Pharmaceutical compositions comprising bispecific antibodies that specifically bind to HGFR and EGFR

Abstract

The present disclosure relates to pharmaceutical compositions comprising bispecific antibodies that specifically bind to HGFR and EGFR. Specifically, the present disclosure relates to pharmaceutical compositions comprising bispecific antibodies that specifically bind to HGFR and EGFR and their use as medicines.

Description

[Technical Field]

[0001] The present disclosure belongs to the field of drug formulations, and specifically relates to pharmaceutical compositions comprising bispecific antibodies that specifically bind to HGFR and EGFR, and their use as medicines. [Background technology]

[0002] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0003] Epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR) are two receptor tyrosine kinases that are highly expressed in various tumor cells, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and head and neck cancer (HNC).

[0004] The EGFR signaling pathway plays a key role in tumor biology by regulating cell proliferation, angiogenesis, and cancer cell metastasis and survival, and dysregulation of this pathway leads to tumorigenesis. HGFR is also linked to the development of many tumors through aberrant signal activation due to overexpression, activating mutations, autocrine or paracrine signaling, or gene amplification. Investigations into the efficacy of cancer treatments have revealed an important relationship between EGFR and the HGFR signaling pathway.

[0005] NSCLC accounts for 83% of all lung cancers, and EGFR-activating mutations are the most common type (10-15% in Caucasians and 50% in Asians). EGFR tyrosine kinase inhibitors (TKIs) have traditionally been the first-line treatment for NSCLC. Despite very high initial response rates (70-80%), drug resistance typically develops within one year. There are two main mechanisms of drug resistance: the emergence of additional EGFR mutations, such as EGFR T790M, and compensatory activation of the EGFR signaling pathway. Both of these factors affect tumor therapeutic efficacy. Summary of the Invention

[0006] The present disclosure provides pharmaceutical compositions comprising bispecific antibodies that specifically bind to HGFR and EGFR, which have therapeutic activity and the additional advantage of good stability.

[0007] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR and a buffering agent, wherein: The bispecific antibody that specifically binds to HGFR and EGFR comprises at least one antigen-binding module 1 that specifically binds to HGFR and at least one antigen-binding module 2 that specifically binds to EGFR; The buffer is a histidine buffer, an acetate buffer, a citrate buffer, a succinate buffer or a phosphate buffer.

[0008] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR and a buffering agent, wherein: The bispecific antibody that specifically binds to HGFR and EGFR comprises at least one antigen-binding module 1 that specifically binds to HGFR and at least one antigen-binding module 2 that specifically binds to EGFR; The antigen binding module 1 comprises a heavy chain variable region M-VH and a light chain variable region M-VL, wherein the M-VH comprises M-HCDR1, M-HCDR2, and M-HCDR3, and the M-VL comprises M-LCDR1, M-LCDR2, and M-LCDR3; and the antigen binding module 2 comprises a heavy chain variable region E-VH and a light chain variable region E-VL, the E-VH comprising E-HCDR1, E-HCDR2, and E-HCDR3, and the E-VL comprising E-LCDR1, E-LCDR2, and E-LCDR3; The E-HCDR1, E-HCDR2, E-HCDR3, E-LCDR1, E-LCDR2 and E-LCDR3 are defined according to the Kabat numbering convention, wherein the E-HCDR1 is represented by SEQ ID NO: 6, the E-HCDR2 is represented by SEQ ID NO: 7, the E-HCDR3 is represented by SEQ ID NO: 8, the E-LCDR1 is represented by SEQ ID NO: 9, the E-LCDR2 is represented by SEQ ID NO: 10, and the E-LCDR3 is represented by SEQ ID NO: 11; and The above M-HCDR1, M-HCDR2, M-HCDR3, M-LCDR1, M-LCDR2 and M-LCDR3 are defined according to the Kabat numbering convention, (i) the M-HCDR1 is represented by SEQ ID NO: 30, the M-HCDR2 is represented by SEQ ID NO: 31, the M-HCDR3 is represented by SEQ ID NO: 32, the M-LCDR1 is represented by SEQ ID NO: 27, the M-LCDR2 is represented by SEQ ID NO: 33, and the M-LCDR3 is represented by SEQ ID NO: 29, or (ii) the M-HCDR1 is represented by SEQ ID NO: 18, the M-HCDR2 is represented by SEQ ID NO: 19, the M-HCDR3 is represented by SEQ ID NO: 20, the M-LCDR1 is represented by SEQ ID NO: 21, the M-LCDR2 is represented by SEQ ID NO: 22, and the M-LCDR3 is represented by SEQ ID NO: 23, or (iii) the M-HCDR1 is represented by SEQ ID NO: 24, the M-HCDR2 is represented by SEQ ID NO: 25, the M-HCDR3 is represented by SEQ ID NO: 26, the M-LCDR1 is represented by SEQ ID NO: 27, the M-LCDR2 is represented by SEQ ID NO: 28, and the M-LCDR3 is represented by SEQ ID NO: 29; The buffer is a histidine buffer, an acetate buffer, a citrate buffer, a succinate buffer or a phosphate buffer.

[0009] In some embodiments, the buffer is a histidine buffer.

[0010] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer.

[0011] In some specific embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0012] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the pH of the pharmaceutical composition is 4.5 to 6.5. In some embodiments, the pharmaceutical composition according to any one of the above, wherein the pH of the pharmaceutical composition is 4.8 to 5.2. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 6.0.

[0013] In some embodiments, the pH of the pharmaceutical composition is 5.0 to 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.5 to 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.5 to 6.0. In some embodiments, the pH of the pharmaceutical composition is 6.0 to 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.8 to 6.2. In some embodiments, the pH of the pharmaceutical composition is about 5.8. In some embodiments, the pH of the pharmaceutical composition is about 6.0. In some embodiments, the pH of the pharmaceutical composition is about 6.2.

[0014] In some embodiments, the pharmaceutical composition according to any one of the above is characterized in that the pH of the pharmaceutical composition is 4.5 to 6.0. In some embodiments, the pharmaceutical composition according to any one of the above is characterized in that the pH of the pharmaceutical composition is 4.5 to 5.5. In some embodiments, the pharmaceutical composition according to any one of the above is characterized in that the pH of the pharmaceutical composition is 4.8 to 5.2. In some embodiments, the pH of the pharmaceutical composition is about 4.8. In some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is about 5.2.

[0015] When a point value is mentioned in this disclosure, it should be understood that the point value includes a margin of error. This margin of error is due to factors such as laboratory environment, operator manipulation, equipment, methodology, and measurement error. For example, when measuring pH, a measured value of approximately 6.0 should be understood to include a margin of error. For example, when measuring a formulation with an industrial pH meter, "approximately 6.0" indicates 6.0±0.2 (i.e., a pH of 5.8 to 6.2).

[0016] In some embodiments, the pH of the pharmaceutical composition is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, or any range between these values. In some embodiments, the pH of the pharmaceutical composition is 4.8. In some embodiments, the pH of the pharmaceutical composition is 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.2. In some embodiments, the pH of the pharmaceutical composition is 5.8. In some embodiments, the pH of the pharmaceutical composition is 6.0. In some embodiments, the pH of the pharmaceutical composition is 6.2.

[0017] Generally, the pH of the pharmaceutical composition obtained by replacing the buffering agent is approximately the same as the pH of the buffering agent. At the same time, as known to those skilled in the art, although pH drift may occur during the drug formulation process, the pH drift of the drug formulation is generally very small (e.g., within ±0.3). In some embodiments, the pH drift of the drug formulation is within ±0.3. In some embodiments, the pH drift of the drug formulation is within ±0.2. In some embodiments, the pH drift of the drug formulation is within ±0.1.

[0018] In some embodiments, in any one of the above pharmaceutical compositions, the bispecific antibody that specifically binds to HGFR and EGFR has a concentration of 1 to 250 mg / mL. In some embodiments, the bispecific antibody that specifically binds to HGFR and EGFR has a concentration of 1 to 200 mg / mL. In some embodiments, the bispecific antibody that specifically binds to HGFR and EGFR has a concentration of 50 to 200 mg / mL. In some embodiments, the bispecific antibody that specifically binds to HGFR and EGFR has a concentration of 80 to 180 mg / mL.

[0019] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 to 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 to 120 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 to 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 to 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 80 to 120 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 90 to 110 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is approximately 100 mg / mL.

[0020] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 80 to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 90 to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 100 to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 120 to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 120 to 180 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 135 to 165 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is approximately 150 mg / mL.

[0021] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 to 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 to 75 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 48 to 72 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 54 to 66 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is about 60 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is about 70 mg / mL.

[0022] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 to 250 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 100 to 250 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 160 to 240 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 180 to 220 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is approximately 200 mg / mL.

[0023] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 45 mg / mL, 48 mg / mL, 50 mg / mL, 54 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 66 mg / mL, 70 mg / mL, 72 mg / mL, 75 mg / mL, 80 mg / mL , 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 135 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 165 mg / mL, 180 mg / mL, 200 mg / mL, 220 mg / mL, 240 mg / mL, or 250 mg / mL, or any range between these point values. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 60 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 70 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 200 mg / mL.

[0024] In some embodiments, the pharmaceutical composition according to any one of the above, further comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from the group consisting of poloxamer (e.g., poloxamer 188, abbreviated as P188), polysorbate (e.g., polysorbate 20, polysorbate 80), poloxamer, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleic sulfobetaine, stearic sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleic sarcosine, stearic sarcosine, linoleic betaine, and myristyl sarcosine. The surfactant may be selected from the group consisting of ethyl betaine, cetyl betaine, lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, and the like. In some embodiments, the surfactant is a polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is polysorbate 80.

[0025] In some embodiments, in the pharmaceutical composition described above, the surfactant has a concentration of 0.01 to 1.0 mg / mL. In some embodiments, the surfactant has a concentration of 0.1 to 0.8 mg / mL. In some embodiments, the surfactant has a concentration of 0.2 to 0.8 mg / mL. In some embodiments, the surfactant has a concentration of 0.3 to 0.8 mg / mL. In some embodiments, the surfactant has a concentration of 0.4 to 0.8 mg / mL. In some embodiments, the surfactant has a concentration of 0.48 to 0.72 mg / mL. In some embodiments, the surfactant has a concentration of 0.54 to 0.66 mg / mL. In some embodiments, the surfactant has a concentration of about 0.6 mg / mL.

[0026] In some embodiments, the concentration of the surfactant is 0.3 to 0.6 mg / mL. In some embodiments, the concentration of the surfactant is 0.4 to 0.6 mg / mL. In some embodiments, the concentration of the surfactant is 0.6 to 0.8 mg / mL. In some embodiments, the concentration of the surfactant is 0.2 to 0.4 mg / mL. In some embodiments, the concentration of the surfactant is 0.2 to 0.3 mg / mL. In some embodiments, the concentration of the surfactant is 0.24 to 0.36 mg / mL. In some embodiments, the concentration of the surfactant is about 0.24 mg / mL. In some embodiments, the concentration of the surfactant is about 0.3 mg / mL.

[0027] In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.24 mg / mL, 0.3 mg / mL, 0.36 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1.0 mg / mL, or any range between these values. In some embodiments, the surfactant concentration is 0.6 mg / mL. In some embodiments, the surfactant concentration is 0.24 mg / mL. In some embodiments, the surfactant concentration is 0.3 mg / mL.

[0028] In some embodiments, the surfactant is 0.6 mg / mL polysorbate 80. In some embodiments, the concentration of the surfactant is 0.24 mg / mL polysorbate 80. In some embodiments, the concentration of the surfactant is 0.3 mg / mL polysorbate 80.

[0029] In some embodiments, the pharmaceutical composition of any one of the above includes a sugar. In some embodiments, the sugar is a sugar of the usual composition (CHO). n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The sugar may be selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc.

[0030] In some embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the sugar is sucrose or trehalose. In some embodiments, the sugar is sucrose.

[0031] In some embodiments, the sugar has a concentration of 10 to 100 mg / mL. In some embodiments, the sugar has a concentration of 20 to 90 mg / mL. In some embodiments, the sugar has a concentration of 20 to 80 mg / mL. In some embodiments, the sugar has a concentration of 30 to 80 mg / mL. In some embodiments, the sugar has a concentration of 50 to 80 mg / mL. In some embodiments, the sugar has a concentration of 60 to 90 mg / mL. In some embodiments, the sugar has a concentration of 60 to 80 mg / mL. In some embodiments, the sugar has a concentration of 67.5 to 82.5 mg / mL. In some embodiments, the sugar has a concentration of 70 to 80 mg / mL. In some embodiments, the sugar has a concentration of about 75 mg / mL.

[0032] In some embodiments, the sugar has a concentration of 56 to 84 mg / mL, 63 to 77 mg / mL, or about 70 mg / mL.

[0033] In some embodiments, the sugar has a concentration of 20 to 50 mg / mL. In some embodiments, the sugar has a concentration of 20 to 40 mg / mL. In some embodiments, the sugar has a concentration of 20 to 35 mg / mL. In some embodiments, the sugar has a concentration of 22.4 to 33.6 mg / mL. In some embodiments, the sugar has a concentration of 25.2 to 30.8 mg / mL. In some embodiments, the sugar has a concentration of about 28 mg / mL.

[0034] In some embodiments, the sugar has a concentration of 30 to 50 mg / mL. In some embodiments, the sugar has a concentration of 30 to 40 mg / mL. In some embodiments, the sugar has a concentration of about 37.5 mg / mL.

[0035] In some embodiments, non-limiting examples of the sugar concentration include 10 mg / mL, 20 mg / mL, 22.4 mg / mL, 25.2 mg / mL, 28 mg / mL, 30 mg / mL, 30.8 mg / mL, 33.6 mg / mL, 35 mg / mL, 37.5 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 56 mg / mL, 55 mg / mL, 60 mg / mL, 63 mg / mL, 65 mg / mL, 67.5 mg / mL, 70 mg / mL, 75 mg / mL, 77 mg / mL, 80 mg / mL, 82.5 mg / mL, 84 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, and any range between these values. In some embodiments, the sugar concentration is 28 mg / mL. In some embodiments, the sugar has a concentration of 37.5 mg / mL, 70 mg / mL, or 75 mg / mL.

[0036] In some embodiments, the sugar is 70-80 mg / mL sucrose, and preferably, the sugar is 75 mg / mL sucrose.

[0037] In some embodiments, the sugar is 63-77 mg / mL sucrose, and preferably, the sugar is 70 mg / mL sucrose.

[0038] In some embodiments, the sugar is 25.2 to 30.8 mg / mL sucrose, and preferably, the sugar is 28 mg / mL sucrose.

[0039] In some embodiments, in the pharmaceutical composition described above, the buffering agent has a concentration of 5 to 100 mM. In some embodiments, the buffering agent has a concentration of 5 to 50 mM. In some embodiments, the buffering agent has a concentration of 10 to 50 mM. In some embodiments, the buffering agent has a concentration of 5 to 30 mM. In some embodiments, the buffering agent has a concentration of 10 to 30 mM. In some embodiments, the buffering agent has a concentration of 15 to 25 mM. In some embodiments, the buffering agent has a concentration of 18 to 22 mM. In some embodiments, the buffering agent has a concentration of 10 to 20 mM. In some embodiments, the buffering agent has a concentration of 10 mM or 20 mM. In some embodiments, the buffering agent has a concentration of about 20 mM.

[0040] In some embodiments, the concentration of the buffering agent is 20 to 30 mM. In some embodiments, the concentration of the buffering agent is 22.5 to 27.5 mM. In some embodiments, the concentration of the buffering agent is about 25 mM.

[0041] In some embodiments, the buffer concentration is 5 to 15 mM. In some embodiments, the buffer concentration is 8 to 12 mM. In some embodiments, the buffer concentration is 9 to 11 mM. In some embodiments, the buffer concentration is about 10 mM.

[0042] In some embodiments, the buffer concentration is 5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 15 mM, 18 mM, 20 mM, 22 mM, 22.5 mM, 25 mM, 27.5 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM, or any range between these values. In some embodiments, the buffer concentration is 10 mM. In some embodiments, the buffer concentration is 20 mM. In some embodiments, the buffer concentration is 25 mM.

[0043] In some embodiments, the buffer is 10 mM or 20 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 10 mM or 20 mM histidine-histidine acetate buffer. In some embodiments, the buffer is 25 mM histidine-histidine hydrochloride buffer.

[0044] In some embodiments, the pharmaceutical composition further comprises an additive. In some embodiments, the additive is methionine, arginine hydrochloride, glycine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, calcium chloride, or disodium ethylenediaminetetraacetate. In some embodiments, the additive is sodium chloride or calcium chloride. In some embodiments, the additive is methionine or arginine hydrochloride. In some embodiments, the additive is arginine hydrochloride. In some embodiments, the additive is methionine.

[0045] In some embodiments, in any one of the pharmaceutical compositions described above, the additive has a concentration of 1 to 300 mM, preferably 25 to 200 mM. In some embodiments, the additive has a concentration of 1 to 200 mM. In some embodiments, the additive has a concentration of 1 to 200 mM. In some embodiments, the additive has a concentration of 1 to 20 mM. In some embodiments, the additive has a concentration of 1 to 20 mM. In some embodiments, the additive has a concentration of 4 to 20 mM. In some embodiments, the additive has a concentration of 8 to 12 mM. In some embodiments, the additive has a concentration of 9 to 11 mM. In some embodiments, the additive has a concentration of about 10 mM.

[0046] In some embodiments, the concentration of the additive is 1 to 10 mM. In some embodiments, the concentration of the additive is 3 to 5 mM. In some embodiments, the concentration of the additive is 3.6 to 4.4 mM. In some embodiments, the concentration of the additive is about 4 mM.

[0047] In some embodiments, the concentration of the additive is 1 to 240 mM. In some embodiments, the concentration of the additive is 80 to 240 mM. In some embodiments, the concentration of the additive is 160 to 240 mM. In some embodiments, the concentration of the additive is 180 to 220 mM. In some embodiments, the concentration of the additive is about 200 mM.

[0048] In some embodiments, the concentration of the additive is 80 to 120 mM, 90 to 110 mM, or about 100 mM.

[0049] In some embodiments, the concentration of the additive is 1 mM, 3 mM, 3.6 mM, 4.4 mM, 5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 20 mM, 25 mM, 30 mM, 50 mM, 75 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 150 mM, 160 mM, 180 mM, 200 mM, 240 mM, 250 mM, 280 mM, or 300 mM, or any range therebetween. In some embodiments, the concentration of the additive is 4 mM. In some embodiments, the concentration of the additive is 10 mM. In some embodiments, the pharmaceutical composition described above, wherein the concentration of the additive is 100 mM. In some embodiments, in any one of the above pharmaceutical compositions, the concentration of the additive is 200 mM.

[0050] In some embodiments, the pharmaceutical composition of any one of the above, wherein the concentration of the additive is 4 mM or 10 mM methionine.

[0051] In some embodiments, in any one of the above pharmaceutical compositions, the concentration of the additive is 100 mM or 200 mM arginine hydrochloride.

[0052] In some embodiments, the pharmaceutical composition of any one of the above, further comprises an enzyme, in some embodiments, the enzyme is hyaluronidase (PH20).

[0053] In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 10 to 3000 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 150 to 2400 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 150 to 2200 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 600 to 2200 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 800 to 2000 U / mL.

[0054] In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 700 to 2400 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 800 to 2400 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 1600 to 2400 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 1800 to 2200 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of approximately 2000 U / mL.

[0055] In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 150 to 2000 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 500 to 1100 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 600 to 1000 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 640 to 960 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 700 to 900 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of 720 to 880 U / mL. In some embodiments, in any one of the above pharmaceutical compositions, the enzyme is present at a concentration of approximately 800 U / mL.

[0056] In some embodiments, the enzyme concentration is 10 U / mL, 50 U / mL, 100 U / mL, 150 U / mL, 300 U / mL, 500 U / mL, 600 U / mL, 640 U / mL, 700 U / mL, 720 U / mL, 800 U / mL, 880 U / mL, 900 U / mL, 960 U / mL, 1000 U / mL, 1100 U / mL, 1200 U / mL, 1500 U / mL, 1600 U / mL, 1800 U / mL, 2000 U / mL, 2200 U / mL, 2400 U / mL, 2500 U / mL, 2800 U / mL, or 3000 U / mL, or any range therebetween. In some embodiments, the pharmaceutical composition described above, wherein the enzyme concentration is 800 U / mL. In some embodiments, the pharmaceutical composition of any one of the above, wherein the enzyme is present at a concentration of 1000 U / mL, or in some embodiments, the pharmaceutical composition of any one of the above, wherein the enzyme is present at a concentration of 2000 U / mL.

[0057] In some embodiments, the pharmaceutical composition of any one of the above, wherein the enzyme is at 1000 U / mL or 2000 U / mL of PH20. In some embodiments, the pharmaceutical composition of any one of the above, wherein the enzyme is at 800 U / mL of PH20.

[0058] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the bispecific antibody that specifically binds to HGFR and EGFR comprises: The antigen binding module 1 comprises a heavy chain variable region M-VH and a light chain variable region M-VL, wherein the M-VH comprises M-HCDR1, M-HCDR2, and M-HCDR3, and the M-VL comprises M-LCDR1, M-LCDR2, and M-LCDR3, The E-HCDR1, E-HCDR2, E-HCDR3, E-LCDR1, E-LCDR2 and E-LCDR3 are defined according to the Kabat numbering convention, wherein the E-HCDR1 is represented by SEQ ID NO: 6, the E-HCDR2 is represented by SEQ ID NO: 7, the E-HCDR3 is represented by SEQ ID NO: 8, the E-LCDR1 is represented by SEQ ID NO: 9, the E-LCDR2 is represented by SEQ ID NO: 10, and the E-LCDR3 is represented by SEQ ID NO: 11; and The above M-HCDR1, M-HCDR2, M-HCDR3, M-LCDR1, M-LCDR2 and M-LCDR3 are defined according to the Kabat numbering convention, (i) the M-HCDR1 is represented by SEQ ID NO: 30, the M-HCDR2 is represented by SEQ ID NO: 31, the M-HCDR3 is represented by SEQ ID NO: 32, the M-LCDR1 is represented by SEQ ID NO: 27, the M-LCDR2 is represented by SEQ ID NO: 33, and the M-LCDR3 is represented by SEQ ID NO: 29, or (ii) the M-HCDR1 is represented by SEQ ID NO: 18, the M-HCDR2 is represented by SEQ ID NO: 19, the M-HCDR3 is represented by SEQ ID NO: 20, the M-LCDR1 is represented by SEQ ID NO: 21, the M-LCDR2 is represented by SEQ ID NO: 22, and the M-LCDR3 is represented by SEQ ID NO: 23, or (iii) the M-HCDR1 is represented by SEQ ID NO: 24, the M-HCDR2 is represented by SEQ ID NO: 25, the M-HCDR3 is represented by SEQ ID NO: 26, the M-LCDR1 is represented by SEQ ID NO: 27, the M-LCDR2 is represented by SEQ ID NO: 28, and the M-LCDR3 is represented by SEQ ID NO: 29; Preferably, (i) The bispecific antibody that specifically binds to HGFR and EGFR comprises two antigen-binding modules 1 that specifically bind to HGFR and one antigen-binding module 2 that specifically binds to EGFR; one antigen-binding module 1 whose M-VH comprises M-HCDR1 represented by SEQ ID NO: 30, M-HCDR2 represented by SEQ ID NO: 31, and M-HCDR3 represented by SEQ ID NO: 32, and whose M-VL comprises M-LCDR1 represented by SEQ ID NO: 27, M-LCDR2 represented by SEQ ID NO: 33, and M-LCDR3 represented by SEQ ID NO: 29; The other antigen binding module 1 has an M-VH comprising an M-HCDR1 represented by SEQ ID NO: 18, an M-HCDR2 represented by SEQ ID NO: 19, and an M-HCDR3 represented by SEQ ID NO: 20, and an M-VL comprising an M-LCDR1 represented by SEQ ID NO: 21, an M-LCDR2 represented by SEQ ID NO: 22, and an M-LCDR3 represented by SEQ ID NO: 23, and The antigen binding module 2 has an E-VH comprising an E-HCDR1 represented by SEQ ID NO: 6, an E-HCDR2 represented by SEQ ID NO: 7, and an E-HCDR3 represented by SEQ ID NO: 8, and an E-VL comprising an E-LCDR1 represented by SEQ ID NO: 9, an E-LCDR2 represented by SEQ ID NO: 10, and an E-LCDR3 represented by SEQ ID NO: 11, or (ii) the bispecific antibody that specifically binds to HGFR and EGFR comprises one antigen-binding module 1 that specifically binds to HGFR and one antigen-binding module 2 that specifically binds to EGFR; The antigen binding module 1 has an M-VH comprising an M-HCDR1 represented by SEQ ID NO: 18, an M-HCDR2 represented by SEQ ID NO: 19, and an M-HCDR3 represented by SEQ ID NO: 20, and an M-VL comprising an M-LCDR1 represented by SEQ ID NO: 21, an M-LCDR2 represented by SEQ ID NO: 22, and an M-LCDR3 represented by SEQ ID NO: 23; and The E-VH of the antigen-binding module 2 comprises an E-HCDR1 represented by SEQ ID NO: 6, an E-HCDR2 represented by SEQ ID NO: 7, and an E-HCDR3 represented by SEQ ID NO: 8, and the E-VL of the antigen-binding module 2 comprises an E-LCDR1 represented by SEQ ID NO: 9, an E-LCDR2 represented by SEQ ID NO: 10, and an E-LCDR3 represented by SEQ ID NO: 11.

[0059] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the bispecific antibody that specifically binds to HGFR and EGFR comprises: (i) the M-VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16, and the M-VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17; or the M-VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12, and the M-VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13; or the M-VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14, and the M-VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15; and / or (ii) the E-VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3, and the E-VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5 or SEQ ID NO:4; Preferably, (i) The bispecific antibody that specifically binds to HGFR and EGFR comprises two antigen-binding modules 1 that specifically bind to HGFR and one antigen-binding module 2 that specifically binds to EGFR; One of the antigen-binding modules 1 has an M-VH represented by SEQ ID NO: 16 and an M-VL represented by SEQ ID NO: 17, The other antigen binding module 1 has M-VH represented by SEQ ID NO: 12 and M-VL represented by SEQ ID NO: 13, and The antigen binding module 2 has an E-VH represented by SEQ ID NO: 3 and an E-VL represented by SEQ ID NO: 5, or (ii) the bispecific antibody that specifically binds to HGFR and EGFR comprises one antigen-binding module 1 that specifically binds to HGFR and one antigen-binding module 2 that specifically binds to EGFR; The antigen-binding module 1 has an M-VH represented by SEQ ID NO: 12 and an M-VL represented by SEQ ID NO: 13, and the antigen-binding module 2 has an E-VH represented by SEQ ID NO: 3 and an E-VL represented by SEQ ID NO: 5.

[0060] In some embodiments, the pharmaceutical composition of any one of the above, wherein: The bispecific antibody that specifically binds to HGFR and EGFR comprises one first chain shown in SEQ ID NO: 34, one second chain shown in SEQ ID NO: 35, one third chain shown in SEQ ID NO: 36, and one fourth chain shown in SEQ ID NO: 37; or It comprises one first strand set forth in SEQ ID NO:38, one second strand set forth in SEQ ID NO:39, one third strand set forth in SEQ ID NO:40, and one fourth strand set forth in SEQ ID NO:41.

[0061] In some embodiments, in any one of the pharmaceutical compositions described above, the bispecific antibody that specifically binds to HGFR and EGFR has a Format 1 or Format 2 structure, the structural schematic of Format 1 is shown in Figure 1, and the structural schematic of Format 2 is shown in Figure 2.

[0062] In some embodiments, in any one of the above pharmaceutical compositions, the bispecific antibody that specifically binds to HGFR and EGFR is a hypofucosylated bispecific antibody, preferably the hypofucosylated bispecific antibody is at least 80%, 85%, 90%, 95% or 100% glycosylated without fucose, more preferably the bispecific antibody has no detectable fucose glycosylation.

[0063] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 10 to 100 mg / mL of sugar; (d) 5 to 100 mM of a buffering agent, and the pH of the pharmaceutical composition is 5.0 to 6.5.

[0064] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 30–80 mg / mL sucrose; (d) 10 to 50 mM of a histidine buffer, and the pH of the pharmaceutical composition is 5.5 to 6.5.

[0065] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 90 to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 10 to 20 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is about 6.0.

[0066] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 90 to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 6.0.

[0067] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 2000 U / mL of PH20; (e) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 6.0.

[0068] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 2000 U / mL of PH20; (e) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.0.

[0069] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 200 mM arginine hydrochloride; (e) 2000 U / mL of PH20; (f) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.0.

[0070] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 100 mM arginine hydrochloride; (e) 2000 U / mL of PH20; (f) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.0.

[0071] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 10 to 100 mg / mL of sugar; (d) 5 to 100 mM of a buffering agent, and the pH of the pharmaceutical composition is 4.5 to 6.5.

[0072] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 5 to 50 mM of a histidine buffer, and the pH of the pharmaceutical composition is 4.8 to 6.2.

[0073] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 80 to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 5 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 5.0 to 6.0.

[0074] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 5 to 50 mM of a histidine buffer, and the pH of the pharmaceutical composition is 5.0 to 6.5.

[0075] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 50 to 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 10 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 5.5 to 6.5.

[0076] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 90 to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 15 to 25 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.8 to 6.2.

[0077] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) about 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) about 0.6 mg / mL polysorbate 80; (c) about 75 mg / mL sucrose; (d) about 20 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 6.0.

[0078] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 6.0.

[0079] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 10 to 100 mg / mL of sugar; (d) 1 to 300 mM of additive; (e) 10 to 3000 U / mL of enzyme; (f) 5 to 100 mM of a buffering agent, and the pH of the pharmaceutical composition is 4.5 to 6.5.

[0080] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 1 to 200 mM methionine; (e) 700 to 2400 U / mL of hyaluronidase; (f) 5 to 50 mM histidine buffer, and the pH of the pharmaceutical composition is 4.8 to 6.2.

[0081] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 80 to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 1 to 20 mM methionine; (e) 1600 to 2400 U / mL of hyaluronidase; (f) 5 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 5.0 to 6.0.

[0082] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 50 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 1 to 200 mM methionine; (e) 150 to 2200 U / mL of hyaluronidase; (f) 5 to 50 mM histidine buffer, and the pH of the pharmaceutical composition is 4.5 to 6.0.

[0083] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 100 to 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 4 to 20 mM methionine; (e) 800 to 2200 U / mL of hyaluronidase; (f) 10 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 4.5 to 5.5.

[0084] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 120 to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 1 to 20 mM methionine; (e) 1600 to 2400 U / mL of hyaluronidase; (f) 20 to 30 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0085] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) about 150 mg / mL of a bispecific antibody that specifically binds HGFR and EGFR; (b) about 0.6 mg / mL polysorbate 80; (c) about 70 mg / mL sucrose; (d) about 10 mM methionine; (e) about 2000 U / mL of hyaluronidase; (f) about 25 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0086] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70 mg / mL sucrose; (d) 10 mM methionine; (e) 2000 U / mL hyaluronidase; (f) 25 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0.

[0087] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 50 to 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.2 to 0.4 mg / mL of polysorbate 80; (c) 20–50 mg / mL sucrose; (d) 1 to 10 mM methionine; (e) 600 to 1000 U / mL of hyaluronidase; (f) 5 to 15 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 4.5 to 5.5.

[0088] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 50 to 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.2 to 0.3 mg / mL polysorbate 80; (c) 20–35 mg / mL sucrose; (d) 1 to 10 mM methionine; (e) 720 to 880 U / mL of hyaluronidase; (f) 8 to 12 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0089] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) about 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) about 0.24 mg / mL polysorbate 80; (c) about 28 mg / mL sucrose; (d) about 4 mM methionine; (e) about 800 U / mL of hyaluronidase; (f) about 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0090] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.24 mg / mL polysorbate 80; (c) 28 mg / mL sucrose; (d) 4 mM methionine; (e) 800 U / mL hyaluronidase; (f) 10 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0.

[0091] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 50 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 80 to 240 mM arginine hydrochloride; (e) 5 to 50 mM histidine buffer, and the pH of the pharmaceutical composition is 4.5 to 6.0.

[0092] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 160 to 240 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 160 to 240 mM arginine hydrochloride; (e) 10 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 4.5 to 5.5.

[0093] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 180 to 220 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 180 to 220 mM arginine hydrochloride; (e) 15 to 25 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0094] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) about 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) about 0.6 mg / mL polysorbate 80; (c) about 75 mg / mL sucrose; (d) about 200 mM arginine hydrochloride; (e) about 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.0.

[0095] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 200 mM arginine hydrochloride; (e) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0.

[0096] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 50 to 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.2 to 0.4 mg / mL of polysorbate 80; (c) 20–50 mg / mL sucrose; (d) 80 to 120 mM arginine hydrochloride; (e) 5 to 15 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 4.5 to 5.5.

[0097] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 80 to 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.2 to 0.4 mg / mL of polysorbate 80; (c) 30–50 mg / mL sucrose; (d) 90 to 110 mM arginine hydrochloride; (e) 15 to 25 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 4.8 to 5.2.

[0098] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) about 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) about 0.3 mg / mL of polysorbate 80; (c) about 37.5 mg / mL sucrose; (d) about 100 mM arginine hydrochloride; (e) about 10 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.0.

[0099] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 100 mM arginine hydrochloride; (e) 10 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0.

[0100] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 10 mM methionine; (e) 2000 U / mL of PH20; (f) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0 or 6.0.

[0101] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 2000 U / mL of PH20; (e) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 6.0.

[0102] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 10 mM methionine; (e) 2000 U / mL of PH20; (f) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0.

[0103] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0.

[0104] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 6.0.

[0105] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 5 mM methionine; (e) 1000 U / mL of PH20; (f) 10 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0 or 6.0.

[0106] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 1000 U / mL of PH20; (e) 10 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 6.0.

[0107] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 5 mM methionine; (e) 1000 U / mL of PH20; (f) 10 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0.

[0108] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 10 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 5.0.

[0109] In some embodiments, the pharmaceutical composition of any one of the above, further comprises the following components: (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 10 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is 6.0.

[0110] In some embodiments, the pharmaceutical composition is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0111] The present disclosure further provides a lyophilized formulation, which is capable of forming any one of the pharmaceutical compositions described above after reconstitution.

[0112] The present disclosure further provides a lyophilized formulation, which is a formulation of any one of the pharmaceutical compositions described above in lyophilized form.

[0113] The present disclosure further provides a method for preparing a lyophilized formulation, comprising lyophilizing any one of the pharmaceutical compositions described above. In some embodiments, the lyophilization described in any one of the above comprises the steps of pre-freezing, primary drying, and secondary drying, in that order.

[0114] The present disclosure further provides a lyophilized preparation obtained by lyophilizing any one of the pharmaceutical compositions described above.

[0115] The present disclosure further provides a reconstituted solution obtained by reconstituting any one of the above-described freeze-dried formulations.

[0116] The present disclosure further provides a reconstituted solution, which is a formulation in a reconstituted form of any one of the freeze-dried formulations described above.

[0117] In some embodiments, the reconstituted solution according to any one of the above has the same components and content as those of the pharmaceutical composition.

[0118] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 1 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 10 to 100 mg / mL of sugar; (d) 5 to 100 mM of a buffering agent, and the pH of the pharmaceutical composition is 5.0 to 6.5.

[0119] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 30–80 mg / mL sucrose; (d) 10 to 50 mM of a histidine buffer, and the pH of the pharmaceutical composition is 5.5 to 6.5.

[0120] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 90 to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 10 to 20 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is about 6.0.

[0121] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 90 to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 6.0.

[0122] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 2000 U / mL of PH20; (e) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 6.0.

[0123] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 2000 U / mL of PH20; (e) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.0.

[0124] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 200 mM arginine hydrochloride; (e) 2000 U / mL of PH20; (f) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.0.

[0125] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 100 mM arginine hydrochloride; (e) 2000 U / mL of PH20; (f) 20 mM histidine-histidine hydrochloride buffer, and the pH of the pharmaceutical composition is about 5.0.

[0126] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 1 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 10 to 100 mg / mL of sugar; (d) 5 to 100 mM of a buffering agent, and the pH of the reconstitution solution is 4.5 to 6.5.

[0127] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 5 to 50 mM of a histidine buffer, and the pH of the pharmaceutical composition is 4.8 to 6.2.

[0128] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 80 to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 5 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 5.0 to 6.0.

[0129] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 5 to 50 mM histidine buffer, and the pH of the reconstitution solution is 5.0 to 6.5.

[0130] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 50 to 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 10 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the reconstitution solution is 5.5 to 6.5.

[0131] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 90 to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 15 to 25 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.8 to 6.2.

[0132] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) about 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) about 0.6 mg / mL polysorbate 80; (c) about 75 mg / mL sucrose; (d) about 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is about 6.0.

[0133] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 6.0.

[0134] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 1 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 10 to 100 mg / mL of sugar; (d) 1 to 300 mM of additive; (e) 10 to 3000 U / mL of enzyme; (f) 5 to 100 mM of a buffering agent, and the pH of the reconstitution solution is 4.5 to 6.5.

[0135] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 1 to 200 mM methionine; (e) 700 to 2400 U / mL of hyaluronidase; (f) 5 to 50 mM histidine buffer, and the pH of the pharmaceutical composition is 4.8 to 6.2.

[0136] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 80 to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 1 to 20 mM methionine; (e) 1600 to 2400 U / mL of hyaluronidase; (f) 5 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the pharmaceutical composition is 5.0 to 6.0.

[0137] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 50 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 1 to 200 mM methionine; (e) 150 to 2200 U / mL of hyaluronidase; (f) 5 to 50 mM histidine buffer, and the pH of the reconstitution solution is 4.5 to 6.0.

[0138] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 100 to 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 4 to 20 mM methionine; (e) 800 to 2200 U / mL of hyaluronidase; (f) 10 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the reconstitution solution is 4.5 to 5.5.

[0139] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 120 to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 1 to 20 mM methionine; (e) 1600 to 2400 U / mL of hyaluronidase; (f) 20 to 30 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 4.8 to 5.2.

[0140] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) about 150 mg / mL of a bispecific antibody that specifically binds HGFR and EGFR; (b) about 0.6 mg / mL polysorbate 80; (c) about 70 mg / mL sucrose; (d) about 10 mM methionine; (e) about 2000 U / mL of hyaluronidase; (f) about 25 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is about 5.0.

[0141] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 70 mg / mL sucrose; (d) 10 mM methionine; (e) 2000 U / mL hyaluronidase; (f) 25 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0.

[0142] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 50 to 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.2 to 0.4 mg / mL of polysorbate 80; (c) 20–50 mg / mL sucrose; (d) 1 to 10 mM methionine; (e) 600 to 1000 U / mL of hyaluronidase; (f) 5 to 15 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the reconstitution solution is 4.5 to 5.5.

[0143] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 50 to 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.2 to 0.3 mg / mL polysorbate 80; (c) 20–35 mg / mL sucrose; (d) 1 to 10 mM methionine; (e) 720 to 880 U / mL of hyaluronidase; (f) 8 to 12 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 4.8 to 5.2.

[0144] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) about 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) about 0.24 mg / mL polysorbate 80; (c) about 28 mg / mL sucrose; (d) about 4 mM methionine; (e) about 800 U / mL of hyaluronidase; (f) about 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is about 5.0.

[0145] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.24 mg / mL polysorbate 80; (c) 28 mg / mL sucrose; (d) 4 mM methionine; (e) 800 U / mL hyaluronidase; (f) 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0.

[0146] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 50 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL polysorbate 80; (c) 20–80 mg / mL sucrose; (d) 80 to 240 mM arginine hydrochloride; (e) 5 to 50 mM histidine buffer, and the pH of the reconstitution solution is 4.5 to 6.0.

[0147] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 160 to 240 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 60–80 mg / mL sucrose; (d) 160 to 240 mM arginine hydrochloride; (e) 10 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the reconstitution solution is 4.5 to 5.5.

[0148] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 180 to 220 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL polysorbate 80; (c) 70–80 mg / mL sucrose; (d) 180 to 220 mM arginine hydrochloride; (e) 15 to 25 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 4.8 to 5.2.

[0149] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) about 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) about 0.6 mg / mL polysorbate 80; (c) about 75 mg / mL sucrose; (d) about 200 mM arginine hydrochloride; (e) about 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is about 5.0.

[0150] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 200 mM arginine hydrochloride; (e) 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0.

[0151] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 50 to 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.2 to 0.4 mg / mL of polysorbate 80; (c) 20–50 mg / mL sucrose; (d) 80 to 120 mM arginine hydrochloride; (e) 5 to 15 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, and the pH of the reconstitution solution is 4.5 to 5.5.

[0152] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 80 to 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.2 to 0.4 mg / mL of polysorbate 80; (c) 30–50 mg / mL sucrose; (d) 90 to 110 mM arginine hydrochloride; (e) 15 to 25 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 4.8 to 5.2.

[0153] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) about 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) about 0.3 mg / mL of polysorbate 80; (c) about 37.5 mg / mL sucrose; (d) about 100 mM arginine hydrochloride; (e) about 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is about 5.0.

[0154] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 100 mM arginine hydrochloride; (e) 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0.

[0155] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 10 mM methionine; (e) 2000 U / mL of PH20; (f) 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0 or 6.0.

[0156] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 2000 U / mL of PH20; (e) 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 6.0.

[0157] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 10 mM methionine; (e) 2000 U / mL of PH20; (f) 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0.

[0158] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0.

[0159] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.6 mg / mL polysorbate 80; (c) 75 mg / mL sucrose; (d) 20 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 6.0.

[0160] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 5 mM methionine; (e) 1000 U / mL of PH20; (f) 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0 or 6.0.

[0161] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 1000 U / mL of PH20; (e) 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 6.0.

[0162] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 5 mM methionine; (e) 1000 U / mL of PH20; (f) 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0.

[0163] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 5.0.

[0164] In some embodiments, the reconstitution solution of any one of the above is a solution comprising the following components: (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.3 mg / mL polysorbate 80; (c) 37.5 mg / mL sucrose; (d) 10 mM histidine-histidine hydrochloride buffer, and the pH of the reconstitution solution is 6.0.

[0165] In some embodiments, the pharmaceutical composition or reconstituted solution described above is an intravenous injection formulation, a subcutaneous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation. In some embodiments, the pharmaceutical composition or reconstituted solution described above is an intravenous injection formulation. In some embodiments, the pharmaceutical composition or reconstituted solution described above is a subcutaneous injection formulation.

[0166] In some embodiments, the pharmaceutical composition or reconstituted solution described above is suitable for intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection. In some embodiments, the pharmaceutical composition or reconstituted solution described above is suitable for intravenous injection. In some embodiments, the pharmaceutical composition or reconstituted solution described above is suitable for subcutaneous injection.

[0167] In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation described in any one of the above is used to prepare a drug for intravenous injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection. In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation described in any one of the above is used to prepare a drug for intravenous injection. In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation described in any one of the above is used to prepare a drug for subcutaneous injection.

[0168] The present disclosure further provides a kit, which comprises at least one container, each container independently containing any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, or any one of the reconstitution solutions described above.

[0169] In some embodiments, the present disclosure further provides a method for diagnosing, treating, or alleviating a condition in a subject, the method comprising administering to the subject an effective amount of any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above.

[0170] In some embodiments, the present disclosure further provides a method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above.

[0171] In some embodiments, the present disclosure further provides any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above for treating or preventing a disease.

[0172] In some embodiments, the present disclosure further provides use of any one of the pharmaceutical compositions described above, any one of the lyophilized formulations described above, any one of the reconstituted solutions described above, or any one of the kits described above in the preparation of a medicament for preventing or treating a disease or condition.

[0173] In some specific embodiments, the disease described in any one of the above is a tumor. In some embodiments, the tumor is selected from lung cancer (including non-small cell lung cancer and small cell lung cancer), breast cancer, pancreatic cancer, colorectal cancer (including colon cancer and rectal cancer), sarcoma, renal cell carcinoma, hepatocellular carcinoma, gastric cancer, ovarian cancer, bladder cancer, head and neck cancer, and glioblastoma. In some embodiments, the tumor is selected from lung cancer, breast cancer, pancreatic cancer, colon cancer, head and neck cancer, gastric cancer, and glioblastoma.

[0174] In some specific embodiments, the lung cancer is non-small cell lung cancer.

[0175] In some specific embodiments, the lung cancer is metastatic non-small cell lung cancer.

[0176] In some specific embodiments, the lung cancer is small cell lung cancer.

[0177] In some specific embodiments, the lung cancer is a human lung adenocarcinoma.

[0178] In some specific embodiments, the lung cancer is gastric cancer.

[0179] In some specific embodiments, the tumor is an EGFR- and / or HGFR-associated tumor. [Brief explanation of the drawings]

[0180] [Figure 1] FIG. 1 is a structural schematic diagram of Format 1. [Figure 2] This is a schematic diagram of the structure of Format 2. [Figure 3]FIG. 1 shows the results of an experiment on the binding activity of antibodies to EGFR CHO-S cells, in which the vertical axis represents mean fluorescence intensity (abbreviated as MFI, hereinafter the same). [Figure 4] FIG. 1 shows the results of an experiment on the binding activity of antibodies to HGFR CHO-S cells. [Figure 5] FIG. 1 shows the results of an experiment on the binding activity of antibodies to H1975-HGF cells. [Figure 6] FIG. 1 shows the results of an experiment on the binding activity of antibodies to MKN-45 cells. [Figure 7] This shows the results of an experiment to inhibit cellular EGFR phosphorylation using an antibody. [Figure 8] This shows the results of an experiment to inhibit cellular HGFR phosphorylation using an antibody. [Figure 9] These are the results of an experiment to inhibit cellular AKT phosphorylation using an antibody. [Figure 10] This shows the results of an experiment to reduce cell surface HGFR using an antibody. [Figure 11] This shows the inhibition of proliferation of SNU-5 cells by the antibody. [Figure 12] These are the results of an ADCC killing experiment on Hs746T cells using antibodies. [Figure 13] This shows the results of an ADCC killing experiment on H292 cells using antibodies. [Figure 14] Inhibition of HCC827 mouse tumor cell growth by antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0181] term In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0182] As used in this disclosure, the singular forms "a," "an," and "the above" include plural referents unless the context clearly dictates otherwise.

[0183] Unless the context clearly indicates otherwise, in the patent specification and claims, the words "comprises," "has," "includes," and the like are to be understood in the sense of "including, but not limited to," rather than in the exclusive or exhaustive sense.

[0184] "Optionally" or "optionally" means that the subsequently described event or circumstance may occur, but may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0185] Those skilled in the art should understand that "about," when used in reference to a reference numerical range, cutoff value, or specific value, can indicate that the value is within one or more standard deviations. Alternatively, "about" can indicate a range of up to 20% difference (i.e., ±20%). Because many of the numerical values ​​used herein were determined experimentally, those skilled in the art should understand that such determinations can and usually do vary between different experiments. Due to this inherent variation, it is believed that the values ​​used herein should not be unduly limited. Thus, the term "about" is used to cover a variation of ±20% or less, a variation of ±10% or less, a variation of ±5% or less, a variation of ±1% or less, a variation of ±0.5% or less, or a variation of ±0.1% or less from the specified value.

[0186] Although the present disclosure provides content ranges or content values, it will be understood by those skilled in the art that the content ranges or content values ​​cover an acceptable margin of error for the specific values ​​measured.

[0187] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0188] "HGFR" should be understood broadly and is intended to cover various forms of HGFR molecules at various stages in the mammalian body, including, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification processes of the HGFR gene (e.g., precursor HGFR, mature HGFR, membrane-expressed HGFR, HGFR splice variants, modified HGFR, or fragments thereof), and the term also covers artificially prepared or in vitro expressed HGFR.

[0189] "EGFR" should be understood broadly and is intended to cover various forms of EGFR molecules at various stages in the mammalian body, including, but not limited to, molecules produced during the processes of amplification, replication, transcription, splicing, processing, translation, and modification of the EGFR gene (e.g., precursor EGFR, mature EGFR, membrane-expressed EGFR, EGFR splice variants, modified EGFR, or fragments thereof), and the term also covers artificially prepared or in vitro expressed EGFR.

[0190] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon bonded to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that act in a manner similar to a naturally occurring amino acid.

[0191] The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments or portions thereof), as long as they exhibit the desired antigen-binding activity. A "native antibody" is a naturally occurring immunoglobulin molecule. For example, a native IgG antibody is a heterotetrameric protein of approximately 150,000 daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain contains one variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains one variable region (VL), also called a variable light domain or light chain variable domain, followed by one constant light domain (light chain constant region, CL).

[0192] The term "bispecific antibody" refers to an antibody (including antibodies or antigen-binding fragments thereof, such as single-chain antibodies) capable of specifically binding to two different antigens or two different antigen epitopes of the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art. Depending on the integrity of the IgG molecule, bispecific antibodies can be classified as IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Depending on the number of antigen-binding regions, bispecific antibodies can be classified as bivalent, trivalent, tetravalent, or higher. Depending on whether the structure is symmetric, bispecific antibodies can be classified as symmetrical bispecific antibodies and asymmetrical bispecific antibodies. Among these, fragment-type bispecific antibodies, such as Fc-fragment-deleted Fab fragments, form bispecific antibodies with relatively low immunogenicity, small molecular weight, and relatively high tumor tissue penetration by linking two or more Fab fragments within a single molecule. Typical antibody structures of this type include F(ab')2, scFv-Fab, and (scFv)2-Fab. Antibodies such as IgG-like bispecific antibodies (e.g., having an Fc fragment) have a relatively large molecular weight, and the Fc fragment contributes to the purification of the antibody and improves its solubility and stability. The Fc portion also binds to the receptor FcRn, potentially increasing the serum half-life of the antibody.Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, and F(ab)4-CrossMAb (Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 1). 18, pages 585-608 (2019), see Chen S1 et al., J Immunol Res. 2019 Feb 11;2019:4516041).

[0193] The term "variable region" or "variable domain" refers to the domain that binds to an antigen in an antigen-binding molecule. Herein, the heavy chain variable region of antigen-binding module 1 that specifically binds to HGFR is designated M-VH and the light chain variable region is designated M-VL. The heavy chain variable region of antigen-binding module 2 that specifically binds to EGFR is designated E-VH and the light chain variable region is designated E-VL. VH and VL each contain four conserved framework regions (FR) and three complementarity-determining regions (CDR). The term "complementarity-determining region" or "CDR" refers to the region that primarily promotes antigen binding in the variable domain, and "framework" or "FR" refers to the variable domain residues excluding the CDR residues. VH contains three CDR regions, HCDR1, HCDR2, and HCDR3, while VL contains three CDR regions, LCDR1, LCDR2, and LCDR3. Herein, the three CDR regions in M-VH are designated M-HCDR1, M-HCDR2, and M-HCDR3, respectively; the three CDR regions in M-VL are designated M-LCDR1, M-LCDR2, and M-LCDR3, respectively; the three CDR regions in E-VH are designated E-HCDR1, E-HCDR2, and E-HCDR3, respectively; and the three CDR regions in E-VL are designated E-LCDR1, E-LCDR2, and E-LCDR3, respectively. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL is sufficient to confer antigen-binding specificity.

[0194] The amino acid sequence boundaries of CDRs can be determined by various known methods, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), and the correspondence between various numbering systems is well known to those skilled in the art. The numbering conventions of the present disclosure are as shown in Table 1 below.

[0195] [Table 1]

[0196] Unless otherwise specified, the variable region and CDR sequences in the examples of the present disclosure are all based on the "Kabat" numbering convention. In specific embodiments, the Kabat numbering convention is used to define amino acid residues, but technical solutions corresponding to other numbering systems are considered equivalent technical solutions.

[0197] The term "antibody fragment" refers to a molecule distinct from an intact antibody, including a portion of the intact antibody that retains the intact antibody's antigen-binding ability. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies composed of antibody fragments.

[0198] Fab refers to a protein consisting of VH and CH1 (Fab heavy chain) and VL and CL (Fab light chain) of an immunoglobulin.

[0199] Fv refers to the antigen-binding domain consisting of VH and VL of an immunoglobulin.

[0200] In some embodiments of the present disclosure, the first Fab has the structure of Fab, and in the substituted Fab, CH1 and CL are replaced with Titin chains or Obscurin chains, respectively.

[0201] The term "Fc region" or "fragment crystallizable region" is intended to define the C-terminal region of an antibody heavy chain, and includes native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two subunits, which may be the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined to extend from the amino acid residue at position Cys226, or from Pro230, to its carboxy terminus. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise specified, the numbering convention for the Fc region is the EU index.

[0202] The term "Titin chain" refers to a 78-118 amino acid fragment of the Titin protein, including a peptide fragment of the Titin Ig-Like 152 domain or a functional variant thereof. The Titin chain can form a dimerized complex with the Obscurin chain.

[0203] The term "Obscurin chain" refers to an 87-117 amino acid fragment of the Obscurin protein, comprising a peptide fragment of the Obscurin Ig-Like 1 domain or a functional variant thereof, or a 78-118 amino acid fragment of the Obscurin-Like 1 protein, comprising a peptide fragment of the Obscurin Ig-Like 1 domain or a functional variant thereof. The Obscurin chain can bind to a Titin chain to form a dimerized complex. The Titin chain and Obscurin chain of the present disclosure can be used to replace CH1 and CL in Fab to form a substituted Fab (Fab-S). This substitution does not affect the binding of the antigen-binding molecule to an antigen.

[0204] The term "antigen binding module" refers to a polypeptide molecule that specifically binds to a target antigen. Antigen binding modules include antibodies and fragments thereof described herein. Specific antigen binding modules include an antibody antigen binding domain, including an antibody heavy chain variable region and an antibody light chain variable region. The term "antigen binding module that specifically binds to HGFR" refers to a module that can bind to HGFR or an epitope thereof with sufficient affinity. In some embodiments, an antigen binding module that specifically binds to HGFR has an equilibrium dissociation constant (KD) of < about 1 μM, < about 100 nM, or < about 10 nM or less, as measured by Biacore. In some embodiments, an antigen binding module that specifically binds to HGFR binds to a conserved epitope in HGFR from different species. The term "antigen binding module that specifically binds to EGFR" refers to a module that can bind to EGFR or an epitope thereof with sufficient affinity so that molecules containing the module can be used as diagnostic and / or therapeutic agents targeting EGFR. In some embodiments, an antigen binding module that specifically binds to EGFR has an equilibrium dissociation constant (KD) of < about 1 μM, < about 100 nM, < about 10 nM, or less, as measured by Biacore. In some embodiments, an antigen binding module that specifically binds to EGFR binds to a conserved epitope in EGFR from different species. The antigen binding module includes an antibody fragment, as defined herein, such as a Fab, a substituted Fab, or an Fv.

[0205] In this context, the ordinal numbers in "antigen-binding module 1," "antigen-binding module 2," "linker 1," and "linker 2" are used solely to distinguish different technical features or chemical entities, and do not limit any order, level, or quantity.

[0206] The term "linker" refers to a linking unit that links two polypeptide fragments. As used herein, linkers appearing in the same or different structural formulae may be the same or different. A linker may be a peptide linker and contain one or more amino acids, typically about 1 to 30, 2 to 24, or 3 to 15 amino acids. Linkers used herein may be the same or different. When "-" appears in a structural formula, it indicates that the units on both sides are directly linked by a covalent bond. When the term "bond" appears in a structural unit, it indicates that the units on both sides of the unit are directly linked.

[0207] Unless otherwise stated, all variable region and CDR sequences in the examples of this disclosure follow the "Kabat" numbering convention.

[0208] A "pharmaceutical composition" refers to a composition containing one or more bispecific antibodies described herein and other ingredients such as physiologically / pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredient(s) to further exert biological activity. In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0209] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or pathology of a medical condition. An effective amount also refers to an amount sufficient to allow or facilitate diagnosis. The effective amount used for a particular subject or veterinary subject can vary depending on factors such as the condition being treated, the subject's overall health, the method, route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0210] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are prepared by well-known conventional methods.

[0211] "Buffer" refers to a buffer that resists changes in pH by the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0212] A "histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, and histidine-histidine sulfate, with histidine-histidine hydrochloride being preferred. Histidine-histidine hydrochloride buffers may be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

[0213] A "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. A preferred citrate buffer is citric acid-sodium citrate.

[0214] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium salt, succinic acid-potassium succinate, succinic acid-calcium salt, etc. A preferred succinate buffer is succinic acid-sodium salt. Illustratively, the succinic acid-sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate salt.

[0215] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include citric acid-disodium hydrogen phosphate, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, etc. A preferred phosphate buffer is citric acid-disodium hydrogen phosphate.

[0216] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. A preferred acetate buffer is acetic acid-sodium acetate.

[0217] A "poloxamer" is a block copolymer of oxirane and epoxypropane that is water-soluble and used as a surfactant in drug formulations. Examples of poloxamers include poloxamer 188 (P188).

[0218] A "lyophilized formulation" refers to a pharmaceutical composition or formulation obtained after a vacuum freeze-drying step is performed on a liquid or solution-form pharmaceutical composition or liquid or solution formulation. Lyophilization typically involves pre-freezing, primary drying, and secondary drying. Pre-freezing involves freezing the product to obtain a crystalline solid. In some embodiments, the pre-freezing temperature is set to -45°C, and the pre-freezing rate is set to 1°C / min. Primary drying, also known as main drying, is the main step in sample freeze-drying. It aims to remove ice from the product while maintaining its shape and minimizing damage to the product. Inappropriate selection of the temperature and vacuum level for primary drying can lead to product collapse. Higher temperatures and vacuum levels increase the efficiency of freeze-drying, but also increase the risk of product collapse. In some embodiments, the temperature for primary drying can be a temperature commonly used in the art, such as -30 to 0°C. Secondary drying, also known as decomposition drying, is the main step in which bound water is removed from the product by applying an ultimate vacuum (0.01 mbar) and increasing the temperature (20 to 40°C). Since most biological products are relatively temperature sensitive, the temperature for secondary drying is often selected at the lower end of the temperature range, e.g., 25°C. The time for lyophilization is related to the freezer, the volume of the lyophilized formulation, and the container of the lyophilized drug. Such time adjustments are well known to those skilled in the art.

[0219] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described herein is water. The pharmaceutical composition described herein can achieve a stable effect, i.e., the antibody therein essentially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition essentially retains its physical and chemical stability and biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are several analytical techniques for measuring protein stability, which can measure stability after storage at a specified temperature for a specified period.

[0220] "Replacement" refers to the replacement of a solvent system that dissolves the antibody protein, for example, replacing a high-salt or high-osmolarity solvent system containing the antibody protein with a buffer system that stabilizes the formulation by physical manipulation methods, such as, but not limited to, ultrafiltration, dialysis, or centrifugation, so that the antibody protein is in a stable formulation.

[0221] A stable formulation is one that shows no significant change when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years. Stable liquid formulations also include liquid formulations that exhibit desired characteristics after storage at temperatures including 25°C for periods including 24 hours, 7 days, 1 month, 3 months, or 6 months. Stable liquid formulations also include liquid formulations that exhibit desired characteristics after storage at temperatures including 40°C for periods including 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include those measured by SEC-HPLC in which the antibody exhibits aggregation or degradation, typically not exceeding about 20% or 10%, and preferably not exceeding about 5%. Visual analysis reveals that the formulation is a pale yellow, almost colorless, or transparent liquid, or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation do not vary by more than ±10%, preferably not exceeding ±5%. The formulation typically exhibits no more than about 10%, preferably not exceeding about 5%, of aggregation.

[0222] An antibody "retains its physical stability" in a drug formulation if it does not exhibit significant increased aggregation, precipitation, and / or denaturation as determined by visual inspection of color and / or clarity or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0223] An antibody "retains its chemical stability" in a drug formulation if it does not undergo significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or cleavage (e.g., assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (e.g., assessed by methods such as peptide mapping coupled with mass spectrometry or MALDI / TOF / MS), deamidation (e.g., assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, or isoaspartic acid content measurement), and isomerization (e.g., assessed by isoaspartic acid content measurement, peptide mapping, etc.).

[0224] An antibody "retains its biological activity" in a drug formulation if the biological activity of the antibody over a given period of time is within a given range of biological activity exhibited at the time the drug formulation was prepared.

[0225] "Administration," "giving," and "treatment," when used on an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" may refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Administration," "giving," and "treatment" also refer to ex vivo and in vitro treatment, e.g., of cells, with a reagent, diagnostic, binding composition, or through another cell. "Treatment," when used on a human, veterinary, or research subject, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0226] "Treatment" refers to providing an internal or external therapeutic agent, including, for example, any one of the reconstituted solutions of the present disclosure, to a patient having one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on those symptoms. Typically, the patient or population being treated is provided with a therapeutic agent in an amount that effectively alleviates one or more disease symptoms, thereby inducing the resolution of those symptoms or inhibiting those symptoms from progressing to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on various factors, including the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional health care providers to assess the severity or progression of the condition. Although an embodiment of the present disclosure (e.g., a method of treatment or product) may be ineffective in alleviating each target disease symptom, it should reduce the target disease symptom in a statistically significant number of patients, as determined by any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0227] The reconstituted solution of the present disclosure can be administered by any suitable means, including parenteral administration, intrapulmonary administration, and intranasal administration, and can be administered intralesionally when localized treatment is required. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses at multiple time points, a bolus dose, and a pulse infusion. In some embodiments, the reconstituted solution of the present disclosure is administered by subcutaneous injection.

[0228] The reconstituted solution of the present disclosure is prepared, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the reagent, the administration method, the administration schedule, and other factors known to medical professionals. Optionally, the reconstituted solution may be formulated with one or more other reagents used in the prevention or treatment of the condition. The effective amount of such other reagents depends on the amount of antigen-binding molecule present in the reconstituted solution, the type of condition or treatment, and other factors. They may be used in the same dosages and via the same routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and via any route empirically / clinically confirmed to be appropriate. The above specification provides details of one or more embodiments of the present disclosure. The present disclosure can be practiced or tested using any methods and materials similar or equivalent to those described herein; however, preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In the specification and claims, singular forms include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the common meaning understood by those of ordinary skill in the art. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate preferred embodiments of the present disclosure. These examples should not be construed in any way as limiting the scope of the present disclosure, which is limited by the claims.

[0229] Example - Preparation and detection of bispecific antibodies that specifically bind to HGFR and EGFR PCT / CN2022 / 105714 (filing date: 2022.07.14, priority patent application number: CN202110794137.9) is incorporated by reference in its entirety into this disclosure.

[0230] Example 1: Preparation of antigen protein and antibody 1.1 Structure of antigenic proteins Human EGFR protein (UniProt Epidermal growth factor receptor, Uniprot number: P00533) was used as a template for EGFR, and a His tag was fused to the EGFR protein to design a His-tagged extracellular domain of human EGFR protein (abbreviated as EGFR-His) for detection in the present invention. The amino acid sequence is as follows: Amino acid sequence of EGFR-His: [ka]

[0231] Human HGFR protein (UniProt Hepatocyte growth factor receptor, Uniprot number: P08581) was used as a template for HGFR. His-tags or Fc-tags were fused to the HGFR protein to design the His-tagged extracellular domain of the HGFR protein (abbreviated as HGFR-His) and the Fc-tagged extracellular domain of the HGFR protein (abbreviated as HGFR-Fc) for detection in the present invention. The amino acid sequences are as follows: Amino acid sequence of HGFR-His: [ka]

[0232] Using human HGF protein (UniProt Hepatocyte growth factor, Uniprot number: P14210) as an HGF template, a His-tagged HGF protein (abbreviated as HGF-His) for detection according to the present disclosure was designed, and its amino acid sequence is as follows: Amino acid sequence of HGF-His: [ka]

[0233] 1.2 Protein purification 1.2.1 Recombinant protein purification steps: The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS. Imidazole was added to a final concentration of 5 mM. A nickel column was equilibrated with PBS containing 5 mM imidazole and washed with 2 to 5 column volumes. The replaced cell supernatant sample was applied to a Ni Sepharose Excel column (GE, 17-3712-02). The column was washed with PBS containing 5 mM imidazole until the A280 reading decreased to baseline. The chromatography column was then washed with PBS + 10 mM imidazole to remove nonspecifically bound heteroproteins, and the flow-through was collected. The target protein was then eluted with PBS containing 300 mM imidazole, and the elution peak was collected. The collected eluate was concentrated and further purified using a Superdex 200 gel chromatograph (GE, 28-9893-35) with PBS as the mobile phase. The dimer peak was removed, and the elution peak was collected. The resulting protein was identified as correct by electrophoresis, peptide mapping, and LC-MS, and then aliquoted for use.

[0234] 1.2.2 Antibody purification steps: The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the supernatant was subjected to affinity chromatography using MabSelect Sure (GE, 17-5438-01). The MabSelect Sure chromatography column was first regenerated with 0.2 M NaOH, washed with pure water, and then equilibrated with PBS. After binding the supernatant, the column was washed with PBS until the A280 reading decreased to baseline. The target protein was eluted with 0.1 M acetate buffer at pH 3.5 and neutralized with 1 M Tris-HCl. After appropriate concentration, the eluted sample was further purified using a Superdex 200 gel chromatograph (GE, 28-9893-35) equilibrated with PBS. The receiving tubes containing the target protein were combined and concentrated to an appropriate concentration. This method was used to purify the antibody of the present invention.

[0235] Example 2. Preparation and identification of bispecific antibodies that specifically bind to HGFR and EGFR Using a molecule that specifically binds to HGFR and a molecule that specifically binds to EGFR, antibodies that specifically bind to HGFR and EGFR were constructed.

[0236] 2.1 Molecules that specifically bind to EGFR The molecule that specifically binds to EGFR can be derived from any suitable antibody, for example, Zalutumumab (abbreviated as Zal) or a variant thereof (for example, Zal.1 obtained by mutating the first amino acid residue of the Zal light chain from A to D), among which: > Amino acid sequence of Zal heavy chain: [ka] > Amino acid sequence of Zal light chain: [ka] > Amino acid sequence of the heavy chain variable region of Zal: [ka] > Amino acid sequence of the light chain variable region of Zal: [ka] The amino acid sequence of the heavy chain variable region of Zal.1 is SEQ ID NO: 3 > Amino acid sequence of the light chain variable region of Zal.1: [ka]

[0237] The sequences of the CDRs of Zal and Zal.1 are shown in Table 2: [Table 2]

[0238] 2.2 Molecules that specifically bind to HGFR Molecules that specifically bind to HGFR can be derived from any suitable antibody, including antibodies such as Onartuzumab (abbreviated as Omab) described in International Publication No. WO2013003680A1 (incorporated herein in its entirety by reference), antibodies such as Ab10 described in International Publication No. WO2016165580A1 (incorporated herein in its entirety by reference), and mutant antibodies of the above antibodies. Among these, the amino acid sequences of the variable regions of Omab and Ab10 are as follows: > Amino acid sequence of the heavy chain variable region of Omab: [ka] > Amino acid sequence of the light chain variable region of Omab: [ka] > Amino acid sequence of the heavy chain variable region of Ab10: [ka] > Amino acid sequence of the light chain variable region of Ab10: [ka] The CDR of Ab10 was mutated to obtain Ab10 mutant antibody Ab10.1, whose constant region is the same as Ab10, and whose variable region amino acid sequence is as follows: > Amino acid sequence of the heavy chain variable region of Ab10.1: [ka] > Amino acid sequence of the light chain variable region of Ab10.1: [ka] Note: In the above sequences specifically binding to the HGFR molecule, the underlined parts are CDR regions identified according to the Kabat numbering system, and the bold italicized parts are mutated amino acid residues.

[0239] CDRs that specifically bind to the HGFR molecule are shown in Table 3: [Table 3]

[0240] 2.3 Bispecific antibodies that specifically bind to HGFR and EGFR Using a molecule that specifically binds to HGFR and a molecule that specifically binds to EGFR, a bispecific antibody having a Format 1 or Format 2 structure that specifically binds to HGFR and EGFR was constructed. The structural schematic of Format 1 is shown in Figure 1, and the structural schematic of Format 2 is shown in Figure 2. Format 1 contains four chains with the following structure, among which: Chain 1 is VH (anti-EGFR antibody)-linker 1-VH (anti-HGFR antibody 1)-IgG1 (CH1)-IgG1Fc (knob), Chain 2 is VL(anti-EGFR antibody)-linker1-VL(anti-HGFR antibody1)-CL; Chain 3 is VH (anti-HGFR antibody 2)-linker 2-Titin chain-IgG1Fc(hole); Chain 4 is the VL(anti-HGFR antibody 2)-linker 2-Obscurin chain.

[0241] Format 2 contains four chains with the following structure, among which: Chain 1 is VH (anti-HGFR antibody)-IgG1 (CH1)-IgG1 Fc (knob); Chain 2 is VL (anti-HGFR antibody)-CL; Chain 3 is VH (anti-EGFR antibody)-linker 2-Titin chain-IgG1Fc(hole); Chain 4 is the VL (anti-EGFR antibody)-linker 2-Obscurin chain; As an example, EM1 with a Format 1 structure and EM2 with a Format 2 structure were constructed using Ab10.1 (anti-HGFR antibody 1), Omab (anti-HGFR antibody 2), Zal.1 (anti-EGFR antibody), human IgG1 heavy chain constant region / kappa light chain constant region, and T.16 (Titin chain) / O.28 (Obscurin chain).

[0242] The amino acid sequences of the four chains of EM1 are as follows: Amino acid sequence of chain 1 of EM1: [ka] Amino acid sequence of chain 2 of EM1: [ka] Amino acid sequence of chain 3 of EM1: [ka] Amino acid sequence of chain 4 of EM1: [ka] The amino acid sequences of the four chains of EM2 are as follows: Amino acid sequence of chain 1 of EM2: [ka] Amino acid sequence of chain 2 of EM2: [ka] Amino acid sequence of chain 3 of EM2: [ka] Amino acid sequence of chain 4 of EM2: [ka] Note: In the above sequence, the single underlined parts are the variable region, the dashed underlined parts are the CL, the dotted underlined parts are the Fc region, the italicized parts are the CH1, the double underlined parts are the linker, and the wavy parts are the Obscurin chain / Titin-T chain.

[0243] Amino acid sequence of linker 1: GGGGSGGGG (SEQ ID NO: 45) Amino acid sequence of linker 2: GGGGS (SEQ ID NO: 46) Amino acid sequence of IgG1Fc(knob): [ka] Amino acid sequence of IgG1Fc(hole): [ka] CL: [ka] T.16 (Titin chain) [ka] O.28 (Obscurin Chain) [ka]

[0244] Test Example Test Example 1: In vitro binding affinity and kinetics experiments using Biacore The affinity of the molecules to be measured for human EGFR (SEQ ID NO: 42) or human HGFR protein (SEQ ID NO: 43) was measured by Biacore T200 (GE) as follows: After affinity capturing antibody molecules using a Protein A biosensor chip, antigen was flowed over the chip surface for 180 seconds, followed by 600 seconds of dissociation. The reaction signal was detected in real time using a Biacore T200 instrument to obtain binding-dissociation curves. After each experimental cycle of dissociation, the biosensor chip was regenerated by washing with 10 mM Gly-HCl (pH 1.5). A 1:1 model was used for data fitting to obtain affinity values ​​for the molecules being measured. In this example, the positive control MCLA was a bispecific antibody that specifically binds to HGFR and EGFR (see PB8532 in WO2019031965A1 for its specific sequence).

[0245] The experimental results are shown in Table 4 and demonstrate that the mutant antibodies and bispecific antibodies constructed in this disclosure all have very strong affinities, with the affinity of Ab10.1 being at least four times that of the parent antibody Ab10.

[0246] [Table 4]

[0247] Test Example 2: In vitro cell binding experiment of antibody The cell-binding activity of the bispecific antibody that specifically binds to HGFR and EGFR was detected by flow cytometry. 6 EGFR CHO-S stably transfected cell lines, HGFR CHO-S stably transfected cell lines, H1975-HGF, or MKN-45 tumor cell lines (cells / mL) were blocked with 1% BSA PBS buffer, followed by incubation for 1 hour with antibody samples diluted at different concentrations (C25 was a negative control, an IgG1 antibody protein unrelated to the target). After two washes, R-PE-goat anti-human (H+L) antibody (Invitrogen, CAT# H10104) was added and incubated for 0.5 hours. After two washes, the fluorescent signal was read using a flow cytometer.

[0248] The experimental results are shown in Figures 3 to 6. As shown by the experimental results, the bispecific antibody according to the present disclosure that specifically binds to HGFR and EGFR has stronger binding ability to tumor cells than the positive antibody MCLA.

[0249] Test Example 3: Blocking experiment of antibodies that antagonize the binding of EGFR and EGF The blocking ability of antibodies against EGF / EGFR was detected by ELISA. Plates were coated with 1 μg / mL rabbit anti-His antibody (GenScript, A01857) and blocked overnight at 4°C. After washing, 2 μg / mL EGFR-His (SEQ ID NO: 42) was added and incubated for 1 hour. After washing, 2 μg / mL EGF-mFc (Acro, EGF-H525b) and antibodies diluted at different concentrations were added and incubated for 1 hour. After washing, horseradish peroxidase-goat anti-mouse IgG antibody (Jackson, 115-035-062) was added and incubated for 1 hour. After washing, the plate was developed with tetramethylbenzidine solution, and finally, stop solution was added, and the OD450 value was measured using a plate reader. In the examples, the positive control JNJ is a bispecific antibody that specifically binds to HGFR and EGFR (JNJ is also known as Amivantamab, and its specific sequence structure is described in WHO Drug Information, 33(2):237-239).

[0250] The experimental results are shown in Table 5. As is clear from the experimental results, the bispecific antibody according to the present disclosure that specifically binds to HGFR and EGFR can effectively block the binding of EGF to EGFR.

[0251] [Table 5]

[0252] Test Example 4: Blocking experiment of antibodies that antagonize the binding of HGFR and HGF The blocking ability of antibodies against HGF / HGFR was detected by ELISA. Plates were coated with 5 μg / mL HGFR-His (sequence see SEQ ID NO: 43) and blocked overnight at 4°C. After washing, 2 μg / mL bio-HGF-His (sequence see SEQ ID NO: 44) and antibodies diluted at different concentrations were added and incubated for 1 hour. After washing, horseradish peroxidase-streptavidin (Jackson, 016-030-084) was added and incubated for 1 hour. After washing, the plate was developed with tetramethylbenzidine solution, and finally, stop solution was added, and the OD450 value was measured using a plate reader.

[0253] The experimental results are shown in Table 6. As shown by the experimental results, the bispecific antibody of the present disclosure that specifically binds to HGFR and EGFR has a stronger maximum blocking ability for blocking the binding of HGFR to HGF than the positive antibody JNJ.

[0254] [Table 6]

[0255] Test Example 5: Experiment on inhibition of cellular EGFR phosphorylation by antibodies H292 cells (also known as NCI-H292, human lung cancer cells (lymph node metastasis), Cell Bank of the Chinese Academy of Sciences, hereafter referred to as NCI-H292) were seeded at 12,000 cells / well into a 96-well plate (Corning, 3599) in RPMI 1640 + 10% FBS medium. The 96-well plate was then cultured at 37°C in a 5% CO2 incubator for 24 hours. The next day, the medium was discarded and replaced with serum-free medium (RPMI 640 + 25 mM HEPES + 0.1 mM NEAA + 1 mM sodium pyruvate) and the cells were starved for 16-20 hours. On the third day, a 5-fold gradient dilution of the antibody was prepared at an initial concentration of 6 μM. The starvation medium was replaced and the cells were cultured in an incubator at 37°C with 5% CO2 for 1 hour. During this time, 50 μL of 100 ng / mL rEGF (R&D, 236-EG-200) was added, and the cells were cultured in an incubator at 37°C with 5% CO2 for 15 minutes. Finally, phosphorylated EGFR was detected according to the instructions of PHOSPHO-EGFR(TYR1068)KITS (Cisbio, 64EG1PEG).

[0256] The experimental results are shown in FIG. 7, and as shown by the experimental results, the bispecific antibody that specifically binds to HGFR and EGFR according to the present disclosure can effectively inhibit cellular EGFR phosphorylation.

[0257] Test Example 6: Experiment on inhibition of cellular HGFR phosphorylation by antibodies H292 cells were seeded at 12,000 cells / well in a 96-well plate (Corning, 3599) in RPMI1640 + 10% FBS medium, and then the 96-well plate was cultured for 24 hours in an incubator at 37°C with 5% CO. The next day, the medium was discarded and replaced with serum-free medium (RPMI640 + 25 mM HEPES + 0.1 mM NEAA + 1 mM sodium pyruvate) and the cells were starved for 16-20 hours. On the third day, a 10-fold gradient dilution of the antibody was prepared at an initial concentration of 1 μM. The starvation medium was replaced and the cells were cultured in an incubator at 37°C with 5% CO2 for 1 hour. During this time, 50 μL of 200 ng / mL rHGF (R&D, 294-HG-005) was added and the cells were cultured in an incubator at 37°C with 5% CO2 for 30 minutes. Finally, phosphorylated HGFR was detected according to the instructions of the pc-Met (Tyr1234 / 1235) Assay Kit (PerkinElmer, ALSU-PCMET-A50).

[0258] The experimental results are shown in FIG. 8. As shown by the experimental results, the bispecific antibody of the present disclosure that specifically binds to HGFR and EGFR can effectively inhibit cellular HGFR phosphorylation, with stronger inhibitory ability than Ab10.1.

[0259] Test Example 7: Cellular AKT phosphorylation inhibition experiment using antibodies H292 cells were seeded at 12,000 cells / well in a 96-well plate (Corning, 3599) in RPMI1640 + 10% FBS medium, and then the 96-well plate was cultured for 24 hours in an incubator at 37°C with 5% CO. The next day, the medium was discarded and replaced with serum-free medium (RPMI640 + 25 mM HEPES + 0.1 mM NEAA + 1 mM sodium pyruvate) and the cells were starved for 16-20 hours. On day 3, a 5-fold gradient dilution of the antibody was prepared at an initial concentration of 4 μM. The starvation medium was replaced and the cells were incubated at 37°C in a 5% CO2 incubator for 1 hour. During this time, 50 μL of 200 ng / mL rHGF (R&D, 294-HG-005) and 50 ng / mL rEGF (R&D, 236-EG-200) were added and mixed evenly. The cells were then incubated at 37°C in a 5% CO2 incubator for another hour. Finally, phosphorylated AKT was detected according to the PHOSPHO-AKT(SER473)KITS (Cisbio, 64AKSPEG) manufacturer's instructions. The intracellular AKT phosphorylation level was measured as the ratio of the fluorescence signal at 665 nm to the fluorescence signal at 620 nm (ratio = fluorescence signal at 665 nm / fluorescence signal at 620 nm × 10). 4 The higher the ratio, the higher the intracellular AKT phosphorylation level.

[0260] The experimental results are shown in FIG. 9. As shown by the experimental results, the bispecific antibody of the present disclosure that specifically binds to HGFR and EGFR can effectively inhibit cellular AKT phosphorylation, and the inhibitory ability is stronger than that of the positive control antibody.

[0261] Test Example 8: Reduction of HGFR expression on cell surface by antibody HCC827 cells (human non-small cell lung cancer cells, ATCC-derived) were taken at 3 × 10 5Cells were seeded at 10 ... After further washing once with PBS, the fluorescent signal value was read using a flow cytometer.

[0262] The experimental results are shown in FIG. 10. As shown by the experimental results, compared to the positive control antibody, the bispecific antibody of the present disclosure that specifically binds to HGFR and EGFR can more effectively reduce HGFR on the cell surface and inhibit the receptor signaling pathway.

[0263] Test Example 9: Growth inhibition experiment of SNU-5 cells by antibodies SNU-5 cells (human gastric cancer cells, ATCC-derived) were seeded at 4000 cells / well into a 96-well plate (Corning, 3903) in IMDM + 5% FBS medium. The plate was then incubated at 37°C, 5% CO2, for 24 hours. Antibodies were diluted 5-fold to 5 μM and added to the 96-well plate. The plate was then incubated at 37°C, 5% CO2, for 5 days. Finally, the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7573) was prepared according to the manufacturer's instructions. 50 μL of CTG was added to each well. The wells were incubated at room temperature for 10 minutes, sealed with a bottom sealing film, and fluorescence intensity (Lum) was detected using a multi-label microwell plate detection system (PerkinElmer, Victor3).

[0264]

number

[0265] The experimental results are shown in FIG. 11. As shown by the experimental results, the bispecific antibody according to the present disclosure that specifically binds to HGFR and EGFR has a stronger ability to inhibit the proliferation of SNU-5 cells than the positive antibody.

[0266] Test Example 10: ADCC killing experiment of antibodies against tumor cells The antibody was prepared at a concentration of 200 nM and diluted 5-fold. Tumor cells, Hs746T cells (human gastric cancer cells, Nanjing Kebai Biotechnology Co., Ltd.) and H292 cells, were harvested and washed once with PBS. Then, the cells were diluted to a cell density of 1 × 10 in a buffer solution (PBS + 10% FBS + 20 mM HEPES). 6After adjusting the cell density to 1 × 10 cells / mL, 3 μL of BATDA reagent (PerkinElmer, AD0116) was added to 2 mL of cells and incubated for 15 min in an incubator at 37°C and 5% CO. After centrifugation, the cells were washed three times with washing buffer (PBS + 20 mM HEPES) and counted to determine a cell density of 1 × 10 cells / mL. 5 NK92-FCGR3A(176V) cells (NK92 cells stably transformed with FCGR3A(176V) (purchased from Nanjing Kebai Biotechnology Co., Ltd.)) were collected and washed once with PBS. The cells were resuspended in medium and counted to a cell density of 5 × 10 5 The concentration was adjusted to cells / mL. Finally, 100 μL of NK92-FCGR3A(176V) cells, 50 μL of diluted antibody, and 50 μL of labeled tumor cells were added to a 96-well plate (Corning, 3788). Simultaneously, a spontaneous release control well containing 100 μL of medium instead of NK92-FCGR3A(176V) cells was set up, a positive control well containing 10 μL of lysis buffer, 50 μL of labeled tumor cells, and 140 μL of medium was set up, and a background control well containing 50 μL of labeled cell supernatant and 150 μL of medium was set up. The 96-well plate was then centrifuged at 300 rpm for 2 minutes and then incubated in an incubator at 37°C with 5% CO2 for 2 hours. 100 μL of the supernatant was transferred to a new 96-well plate (Corning, 3788) and centrifuged at 300 g for 5 min. 20 μL was then transferred to a detection plate (PerkinElmer, AD0116), and 200 μL of europium solution (PerkinElmer, AD0116) was added. The plate was incubated at room temperature for 15 min with shaking, and TRF was finally detected. EM1(afuc) is the defucosylated EM1, and EM2(afuc) is the defucosylated EM2.

[0267]

number

[0268] The experimental results are shown in Figures 12 and 13. As shown by the experimental results, the bispecific antibodies that specifically bind to HGFR and EGFR according to the present disclosure can significantly improve their ADCC killing ability after being defucosylated.

[0269] Test Example 11: In vivo efficacy test of antibody in mouse H1975-HGF model H1975-HGF cells (H1975 cells stably transformed with human HGF (human lung adenocarcinoma cells, ATCC)) (5 ​​× 10 6 ) were inoculated subcutaneously into the right flank of female CD1 nude mice (Weitong Lihua).

[0270] The tumor volume was 189 mm 3 After this, the animals were randomly assigned to groups of 10 animals each based on tumor volume. These groups included the C25-3mpk group (a negative control, an IgG1 antibody protein unrelated to the target, at a dose of 3 mg / kg), the JNJ-3mpk group (a positive control, at a dose of 3 mg / kg), and the EM1-3.5mpk group (an EM1 test group, at a dose of 3.5 mg / kg). From day 0, the animals were administered the drug twice weekly via intraperitoneal injection for 2 to 3 consecutive weeks. Tumor volumes were measured twice weekly, and the mice were weighed and recorded. Data were statistically analyzed using a t-test. C25-3mpk, JNJ-3mpk, and EM1-3.5mpk were administered at equimolar doses.

[0271]

number

[0272] The experimental results are shown in Table 7. As shown by the experimental results, the bispecific antibodies that specifically bind to HGFR and EGFR according to the present disclosure can significantly inhibit tumor cell growth. On day 21, the equimolar group of bispecific antibodies that specifically bind to HGFR and EGFR according to the present disclosure had mean tumor volumes that were less than half of the positive control, and had stronger tumor-inhibiting efficacy than the positive control antibody JNJ.

[0273] [Table 7]

[0274] Test Example 12: In vivo efficacy test of antibody in mouse HCC827 model HCC827 cells (human non-small cell lung cancer cells, derived from ATCC) (8.555×10 6 1) were inoculated subcutaneously into the right flank of NUNU female mice (Weitong Lihua).

[0275] Tumor volume 200mm 3 After the tumor volume reached 10, the animals were randomly assigned to groups of 10 each. These groups included the C25-3mpk group (a negative control where C25 is an IgG1 antibody protein unrelated to the target, dose 3 mg / kg), the MCLA-3mpk group (a positive control, dose 3 mg / kg), the EM1-3.5mpk group (a high-dose test group for EM1, dose 3.5 mg / kg), the EM1-1.17mpk group (a low-dose test group for EM1, dose 1.17 mg / kg), and the EM2-3mpk group (an EM2 test group, dose 3 mg / kg). From day 0, the animals were administered intraperitoneally twice weekly for 2–3 consecutive weeks. Tumor volumes were measured twice weekly, and the mice were weighed and recorded. The C25-3mpk, MCLA-3mpk, EM1-3.5mpk, and EM2-3mpk groups were equimolar.

[0276] The experimental results are shown in FIG. 14. As shown by the experimental results, the bispecific antibody of the present disclosure that specifically binds to HGFR and EGFR can significantly inhibit tumor cell growth, and its tumor-inhibiting effect is stronger than that of the positive control antibody.

[0277] Preparation Example - Bispecific antibody formulation that specifically binds to HGFR and EGFR SEC molecular exclusion chromatography: This is an analytical method that separates solutes based on the correlation between the pore size of gel pores and the coil size of polymer sample molecules.

[0278] SEC% (SEC monomer content percentage) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas). ΔSEC% = SEC% of formulation after stability experiment - SEC% of formulation before stability experiment.

[0279] SEC measurement equipment: Agilent HPLC 1260, Column: Waters, BioResolve™ SEC mAb 200 Å, 2.5 μm, 7.8 × 300 mm.

[0280] NR-CE Capillary Gel Electrophoresis: This is electrophoresis, which is carried out by moving a gel as a support medium in a capillary, and is a method of separating samples according to their molecular weight at a constant voltage.

[0281] NR-CE%=A main peak / A total×100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas).

[0282] ΔNR-CE% = NR-CE% of formulation after stability experiment - NR-CE% of formulation before stability experiment.

[0283] Equipment for CE measurement: Beckman capillary electrophoresis apparatus, model number PA800 plus.

[0284] IEC ion exchange chromatography: This is a type of chromatography that uses an ion exchange resin or a chemically bonded ion exchange agent as the stationary phase and achieves separation by utilizing the difference in ion exchange capacity or selectivity coefficient of the components to be separated.

[0285] IEC% = A neutral peak area / A total area × 100% (A total area is the sum of the acidic peak, neutral peak and basic peak areas). ΔIEC% = IEC% of formulation after stability experiment - IEC% of formulation before stability experiment.

[0286] Equipment for IEC measurements: Agilent HPLC 1260.

[0287] Osmolality measurement: Osmotic pressure was measured using the freezing point method. Based on the fact that the freezing point depression value is directly proportional to the molar concentration of the solution, a highly sensitive temperature measuring element was used to measure the freezing point of the solution and convert it into osmotic pressure using an electrical quantity.

[0288] Osmolality measuring device: Loser, model number OM815.

[0289] protein The protein employed in the following examples is the HGFR-EGFR bispecific antibody EM1 (hereinafter abbreviated as "protein").

[0290] Equipment for measuring protein concentration: UV-visible spectrophotometer, model number: Nano Drop oneC, light path 1 mm.

[0291] Formulation Example 1. Screening of Different Ionic Strengths Formulations of 100 mg / mL protein, 80 mg / mL sucrose, and 0.4 mg / mL polysorbate 80 (PS80) were prepared using the buffer systems shown in Table 8. Forced degradation studies were performed on the samples (4 weeks at 40°C) to investigate the effect of different buffer systems on protein stability using SEC and NR-CE as evaluation indices.

[0292] The results are shown in Table 8. SEC data shows no significant difference between the two. NR-CE data shows that the 20 mM ionic strength formulation is superior to the 50 mM ionic strength formulation.

[0293] [Table 8]

[0294] Formulation Example 2. pH and buffer system screening Formulations of 100 mg / mL protein, 80 mg / mL sucrose, and 0.4 mg / mL polysorbate 80 (PS80) were prepared using the buffer systems shown in Table 9. Forced degradation studies were performed on samples (40°C for 4 weeks) to examine the effect of different buffer systems on protein stability using appearance, SEC, and NR-CE as evaluation indices.

[0295] The results are shown in Table 9. The appearance data showed that the formulations containing His-HCl and His-AA had superior appearance to the other groups. The SEC and NR-CE data showed that after 4 weeks at 40°C, the formulations containing His-HCl (pH 6.0 or pH 6.5) had superior monomer purity to the other groups.

[0296] [Table 9]

[0297] Note: AA stands for acetic acid-sodium acetate, His-AA stands for histidine-histidine acetate, CA stands for citric acid-sodium citrate, SA stands for succinic acid-sodium succinate, PB stands for disodium hydrogen phosphate-sodium dihydrogen phosphate, and so on.

[0298] Formulation Example 3. Screening of sugar species Formulations of 100 mg / mL protein and 0.6 mg / mL PS80 containing various sugars as shown in Table 10 were prepared in 20 mM His-HCl buffer (pH 6.0). Forced degradation studies were performed on the samples (40°C for 4 weeks) to examine the effect of different sugar species on protein stability using appearance, SEC, and NR-CE as evaluation indices.

[0299] The results are shown in Table 10, and as shown by the data in the appearance and purity sections, the formulations containing sucrose are superior to the formulations containing trehalose after 4 weeks at 40°C.

[0300] [Table 10]

[0301] Formulation Example 4. Surfactant Screening Formulations were prepared using 20 mM His-HCl buffer (pH 6.0) containing 100 mg / mL protein, 75 mg / mL sucrose, and the surfactants listed in Table 11. Forced degradation studies were performed on the samples (25°C, 300 rpm shaking for 7 days, followed by 40°C incubation for 4 weeks) to examine the effect of different surfactants on protein stability using SEC and NR-CE as evaluation indices.

[0302] The results are shown in Table 11. According to the appearance data, after 7 days of shaking, the formulation containing 0.4 mg / mL PS80 had a worse appearance than the other formulations. After 4 weeks at 40°C, the formulation containing 0.6 mg / mL poloxamer 188 (P188) had a worse appearance than the other formulations. According to the NR-CE data, after 4 weeks at 40°C, the formulation containing 0.6 mg / mL poloxamer 188 (P188) was worse than the other formulations, the formulation containing 0.4 mg / mL PS80 was slightly better, and there was no significant difference between the formulations containing 0.6 mg / mL PS80 and 0.8 mg / mL PS80.

[0303] [Table 11]

[0304] Note: Shaking D7, i.e., shaking for 7 days, 40°C W4, i.e., leaving at 40°C for 4 weeks.

[0305] Formulation Example 5. Lyophilization The protein samples in Table 12 were prepared and freeze-dried, and the freeze-drying parameters are shown in Table 13: [Table 12] [Table 13]

[0306] *: Primary and secondary drying times are adjusted according to specific circumstances.

[0307] In terms of appearance of the two freeze-dried finished products, the solid powders were both intact, with no obvious disintegration, and there was no significant difference in the appearance of the solid powders.

[0308] Formulation Example 6. Freeze-drying stability 1 A formulation of 37.5 mg / mL sucrose, 0.3 mg / mL PS80, and 50 mg / mL protein sample in 10 mM His-HCl pH 6.0 was prepared and screened for freeze-drying process using 20 mL vials with 20 mm freeze-drying rubber stoppers. The freeze-drying parameters are shown in Table 14. The pre- and post-freeze-drying and high-temperature stability of the final processed sample are shown in Table 15.

[0309] [Table 14]

[0310] *: Primary and secondary drying times are adjusted according to specific circumstances.

[0311] [Table 15]

[0312] As a result, the appearance was transparent before freeze-drying, and after freeze-drying the solid powder was complete and without obvious disintegration, and was transparent after redissolution. The results at high temperature (40°C) showed that there were no significant changes in SEC, NR-CE and IEC under the conditions of 40°C 4W, indicating good stability.

[0313] Formulation Example 7. Relationship between viscosity and pH Proteins prepared with 20 mM His-HCl pH 5.0 and 20 mM His-HCl pH 6.0 were concentrated to different concentrations, and their viscosities at 25° C. were measured. The results are shown in Table 16. In the His-HCl system, the viscosity at pH 5.0 was lower than that at pH 6.0.

[0314] [Table 16]

[0315] Note: N / A indicates that the viscosity of the sample was too high to measure.

[0316] Formulation Example 8. Freeze-drying stability 2 (subcutaneous formulation) The protein samples in Table 17 were prepared and lyophilized, and the lyophilization parameters are shown in Table 14: [Table 17] [Table 18] [Table 19]

[0317] As a result, the appearance was transparent before freeze-drying, and after freeze-drying, the solid powder was complete and without obvious disintegration. After reconstitution, it was transparent. After reconstitution, the results of 24 hours at 25°C showed no significant changes in SEC, NR-CE, and IEC, indicating relatively good stability.

[0318] Formulation Example 9. Freeze-drying stability 3 (subcutaneous formulation) The protein samples in Table 20 were prepared and freeze-dried, and the freeze-drying parameters are shown in Table 14: [Table 20] [Table 21] [Table 22]

[0319] The results showed that the appearance was transparent before freeze-drying, the solid powder was complete and without obvious disintegration after freeze-drying, and was transparent after redissolution. The high-temperature results showed that there was no significant change in SEC and enzyme activity at 40°C and 4W, indicating that the stability was relatively good.

[0320] Formulation Example 10. Freeze-drying stability 4 (subcutaneous formulation) The protein samples in Table 23 were prepared and lyophilized, and the lyophilization parameters are shown in Table 14: [Table 23] [Table 24] [Table 25]

[0321] Note: 40°C 4W indicates that the product is left at 40°C for 4 weeks under the specified conditions, 2-8°C M6 indicates that the product is left at 4°C for 6 months under the specified conditions, and 25°C M6 indicates that the product is left at 25°C for 6 months under the specified conditions.

[0322] The results showed that the appearance was transparent before freeze-drying, and the solid powder was complete and without obvious disintegration after freeze-drying. After reconstitution, the powder was transparent. The results of 40°C 4W, 25°C M6, and 2-8°C M6 showed that there were no significant changes in purity and enzyme activity, indicating relatively good stability.

[0323] Formulation Example 11. Freeze-drying of final formulation 5 (subcutaneous formulation) The protein samples in Table 26 were prepared and lyophilized, and the lyophilization parameters are shown in Table 14: [Table 26]

[0324] After lyophilization, the appearance of the lyophilized product is clear and transparent, with no obvious collapse. The formulation after reconstitution is 150 mg / mL protein, 25 mM His-HCl at pH 5.0, 70 mg / mL sucrose, 10 mM methionine, 0.6 mg / mL PS80, and 2000 U / mL hyaluronidase.

Claims

1. A pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR and a buffer, wherein: The bispecific antibody that specifically binds to HGFR and EGFR comprises at least one antigen-binding module 1 that specifically binds to HGFR and at least one antigen-binding module 2 that specifically binds to EGFR; The antigen binding module 1 comprises a heavy chain variable region M-VH and a light chain variable region M-VL, wherein the M-VH comprises M-HCDR1, M-HCDR2, and M-HCDR3, and the M-VL comprises M-LCDR1, M-LCDR2, and M-LCDR3; and the antigen binding module 2 comprises a heavy chain variable region E-VH and a light chain variable region E-VL, the E-VH comprising E-HCDR1, E-HCDR2, and E-HCDR3, and the E-VL comprising E-LCDR1, E-LCDR2, and E-LCDR3; The E-HCDR1, E-HCDR2, E-HCDR3, E-LCDR1, E-LCDR2 and E-LCDR3 are defined according to the Kabat numbering convention, wherein the E-HCDR1 is represented by SEQ ID NO: 6, the E-HCDR2 is represented by SEQ ID NO: 7, the E-HCDR3 is represented by SEQ ID NO: 8, the E-LCDR1 is represented by SEQ ID NO: 9, the E-LCDR2 is represented by SEQ ID NO: 10, and the E-LCDR3 is represented by SEQ ID NO: 11; and The M-HCDR1, M-HCDR2, M-HCDR3, M-LCDR1, M-LCDR2 and M-LCDR3 are defined according to the Kabat numbering convention, wherein: (i) the M-HCDR1 is represented by SEQ ID NO: 30, the M-HCDR2 is represented by SEQ ID NO: 31, the M-HCDR3 is represented by SEQ ID NO: 32, the M-LCDR1 is represented by SEQ ID NO: 27, the M-LCDR2 is represented by SEQ ID NO: 33, and the M-LCDR3 is represented by SEQ ID NO: 29; or (ii) the M-HCDR1 is represented by SEQ ID NO: 18, the M-HCDR2 is represented by SEQ ID NO: 19, the M-HCDR3 is represented by SEQ ID NO: 20, the M-LCDR1 is represented by SEQ ID NO: 21, the M-LCDR2 is represented by SEQ ID NO: 22, and the M-LCDR3 is represented by SEQ ID NO: 23; or (iii) the M-HCDR1 is represented by SEQ ID NO: 24, the M-HCDR2 is represented by SEQ ID NO: 25, the M-HCDR3 is represented by SEQ ID NO: 26, the M-LCDR1 is represented by SEQ ID NO: 27, the M-LCDR2 is represented by SEQ ID NO: 28, and the M-LCDR3 is represented by SEQ ID NO: 29; the buffer is a histidine buffer, an acetate buffer, a citrate buffer, a succinate buffer, or a phosphate buffer; Preferably, the buffer is a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer. Pharmaceutical compositions.

2. the pH of the pharmaceutical composition is 4.5 to 6.5; Preferably, the pH of the pharmaceutical composition is 4.8 to 6.2; More preferably, the pH of the pharmaceutical composition is 5.0 to 6.

0. The pharmaceutical composition of claim 1.

3. the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 to 250 mg / mL; Preferably, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 to 200 mg / mL; More preferably, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 to 200 mg / mL; Most preferably, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 80 to 180 mg / mL. The pharmaceutical composition according to claim 1 or 2.

4. the pharmaceutical composition comprises a surfactant; Preferably, the surfactant is a polysorbate or a poloxamer; More preferably, the surfactant is polysorbate 80. The pharmaceutical composition according to any one of claims 1 to 3.

5. the surfactant has a concentration of 0.01 to 1.0 mg / mL; Preferably, the concentration of the surfactant is 0.1 to 0.8 mg / mL; More preferably, the concentration of the surfactant is 0.4 to 0.8 mg / mL. The pharmaceutical composition according to claim 4.

6. the pharmaceutical composition comprises a sugar; Preferably, the sugar is sucrose, trehalose, mannitol or sorbitol; More preferably, the sugar is sucrose. The pharmaceutical composition according to any one of claims 1 to 5.

7. the sugar has a concentration of 10 to 100 mg / mL; Preferably, the sugar has a concentration of 20 to 80 mg / mL; More preferably, the sugar concentration is 60 to 80 mg / mL. The pharmaceutical composition according to claim 6.

8. The concentration of the buffer is 5 to 100 mM; Preferably, the concentration of the buffer is 5 to 50 mM; More preferably, the concentration of the buffer is 5 to 30 mM. The pharmaceutical composition according to any one of claims 1 to 7.

9. The pharmaceutical composition further comprises an additive, preferably, the additive is methionine, arginine hydrochloride, glycine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, calcium chloride, or disodium ethylenediaminetetraacetate, more preferably, the additive is methionine or arginine hydrochloride, and most preferably, the additive is methionine. The pharmaceutical composition according to any one of claims 1 to 8.

10. The concentration of the additive is 1 to 300 mM, preferably 1 to 200 mM, and more preferably 1 to 20 mM. The pharmaceutical composition of claim 9.

11. The pharmaceutical composition further comprises an enzyme, preferably, the enzyme is hyaluronidase. The pharmaceutical composition according to any one of claims 1 to 10.

12. The enzyme concentration is 10 to 3000 U / mL, preferably 150 to 2400 U / mL, more preferably 700 to 2400 U / mL, more preferably 1600 to 2400 U / mL; The pharmaceutical composition of claim 11.

13. The bispecific antibody that specifically binds to HGFR and EGFR includes: (i) the bispecific antibody that specifically binds to HGFR and EGFR comprises two antigen-binding modules 1 that specifically bind to HGFR and one antigen-binding module 2 that specifically binds to EGFR; one antigen-binding module 1, whose M-VH comprises M-HCDR1 represented by SEQ ID NO: 30, M-HCDR2 represented by SEQ ID NO: 31, and M-HCDR3 represented by SEQ ID NO: 32, and whose M-VL comprises M-LCDR1 represented by SEQ ID NO: 27, M-LCDR2 represented by SEQ ID NO: 33, and M-LCDR3 represented by SEQ ID NO: 29; The other antigen binding module 1 has an M-VH comprising an M-HCDR1 represented by SEQ ID NO: 18, an M-HCDR2 represented by SEQ ID NO: 19, and an M-HCDR3 represented by SEQ ID NO: 20, and an M-VL comprising an M-LCDR1 represented by SEQ ID NO: 21, an M-LCDR2 represented by SEQ ID NO: 22, and an M-LCDR3 represented by SEQ ID NO: 23; and The antigen binding module 2 has an E-VH comprising an E-HCDR1 represented by SEQ ID NO: 6, an E-HCDR2 represented by SEQ ID NO: 7, and an E-HCDR3 represented by SEQ ID NO: 8, and an E-VL comprising an E-LCDR1 represented by SEQ ID NO: 9, an E-LCDR2 represented by SEQ ID NO: 10, and an E-LCDR3 represented by SEQ ID NO: 11; or (ii) the bispecific antibody that specifically binds to HGFR and EGFR comprises one antigen-binding module 1 that specifically binds to HGFR and one antigen-binding module 2 that specifically binds to EGFR; The antigen binding module 1 has an M-VH comprising an M-HCDR1 represented by SEQ ID NO: 18, an M-HCDR2 represented by SEQ ID NO: 19, and an M-HCDR3 represented by SEQ ID NO: 20, and an M-VL comprising an M-LCDR1 represented by SEQ ID NO: 21, an M-LCDR2 represented by SEQ ID NO: 22, and an M-LCDR3 represented by SEQ ID NO: 23; and The antigen binding module 2 has an E-VH comprising an E-HCDR1 represented by SEQ ID NO: 6, an E-HCDR2 represented by SEQ ID NO: 7, and an E-HCDR3 represented by SEQ ID NO: 8, and an E-VL comprising an E-LCDR1 represented by SEQ ID NO: 9, an E-LCDR2 represented by SEQ ID NO: 10, and an E-LCDR3 represented by SEQ ID NO:

11. The pharmaceutical composition according to any one of claims 1 to 12.

14. The bispecific antibody that specifically binds to HGFR and EGFR includes: (i) the M-VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 16, and the M-VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17; or the M-VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12, and the M-VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13; or the M-VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14, and the M-VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15; or (ii) the E-VH comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3, and the E-VL comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5 or SEQ ID NO:4; Preferably, (i) the bispecific antibody that specifically binds to HGFR and EGFR comprises two antigen-binding modules 1 that specifically bind to HGFR and one antigen-binding module 2 that specifically binds to EGFR; One of the antigen-binding modules 1 has M-VH represented by SEQ ID NO: 16 and M-VL represented by SEQ ID NO: 17, The other antigen binding module 1 has M-VH represented by SEQ ID NO: 12 and M-VL represented by SEQ ID NO: 13, and The antigen binding module 2 has an E-VH represented by SEQ ID NO: 3 and an E-VL represented by SEQ ID NO: 5; or (ii) the bispecific antibody that specifically binds to HGFR and EGFR comprises one antigen-binding module 1 that specifically binds to HGFR and one antigen-binding module 2 that specifically binds to EGFR; The antigen binding module 1 has an M-VH represented by SEQ ID NO: 12 and an M-VL represented by SEQ ID NO: 13, and the antigen binding module 2 has an E-VH represented by SEQ ID NO: 3 and an E-VL represented by SEQ ID NO:

5. The pharmaceutical composition of claim 13.

15. the bispecific antibody that specifically binds to HGFR and EGFR comprises one first chain set forth in SEQ ID NO: 34, one second chain set forth in SEQ ID NO: 35, one third chain set forth in SEQ ID NO: 36, and one fourth chain set forth in SEQ ID NO: 37; or one first strand represented by SEQ ID NO: 38, one second strand represented by SEQ ID NO: 39, one third strand represented by SEQ ID NO: 40, and one fourth strand represented by SEQ ID NO: 41; 15. The pharmaceutical composition according to claim 13 or 14.

16. The following components: (a) 1 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 10 to 100 mg / mL of a sugar; (d) 5 to 100 mM of a buffering agent, wherein the pH of the pharmaceutical composition is 4.5 to 6.5; Preferably, the pharmaceutical composition comprises the following ingredients: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL of polysorbate 80; and (c) 20-80 mg / mL sucrose; (d) 5 to 50 mM histidine buffer, wherein the pH of the pharmaceutical composition is 4.8 to 6.2; More preferably, the pharmaceutical composition comprises the following ingredients: (a) 80-180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL of polysorbate 80; and (c) 60-80 mg / mL sucrose; (d) 5 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.

0. The pharmaceutical composition according to any one of claims 1 to 8 and 13 to 15.

17. The following components: (a) 1 to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.01 to 1.0 mg / mL of a surfactant; (c) 10 to 100 mg / mL of a sugar; (d) 1 to 300 mM of an additive; (e) 10 to 3000 U / mL of an enzyme; (f) 5 to 100 mM of a buffering agent, wherein the pH of the pharmaceutical composition is 4.5 to 6.5; Preferably, the pharmaceutical composition comprises the following ingredients: (a) 1 to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.1 to 0.8 mg / mL of polysorbate 80; and (c) 20-80 mg / mL sucrose; (d) 1 to 200 mM methionine; (e) 700 to 2400 U / mL of hyaluronidase; (f) 5 to 50 mM histidine buffer, wherein the pH of the pharmaceutical composition is 4.8 to 6.2; More preferably, the pharmaceutical composition comprises the following ingredients: (a) 80-180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR; (b) 0.4 to 0.8 mg / mL of polysorbate 80; and (c) 60-80 mg / mL sucrose; (d) 1 to 20 mM methionine; (e) 1600 to 2400 U / mL of hyaluronidase; (f) 5 to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.

0. The pharmaceutical composition according to any one of claims 1 to 15.

18. After reconstitution, the pharmaceutical composition according to any one of claims 1 to 17 can be formed. Lyophilized formulation.

19. 18. The method of claim 1, further comprising the step of lyophilizing the pharmaceutical composition of claim 1. Methods for preparing lyophilized formulations.

20. 20. Obtained by the method of claim 19. Lyophilized formulation.

21. 21. The freeze-dried formulation according to claim 18 or 20, which is prepared by reconstituting the freeze-dried formulation. Reconstitution solution.

22. an intravenous injection formulation, a subcutaneous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation, preferably an intravenous injection formulation or a subcutaneous injection formulation; A pharmaceutical composition according to any one of claims 1 to 17 or a reconstituted solution according to claim 21.

23. A method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 17, or the lyophilized formulation according to claim 18 or 20, or the reconstituted solution according to claim 21; Preferably, the disease is a tumor, more preferably the tumor is selected from lung cancer, breast cancer, pancreatic cancer, colorectal cancer, sarcoma, renal cell carcinoma, hepatocellular carcinoma, gastric cancer, ovarian cancer, bladder cancer, head and neck cancer and glioblastoma; method.

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