain, or in the constant region of the antibody. In some embodiments, the cysteine ​​is typical. Any cysteine ​​residue that does not participate in maintaining the correct conformation of the antibody can also be substituted (typically with serine) to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bonds can be added to the antibody to improve its stability, especially when the antibody is an antibody fragment such as an Fv fragment.

[0355] Antibodies can also be modified, for example, in variable domains of the heavy and / or light chains, to alter their binding properties. Changes in the variable regions can alter binding affinity and / or specificity. In some embodiments, no more than one to five conserved amino acid substitutions are made within the CDR domain. In other embodiments, no more than one to three conserved amino acid substitutions are made within the CDR domain. For example, mutations can be made in one or more CDR regions to increase or decrease the antibody's K+ affinity for IL-6. D Increase or decrease k off This can alter the binding specificity of the antibody. Site-directed mutagenesis is well-known in the art. See, for example, Sambrook et al. and Ausubel et al. above.

[0356] According to one aspect, the IgG domain of the IL-6 antagonist antibody may be IgG1, IgG2, IgG3, or IgG4. According to another aspect, the IgG domain may be a complex domain, wherein the constant region is formed by one or more of the aforementioned isotypes (e.g., a CH1 region from IgG2 or IgG4, and a hinge region, CH2, and CH3 region from IgG1). In selecting the isotype, it is known in the art that human isotypes IgG1 and IgG3 have complement-mediated cytotoxicity, while human isotypes IgG2 and IgG4 have weaker or no complement-mediated cytotoxicity. In some embodiments, the isotype of the IL-6 antagonist antibody is IgG1.

[0357] The light chain constant region can be human lambda or kappa type. In some embodiments, the IL-6 antagonist antibody has a human kappa light chain constant region.

[0358] Human constant regions exhibit both allotropic and allotropic variations among individuals, meaning that the polymorphic locations of constant regions may differ between individuals. Allotropic regions differ from allotropic regions in that serum recognizing an allotype binds to one or more non-polymorphic regions of other allotypes. The term "human constant region" includes any arrangement of residues having any natural allotype or occupying a polymorphic location in a natural allotype, or up to 3, 5, or 10 substitutions (described below) made to reduce or increase effector function.

[0359] One or more amino acids at the amino or carboxyl terminus of the light chain and / or heavy chain (e.g., C-terminal lysine of the heavy chain) may be missing or derivatized in part or all of the molecule.

[0360] Substitutions can be made in the constant region to reduce or increase effector functions, such as complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) (see, for example, Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA103:4005, 2006), or to prolong the half-life in humans (see, for example, Hinton et al., J. Biol. Chem.279:6213, 2004).

[0361] In some embodiments, the IL-6 antagonist antibodies provided herein include one or more substitutions that reduce complement-mediated cytotoxicity. Depending on the nature of the mutation, the reduction in complement-mediated cytotoxicity can be achieved with or without reducing Fc receptor binding. Antibodies with reduced complement-mediated cytotoxicity but little or no reduction in Fc receptor binding allow the desired effect of Fc-mediated iC3b phagocytosis to be achieved without activating complement, which may cause side effects. Exemplary mutations known to reduce complement-mediated cytotoxicity in human constant regions include mutations at sites numbered EU 241, 264, 265, 270, 296, 297, 322, 329, and 331. Mutations at sites 318, 320, and 322 have been reported to reduce complement activation in mouse antibodies. Alanine is a preferred residue occupying these sites in the mutation constant region. Some exemplary human mutations that have been used include F241A, V264A, D265A, V296A, N297A, K322A, and P331S in human IgG3, and D270A or E, N297Q, K322A, P329A, and P331S (EU number) in human IgG1.

[0362] As elsewhere, the EU numbering scheme is used to number the amino acids in the constant region of the antibody. When residues in the variable region are referred to herein (unless otherwise specified), residue numbering is based on the variable domain (or, if specified, on SEQ ID NO). Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce affinity for the Fcγ receptor (particularly the FcγRI receptor) and decrease complement binding and activation (see, e.g., US 6,624,821, WO / 2009 / 052439). Alanine substitutions at positions 234, 235, and 237 reduce effector function, particularly in the context of human IgG1. Optionally, positions 234, 236, and / or 237 in human IgG2 are substituted with alanine, and position 235 is substituted with glutamine (see, e.g., US 5,624,821) to reduce Fc receptor binding. Exemplary substitutions for increasing half-life include Gln at position 250 and / or Leu at position 428. An aspect is also provided where the presented anti-IL-6 antibody has a human IgG1 isotype, preferably having at least one mutation in the constant region. Preferably, the mutation reduces complement fixation or activation caused by the constant region. A particularly preferred aspect is also provided where the antibody has one or more mutations at the EU-numbered sites E233, L234, L235, G236, G237, A327, A330, and P331. More preferably, these mutations constitute one or more of the following EU-numbered mutations: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S. In the most preferred embodiment, human IgG1 has the following EU-numbered mutations: L234A, L235A, and G237A.

[0363] Coupled The half-life of IL-6 antagonist antibodies and / or IL-6 antibody VEGF traps can be extended by attaching a “half-life extension moiety” or “half-life extension group”, which are used interchangeably in this document. The latter refers to one or more chemical groups attached to one or more amino acid side chain functional groups (such as -SH, -OH, -COOH, -CONH2, -NH2) or one or more N- and / or O-glycan structures, which can increase the in vivo circulating half-life of the protein / peptide when coupled to these proteins / peptides. Examples of extended half-life include polymers described herein, particularly polymers of zwitterionic monomers, such as HEMA-phosphorylcholine, PEG, biocompatible fatty acids and their derivatives, hydroxyalkyl starch (HAS) (e.g., hydroxyethyl starch (HES)), polyethylene glycol (PEG), poly(glycine-serine) (HAP), hyaluronic acid (HA), heparin polymers (HEP), Fleximers (flexible polymers), dextran, polysialic acid (PSA), Fc domains, transferrin, albumin, elastin-like peptides (ELP), XTEN polymers, PAS polymers, PA polymers, albumin-binding peptides, CTP peptides, FcRn-binding peptides, and any combination thereof.

[0364] In some embodiments, the antibody is coupled to a phosphorylcholine-containing polymer. In some embodiments, the antibody is coupled to a polymer containing poly(acryloyloxyethyl phosphorylcholine), such as an acrylic polymer containing at least one acryloyloxyethyl phosphorylcholine monomer (e.g., 2-methacryloyloxyethyl phosphorylcholine, i.e., 2-methacryloyl-2'-trimethylammonium ethyl phosphate).

[0365] In some embodiments, the antibody and / or antibody-VEGF trap fusion protein is coupled to a water-soluble polymer, which is a polymer soluble in water. The amount of light transmitted through the water-soluble polymer solution should be at least about 75% of the light transmittance of the same solution after filtration, more preferably at least about 95%. By weight, the water-soluble polymer or fragment thereof is...

Claims

1. A pharmaceutical preparation comprising: A fusion protein conjugate, wherein the fusion protein conjugate comprises a first fusion protein conjugated to a phosphoric choline polymer; An uncoupled fusion protein, wherein the uncoupled fusion protein comprises a second fusion protein uncoupled to a phosphocholine polymer; Buffer solution, wherein the buffer solution contains histidine; and Surfactants, in, The first fusion protein and the second fusion protein each comprise: CDRH3, which has at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and VEGF capture body, The content of the uncoupled fusion protein in the formulation is 10% to 60% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the uncoupled fusion protein.

2. The formulation according to claim 1, wherein, The VEGF trap has at least 80% sequence identity with SEQ ID NO:

114.

3. The formulation according to claim 1, wherein, The first fusion protein and the second fusion protein each comprise: A light chain having at least 80% identity with the sequence of SEQ ID NO: 169; and Heavy chains that have at least 80% identity with the sequence of SEQ ID NO:

170.

4. The formulation according to claim 3, wherein, The fusion protein conjugate comprises the following structure: Equation (17A) in: Each heavy chain of the first fusion protein is represented by the letter H, and each light chain of the first fusion protein is represented by the letter L; The polymer is bonded to the heavy chain via a thiol group (EU number) of C443, and this bond is shown on one of the heavy chains; PC is The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) -H; c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is approximately 2500 ± 15%.

5. The formulation according to claim 1, wherein, The first fusion protein and the second fusion protein each comprise an antagonistic IL-6 antibody or an antigen-binding fragment thereof, wherein the antagonistic IL-6 antibody or antigen-binding fragment thereof comprises: A heavy chain amino acid variable region comprising a heavy chain, said heavy chain containing at least one sequence of SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and The light chain amino acid variable region comprises a light chain containing at least one sequence of SEQ ID NO: 91-93, 28-30.

6. The formulation according to claim 1, wherein, The first fusion protein and the second fusion protein each contain an antagonistic IL-6 antibody or a fragment thereof, wherein the antagonistic IL-6 antibody or fragment thereof contains: a heavy chain variable region (VH) comprising three complementarity-determining regions: VH(CDR1), VHCDR2, and VH CDR3 having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 256; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 91-93.

7. The formulation according to claim 1, wherein, The first fusion protein and the second fusion protein each comprise an antagonistic antibody or a fragment thereof that binds to IL-6, said antibody comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; and At least one of the following mutations (EU number): L234A, L235A, and G237A, The first fusion protein contains at least one of the following mutations (EU number): Q347C or L443C, and optionally, the second fusion protein contains at least one of the following mutations (EU number): Q347C or L443C.

8. The formulation according to claim 1, wherein, The first fusion protein comprises an antagonistic IL-6 antibody or its antigen-binding fragment, wherein the VEGF trap is located at one of the following: The N-terminus of the heavy chain containing IL-6 VH; or Between the hinge region of the heavy chain containing IL-6 VH and the downstream of the CH1 structural domain.

9. The formulation according to claim 1, wherein, The surfactant comprises polysorbate 20, polysorbate 80, or poloxamer 188.

10. The formulation according to claim 1, wherein, The surfactant is present in a concentration of 0.01% (w / w) to 0.1% (w / w) in the formulation.

11. The formulation according to claim 1, wherein, The buffer solution contains histidine acetate.

12. The formulation according to claim 11, wherein, The concentration of histidine acetate is from 1 mM to 60 mM.

13. The formulation according to claim 12, wherein, The concentration of the histidine acetate is approximately 15 mM.

14. The formulation according to claim 1, wherein, The pH value of the preparation is 4.9 to 6.

2.

15. The formulation according to claim 1, wherein, The pH value of the preparation is 5.6 to 5.

9.

16. The formulation according to claim 1, wherein, The pH value of the formulation is approximately 5.

6.

17. The formulation according to claim 1, wherein, The total concentration of the first fusion protein and the second fusion protein is 40 mg / mL to 60 mg / mL.

18. The formulation according to claim 1, wherein, The total concentration of the first fusion protein and the second fusion protein is approximately 50 mg / mL.

19. A pharmaceutical preparation comprising: Fusion protein conjugates, comprising: The first fusion protein contains: A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 170, with or without a C-terminal lysine; and A light chain comprising the amino acid sequence shown in SEQ ID NO: 169; and Phosphocholine-containing polymers coupled to the heavy chain, in, The fusion protein conjugate comprises the following structure: Equation (17A) in: Each heavy chain of the first fusion protein is represented by the letter H, and each light chain of the first fusion protein is represented by the letter L; The polymer is bonded to the heavy chain of the first fusion protein via a thiol group of C443 (EU number), and this bond is shown on one of the heavy chains; PC is The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) -H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and The numbers n1, n2, n3, n4, n5, n6, n7, n8, and n9 are either the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is approximately 2500 ± 15%; An uncoupled fusion protein comprising a second fusion protein uncoupled to a phosphocholine-containing polymer, said uncoupled fusion protein comprising: A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 170, having or lacking a C-terminal lysine, or a variant thereof having L449 as numbered according to SEQ ID NO: 170; and A light chain containing the amino acid sequence shown in SEQ ID NO: 169, The total concentration of the first fusion protein and the second fusion protein is 40 mg / mL to 60 mg / mL. The uncoupled fusion protein in the formulation is approximately 30% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the uncoupled fusion protein. Histidine acetate, in concentrations ranging from 1 mM to 60 mM; and The surfactant is selected from polysorbate 20, polysorbate 80, and poloxamer 188, wherein the surfactant is present in the formulation at a concentration of 0.01% (w / w) to 0.1% (w / w). The pH value of the preparation is 4.9 to 6.

2.

20. The formulation according to claim 19, comprising or primarily comprising the following components: The fusion protein conjugate; The uncoupled fusion protein, The total concentration of the first fusion protein and the second fusion protein is 40 mg / mL to 60 mg / mL. The uncoupled fusion protein in the formulation is approximately 30% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the uncoupled fusion protein. Histidine acetate, in concentrations ranging from 1 mM to 60 mM; and The surfactant is present in a concentration of 0.01% (w / w) to 0.1% (w / w) in the formulation.

21. A pharmaceutical formulation comprising: A pharmaceutically effective amount of the fusion protein, wherein the concentration of the fusion protein is approximately 50 mg / mL; A phosphoric choline-containing polymer, wherein the fusion protein is coupled to or not coupled to the polymer; Histidine acetate, with a concentration of approximately 15 mM; and A surfactant, at a concentration of approximately 0.025% (w / w), wherein the surfactant comprises polysorbate 20, polysorbate 80, and / or poloxamer 188. in, The pH value of the formulation is approximately 5.

6. The uncoupled fusion protein in the formulation comprises approximately 30% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the uncoupled fusion protein. The fusion protein comprises: A light chain comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 169; and A heavy chain containing an amino acid sequence having at least 80% identity with SEQ ID NO:

170. The fusion protein conjugate comprises the following structure: Equation (17A) in: Each heavy chain of the fusion protein is represented by the letter H, and each light chain of the fusion protein is represented by the letter L; The polymer is bonded to the heavy chain via a thiol group (EU number) of C443, and this bond is shown on one of the heavy chains; PC is The curve represents the connection point with the remainder of the polymer, where X is a) -OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) -H; or c) any halogen, including -Br, -Cl, or -I; d) -SCN; or e) -NCS; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is approximately 2500 ± 15%.

22. The formulation according to claim 21, comprising or substantially comprising the following: The pharmaceutically effective amount of the fusion protein, wherein the concentration of the fusion protein is approximately 50 mg / mL; The phosphoric choline-containing polymer, wherein the fusion protein is coupled to or not coupled to the polymer; Histidine acetate, with a concentration of approximately 15 mM; and The surfactant has a concentration of approximately 0.025% (w / w). The uncoupled fusion protein in the formulation is approximately 30% of the total molar amount of the fusion protein conjugate and the uncoupled fusion protein, wherein the total molar amount is the sum of the molar amount of the fusion protein conjugate and the molar amount of the uncoupled fusion protein.

23. The formulation according to any one of the preceding claims, wherein, The formulation is stable for at least 4 weeks when stored at approximately 37°C.

24. The formulation according to any one of the preceding claims, wherein, The formulation is stable for about 2 months when stored at about 37°C.

25. The formulation according to any one of the preceding claims, wherein, The formulation is essentially turbid.

26. The formulation according to any one of the preceding claims, wherein, The formulation showed essentially no turbidity after being stored at approximately 37°C for at least 4 weeks.

27. The formulation according to any one of the preceding claims, wherein, The formulation showed virtually no turbidity after being stored at approximately 37°C for about 2 months.

28. The formulation according to any one of the preceding claims is a formulation for intravitreal injection.

29. A pharmaceutical preparation comprising: Fusion proteins, which include: CDRH3, which has at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and VEGF capture entity; and Buffer solution, in, The pH value of the pharmaceutical preparation is 4.5 to 6.

8. Optionally, the formulation comprises a surfactant.

30. The formulation according to claim 29, wherein, The VEGF trap has at least 80% sequence identity with SEQ ID NO:

114.

31. The formulation according to claim 29, wherein, The fusion protein comprises an anti-IL-6 antibody or an antigen-binding fragment thereof, the anti-IL-6 antibody or antigen-binding fragment thereof comprising: a) A heavy chain amino acid variable region comprising a heavy chain having a sequence having at least one of SEQ ID NO: 7-13, 19-27, 89, 90, 256-262; and b) A light chain amino acid variable region comprising a light chain having a sequence of at least one of SEQ ID NO: 91-93, 28-30.

32. The formulation according to claim 29, wherein, The fusion protein comprises an anti-IL-6 antibody or an antigen-binding fragment thereof, the anti-IL-6 antibody or antigen-binding fragment thereof comprising: The heavy chain variable region (VH) contains three complementarity-determining regions: VH CDR1, VH CDR2, and VH CDR3, which have amino acid sequences selected from the CDRs listed in SEQ ID NO: 256; The light chain variable region (VL) comprises VLCDR1, VL CDR2, and VL CDR3 having an amino acid sequence selected from the CDRs listed in SEQ ID NO: 91-93.

33. The formulation according to claim 29, wherein, The fusion protein comprises an anti-IL-6 antibody or an antigen-binding fragment thereof, the anti-IL-6 antibody or antigen-binding fragment thereof comprising: CDRH1, which is CDRH1 in SEQ ID NO: 172; CDRH2, which is CDRH2 in SEQ ID NO: 173; CDRL1, which is CDRL1 in SEQ ID NO: 199; CDRL2, which is CDRL2 in SEQ ID NO: 200; CDRL3, which is CDRL3 in SEQ ID NO: 201; At least one of the following mutations (EU number): L234A, L235A, and G237A, Optionally, the anti-IL-6 antibody or its antigen-binding fragment contains at least one of the following mutations (EU number): Q347C or L443C.

34. The formulation according to claim 29, wherein, The VEGF trap is located at one of the following two locations: a) The N-terminus of the heavy chain containing IL-6 VH; or b) Between the hinge region of the heavy chain containing IL-6 VH and the downstream of the CH1 structural domain.

35. The formulation according to claim 29, wherein, The fusion protein comprises: A light chain containing SEQ ID NO: 169, or a sequence having at least 80% identity with it; and The heavy chain containing SEQ ID NO: 170 (with or without a C-terminal lysine), or a sequence that is at least 80% identical to it.

36. The formulation according to claim 29, wherein, The buffer solution is selected from sodium acetate, histidine acetate, and histidine hydrochloride.

37. The formulation according to claim 36, wherein, The concentration of the buffer solution is from 10 mM to 50 mM.

38. The formulation according to claim 29, comprising the surfactant.

39. The formulation according to claim 38, wherein, The surfactant is selected from polysorbate 20, polysorbate 80, and poloxamer 188.

40. The formulation according to claim 38, wherein, The surfactant content is from 0.01% (w / w) to 0.1% (w / w).

41. The formulation according to claim 29, wherein, The pH value of the formulation is approximately 5.

7.

42. The formulation according to claim 29, wherein, The pH value of the formulation is approximately 6.

3.

43. The formulation according to claim 29, comprising carbohydrates.

44. The formulation according to claim 43, wherein, The carbohydrate is sucrose or trehalose.

45. The formulation according to claim 44, wherein, The concentration of the carbohydrate is at most 20% (w / v).

46. ​​The formulation according to claim 29, wherein, The concentration of the fusion protein is from 20 mg / mL to 200 mg / mL.

47. A pharmaceutical preparation comprising: Fusion protein, the fusion protein comprising: CDRH3, which has at least 80% identity with the amino acid sequence (QAWGYYALDI) of SEQ ID NO: 174; and VEGF capture body; 10 mM to 50 mM sodium acetate or histidine acetate; 0 to 0.1% (w / w) of polysorbate 20; and 0 to 8% (w / v) sucrose or trehalose, in, The pH range of the pharmaceutical preparation is 4.5 to 6.8, and the concentration of the fusion protein is 20 mg / mL to 200 mg / mL.

48. A pharmaceutical preparation comprising: Fusion protein, the fusion protein comprising: A light chain containing SEQ ID NO: 169, or a sequence having at least 80% identity with it; and The heavy chain containing SEQ ID NO: 170 (with or without a C-terminal lysine), or a sequence that is at least 80% identical to it; Sodium acetate at approximately 15 mM; Approximately 0.025% (w / w) of polysorbate 20; and Approximately 5.2% (w / v) sucrose, in, The pH of the pharmaceutical preparation is approximately 5.7, and the content of the fusion protein is approximately 100 mg / mL.

49. A pharmaceutical preparation comprising: Fusion protein, the fusion protein comprising: A light chain containing SEQ ID NO: 169, or a sequence having at least 80% identity with it; and The heavy chain containing SEQ ID NO: 170 (with or without a C-terminal lysine), or a sequence that is at least 80% identical to it; Approximately 15 mM of histidine acetate; Approximately 0.025% (w / w) of polysorbate 20; and Approximately 5.2% (w / v) sucrose, in, The pH value of the pharmaceutical preparation is approximately 6.3, and the content of the fusion protein is approximately 100 mg / mL.

50. The formulation according to any one of claims 29-49, wherein, The fusion protein contains a heavy chain, which contains L443 (EU number) in the Fc domain.

51. The formulation according to any one of claims 29-49, wherein, The fusion protein comprises a heavy chain containing a variant of SEQ ID NO:170 (with or without a C-terminal lysine), the variant having L449 according to SEQ ID NO:

170.

52. The formulation according to any one of claims 29-51, wherein, The formulation is stable for at least 4 weeks when stored at approximately 37°C.

53. The formulation according to any one of claims 29-52, wherein, The formulation is stable for about 2 months when stored at about 37°C.

54. The formulation according to any one of claims 29-53, wherein it is a formulation for intravitreal injection.

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