Pharmaceutical composition containing bispecific antibody specifically binding to HGFR and EGFR

Abstract

The present disclosure relates to a pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR. In particular, the present disclosure relates to a pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR and its use as a medicament.

Description

A pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR Technical Field

[0001] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR, and its use as a drug. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

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

[0004] The EGFR signaling pathway plays a crucial role in tumor biology by regulating cell proliferation, angiogenesis, and the metastasis and survival of cancer cells. Dysregulation of this pathway can lead to tumorigenesis. HGFR is also implicated in the development of many tumors due to aberrant signaling activation caused by overexpression, activating mutations, autocrine or paracrine signaling, or gene amplification. Investigations into cancer treatment efficacy have revealed a significant connection between the EGFR and HGFR signaling pathways.

[0005] NSCLC accounts for 83% of all lung cancers, and activating EGFR mutations are a common type (10-15% in whites and 50% in Asians). EGFR tyrosine kinase inhibitors (TKIs) have always been the first-line treatment for NSCLC. Although the initial response rate is high (70-80%), resistance usually develops within a year. There are two main mechanisms of resistance: one is the emergence of another EGFR mutation, such as EGFR T790M; the other is compensatory activation of the HGFR signaling pathway. These factors all affect the treatment effect of tumors.

[0006] Summary of the Invention

[0007] The present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR, which has therapeutic activity and the advantages of good stability.

[0008] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR and a buffer, wherein:

[0009] 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;

[0010] The buffer is histidine buffer, acetate buffer, citrate buffer, succinate buffer or phosphate buffer.

[0011] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a bispecific antibody that specifically binds to HGFR and EGFR and a buffer, wherein:

[0012] 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;

[0013] 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

[0014] The antigen binding module 2 comprises a heavy chain variable region E-VH and a light chain variable region E-VL, wherein the E-VH comprises E-HCDR1, E-HCDR2 and E-HCDR3, and the E-VL comprises E-LCDR1, E-LCDR2 and E-LCDR3.

[0015] The E-HCDR1, E-HCDR2, E-HCDR3, E-LCDR1, E-LCDR2 and E-LCDR3 are defined according to the Kabat numbering convention, the E-HCDR1 is shown in SEQ ID NO: 6, the E-HCDR2 is shown in SEQ ID NO: 7, the E-HCDR3 is shown in SEQ ID NO: 8, the E-LCDR1 is shown in SEQ ID NO: 9, the E-LCDR2 is shown in SEQ ID NO: 10, and the E-LCDR3 is shown in SEQ ID NO: 11; and

[0016] The M-HCDR1, M-HCDR2, M-HCDR3, M-LCDR1, M-LCDR2 and M-LCDR3 are defined according to the Kabat numbering convention, wherein:

[0017] (i) the M-HCDR1 is shown in SEQ ID NO: 30, the M-HCDR2 is shown in SEQ ID NO: 31, the M-HCDR3 is shown in SEQ ID NO: 32, the M-LCDR1 is shown in SEQ ID NO: 27, the M-LCDR2 is shown in SEQ ID NO: 33, and the M-LCDR3 is shown in SEQ ID NO: 29, or

[0018] (ii) the M-HCDR1 comprises SEQ ID NO: 18, the M-HCDR2 comprises SEQ ID NO: 19, the M-HCDR3 comprises SEQ ID NO: 20, the M-LCDR1 comprises SEQ ID NO: 21, the M-LCDR2 comprises SEQ ID NO: 22, and the M-LCDR3 comprises SEQ ID NO: 23, or

[0019] (iii) the M-HCDR1 is set forth in SEQ ID NO: 24, the M-HCDR2 is set forth in SEQ ID NO: 25, the M-HCDR3 is set forth in SEQ ID NO: 26, the M-LCDR1 is set forth in SEQ ID NO: 27, the M-LCDR2 is set forth in SEQ ID NO: 28, and the M-LCDR3 is set forth in SEQ ID NO: 29;

[0020] The buffer is histidine buffer, acetate buffer, citrate buffer, succinate buffer or phosphate buffer.

[0021] In some embodiments, the buffer is a histidine buffer.

[0022] In some embodiments, the buffer is a histidine-histidine hydrochloride buffer or a histidine-histidine acetate buffer.

[0023] In some specific embodiments, the buffer is histidine-histidine hydrochloride buffer.

[0024] In some embodiments, the pharmaceutical composition of any one of the above items, wherein the pH of the pharmaceutical composition is 4.5 to 6.5. In some embodiments, the pharmaceutical composition of any one of the above items, 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.

[0025] In some embodiments, the pH of the pharmaceutical composition is between 5.0 and 6.5. In some embodiments, the pH of the pharmaceutical composition is between 5.5 and 6.5. In some embodiments, the pH of the pharmaceutical composition is between 5.5 and 6.0. In some embodiments, the pH of the pharmaceutical composition is between 6.0 and 6.5. In some embodiments, the pharmaceutical composition of any of the above, wherein the pH of the pharmaceutical composition is between 5.8 and 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.

[0026] In some embodiments, the pharmaceutical composition of any of the above items, wherein the pH of the pharmaceutical composition is 4.5 to 6.0. In some embodiments, the pharmaceutical composition of any of the above items, wherein the pH of the pharmaceutical composition is 4.5 to 5.5. In some embodiments, the pharmaceutical composition of any of the above items, wherein 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.

[0027] 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, human operation, instrumentation, methodology, and measurement error. Taking pH as an example, when a measured value is about 6.0, it should be understood that it includes a margin of error. As an example, when a formulation is measured using an industrial pH meter, "about 6.0" means 6.0 ± 0.2 (i.e., a pH of 5.8 to 6.2).

[0028] 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 therebetween. 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.

[0029] Typically, the pH of the pharmaceutical composition obtained by replacing the buffer is almost the same as the buffer pH. Meanwhile, it is well known to those skilled in the art that, during the process of pharmaceutical preparation, there may sometimes be a pH drift, but the drift of the pH of the pharmaceutical preparation is generally very small (e.g., within the range of ± 0.3). In some embodiments, the drift of the pH of the pharmaceutical preparation is within the range of ± 0.3. In some embodiments, the drift of the pH of the pharmaceutical preparation is within the range of ± 0.2. In some embodiments, the drift of the pH of the pharmaceutical preparation is within the range of ± 0.1.

[0030] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 mg / mL to 250 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 mg / mL to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 mg / mL to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 80 mg / mL to 180 mg / mL.

[0031] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 mg / mL to 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 mg / mL to 120 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 1 mg / mL to 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 mg / mL to 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 80 mg / mL to 120 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 90 mg / mL to 110 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is about 100 mg / mL.

[0032] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 80 mg / mL to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 90 mg / mL to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 100 mg / mL to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 120 mg / mL to 200 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 120 mg / mL to 180 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 135 mg / mL to 165 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is about 150 mg / mL.

[0033] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 mg / mL to 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 mg / mL to 75 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 48 mg / mL to 72 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 54 mg / mL 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.

[0034] In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 50 mg / mL to 250 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 100 mg / mL to 250 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 160 mg / mL to 240 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is 180 mg / mL to 220 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds to HGFR and EGFR is about 200 mg / mL.

[0035] 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, In some embodiments, the concentration of the bispecific antibody that specifically binds HGFR and EGFR is 60 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds HGFR and EGFR is 70 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds HGFR and EGFR is 100 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds HGFR and EGFR is 150 mg / mL. In some embodiments, the concentration of the bispecific antibody that specifically binds HGFR and EGFR is 200 mg / mL.

[0036] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from poloxamer (e.g., poloxamer 188, abbreviated as P188), polysorbate (e.g., polysorbate 20, polysorbate 80), polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-sweet In some embodiments, the surfactant is a polysorbate or a poloxamer. In some embodiments, the surfactant is a polysorbate 80, a polysorbate 20, or a poloxamer 188. In some embodiments, the surfactant is a polysorbate 80.

[0037] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the surfactant concentration is 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the surfactant concentration is 0.1 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.3 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.4 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.48 mg / mL to 0.72 mg / mL. In some embodiments, the surfactant concentration is 0.54 mg / mL to 0.66 mg / mL. In some embodiments, the surfactant concentration is about 0.6 mg / mL.

[0038] In some embodiments, the surfactant concentration is between 0.3 mg / mL and 0.6 mg / mL. In some embodiments, the surfactant concentration is between 0.4 mg / mL and 0.6 mg / mL. In some embodiments, the surfactant concentration is between 0.6 mg / mL and 0.8 mg / mL. In some embodiments, the surfactant concentration is between 0.2 mg / mL and 0.4 mg / mL. In some embodiments, the surfactant concentration is between 0.2 mg / mL and 0.3 mg / mL. In some embodiments, the surfactant concentration is between 0.24 mg / mL and 0.36 mg / mL. In some embodiments, the surfactant concentration is about 0.24 mg / mL. In some embodiments, the surfactant concentration is about 0.3 mg / mL.

[0039] 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 point 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.

[0040] In some embodiments, the surfactant is 0.6 mg / mL polysorbate 80. In some embodiments, the surfactant concentration is 0.24 mg / mL polysorbate 80. In some embodiments, the surfactant concentration is 0.3 mg / mL polysorbate 80.

[0041] In some embodiments, the pharmaceutical composition as described in any one of the above items comprises sugar. In some embodiments, the sugar is selected from the conventional composition (CH2O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. The sugar can be selected from sucrose, trehalose, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like.

[0042] 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.

[0043] In some embodiments, the concentration of the sugar is between 10 mg / mL and 100 mg / mL. In some embodiments, the concentration of the sugar is between 20 mg / mL and 90 mg / mL. In some embodiments, the concentration of the sugar is between 20 mg / mL and 80 mg / mL. In some embodiments, the concentration of the sugar is between 30 mg / mL and 80 mg / mL. In some embodiments, the concentration of the sugar is between 50 mg / mL and 80 mg / mL. In some embodiments, the concentration of the sugar is between 60 mg / mL and 90 mg / mL. In some embodiments, the concentration of the sugar is between 60 mg / mL and 80 mg / mL. In some embodiments, the concentration of the sugar is between 67.5 mg / mL and 82.5 mg / mL. In some embodiments, the concentration of the sugar is between 70 mg / mL and 80 mg / mL. In some embodiments, the concentration of the sugar is about 75 mg / mL.

[0044] In some embodiments, the concentration of the saccharide is between 56 mg / mL and 84 mg / mL. In some embodiments, the concentration of the saccharide is between 63 mg / mL and 77 mg / mL. In some embodiments, the concentration of the saccharide is about 70 mg / mL.

[0045] In some embodiments, the concentration of the sugar is between 20 mg / mL and 50 mg / mL. In some embodiments, the concentration of the sugar is between 20 mg / mL and 40 mg / mL. In some embodiments, the concentration of the sugar is between 20 mg / mL and 35 mg / mL. In some embodiments, the concentration of the sugar is between 22.4 mg / mL and 33.6 mg / mL. In some embodiments, the concentration of the sugar is between 25.2 mg / mL and 30.8 mg / mL. In some embodiments, the concentration of the sugar is about 28 mg / mL.

[0046] In some embodiments, the concentration of the saccharide is 30 mg / mL to 50 mg / mL. In some embodiments, the concentration of the saccharide is 30 mg / mL to 40 mg / mL. In some embodiments, the concentration of the saccharide is about 37.5 mg / mL.

[0047] In some embodiments, the concentration of the sugar is non-limitingly 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 point values. In some embodiments, the concentration of the sugar is 28 mg / mL. In some embodiments, the concentration of the sugar is 37.5 mg / mL. In some embodiments, the sugar is present at a concentration of 70 mg / mL. In some embodiments, the sugar is present at a concentration of 75 mg / mL.

[0048] In some embodiments, the sugar is 70 mg / mL to 80 mg / mL of sucrose; preferably, the sugar is 75 mg / mL of sucrose.

[0049] In some embodiments, the sugar is 63 mg / mL to 77 mg / mL of sucrose; preferably, the sugar is 70 mg / mL of sucrose.

[0050] In some embodiments, the sugar is 25.2 mg / mL to 30.8 mg / mL of sucrose; preferably, the sugar is 28 mg / mL of sucrose.

[0051] In some embodiments, the pharmaceutical composition as described in any of the above, the concentration of the buffer is 5mM to 100mM. In some embodiments, the concentration of the buffer is 5mM to 50mM. In some embodiments, the concentration of the buffer is 10mM to 50mM. In some embodiments, the concentration of the buffer is 5mM to 30mM. In some embodiments, the concentration of the buffer is 10mM to 30mM. In some embodiments, the concentration of the buffer is 15mM to 25mM. In some embodiments, the concentration of the buffer is 18mM to 22mM. In some embodiments, the concentration of the buffer is 10mM to 20mM. In some embodiments, the concentration of the buffer is 10mM or 20mM. In some embodiments, the concentration of the buffer is about 20mM.

[0052] In some embodiments, the concentration of the buffer is 20 mM to 30 mM. In some embodiments, the concentration of the buffer is 22.5 mM to 27.5 mM. In some embodiments, the concentration of the buffer is about 25 mM.

[0053] In some embodiments, the concentration of the buffer is 5 mM to 15 mM. In some embodiments, the concentration of the buffer is 8 mM to 12 mM. In some embodiments, the concentration of the buffer is 9 mM to 11 mM. In some embodiments, the concentration of the buffer is about 10 mM.

[0054] In some embodiments, the concentration of the buffer is 5mM, 8mM, 9mM, 10mM, 11mM, 12mM, 15mM, 18mM, 20mM, 22mM, 22.5mM, 25mM, 27.5mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, and any ranges between these point values. In some embodiments, the concentration of the buffer is 10mM. In some embodiments, the concentration of the buffer is 20mM. In some embodiments, the concentration of the buffer is 25mM.

[0055] 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.

[0056] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the pharmaceutical composition further comprises an excipient. In some embodiments, the excipient is methionine, arginine hydrochloride, glycine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, calcium chloride, or disodium edetate. In some embodiments, the excipient is sodium chloride or calcium chloride. In some embodiments, the excipient is methionine or arginine hydrochloride. In some embodiments, the excipient is arginine hydrochloride. In some embodiments, the excipient is methionine.

[0057] In some embodiments, the pharmaceutical composition of any of the above, wherein the concentration of the excipient is 1 mM to 300 mM; preferably, the concentration of the excipient is 25 mM to 200 mM. In some embodiments, the concentration of the excipient is 1 mM to 200 mM. In some embodiments, the concentration of the excipient is 1 mM to 20 mM. In some embodiments, the concentration of the excipient is 4 mM to 20 mM. In some embodiments, the concentration of the excipient is 8 mM to 12 mM. In some embodiments, the concentration of the excipient is 9 mM to 11 mM. In some embodiments, the concentration of the excipient is about 10 mM.

[0058] In some embodiments, the concentration of the excipient is 1 mM to 10 mM. In some embodiments, the concentration of the excipient is 3 mM to 5 mM. In some embodiments, the concentration of the excipient is 3.6 mM to 4.4 mM. In some embodiments, the concentration of the excipient is about 4 mM.

[0059] In some embodiments, the concentration of the excipient is 1 mM to 240 mM. In some embodiments, the concentration of the excipient is 80 mM to 240 mM. In some embodiments, the concentration of the excipient is 160 mM to 240 mM. In some embodiments, the concentration of the excipient is 180 mM to 220 mM. In some embodiments, the concentration of the excipient is about 200 mM.

[0060] In some embodiments, the concentration of the excipient is 80 mM to 120 mM. In some embodiments, the concentration of the excipient is 90 mM to 110 mM. In some embodiments, the concentration of the excipient is about 100 mM.

[0061] In some embodiments, the concentration of adjuvant is 1mM, 3mM, 3.6mM, 4.4mM, 5mM, 8mM, 9mM, 10mM, 11mM, 12mM, 20mM, 25mM, 30mM, 50mM, 75mM, 80mM, 90mM, 100mM, 110mM, 120mM, 150mM, 160mM, 180mM, 200mM, 240mM, 250mM, 280mM or 300mM, and any range between these point values. In some embodiments, the concentration of the adjuvant is 4mM. In some embodiments, the concentration of the adjuvant is 10mM. In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the concentration of the adjuvant is 100mM. In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the concentration of the adjuvant is 200mM.

[0062] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the concentration of the excipient is 4 mM or 10 mM methionine.

[0063] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the concentration of the excipient is 100 mM or 200 mM arginine hydrochloride.

[0064] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the pharmaceutical composition further comprises an enzyme. In some embodiments, the enzyme is hyaluronidase (PH20).

[0065] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 10 U / mL to 3000 U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 150 U / mL to 2400 U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 150 U / mL to 2200 U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 600 U / mL to 2200 U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 800 U / mL to 2000 U / mL.

[0066] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 700U / mL to 2400U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 800U / mL to 2400U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 1600U / mL to 2400U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 1800U / mL to 2200U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is about 2000U / mL.

[0067] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 150U / mL to 2000U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 500U / mL to 1100U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 600U / mL to 1000U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 640U / mL to 960U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 700U / mL to 900U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is 720U / mL to 880U / mL. In some embodiments, the pharmaceutical composition as described in any of the above, wherein the concentration of the enzyme is about 800U / mL.

[0068] In some embodiments, the concentration of the enzyme 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, and any ranges between these points. In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the concentration of the enzyme is 800 U / mL. In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the concentration of the enzyme is 1000 U / mL. In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the concentration of the enzyme is 2000 U / mL.

[0069] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the enzyme is 1000U / mL or 2000U / mL of PH20. In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the enzyme is 800U / mL of PH20.

[0070] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the bispecific antibody that specifically binds to HGFR and EGFR,

[0071] 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, wherein:

[0072] The E-HCDR1, E-HCDR2, E-HCDR3, E-LCDR1, E-LCDR2 and E-LCDR3 are defined according to the Kabat numbering convention, the E-HCDR1 is shown in SEQ ID NO: 6, the E-HCDR2 is shown in SEQ ID NO: 7, the E-HCDR3 is shown in SEQ ID NO: 8, the E-LCDR1 is shown in SEQ ID NO: 9, the E-LCDR2 is shown in SEQ ID NO: 10, and the E-LCDR3 is shown in SEQ ID NO: 11; and

[0073] The M-HCDR1, M-HCDR2, M-HCDR3, M-LCDR1, M-LCDR2 and M-LCDR3 are defined according to the Kabat numbering convention, wherein:

[0074] (i) the M-HCDR1 is shown in SEQ ID NO: 30, the M-HCDR2 is shown in SEQ ID NO: 31, the M-HCDR3 is shown in SEQ ID NO: 32, the M-LCDR1 is shown in SEQ ID NO: 27, the M-LCDR2 is shown in SEQ ID NO: 33, and the M-LCDR3 is shown in SEQ ID NO: 29, or

[0075] (ii) the M-HCDR1 comprises SEQ ID NO: 18, the M-HCDR2 comprises SEQ ID NO: 19, the M-HCDR3 comprises SEQ ID NO: 20, the M-LCDR1 comprises SEQ ID NO: 21, the M-LCDR2 comprises SEQ ID NO: 22, and the M-LCDR3 comprises SEQ ID NO: 23, or

[0076] (iii) the M-HCDR1 is set forth in SEQ ID NO: 24, the M-HCDR2 is set forth in SEQ ID NO: 25, the M-HCDR3 is set forth in SEQ ID NO: 26, the M-LCDR1 is set forth in SEQ ID NO: 27, the M-LCDR2 is set forth in SEQ ID NO: 28, and the M-LCDR3 is set forth in SEQ ID NO: 29;

[0077] Preferably,

[0078] (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,

[0079] One of the antigen binding moieties 1, whose M-VH has: M-HCDR1 as shown in SEQ ID NO: 30, M-HCDR2 as shown in SEQ ID NO: 31, and M-HCDR3 as shown in SEQ ID NO: 32, and whose M-VL has: M-LCDR1 as shown in SEQ ID NO: 27, M-LCDR2 as shown in SEQ ID NO: 33, and M-LCDR3 as shown in SEQ ID NO: 29;

[0080] another antigen binding moiety 1, whose M-VH has: M-HCDR1 as shown in SEQ ID NO: 18, M-HCDR2 as shown in SEQ ID NO: 19, and M-HCDR3 as shown in SEQ ID NO: 20, and whose M-VL has: M-LCDR1 as shown in SEQ ID NO: 21, M-LCDR2 as shown in SEQ ID NO: 22, and M-LCDR3 as shown in SEQ ID NO: 23; and

[0081] The antigen binding moiety 2, whose E-VH has: E-HCDR1 as shown in SEQ ID NO: 6, E-HCDR2 as shown in SEQ ID NO: 7 and E-HCDR3 as shown in SEQ ID NO: 8, and its E-VL has: E-LCDR1 as shown in SEQ ID NO: 9, E-LCDR2 as shown in SEQ ID NO: 10 and E-LCDR3 as shown in SEQ ID NO: 11; or

[0082] (ii) the bispecific antibody that specifically binds to HGFR and EGFR comprises an antigen-binding module 1 that specifically binds to HGFR and an antigen-binding module 2 that specifically binds to EGFR,

[0083] The antigen binding moiety 1, whose M-VH has: M-HCDR1 as shown in SEQ ID NO: 18, M-HCDR2 as shown in SEQ ID NO: 19 and M-HCDR3 as shown in SEQ ID NO: 20, and its M-VL has: M-LCDR1 as shown in SEQ ID NO: 21, M-LCDR2 as shown in SEQ ID NO: 22 and M-LCDR3 as shown in SEQ ID NO: 23; and

[0084] The antigen binding module 2, its E-VH has: E-HCDR1 as shown in SEQ ID NO: 6, E-HCDR2 as shown in SEQ ID NO: 7 and E-HCDR3 as shown in SEQ ID NO: 8, and its E-VL has: E-LCDR1 as shown in SEQ ID NO: 9, E-LCDR2 as shown in SEQ ID NO: 10 and E-LCDR3 as shown in SEQ ID NO: 11.

[0085] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody specifically binding to HGFR and EGFR, wherein:

[0086] (i) the M-VH comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 16, and the M-VL comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 17; or

[0087] 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

[0088] 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

[0089] (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;

[0090] Preferably,

[0091] (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,

[0092] wherein the M-VH of one antigen binding moiety 1 is represented by SEQ ID NO: 16, and the M-VL is represented by SEQ ID NO: 17;

[0093] The M-VH of another antigen binding moiety 1 is shown in SEQ ID NO: 12, and the M-VL is shown in SEQ ID NO: 13; and

[0094] The E-VH of the antigen binding moiety 2 is shown in SEQ ID NO: 3, and the E-VL is shown in SEQ ID NO: 5; or

[0095] (ii) the bispecific antibody that specifically binds to HGFR and EGFR comprises an antigen-binding module 1 that specifically binds to HGFR and an antigen-binding module 2 that specifically binds to EGFR,

[0096] The M-VH of the antigen binding moiety 1 is shown as SEQ ID NO: 12, and the M-VL is shown as SEQ ID NO: 13, and the E-VH of the antigen binding moiety 2 is shown as SEQ ID NO: 3, and the E-VL is shown as SEQ ID NO: 5.

[0097] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein:

[0098] The bispecific antibody that specifically binds to HGFR and EGFR comprises a first chain as shown in SEQ ID NO: 34, a second chain comprising SEQ ID NO: 35, a third chain comprising SEQ ID NO: 36, and a fourth chain comprising SEQ ID NO: 37; or

[0099] It has a first strand as shown in SEQ ID NO:38, a second strand as shown in SEQ ID NO:39, a third strand as shown in SEQ ID NO:40, and a fourth strand as shown in SEQ ID NO:41.

[0100] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody that specifically binds to HGFR and EGFR has a Format 1 or Format 2 structure, the structural schematic diagram of Format 1 is shown in Figure 1, and the structural schematic diagram of Format 2 is shown in Figure 2.

[0101] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the bispecific antibody that specifically binds to HGFR and EGFR is a low-fucosylated bispecific antibody; preferably, the low-fucosylated bispecific antibody is an antibody that is at least 80%, 85%, 90%, 95% or 100% unmodified by fucosylation; more preferably, no fucosylation modification is detected on the bispecific antibody.

[0102] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0103] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0104] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0105] (c) 10 mg / mL to 100 mg / mL of sugar, and

[0106] (d) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0107] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0108] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0109] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0110] (c) 30 mg / mL to 80 mg / mL of sucrose, and

[0111] (d) 10 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 5.5 to 6.5.

[0112] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0113] (a) 90 mg / mL to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0114] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0115] (c) 70 mg / mL to 80 mg / mL of sucrose, and

[0116] (d) 10 mM to 20 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is about 6.0.

[0117] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0118] (a) 90 mg / mL to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0119] (b) 0.6 mg / mL of polysorbate 80,

[0120] (c) 70 mg / mL to 80 mg / mL of sucrose, and

[0121] (d) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 6.0.

[0122] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0123] (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0124] (b) 0.6 mg / mL of polysorbate 80,

[0125] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0126] (d) 2000 U / mL of PH20, and

[0127] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 6.0.

[0128] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0129] (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0130] (b) 0.6 mg / mL of polysorbate 80,

[0131] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0132] (d) 2000 U / mL of PH20, and

[0133] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0134] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0135] (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0136] (b) 0.6 mg / mL of polysorbate 80,

[0137] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0138] (d) 200 mM arginine hydrochloride,

[0139] (e) 2000 U / mL of PH20, and

[0140] (f) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0141] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0142] (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0143] (b) 0.6 mg / mL of polysorbate 80,

[0144] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0145] (d) 100 mM arginine hydrochloride,

[0146] (e) 2000 U / mL of PH20, and

[0147] (f) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0148] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0149] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0150] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0151] (c) 10 mg / mL to 100 mg / mL of sugar, and

[0152] (d) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.5.

[0153] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0154] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0155] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0156] (c) 20 mg / mL to 80 mg / mL of sucrose, and

[0157] (d) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 4.8 to 6.2.

[0158] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0159] (a) 80 mg / mL to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0160] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0161] (c) 60 mg / mL to 80 mg / mL of sucrose, and

[0162] (d) 5 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0163] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0164] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0165] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0166] (c) 20 mg / mL to 80 mg / mL of sucrose, and

[0167] (d) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 5.0 to 6.5.

[0168] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0169] (a) 50 mg / mL to 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0170] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0171] (c) 60 mg / mL to 80 mg / mL of sucrose, and

[0172] (d) 10 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 5.5 to 6.5.

[0173] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0174] (a) 90 mg / mL to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0175] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0176] (c) 70 mg / mL to 80 mg / mL of sucrose, and

[0177] (d) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.8 to 6.2.

[0178] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0179] (a) about 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0180] (b) about 0.6 mg / mL of polysorbate 80,

[0181] (c) about 75 mg / mL of sucrose, and

[0182] (d) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 6.0.

[0183] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0184] (a) 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0185] (b) 0.6 mg / mL of polysorbate 80,

[0186] (c) 75 mg / mL sucrose, and

[0187] (d) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 6.0.

[0188] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0189] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0190] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0191] (c) 10 mg / mL to 100 mg / mL of sugar,

[0192] (d) 1 mM to 300 mM excipients,

[0193] (e) 10 U / mL to 3000 U / mL of enzyme, and

[0194] (f) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.5.

[0195] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0196] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0197] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0198] (c) 20 mg / mL to 80 mg / mL of sucrose,

[0199] (d) 1 mM to 200 mM methionine,

[0200] (e) 700 U / mL to 2400 U / mL of hyaluronidase, and

[0201] (f) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 4.8 to 6.2.

[0202] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0203] (a) 80 mg / mL to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0204] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0205] (c) 60 mg / mL to 80 mg / mL of sucrose,

[0206] (d) 1 mM to 20 mM methionine,

[0207] (e) 1600 U / mL to 2400 U / mL of hyaluronidase, and

[0208] (f) 5 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0209] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0210] (a) 50 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0211] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0212] (c) 20 mg / mL to 80 mg / mL of sucrose,

[0213] (d) 1 mM to 200 mM methionine,

[0214] (e) 150 U / mL to 2200 U / mL of hyaluronidase, and

[0215] (f) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 4.5 to 6.0.

[0216] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0217] (a) 100 mg / mL to 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0218] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0219] (c) 60 mg / mL to 80 mg / mL of sucrose,

[0220] (d) 4 mM to 20 mM methionine,

[0221] (e) 800 U / mL to 2200 U / mL of hyaluronidase, and

[0222] (f) 10 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0223] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0224] (a) 120 mg / mL to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0225] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0226] (c) 60 mg / mL to 80 mg / mL of sucrose,

[0227] (d) 1 mM to 20 mM methionine,

[0228] (e) 1600 U / mL to 2400 U / mL of hyaluronidase, and

[0229] (f) 20 mM to 30 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0230] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0231] (a) about 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0232] (b) about 0.6 mg / mL of polysorbate 80,

[0233] (c) about 70 mg / mL of sucrose,

[0234] (d) about 10 mM methionine,

[0235] (e) about 2000 U / mL of hyaluronidase, and

[0236] (f) about 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0237] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0238] (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0239] (b) 0.6 mg / mL of polysorbate 80,

[0240] (c) 70 mg / mL sucrose,

[0241] (d) 10 mM methionine,

[0242] (e) 2000 U / mL of hyaluronidase, and

[0243] (f) 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0244] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0245] (a) 50 mg / mL to 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0246] (b) 0.2 mg / mL to 0.4 mg / mL of polysorbate 80,

[0247] (c) 20 mg / mL to 50 mg / mL of sucrose,

[0248] (d) 1 mM to 10 mM methionine,

[0249] (e) 600 U / mL to 1000 U / mL of hyaluronidase, and

[0250] (f) 5 mM to 15 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0251] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0252] (a) 50 mg / mL to 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0253] (b) 0.2 mg / mL to 0.3 mg / mL of polysorbate 80,

[0254] (c) 20 mg / mL to 35 mg / mL of sucrose,

[0255] (d) 1 mM to 10 mM methionine,

[0256] (e) 720 U / mL to 880 U / mL of hyaluronidase, and

[0257] (f) 8 mM to 12 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0258] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0259] (a) about 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0260] (b) about 0.24 mg / mL of polysorbate 80,

[0261] (c) about 28 mg / mL of sucrose,

[0262] (d) about 4 mM methionine,

[0263] (e) about 800 U / mL of hyaluronidase, and

[0264] (f) about 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0265] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0266] (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0267] (b) 0.24 mg / mL of polysorbate 80,

[0268] (c) 28 mg / mL sucrose,

[0269] (d) 4 mM methionine,

[0270] (e) 800 U / mL of hyaluronidase, and

[0271] (f) 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0272] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0273] (a) 50 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0274] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0275] (c) 20 mg / mL to 80 mg / mL of sucrose,

[0276] (d) 80 mM to 240 mM arginine hydrochloride, and

[0277] (e) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 4.5 to 6.0.

[0278] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0279] (a) 160 mg / mL to 240 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0280] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0281] (c) 60 mg / mL to 80 mg / mL of sucrose,

[0282] (d) 160 mM to 240 mM arginine hydrochloride, and

[0283] (e) 10 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0284] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0285] (a) 180 mg / mL to 220 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0286] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0287] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0288] (d) 180 mM to 220 mM arginine hydrochloride, and

[0289] (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0290] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0291] (a) about 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0292] (b) about 0.6 mg / mL of polysorbate 80,

[0293] (c) about 75 mg / mL of sucrose,

[0294] (d) about 200 mM arginine hydrochloride, and

[0295] (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0296] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0297] (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0298] (b) 0.6 mg / mL of polysorbate 80,

[0299] (c) 75 mg / mL sucrose,

[0300] (d) 200 mM arginine hydrochloride, and

[0301] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0302] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0303] (a) 50 mg / mL to 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0304] (b) 0.2 mg / mL to 0.4 mg / mL of polysorbate 80,

[0305] (c) 20 mg / mL to 50 mg / mL of sucrose,

[0306] (d) 80 mM to 120 mM arginine hydrochloride, and

[0307] (e) 5 mM to 15 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 4.5 to 5.5.

[0308] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0309] (a) 80 mg / mL to 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0310] (b) 0.2 mg / mL to 0.4 mg / mL of polysorbate 80,

[0311] (c) 30 mg / mL to 50 mg / mL of sucrose,

[0312] (d) 90 mM to 110 mM arginine hydrochloride, and

[0313] (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0314] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0315] (a) about 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0316] (b) about 0.3 mg / mL of polysorbate 80,

[0317] (c) about 37.5 mg / mL of sucrose,

[0318] (d) about 100 mM arginine hydrochloride, and

[0319] (e) about 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0320] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0321] (a) 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0322] (b) 0.3 mg / mL of polysorbate 80,

[0323] (c) 37.5 mg / mL sucrose,

[0324] (d) 100 mM arginine hydrochloride, and

[0325] (e) 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0326] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0327] (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0328] (b) 0.6 mg / mL of polysorbate 80,

[0329] (c) 75 mg / mL sucrose,

[0330] (d) 10 mM methionine,

[0331] (e) 2000 U / mL of PH20, and

[0332] (f) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0 or 6.0.

[0333] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0334] (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0335] (b) 0.6 mg / mL of polysorbate 80,

[0336] (c) 75 mg / mL sucrose,

[0337] (d) 2000 U / mL of PH20, and

[0338] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 6.0.

[0339] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0340] (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0341] (b) 0.6 mg / mL of polysorbate 80,

[0342] (c) 75 mg / mL sucrose,

[0343] (d) 10 mM methionine,

[0344] (e) 2000 U / mL of PH20, and

[0345] (f) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0346] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0347] (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0348] (b) 0.6 mg / mL of polysorbate 80,

[0349] (c) 75 mg / mL sucrose, and

[0350] (d) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0351] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0352] (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0353] (b) 0.6 mg / mL of polysorbate 80,

[0354] (c) 75 mg / mL sucrose, and

[0355] (d) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 6.0.

[0356] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0357] (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0358] (b) 0.3 mg / mL of polysorbate 80,

[0359] (c) 37.5 mg / mL sucrose,

[0360] (d) 5 mM methionine,

[0361] (e) 1000 U / mL of PH20, and

[0362] (f) 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0 or 6.0.

[0363] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0364] (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0365] (b) 0.3 mg / mL of polysorbate 80,

[0366] (c) 37.5 mg / mL sucrose,

[0367] (d) 1000 U / mL of PH20, and

[0368] (e) 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 6.0.

[0369] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0370] (a) 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0371] (b) 0.3 mg / mL of polysorbate 80,

[0372] (c) 37.5 mg / mL sucrose,

[0373] (d) 5 mM methionine,

[0374] (e) 1000 U / mL of PH20, and

[0375] (f) 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0376] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0377] (a) 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0378] (b) 0.3 mg / mL of polysorbate 80,

[0379] (c) 37.5 mg / mL of sucrose, and

[0380] (d) 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 5.0.

[0381] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0382] (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0383] (b) 0.3 mg / mL of polysorbate 80,

[0384] (c) 37.5 mg / mL of sucrose, and

[0385] (d) 10 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is 6.0.

[0386] In some embodiments, the pharmaceutical composition as described above is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0387] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the pharmaceutical compositions described above after reconstitution.

[0388] The present disclosure also provides a lyophilized preparation, which is a lyophilized preparation of the pharmaceutical composition as described in any one of the above items.

[0389] The present disclosure also provides a method for preparing a lyophilized formulation, comprising the step of freeze-drying the pharmaceutical composition as described in any one of the above items. In some embodiments, the freeze-drying as described in any one of the above items sequentially comprises the steps of pre-freezing, primary drying, and secondary drying.

[0390] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.

[0391] The present disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted the lyophilized preparation as described in any one of the above items.

[0392] The present disclosure also provides a reconstituted solution, which is a reconstituted preparation of any of the above lyophilized preparations.

[0393] In some embodiments, the components and contents of the reconstituted solution as described in any of the above items are the same as those of the aforementioned pharmaceutical composition.

[0394] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0395] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0396] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0397] (c) 10 mg / mL to 100 mg / mL of sugar, and

[0398] (d) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0399] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0400] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0401] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0402] (c) 30 mg / mL to 80 mg / mL of sucrose, and

[0403] (d) 10 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 5.5 to 6.5.

[0404] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0405] (a) 90 mg / mL to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0406] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0407] (c) 70 mg / mL to 80 mg / mL of sucrose, and

[0408] (d) 10 mM to 20 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is about 6.0.

[0409] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0410] (a) 90 mg / mL to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0411] (b) 0.6 mg / mL of polysorbate 80,

[0412] (c) 70 mg / mL to 80 mg / mL of sucrose, and

[0413] (d) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 6.0.

[0414] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0415] (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0416] (b) 0.6 mg / mL of polysorbate 80,

[0417] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0418] (d) 2000 U / mL of PH20, and

[0419] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 6.0.

[0420] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0421] (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0422] (b) 0.6 mg / mL of polysorbate 80,

[0423] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0424] (d) 2000 U / mL of PH20, and

[0425] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0426] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0427] (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0428] (b) 0.6 mg / mL of polysorbate 80,

[0429] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0430] (d) 200 mM arginine hydrochloride,

[0431] (e) 2000 U / mL of PH20, and

[0432] (f) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0433] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0434] (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0435] (b) 0.6 mg / mL of polysorbate 80,

[0436] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0437] (d) 100 mM arginine hydrochloride,

[0438] (e) 2000 U / mL of PH20, and

[0439] (f) 20 mM histidine-histidine hydrochloride buffer; the pH of the pharmaceutical composition is about 5.0.

[0440] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0441] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0442] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0443] (c) 10 mg / mL to 100 mg / mL of sugar, and

[0444] (d) a 5 mM to 100 mM buffer, wherein the pH of the reconstituted solution is 4.5 to 6.5.

[0445] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0446] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0447] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0448] (c) 20 mg / mL to 80 mg / mL of sucrose, and

[0449] (d) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 4.8 to 6.2.

[0450] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0451] (a) 80 mg / mL to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0452] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0453] (c) 60 mg / mL to 80 mg / mL of sucrose, and

[0454] (d) 5 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0455] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0456] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0457] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0458] (c) 20 mg / mL to 80 mg / mL of sucrose, and

[0459] (d) 5 mM to 50 mM histidine buffer; the pH of the reconstituted solution is 5.0 to 6.5.

[0460] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0461] (a) 50 mg / mL to 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0462] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0463] (c) 60 mg / mL to 80 mg / mL of sucrose, and

[0464] (d) 10 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the reconstituted solution is 5.5 to 6.5.

[0465] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0466] (a) 90 mg / mL to 110 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0467] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0468] (c) 70 mg / mL to 80 mg / mL of sucrose, and

[0469] (d) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.8 to 6.2.

[0470] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0471] (a) about 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0472] (b) about 0.6 mg / mL of polysorbate 80,

[0473] (c) about 75 mg / mL of sucrose, and

[0474] (d) about 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is about 6.0.

[0475] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0476] (a) 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0477] (b) 0.6 mg / mL of polysorbate 80,

[0478] (c) 75 mg / mL sucrose, and

[0479] (d) 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 6.0.

[0480] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0481] (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0482] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0483] (c) 10 mg / mL to 100 mg / mL of sugar,

[0484] (d) 1 mM to 300 mM excipients,

[0485] (e) 10 U / mL to 3000 U / mL of enzyme, and

[0486] (f) a 5 mM to 100 mM buffer, wherein the pH of the reconstituted solution is 4.5 to 6.5.

[0487] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0488] (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0489] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0490] (c) 20 mg / mL to 80 mg / mL of sucrose,

[0491] (d) 1 mM to 200 mM methionine,

[0492] (e) 700 U / mL to 2400 U / mL of hyaluronidase, and

[0493] (f) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 4.8 to 6.2.

[0494] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0495] (a) 80 mg / mL to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0496] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0497] (c) 60 mg / mL to 80 mg / mL of sucrose,

[0498] (d) 1 mM to 20 mM methionine,

[0499] (e) 1600 U / mL to 2400 U / mL of hyaluronidase, and

[0500] (f) 5 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.0.

[0501] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0502] (a) 50 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0503] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0504] (c) 20 mg / mL to 80 mg / mL of sucrose,

[0505] (d) 1 mM to 200 mM methionine,

[0506] (e) 150 U / mL to 2200 U / mL of hyaluronidase, and

[0507] (f) 5 mM to 50 mM histidine buffer; the pH of the reconstituted solution is 4.5 to 6.0.

[0508] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0509] (a) 100 mg / mL to 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0510] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0511] (c) 60 mg / mL to 80 mg / mL of sucrose,

[0512] (d) 4 mM to 20 mM methionine,

[0513] (e) 800 U / mL to 2200 U / mL of hyaluronidase, and

[0514] (f) 10 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the reconstituted solution is 4.5 to 5.5.

[0515] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0516] (a) 120 mg / mL to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0517] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0518] (c) 60 mg / mL to 80 mg / mL of sucrose,

[0519] (d) 1 mM to 20 mM methionine,

[0520] (e) 1600 U / mL to 2400 U / mL of hyaluronidase, and

[0521] (f) 20 mM to 30 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 4.8 to 5.2.

[0522] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0523] (a) about 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0524] (b) about 0.6 mg / mL of polysorbate 80,

[0525] (c) about 70 mg / mL of sucrose,

[0526] (d) about 10 mM methionine,

[0527] (e) about 2000 U / mL of hyaluronidase, and

[0528] (f) about 25 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is about 5.0.

[0529] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0530] (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0531] (b) 0.6 mg / mL of polysorbate 80,

[0532] (c) 70 mg / mL sucrose,

[0533] (d) 10 mM methionine,

[0534] (e) 2000 U / mL of hyaluronidase, and

[0535] (f) 25 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0.

[0536] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0537] (a) 50 mg / mL to 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0538] (b) 0.2 mg / mL to 0.4 mg / mL of polysorbate 80,

[0539] (c) 20 mg / mL to 50 mg / mL of sucrose,

[0540] (d) 1 mM to 10 mM methionine,

[0541] (e) 600 U / mL to 1000 U / mL of hyaluronidase, and

[0542] (f) 5 mM to 15 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the reconstituted solution is 4.5 to 5.5.

[0543] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0544] (a) 50 mg / mL to 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0545] (b) 0.2 mg / mL to 0.3 mg / mL of polysorbate 80,

[0546] (c) 20 mg / mL to 35 mg / mL of sucrose,

[0547] (d) 1 mM to 10 mM methionine,

[0548] (e) 720 U / mL to 880 U / mL of hyaluronidase, and

[0549] (f) 8 mM to 12 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 4.8 to 5.2.

[0550] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0551] (a) about 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0552] (b) about 0.24 mg / mL of polysorbate 80,

[0553] (c) about 28 mg / mL of sucrose,

[0554] (d) about 4 mM methionine,

[0555] (e) about 800 U / mL of hyaluronidase, and

[0556] (f) about 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is about 5.0.

[0557] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0558] (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0559] (b) 0.24 mg / mL of polysorbate 80,

[0560] (c) 28 mg / mL sucrose,

[0561] (d) 4 mM methionine,

[0562] (e) 800 U / mL of hyaluronidase, and

[0563] (f) 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0.

[0564] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0565] (a) 50 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR,

[0566] (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80,

[0567] (c) 20 mg / mL to 80 mg / mL of sucrose,

[0568] (d) 80 mM to 240 mM arginine hydrochloride, and

[0569] (e) 5 mM to 50 mM histidine buffer; the pH of the reconstituted solution is 4.5 to 6.0.

[0570] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0571] (a) 160 mg / mL to 240 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0572] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0573] (c) 60 mg / mL to 80 mg / mL of sucrose,

[0574] (d) 160 mM to 240 mM arginine hydrochloride, and

[0575] (e) 10 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the reconstituted solution is 4.5 to 5.5.

[0576] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0577] (a) 180 mg / mL to 220 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0578] (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80,

[0579] (c) 70 mg / mL to 80 mg / mL of sucrose,

[0580] (d) 180 mM to 220 mM arginine hydrochloride, and

[0581] (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 4.8 to 5.2.

[0582] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0583] (a) about 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0584] (b) about 0.6 mg / mL of polysorbate 80,

[0585] (c) about 75 mg / mL of sucrose,

[0586] (d) about 200 mM arginine hydrochloride, and

[0587] (e) about 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is about 5.0.

[0588] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0589] (a) 200 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0590] (b) 0.6 mg / mL of polysorbate 80,

[0591] (c) 75 mg / mL sucrose,

[0592] (d) 200 mM arginine hydrochloride, and

[0593] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0.

[0594] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0595] (a) 50 mg / mL to 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0596] (b) 0.2 mg / mL to 0.4 mg / mL of polysorbate 80,

[0597] (c) 20 mg / mL to 50 mg / mL of sucrose,

[0598] (d) 80 mM to 120 mM arginine hydrochloride, and

[0599] (e) 5 mM to 15 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the reconstituted solution is 4.5 to 5.5.

[0600] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0601] (a) 80 mg / mL to 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0602] (b) 0.2 mg / mL to 0.4 mg / mL of polysorbate 80,

[0603] (c) 30 mg / mL to 50 mg / mL of sucrose,

[0604] (d) 90 mM to 110 mM arginine hydrochloride, and

[0605] (e) 15 mM to 25 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 4.8 to 5.2.

[0606] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0607] (a) about 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0608] (b) about 0.3 mg / mL of polysorbate 80,

[0609] (c) about 37.5 mg / mL of sucrose,

[0610] (d) about 100 mM arginine hydrochloride, and

[0611] (e) about 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is about 5.0.

[0612] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0613] (a) 100 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0614] (b) 0.3 mg / mL of polysorbate 80,

[0615] (c) 37.5 mg / mL sucrose,

[0616] (d) 100 mM arginine hydrochloride, and

[0617] (e) 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0.

[0618] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0619] (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0620] (b) 0.6 mg / mL of polysorbate 80,

[0621] (c) 75 mg / mL sucrose,

[0622] (d) 10 mM methionine,

[0623] (e) 2000 U / mL of PH20, and

[0624] (f) 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0 or 6.0.

[0625] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0626] (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0627] (b) 0.6 mg / mL of polysorbate 80,

[0628] (c) 75 mg / mL sucrose,

[0629] (d) 2000 U / mL of PH20, and

[0630] (e) 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 6.0.

[0631] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0632] (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0633] (b) 0.6 mg / mL of polysorbate 80,

[0634] (c) 75 mg / mL sucrose,

[0635] (d) 10 mM methionine,

[0636] (e) 2000 U / mL of PH20, and

[0637] (f) 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0.

[0638] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0639] (a) 150 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0640] (b) 0.6 mg / mL of polysorbate 80,

[0641] (c) 75 mg / mL sucrose, and

[0642] (d) 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0.

[0643] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0644] (a) 120 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0645] (b) 0.6 mg / mL of polysorbate 80,

[0646] (c) 75 mg / mL sucrose, and

[0647] (d) 20 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 6.0.

[0648] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0649] (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0650] (b) 0.3 mg / mL of polysorbate 80,

[0651] (c) 37.5 mg / mL sucrose,

[0652] (d) 5 mM methionine,

[0653] (e) 1000 U / mL of PH20, and

[0654] (f) 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0 or 6.0.

[0655] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0656] (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0657] (b) 0.3 mg / mL of polysorbate 80,

[0658] (c) 37.5 mg / mL sucrose,

[0659] (d) 1000 U / mL of PH20, and

[0660] (e) 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 6.0.

[0661] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0662] (a) 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0663] (b) 0.3 mg / mL of polysorbate 80,

[0664] (c) 37.5 mg / mL sucrose,

[0665] (d) 5 mM methionine,

[0666] (e) 1000 U / mL of PH20, and

[0667] (f) 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0.

[0668] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0669] (a) 75 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0670] (b) 0.3 mg / mL of polysorbate 80,

[0671] (c) 37.5 mg / mL of sucrose, and

[0672] (d) 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 5.0.

[0673] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0674] (a) 60 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR,

[0675] (b) 0.3 mg / mL of polysorbate 80,

[0676] (c) 37.5 mg / mL of sucrose, and

[0677] (d) 10 mM histidine-histidine hydrochloride buffer; the pH of the reconstituted solution is 6.0.

[0678] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation, or an intramuscular injection preparation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is an intravenous injection preparation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is a subcutaneous injection preparation.

[0679] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for intravenous injection. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any of the above items is suitable for subcutaneous injection.

[0680] In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation as described in any of the above items 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 as described in any of the above items is used to prepare a drug for intravenous injection. In some embodiments, the pharmaceutical composition, reconstituted solution, or lyophilized formulation as described in any of the above items is used to prepare a drug for subcutaneous injection.

[0681] The present disclosure also provides a drug kit comprising at least one container, each container independently containing the pharmaceutical composition as described in any one of the above items, the lyophilized formulation as described in any one of the above items, or the reconstituted solution as described in any one of the above items.

[0682] In some embodiments, the present disclosure also provides a method for diagnosing, treating, or alleviating a condition in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition, lyophilized formulation, reconstituted solution, or kit as described above.

[0683] In some embodiments, the present disclosure also provides a method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition as described in any one of the above, the lyophilized formulation as described in any one of the above, the reconstituted solution as described in any one of the above, or the drug kit as described in any one of the above.

[0684] In some embodiments, the present disclosure further provides the pharmaceutical composition as described in any of the above, the lyophilized formulation as described in any of the above, the reconstituted solution as described in any of the above, or the drug kit as described in any of the above, for use in treating or preventing a disease.

[0685] In one aspect, the present disclosure also provides use of the pharmaceutical composition as described in any of the preceding items, the lyophilized formulation as described in any of the preceding items, the reconstituted solution as described in any of the preceding items, or the kit as described in any of the preceding items in the preparation of a medicament for preventing or treating a disease or condition.

[0686] In some specific embodiments, the disease described in any 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.

[0687] In some specific embodiments, the lung cancer is non-small cell lung cancer.

[0688] In some specific embodiments, the lung cancer is metastatic non-small cell lung cancer.

[0689] In some specific embodiments, the lung cancer is small cell lung cancer.

[0690] In some specific embodiments, the lung cancer is human lung adenocarcinoma.

[0691] In some specific embodiments, the lung cancer is gastric cancer.

[0692] In some specific embodiments, the tumor is an EGFR and / or HGFR-related tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0693] Figure 1: Schematic diagram of the structure of Format1.

[0694] Figure 2: Schematic diagram of the structure of Format2.

[0695] Figure 3: Graph showing the results of an antibody binding activity assay with EGFR CHO-S cells, wherein the ordinate represents mean fluorescence intensity (MFI, the same below).

[0696] Figure 4: Antibody binding activity assay results for HGFR CHO-S cells.

[0697] Figure 5: Antibody binding activity assay results for H1975-HGF cells.

[0698] Figure 6: Antibody binding activity assay results for MKN-45 cells.

[0699] Figure 7: Antibody inhibition experiment results on cell EGFR phosphorylation.

[0700] FIG8 : The results of the antibody inhibition experiment on cell HGFR phosphorylation.

[0701] FIG9 : The results of the antibody inhibition experiment on cellular AKT phosphorylation.

[0702] FIG10 : Experimental results of antibody reduction of cell surface HGFR.

[0703] FIG11 : Antibodies inhibit the proliferation of SNU-5 cells.

[0704] FIG12 : ADCC killing test results of antibodies on Hs746T cells.

[0705] FIG13 : ADCC killing test results of antibodies on H292 cells.

[0706] FIG. 14 : Antibodies inhibit the growth of HCC827 mouse tumor cells. DETAILED DESCRIPTION

[0707] the term

[0708] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0709] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0710] Unless the context clearly requires otherwise, in the patent specification and claims, the words "comprising," "having," "including," and the like should be construed in the sense of "including but not limited to," rather than in an exclusive or exhaustive sense.

[0711] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not.

[0712] It will be understood by those skilled in the art that when reference is made to a numerical range, a cutoff value, or a specific value, "about" can be expressed as being within one or more than one standard deviation. Alternatively, "about" can be expressed as being within a range of up to 20% (i.e., ± 20%). Since many numerical values ​​used herein are determined experimentally, it will be understood by those skilled in the art that such determinations can differ between different experiments and typically differ between different experiments. Due to this inherent difference, it is believed that the values ​​used herein should not be overly limited. Therefore, the term "about" is used to encompass variations of ± 20% or less, ± 10% or less, ± 5% or less, ± 1% or less, ± 0.5% or less, or ± 0.1% or less from a specified value.

[0713] Although the present disclosure provides content ranges or content values, persons of ordinary skill in the art understand that the content ranges or content values ​​encompass an acceptable error range for the specific values ​​determined.

[0714] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0715] "HGFR" should be understood broadly and is intended to encompass various forms of HGFR molecules at various stages in mammals, such as, but not limited to, molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the HGFR gene (e.g., precursor HGFR, mature HGFR, membrane-expressed HGFR, HGFR splice variants, modified HGFR, or fragments thereof); the term also encompasses HGFR artificially prepared or expressed in vitro.

[0716] "EGFR" should be understood broadly and is intended to cover various forms of EGFR molecules at various stages in the mammalian body, such as but not limited to molecules produced during the 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); the term also covers artificially prepared or in vitro expressed EGFR.

[0717] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as 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 naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, a carboxyl 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 naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0718] The term "antibody" is used in the broadest sense and covers various 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 antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Native antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric proteins of approximately 150,000 daltons, composed of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0719] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures. According to the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. According to the number of antigen-binding regions, it can be divided into bispecific antibodies of divalent, trivalent, tetravalent or more valence. According to whether the structure is symmetrical, it can be divided into bispecific antibodies of symmetrical structure and bispecific antibodies of asymmetrical structure. Among them, fragment-type bispecific antibodies, such as Fab fragments lacking Fc fragments, form bispecific antibodies by combining two or more Fab fragments into one molecule. It has low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab′)2, scFv-Fab, (scFv)2-Fab, etc. IgG-like bispecific antibodies (e.g., with an Fc fragment) have a relatively large molecular weight. The Fc fragment helps purify the antibody and improve its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 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, F(ab)4-CrossMAb and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11;2019:4516041).

[0720] The term "variable region" or "variable domain" refers to the domain of an antigen-binding molecule that binds to an antigen. Herein, the heavy chain variable region in the antigen-binding moiety 1 that specifically binds to HGFR is labeled M-VH, and the light chain variable region is labeled M-VL; the heavy chain variable region in the antigen-binding moiety 2 that specifically binds to EGFR is labeled E-VH, and the light chain variable region is labeled E-VL. VH and VL each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; 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; the three CDR regions in M-VL are designated M-LCDR1, M-LCDR2, and M-LCDR3; the three CDR regions in E-VH are designated E-HCDR1, E-HCDR2, and E-HCDR3; and the three CDR regions in E-VL are designated E-LCDR1, E-LCDR2, and E-LCDR3. Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0721] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "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), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc. The correspondence between various numbering systems is well known to those skilled in the art. The numbering convention of the present disclosure is shown in Table 1 below.

[0722] Table 1. Relationships between CDR numbering systems

[0723] Unless otherwise indicated, the variable region and CDR sequences in the disclosed embodiments are all subject to the "Kabat" numbering convention. Although the Kabat numbering convention is used to define amino acid residues in specific embodiments, corresponding technical solutions provided by other numbering systems will be considered equivalent technical solutions.

[0724] The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that retains the antigen-binding ability of the intact antibody. 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 formed from antibody fragments.

[0725] Fab refers to a protein composed of VH and CH1 (Fab heavy chain) and VL and CL (Fab light chain) of an immunoglobulin.

[0726] Fv refers to the antigen-binding domain composed of the VH and VL of an immunoglobulin.

[0727] In the present disclosure, in some embodiments, the first Fab has a structure of Fab. In the replaced Fab, CH1 and CL are replaced by titin chain or obscurin chain, respectively.

[0728] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including native Fc regions and modified Fc regions. In some embodiments, the Fc region comprises two identical or different subunits. In some embodiments, the Fc region of the human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise indicated, the numbering convention for the Fc region is the EU index.

[0729] The term "Titin chain" refers to a 78-118 amino acid fragment of the titin protein, which comprises a peptide segment of the titin Ig-like 152 domain or a functional variant thereof. The titin chain is capable of forming a dimerization complex with the obscurin chain.

[0730] The term "obscurin chain" refers to an 87-117 amino acid fragment of the obscurin protein that contains a peptide fragment of the obscurin Ig-like 1 domain or a functional variant thereof; or refers to a 78-118 amino acid fragment of the obscurin-like 1 protein that contains a peptide fragment of the obscurin-like Ig-like 1 domain or a functional variant thereof. The obscurin chain is capable of binding to the titin chain to form a dimerization complex. The titin chain and obscurin chain disclosed herein can be used to replace the CH1 and CL in Fab to form a replaced Fab (Fab-S). This replacement does not affect the binding of the antigen-binding molecule to the antigen.

[0731] The term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to a target antigen. Antigen binding moieties include antibodies and fragments thereof as described herein. Specific antigen binding moieties include the antigen binding domain of an antibody, which comprises an antibody heavy chain variable region and an antibody light chain variable region. The term "antigen binding moiety that specifically binds to HGFR" refers to a module that can bind to HGFR or its epitope with sufficient affinity. In certain embodiments, the antigen binding moiety that specifically binds to HGFR has the following equilibrium dissociation constant (KD): <about 1 μM, <about 100 nM or <about 10 nM or less, which is measured by the Biacore method. In certain embodiments, the antigen binding moiety that specifically binds to HGFR binds to a conserved epitope in HGFR from different species. The term "antigen binding moiety that specifically binds to EGFR" refers to a module that can bind to EGFR or its epitope with sufficient affinity so that the molecule containing the module can be used as a diagnostic and / or therapeutic agent targeting EGFR. In certain embodiments, the antigen binding moiety 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 certain embodiments, the antigen binding moiety that specifically binds to EGFR binds to a conserved epitope in EGFR from different species. The antigen binding moiety comprises an antibody fragment as defined herein, such as a Fab, a replaced Fab, or a Fv.

[0732] In this context, the ordinal numbers in "antigen binding module 1", "antigen binding module 2", "linker 1" and "linker 2" are only used to distinguish different technical features and chemical entities; they do not limit any order, level or quantity.

[0733] The term "linker" refers to a connecting unit that connects two polypeptide fragments. In this article, the linkers appearing in the same or different structural formulas can be the same or different. The linker can be a peptide linker that contains one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used in this article can be the same or different. When "-" appears in the structural formula, it means that the units on both sides are directly connected by a covalent bond. When the term "bond" appears in a structural unit, it means that the units on both sides of the unit are directly connected.

[0734] Unless otherwise specified, the variable region and CDR sequences in the disclosed examples are all numbered using the "Kabat" convention.

[0735] A "pharmaceutical composition" refers to a composition containing one or more bispecific antibodies described herein, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and enabling its biological activity. As used herein, the terms "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0736] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0737] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react 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 types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods.

[0738] "Buffer" refers to a buffer that tolerates changes in pH through 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.

[0739] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, histidine-histidine sulfate, and the like, with histidine-histidine hydrochloride buffer being preferred. Histidine-histidine hydrochloride buffer can be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

[0740] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0741] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0742] "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, and the like. A preferred phosphate buffer is citric acid-disodium hydrogen phosphate.

[0743] An "acetate buffer" is a buffer comprising 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, and the like. A preferred acetate buffer is acetic acid-sodium acetate.

[0744] "Poloxamer" is a block copolymer of ethylene oxide and propylene oxide that is water-soluble and used as a surfactant in pharmaceutical formulations. Examples of poloxamers include poloxamer 188 (P188).

[0745] A "lyophilized preparation" refers to a pharmaceutical composition in liquid or solution form, or the preparation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step. Generally, freeze-drying involves pre-freezing, primary drying, and secondary drying. The purpose of pre-freezing is to freeze the product to obtain a crystalline solid. In some embodiments, the pre-freezing temperature is set at -45°C, and the pre-freezing rate is set at 1°C / min. Primary drying, also known as main drying, is the primary stage of freeze-drying the sample. Its purpose is to remove ice from the product while maintaining its shape and minimizing damage. Improper selection of the primary drying temperature and vacuum level can lead to product collapse. Higher temperatures and vacuum levels accelerate freeze-drying efficiency, but also increase the risk of product collapse. In some embodiments, the primary drying temperature can be conventional in the art, such as -30°C to 0°C. Secondary drying, also known as desorption drying, is the primary step in removing bound water from the product by applying an extreme vacuum (0.01 mbar) and increasing the temperature (20°C to 40°C). Because most biological products are temperature-sensitive, the secondary drying temperature is often selected at the lower end of the temperature range, such as 25°C. The time of freeze drying is related to the freezer, the dosage of the freeze-dried preparation, and the container of the freeze-dried medicine. The adjustment of this time is well known to those skilled in the art.

[0746] Unless otherwise specified, the pharmaceutical compositions described herein are in solution form, and the solvent therein is water. The pharmaceutical compositions described herein can achieve a stable effect: the antibodies therein substantially retain their physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability as well as their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. There are currently a variety of analytical techniques for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0747] "Displacement" refers to the replacement of the solvent system in which the antibody protein is dissolved. For example, a high-salt or hypertonic solvent system containing the antibody protein is replaced by a physical manipulation using a buffer system that stabilizes the formulation, thereby stabilizing the antibody protein in the formulation. Such physical manipulations include, but are not limited to, ultrafiltration, dialysis, or centrifugation.

[0748] A stable formulation is one in which no significant changes are observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for a period of time, including 24 hours, 7 days, 1 month, 3 months, or 6 months. Also included are formulations that exhibit the desired characteristics after storage at 40°C for a period of time, including 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include: typically no more than about 20% or 10%, preferably no more than about 5%, of the antibody aggregates or degrades as measured by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation vary by no more than ±10%, preferably no more than ±5%. The formulation typically exhibits no more than about 10%, preferably no more than about 5%, of aggregation.

[0749] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured 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).

[0750] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0751] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.

[0752] "Administer," "give," and "treat," as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0753] "Treatment" means administering an internal or external therapeutic agent, such as a reconstituted solution comprising any of the present disclosures, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered to a patient or population treated in an amount effective to alleviate one or more symptoms of a disease to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0754] The reconstituted solution of the present disclosure can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection. A variety of dosing time schemes are contemplated herein, including but not limited to, single or multiple administrations at multiple time points, push administration and pulse infusion. In some embodiments, the reconstituted solution of the present disclosure is administered by subcutaneous injection.

[0755] The reconstituted solution of the present disclosure will be formulated, dosed and applied in a manner consistent with good medical practice. Factors considered 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 agent, the method of administration, the time schedule for administration, and other factors known to medical practitioners. Optionally, the reconstituted solution can also be formulated together with one or more other agents for preventing or treating the condition. The effective amount of such other agents depends on the amount of the antigen binding molecules present in the reconstituted solution, the type of condition or treatment, and other factors. It can be used with the same dosage and administration route as described herein, or with about 1 to 99% of the dosage described herein, or with any dosage, and is determined to be suitable by experience / clinical use. Details of one or more embodiments of the present disclosure are set forth in the above description. Although any method and material similar or identical to that described herein can be used to implement or test the present disclosure, preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will be apparent from the description and claims. In the description and claims, unless otherwise clearly indicated in the context, the singular form includes the case of plural referents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference. The following examples are presented to more fully illustrate the preferred embodiments of the present disclosure. These examples should not be construed in any way to limit the scope of this disclosure, which is defined by the claims.

[0756] Example - Preparation and detection of bispecific antibodies specifically binding to HGFR and EGFR

[0757] PCT / CN2022 / 105714 (application date: 2022.07.14; priority patent application number: CN202110794137.9) is incorporated into this disclosure in its entirety by reference.

[0758] Example 1: Preparation of antigenic proteins and antibodies

[0759] 1.1 Antigen protein structure

[0760] Human EGFR protein (UniProt Epidermal growth factor receptor, UniProt No.: P00533) was used as the template for EGFR. A His tag was fused to the EGFR protein to design the extracellular domain of human EGFR protein with a His tag (abbreviated as EGFR-His) for detection in the present invention. The amino acid sequence is as follows:

[0761] Amino acid sequence of EGFR-His:

[0762] Note: The underlined part is the extracellular domain of EGFR protein; the italicized part is the His tag.

[0763] Human HGFR protein (UniProt Hepatocyte growth factor receptor, UniProt No.: P08581) was used as a template for HGFR. A His tag or Fc tag was fused to the HGFR protein to design the extracellular domain of HGFR protein with a His tag (abbreviated as HGFR-His) and the extracellular domain of HGFR protein with an Fc tag (abbreviated as HGFR-Fc) for detection in the present invention. The amino acid sequences are as follows:

[0764] Amino acid sequence of HGFR-His:

[0765] Note: The underlined part is the extracellular domain of HGFR protein; the italicized part is the His tag.

[0766] Using human HGF protein (UniProt Hepatocyte growth factor, UniProt No.: P14210) as a template for HGF, a His-tagged HGF protein (abbreviated as HGF-His) for detection in this disclosure was designed. Its amino acid sequence is as follows:

[0767] Amino acid sequence of HGF-His:

[0768] Note: The underlined part is HGF protein; the italicized part is His tag.

[0769] 1.2 Protein purification

[0770] 1.2.1 Recombinant protein purification steps:

[0771] The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS, and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with a PBS solution containing 5mM imidazole and rinsed for 2-5 column volumes. The replaced cell supernatant sample was loaded onto a Ni Sepharose Excel column (GE, 17-3712-02). The column was rinsed with a PBS solution containing 5mM imidazole until the A280 reading dropped to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound proteins, and the effluent was collected. The target protein was then eluted with a PBS solution containing 300mM imidazole, and the elution peak was collected. The collected eluate was concentrated and further purified using gel chromatography Superdex200 (GE, 28-9893-35) with PBS as the mobile phase. The aggregate 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.

[0772] 1.2.2 Antibody purification steps:

[0773] The supernatant sample of cell expression was subjected to high-speed centrifugation to remove impurities, and the supernatant was subjected to MabSelect Sure (GE, 17-5438-01) affinity chromatography. The MabSelect Sure chromatography column was first regenerated with 0.2M NaOH, rinsed with pure water, and then equilibrated with PBS. After the supernatant was combined, it was washed with PBS until the A280 reading dropped to baseline. The target protein was eluted with 0.1M acetate buffer at pH 3.5 and neutralized with 1M Tris-HCl. After the eluted sample was appropriately concentrated, it was further purified using a PBS-equilibrated gel chromatography Superdex200 (GE, 28-9893-35). The target protein was collected and concentrated to an appropriate concentration in the receiving tube. This method is used to purify the antibodies involved in the present invention.

[0774] Example 2. Preparation and identification of bispecific antibodies that specifically bind to HGFR and EGFR

[0775] Antibodies that specifically bind to HGFR and EGFR are constructed using molecules that specifically bind to HGFR and EGFR.

[0776] 2.1 Molecules that specifically bind to EGFR

[0777] The molecule that specifically binds to EGFR can be derived from any suitable antibody, such as Zalutumumab (abbreviated as Zal) or its variants (such as Zal.1, which is obtained by mutating the first amino acid residue of the Zal light chain from A to D), wherein:

[0778] >Amino acid sequence of Zal heavy chain:

[0779] >Amino acid sequence of Zal light chain:

[0780] >Amino acid sequence of the heavy chain variable region of Zal:

[0781] >Amino acid sequence of the light chain variable region of Zal:

[0782] The amino acid sequence of the heavy chain variable region of Zal.1 is SEQ ID NO: 3

[0783] >Amino acid sequence of the light chain variable region of Zal.1:

[0784] The CDR sequences of Zal and Zal.1 are shown in Table 2:

[0785] Table 2. CDRs that specifically bind to EGFR molecules

[0786] Note: The above CDRs are confirmed according to the Kabat numbering system.

[0787] 2.2 Molecules that specifically bind to HGFR

[0788] Molecules that specifically bind to HGFR can be derived from any suitable antibody, including antibodies such as Onartuzumab (Omab) described in International Publication No. WO2013003680A1 (incorporated herein by reference in its entirety); Ab10 described in International Publication No. WO2016165580A1 (incorporated herein by reference in its entirety); and mutants of the aforementioned antibodies. The amino acid sequences of the variable regions of Omab and Ab10 are as follows:

[0789] >Amino acid sequence of the heavy chain variable region of Omab:

[0790] >Amino acid sequence of the light chain variable region of Omab:

[0791] >Amino acid sequence of the heavy chain variable region of Ab10:

[0792] >Amino acid sequence of the light chain variable region of Ab10:

[0793] By mutating the CDR of Ab10, a mutant antibody of Ab10 was obtained: Ab10.1; the constant region of Ab10.1 is the same as Ab10, and the amino acid sequence of the variable region of Ab10.1 is as follows:

[0794] >Amino acid sequence of the heavy chain variable region of Ab10.1:

[0795] >Amino acid sequence of the light chain variable region of Ab10.1:

[0796] Note: In the above sequence that specifically binds to the HGFR molecule, the underlined portion is the CDR region confirmed according to the Kabat numbering system, and the italicized bold portion is the mutated amino acid residue.

[0797] The CDRs that specifically bind to HGFR molecules are shown in Table 3:

[0798] Table 3. CDRs that specifically bind to HGFR molecules

[0799] Note: The above CDRs are confirmed according to the Kabat numbering system.

[0800] 2.3 Bispecific antibodies that specifically bind to HGFR and EGFR

[0801] Using molecules that specifically bind to HGFR and molecules that specifically bind to EGFR, bispecific antibodies with Format 1 or Format 2 structures that specifically bind to HGFR and EGFR are constructed. The structural diagram of Format 1 is shown in Figure 1, and the structural diagram of Format 2 is shown in Figure 2.

[0802] Format1 contains four chains with the following structure:

[0803] Chain 1 is:

[0804] VH (anti-EGFR antibody)-linker 1-VH (anti-HGFR antibody 1)-IgG1 (CH1)-IgG1Fc (knob),

[0805] Chain 2 is: VL (anti-EGFR antibody)-linker 1-VL (anti-HGFR antibody 1)-CL,

[0806] Chain 3 is: VH (anti-HGFR antibody 2)-linker 2-Titin chain-IgG1Fc (hole),

[0807] Chain 4 is: VL (anti-HGFR antibody 2)-Linker 2-Obscurin chain.

[0808] Format2 contains four chains with the following structure:

[0809] Chain 1 is: VH (anti-HGFR antibody)-IgG1 (CH1)-IgG1Fc (knob),

[0810] Chain 2 is: VL (anti-HGFR antibody)-CL,

[0811] Chain 3 is: VH (anti-EGFR antibody)-linker 2-Titin chain-IgG1Fc (hole),

[0812] Chain 4 is: VL (anti-EGFR antibody)-Linker 2-Obscurin chain;

[0813] Exemplarily, 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) were used to construct EM1 with Format1 structure and EM2 with Format2 structure.

[0814] The amino acid sequences of the four chains of EM1 are as follows:

[0815] Amino acid sequence of chain 1 of EM1:

[0816] Amino acid sequence of chain 2 of EM1:

[0817] Amino acid sequence of chain 3 of EM1:

[0818] Amino acid sequence of chain 4 of EM1:

[0819] The amino acid sequences of the four chains of EM2 are shown below:

[0820] Amino acid sequence of chain 1 of EM2:

[0821] Amino acid sequence of chain 2 of EM2:

[0822] Amino acid sequence of chain 3 of EM2:

[0823] Amino acid sequence of chain 4 of EM2:

[0824] Note: In the above sequence, the single underlined part is the variable region, the dotted underlined part is the CL, the dotted underlined part is the Fc region, the italic part is CH1, the double underlined part is the linker, and the wavy part is the obscurin chain / Titin-T chain.

[0825] Amino acid sequence of linker 1: GGGGSGGGG (SEQ ID NO: 45)

[0826] Amino acid sequence of linker 2: GGGGS (SEQ ID NO: 46)

[0827] Amino acid sequence of IgG1Fc (knob):

[0828] Amino acid sequence of IgG1Fc(hole):

[0829] CL:

[0830] T.16 (Titin Chain)

[0831] O.28 (Obscurin chain)

[0832] Test Case

[0833] Test Example 1: Biacore detection of in vitro binding affinity and kinetics experiments

[0834] The affinity of the test molecule to human EGFR (SEQ ID NO: 42) or human HGFR protein (SEQ ID NO: 43) was determined by Biacore T200 (GE) as follows:

[0835] Antibody molecules were affinity-captured using a Protein A biosensor chip. The antigen was then passed over the chip surface for 180 seconds, followed by 600 seconds of dissociation. Binding and dissociation curves were obtained using a Biacore T200 instrument to monitor the reaction signal in real time. After each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Gly-HCl (pH 1.5). A 1:1 model was used for data fitting to obtain affinity values ​​for the test molecules. In this example, the positive control, MCLA, is a bispecific antibody that specifically binds to HGFR and EGFR (for the specific sequence, see PB8532 in WO2019031965A1).

[0836] The experimental results are shown in Table 4. The experimental results show that the mutant antibodies and bispecific antibodies constructed in the present disclosure have very strong affinities, and the affinity of Ab10.1 is at least 4 times that of the parent antibody Ab10.

[0837] Table 4: In vitro affinity of the antibodies of the present invention to human EGFR or human HGFR

[0838] Test Example 2: In vitro cell binding assay of antibodies

[0839] The cell binding activity of the bispecific antibody that specifically binds to HGFR and EGFR was detected by flow cytometry. 6 After blocking cells with 1% BSA / PBS buffer (100 μg / mL) of EGFR CHO-S stably transfected cells, HGFR CHO-S stably transfected cells, H1975-HGF, or MKN-45 tumor cell lines, cells were added with different diluted antibody samples (C25 was a negative control, which is an IgG1 antibody protein against an irrelevant target, the same below) and incubated for 1 hour. After washing twice, R-PE-goat anti-human (H+L) antibody (Invitrogen, CAT#H10104) was added and incubated for 0.5 hours. After washing twice, fluorescence signal values ​​were read using a flow cytometer.

[0840] The experimental results are shown in Figures 3 to 6, which show that the bispecific antibody specifically binding to HGFR and EGFR disclosed herein has a stronger binding ability to tumor cells than the positive antibody MCLA.

[0841] Test Example 3: Antibody blocking experiment of EGFR binding to EGF

[0842] The blocking ability of the antibody against EGF / EGFR was tested by ELISA. Coat with 1 μg / mL rabbit anti-His antibody (GenScript, A01857) and block at 4°C overnight. After washing the plate, add 2 μg / mL EGFR-His (SEQ ID NO: 42) and incubate for 1 hour. After washing the plate, add 2 μg / mL EGF-mFc (Acro, EGF-H525b) and antibodies diluted at different concentrations and incubate for 1 hour. After washing the plate, add horseradish peroxidase-goat anti-mouse IgG antibody (Jackson, 115-035-062) and incubate for 1 hour. After washing the plate, add tetramethylbenzidine solution for color development, and finally add stop solution, and measure the OD450 value on a microplate reader. In the examples, the positive control JNJ is a bispecific antibody that specifically binds to HGFR and EGFR (JNJ is also called Amivantamab, for its specific sequence structure, see WHO Drug Information, 33(2): 237-239).

[0843] The experimental results are shown in Table 5. The experimental results show that the bispecific antibody specifically binding to HGFR and EGFR disclosed herein can effectively block the binding of EGF to EGFR.

[0844] Table 5. Experimental results of bispecific antigen binding molecules blocking the binding of EGF to EGFR

[0845] Test Example 4: Antibody antagonism blocking experiment of HGFR binding to HGF

[0846] The blocking ability of the antibody against HGF / HGFR was tested by ELISA. 5 μg / mL HGFR-His (sequence see SEQ ID NO: 43) was coated and blocked overnight at 4°C. After washing, 2 μg / mL bio-HGF-His (sequence see SEQ ID NO: 44) and different concentrations of diluted antibodies 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, tetramethylbenzidine solution was added for color development, and finally, stop solution was added and the OD450 value was measured on a microplate reader.

[0847] The experimental results are shown in Table 6, which show that the maximum blocking ability of the bispecific antibody specifically binding to HGFR and EGFR disclosed herein to block HGFR binding to HGF is stronger than that of the positive antibody JNJ.

[0848] Table 6. Results of experiments in which bispecific antigen-binding molecules blocked the binding of HGF to HGFR

[0849] Test Example 5: Antibody Inhibition Experiment on Cellular EGFR Phosphorylation

[0850] H292 cells (also known as NCI-H292, human lung cancer cells (lymph node metastasis), cell bank of the Chinese Academy of Sciences, hereinafter referred to as the same) were plated at 12,000 cells / well in a 96-well plate (Corning, 3599) in RPMI1640 + 10% FBS. The 96-well plates were then incubated in a 37°C, 5% CO2 incubator for 24 hours. 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) for 16-20 hours. On the third day, the antibody was prepared with an initial concentration of 6 μM and diluted 5-fold. The starvation medium was replaced and the cells were cultured in a 37°C, 5% CO2 incubator for 1 hour. During this period, 50 μL of 100 ng / mL rEGF (R&D, 236-EG-200) was added and the cells were cultured in a 37°C, 5% CO2 incubator for another 15 minutes. Finally, phosphorylated EGFR was detected according to the instructions of PHOSPHO-EGFR (TYR1068) KITS (Cisbio, 64EG1PEG).

[0851] The experimental results are shown in FIG7 , which show that the bispecific antibody disclosed herein that specifically binds to HGFR and EGFR can effectively inhibit cellular EGFR phosphorylation.

[0852] Test Example 6: Antibody Inhibition Experiment on Cellular HGFR Phosphorylation

[0853] H292 cells were plated at 12,000 cells / well in a 96-well plate (Corning, 3599) in RPMI1640 + 10% FBS. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours. 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) for 16-20 hours. On the third day, the antibody was prepared with an initial concentration of 1 μM and diluted 10-fold. The starvation medium was replaced and the cells were cultured in a 37°C, 5% CO2 incubator for 1 hour. During this period, 50 μL of 200 ng / mL rHGF (R&D, 294-HG-005) was added and cultured in a 37°C, 5% CO2 incubator for 30 minutes. Finally, phosphorylated HGFR was detected according to the instructions of the pc-Met (Tyr1234 / 1235) Assay Kit (PerkinElmer, ALSU-PCMET-A50).

[0854] The experimental results are shown in FIG8 , which show that the bispecific antibody disclosed herein that specifically binds to HGFR and EGFR can effectively inhibit cellular HGFR phosphorylation, and its inhibitory ability is stronger than that of Ab10.1.

[0855] Test Example 7: Antibody Inhibition Experiment on Cellular AKT Phosphorylation

[0856] H292 cells were plated at 12,000 cells / well in a 96-well plate (Corning, 3599) in RPMI1640 + 10% FBS. The plates were then incubated at 37°C in a 5% CO2 incubator for 24 hours. 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) for 16-20 hours. On the third day, the antibody was prepared at an initial concentration of 4 μM and serially diluted 5-fold. The starvation medium was replaced and the cells were cultured in a 37°C, 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. The cells were cultured in a 37°C, 5% CO2 incubator for another 1 hour. Finally, phosphorylated AKT was detected according to the instructions of PHOSPHO-AKT (SER473) KITS (Cisbio, 64AKSPEG). The level of intracellular AKT phosphorylation was reflected by the ratio of the fluorescence signal value at 665 nm to the fluorescence signal value at 620 nm (ratio = fluorescence signal value at 665 nm / fluorescence signal value at 620 nm * 10). 4 , the higher the ratio, the higher the intracellular AKT phosphorylation level.

[0857] The experimental results are shown in FIG9 , which show that the bispecific antibody disclosed herein that specifically binds to HGFR and EGFR can effectively inhibit cellular AKT phosphorylation, and its inhibitory ability is stronger than that of the positive control antibody.

[0858] Test Example 8: Antibodies reduce HGFR expression on the cell surface

[0859] HCC827 cells (human non-small cell lung cancer cells, from ATCC) were cultured at 3×10 5Cells were plated in 6-well plates in RPMI 1640 medium plus 10% FBS and incubated at 37°C in a 5% CO2 incubator for 24 hours. Antibodies were prepared to a concentration of 200 nM and treated for 18 hours. After treatment, cells were washed once with PBS and harvested with 500 μL of enzyme-free cell dissociation buffer (Gibco, #13151). The cells were separated by centrifugation from the enzyme-free cell dissociation buffer. Immunofluorescence staining was performed using a goat anti-human HGFR antibody (R&D systems, AF276) for 1 hour at 4°C. Subsequently, the cells were washed twice with PBS containing 2% FBS. ALEXA488-donkey anti-goat secondary antibody (Thermo Fisher, A-11055) was added and reacted for 1 hour at 4°C. After washing twice, the cells were mounted with BD Cytofix (BD, #554655). After washing once with PBS, fluorescence signals were read using a flow cytometer.

[0860] The experimental results are shown in FIG10 , which show that compared with the positive control antibody, the bispecific antibody disclosed herein that specifically binds to HGFR and EGFR can more effectively reduce HGFR on the cell surface and inhibit the receptor signaling pathway.

[0861] Test Example 9: Antibody inhibition experiment on SNU-5 cell proliferation

[0862] SNU-5 cells (human gastric cancer cells, from ATCC) were plated at 4000 cells / well in a 96-well plate (Corning, 3903) in IMDM + 5% FBS. The 96-well plate was then incubated at 37°C, 5% CO2 for 24 hours. Antibodies were prepared to a concentration of 5 μM, diluted 5-fold, and added to the 96-well plate. The plates were then incubated at 37°C, 5% CO2 for 5 days. Finally, the antibodies were prepared according to the manufacturer's instructions. Luminescent Cell Viability Assay (Promega, G7573) was used. 50 μL CTG was added to each well and incubated at room temperature for 10 min. After sealing with a sealing film, the fluorescence intensity (Luminescence, abbreviated as Lum) was detected using a multi-label microplate detection system (PerkinElmer, victor3).

[0863] Calculation formula: Lum = measured value - culture medium reading;

[0864] % inhibition = (Lum0 - Lum) / Lum0*100%;

[0865] Lum0 is the fluorescence intensity of the well with an antibody concentration of 0, and Lum is the fluorescence intensity of the well with each antibody dilution concentration.

[0866] The experimental results are shown in FIG11 , which show that the bispecific antibody specifically binding to HGFR and EGFR disclosed herein has a stronger ability to inhibit the proliferation of SNU-5 cells than the positive antibody.

[0867] Test Example 10: Antibody-to-tumor cell ADCC killing experiment

[0868] The antibody was prepared to 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 collected, washed once with PBS, and the cell density was adjusted to 1×10 with buffer (PBS + 10% FBS + 20mM HEPES). 6 10 cells / mL, then 3 μL BATDA reagent (PerkinElmer, AD0116) was added to 2 mL of cells and incubated at 37°C, 5% CO2 incubator for 15 min. After centrifugation, the cells were washed three times with rinsing buffer (PBS + 20 mM HEPES) and counted to adjust the cell density to 1×10 5 Collect NK92-FCGR3A(176V) cells (NK92 cells stably transformed with FCGR3A(176V) (purchased from Nanjing Kebai Biotechnology Co., Ltd.)), wash once with PBS, resuspend the cells in culture medium, count the cells, and adjust the cell density to 5×10 5 / mL. Finally, 100 μL NK92--FCGR3A (176V) cells, 50 μL diluted antibody, and 50 μL labeled tumor cells were added to a 96-well plate (corning, 3788). At the same time, a spontaneous control release well was set up, which did not contain NK92--FCGR3A (176V) cells but contained 100 μL of culture medium; a positive control well was set up, which contained 10 μL lysis buffer + 50 μL labeled tumor cells + 140 μL culture medium; a background control well was set up, which contained 50 μL labeled cell supernatant + 150 μL culture medium. The 96-well plate was then centrifuged at 300 rpm for 2 minutes and incubated in a 37°C, 5% CO2 incubator for 2 hours. Transfer 100 μL of the supernatant to a new 96-well plate (corning, 3788) and centrifuge at 300 g for 5 minutes. Transfer 20 μL to an assay plate (PerkinElmer, AD0116). Add 200 μL of europium solution (PerkinElmer, AD0116) and incubate at room temperature with shaking for 15 minutes. Finally, measure TRF. EM1(afuc) represents defucosylated EM1, and EM2(afuc) represents defucosylated EM2.

[0869] Calculation formula: specific release % = (experimental release - spontaneous release) / (maximum release - spontaneous release) * 100.

[0870] The experimental results are shown in Figures 12 and 13, which show that the ADCC killing ability of the bispecific antibody specifically binding to HGFR and EGFR disclosed herein can be significantly improved after defucosylation.

[0871] Test Example 11: In vivo efficacy study of the antibody in the mouse H1975-HGF model

[0872] H1975-HGF cells (H1975 cells stably transfected with human HGF (human lung adenocarcinoma cells, ATCC)) (5×10 6 The cells were inoculated subcutaneously in the right flank of CD1 nude female mice (Vitamin B).

[0873] When the tumor size is 189 mm 3 At the same time, the animals were randomly divided into groups according to the size of the tumor, with 10 animals in each group, including the C25-3mpk group (negative control, which is an IgG1 antibody protein of an unrelated target, with a dosage of 3 mg / kg), the JNJ-3mpk group (positive control, with a dosage of 3 mg / kg) and the EM1-3.5mpk group (EM1 test group, with a dosage of 3.5 mg / kg). Starting from the animal group (0 day), the drug was administered twice a week by intraperitoneal injection for 2 to 3 consecutive weeks. The tumor volume was measured twice a week, the mice were weighed, and the data were recorded. The data were statistically analyzed by T-test. Among them, C25-3mpk, JNJ-3mpk, and EM1-3.5mpk were equimolar.

[0874] Tumor volume (T) calculation formula: T = 1 / 2 length × (short) 2 ;

[0875] Relative tumor proliferation rate T / C% = (T-T0) / (C-C0) × 100%;

[0876] Tumor inhibition rate TGI% = 1-T / C%;

[0877] Wherein, T and T0 are the tumor volumes of the drug-treated group, wherein T0 is the tumor volume at the beginning of the experiment; C and C0 are the tumor volumes of the negative control group, wherein C0 is the tumor volume of the negative control group at the beginning of the experiment.

[0878] The experimental results are shown in Table 7, which show that the bispecific antibody that specifically binds to HGFR and EGFR disclosed herein can significantly inhibit the growth of tumor cells. On day 21, the average tumor volume of the equimolar bispecific antibody group that specifically binds to HGFR and EGFR disclosed herein was less than half of that of the positive control, and the tumor inhibitory efficacy was stronger than that of the positive control antibody JNJ.

[0879] Table 7. In vivo efficacy of antibodies in the mouse H1975-HGF model

[0880] Test Example 12: In vivo efficacy study of antibodies in the mouse HCC827 model

[0881] HCC827 cells (human non-small cell lung cancer cells, from ATCC) (8.555×10 6 The cells were inoculated subcutaneously in the right flank of NUNU female mice (Weitonglihua).

[0882] When the tumor size is 200 mm 3 At the time of the study, animals were randomly divided into groups according to tumor size, with 10 animals in each group, including the C25-3mpk group (negative control, in which C25 is an IgG1 antibody protein with an irrelevant target, and the dose was 3 mg / kg), the MCLA-3mpk group (positive control, and the dose was 3 mg / kg), the EM1-3.5mpk group (EM1 high-dose test group, and the dose was 3.5 mg / kg), the EM1-1.17mpk group (EM1 low-dose test group, and the dose was 1.17 mg / kg), and the EM2-3mpk group (EM2 test group, and the dose was 3 mg / kg). Starting from the animal grouping (day 0), the drug was administered twice a week by intraperitoneal injection for 2 to 3 consecutive weeks. The tumor volume was measured twice a week, the mice were weighed, and the data were recorded. Among them, C25-3mpk, MCLA-3mpk, EM1-3.5mpk, and EM2-3mpk were in equimolar amounts.

[0883] The experimental results are shown in FIG14 , which show that the bispecific antibody disclosed herein that specifically binds to HGFR and EGFR can significantly inhibit the growth of tumor cells, and the tumor inhibition effect is stronger than that of the positive control antibody.

[0884] Preparation Example - Bispecific Antibody Preparation Specific for HGFR and EGFR

[0885] SEC size exclusion chromatography:

[0886] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0887] SEC% (SEC monomer content percentage) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, A total is the sum of all peak areas). ΔSEC% = SEC% of the formulation after the stability experiment - SEC% of the formulation before the stability experiment.

[0888] SEC measurement instrument: Agilent HPLC 1260;

[0889] Column: Waters, BioResolve™ SEC mAb 2.5μm, 7.8×300mm.

[0890] NR-CE capillary gel electrophoresis:

[0891] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0892] NR-CE%=Amainpeak / Atotal×100% (Amainpeak is the peak area of ​​the main peak in the sample, and Atotal is the sum of all peak areas).

[0893] ΔNR-CE%=NR-CE% of the formulation after the stability experiment-NR-CE% of the formulation before the stability experiment.

[0894] CE measurement instrument: Beckman capillary electrophoresis instrument, model PA800plus.

[0895] IEC ion exchange chromatography:

[0896] A chromatographic method that uses ion exchange resin or chemically bonded ion exchanger as the stationary phase and utilizes the difference in ion exchange capacity or selectivity coefficient of the separated components to achieve separation.

[0897] 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 the preparation after the stability test - IEC% of the preparation before the stability test.

[0898] IEC measurement instrument: Agilent HPLC 1260.

[0899] Osmolality determination:

[0900] The freezing point method for determining osmotic pressure is based on the fact that the freezing point depression value is directly proportional to the molar concentration of the solution. A highly sensitive temperature sensing element is used to determine the freezing point of the solution and convert the electrical quantity into osmotic pressure.

[0901] Instrument for osmotic pressure measurement: Loser, model OM815.

[0902] protein

[0903] The protein used in the following examples is the HGFR-EGFR bispecific antibody EM1 (hereinafter referred to as "protein").

[0904] The protein concentration was determined using a UV-visible spectrophotometer, model: Nano Drop oneC, with an optical path length of 1 mm.

[0905] Formulation Example 1. Screening of different ionic strengths

[0906] Formulations containing 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 (40°C for 4 weeks) were conducted to investigate the effects of different buffer systems on protein stability, using SEC and NR-CE as evaluation metrics.

[0907] The results are shown in Table 8. SEC data showed no significant difference between the two groups. NR-CE data showed that the 20 mM ionic strength group was superior to the 50 mM ionic strength group.

[0908] Table 8 Screening results of different ionic strengths

[0909] Note: His-HCl stands for histidine-histidine hydrochloride; 40℃ W4: 40℃ for 4 weeks, the same below.

[0910] Formulation Example 2. pH and buffer system screening

[0911] Formulations containing 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 (40°C for 4 weeks) were conducted to investigate the effects of different buffer systems on protein stability, using appearance, SEC, and NR-CE as evaluation criteria.

[0912] The results are shown in Table 9. Appearance data showed that the appearance of the preparations containing His-HCl and His-AA was superior to that of the other groups. SEC and NR-CE data showed that after 4 weeks at 40°C, the monomer purity of the preparations containing His-HCl (pH 6.0 or pH 6.5) was superior to that of the other groups.

[0913] Table 9 pH and buffer system screening results

[0914] 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; the same below.

[0915] Formulation Example 3. Sugar Type Screening

[0916] Formulations containing 100 mg / mL protein, the various sugars listed in Table 10, and 0.6 mg / mL PS80 were prepared in 20 mM His-HCl buffer (pH 6.0). Forced degradation studies (40°C for 4 weeks) were performed to investigate the effects of various sugar types on protein stability, using appearance, SEC, and NR-CE as evaluation criteria.

[0917] The results are shown in Table 10. The data of appearance and purity showed that after being placed at 40°C for 4 weeks, the preparation group containing sucrose was superior to the preparation group containing trehalose.

[0918] Table 10 Sugar type screening results

[0919] Formulation Example 4. Surfactant Screening

[0920] Formulations containing 100 mg / mL protein, 75 mg / mL sucrose, and the surfactants listed in Table 11 were prepared in 20 mM His-HCl buffer (pH 6.0). Forced degradation studies (25°C, 300 rpm shaking for 7 days; 40°C for 4 weeks) were performed to investigate the effects of different surfactants on protein stability using SEC and NR-CE.

[0921] The results are shown in Table 11. Appearance data showed that after 7 days of shaking, the appearance of the formulation containing 0.4 mg / mL PS80 was worse than that of the other formulations. After 4 weeks at 40°C, the appearance of the formulation containing 0.6 mg / mL poloxamer 188 (P188) was worse than that of the other formulations. NR-CE data showed that after 4 weeks at 40°C, the formulation containing 0.6 mg / mL poloxamer 188 (P188) was worse than that of the other formulations, while the formulation containing 0.4 mg / mL PS80 was slightly better. There was no significant difference between the formulations containing 0.6 mg / mL PS80 and 0.8 mg / mL PS80.

[0922] Table 11 Surfactant screening results

[0923] Note: Shake D7: shake for 7 days; 40℃ W4: place at 40℃ for 4 weeks.

[0924] Formulation Example 5. Lyophilization

[0925] The protein samples in Table 12 were prepared and lyophilized. The lyophilization parameters are shown in Table 13:

[0926] Table 12 Protein lyophilized preparation prescription design table

[0927] Table 13 Freeze-drying parameters for protein freeze-drying process development

[0928] *: The time of primary drying and secondary drying shall be adjusted according to the specific situation.

[0929] The appearance of the two freeze-dried finished products showed that the powder cakes were intact, without obvious collapse, and there was no significant difference in the appearance of the powder cakes.

[0930] Formulation Example 6. Freeze-dried stability 1

[0931] A formulation containing 37.5 mg / mL sucrose, 0.3 mg / mL PS80, and 50 mg / mL protein sample was prepared using 10 mM His-HCl (pH 6.0). Lyophilization process screening was performed using 20 mL vials and 20 mm lyophilization stoppers. Lyophilization parameters are shown in Table 14. The stability of the final process sample before and after lyophilization and at high temperature is shown in Table 15.

[0932] Table 14 Lyophilization parameters for protein lyophilization process development

[0933] *: The time of primary drying and secondary drying shall be adjusted according to the specific situation.

[0934] Table 15 Product stability data after freeze-drying

[0935] The results showed that the appearance was clear before freeze-drying, the powder was intact without obvious collapse after freeze-drying, and it was clear after re-dissolution; the high temperature (40℃) results showed that there was no significant change in SEC, NR-CE and IEC under 40℃ 4W conditions, and the stability was good.

[0936] Formulation Example 7. Relationship between Viscosity and pH

[0937] 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 were measured at 25° C. 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.

[0938] Table 16 Relationship between viscosity and pH

[0939] Note: N / A means that the sample could not be prepared due to its high viscosity.

[0940] Formulation Example 8. Freeze-dried stability 2 (subcutaneous preparation)

[0941] The protein samples in Table 17 were prepared and lyophilized. The lyophilization parameters are shown in Table 14:

[0942] Table 17 Protein lyophilized preparation prescription design table

[0943] Table 18 Protein reconstitution formula

[0944] Table 19 Product stability data after freeze-drying

[0945] The results showed that the appearance was clear before freeze-drying, the powder was intact without obvious collapse after freeze-drying, and it was clear after re-dissolution; the results after re-dissolution at 25℃ for 24 hours showed that there was no significant change in SEC, NR-CE and IEC, and the stability was good.

[0946] Formulation Example 9. Freeze-dried stability 3 (subcutaneous preparation)

[0947] The protein samples in Table 20 were prepared and lyophilized. The lyophilization parameters are shown in Table 14:

[0948] Table 20 Protein lyophilized preparation prescription design table

[0949] Table 21 Protein reconstitution formula

[0950] Table 22 Product stability data after freeze-drying

[0951] The results showed that the appearance was clear before freeze-drying, the powder was intact without obvious collapse after freeze-drying, and it was clear after re-dissolution; the high temperature results showed that there was no significant change in SEC and enzyme activity under 40℃4W conditions, and the stability was good.

[0952] Formulation Example 10. Freeze-dried stability 4 (subcutaneous preparation)

[0953] The protein samples in Table 23 were prepared and lyophilized. The lyophilization parameters are shown in Table 14:

[0954] Table 23 Protein lyophilized preparation prescription design table

[0955] Table 24 Protein reconstitution formula

[0956] Table 25 Product stability data after freeze-drying

[0957] Note: 40℃4W means that the product was placed at 40℃ for 4 weeks; 2-8℃M6 means that the product was placed at 4℃ for 6 months; 25℃M6 means that the product was placed at 25℃ for 6 months.

[0958] The results showed that the appearance was clear before freeze-drying, the powder was intact without obvious collapse after freeze-drying, and it was clear after re-dissolution; the results of 40℃4W, 25℃M6 and 2-8℃M6 showed that there was no significant change in purity and enzyme activity, and the stability was good.

[0959] Preparation Example 11. Final Formula Lyophilization 5 (Subcutaneous Preparation)

[0960] The protein samples in Table 26 were prepared and lyophilized. The lyophilization parameters are shown in Table 14:

[0961] Table 26 Protein lyophilized preparation prescription design table

[0962] Freeze-dried powder cakes were intact and showed no visible collapse. The reconstituted formulation contained 150 mg / mL protein, 25 mM His-HCl (pH 5.0), 70 mg / mL sucrose, 10 mM methionine, 0.6 mg / mL PS800, and 2000 U / mL hyaluronidase. The powder cakes were clear and transparent after reconstitution.

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, wherein the E-VH comprises E-HCDR1, E-HCDR2 and E-HCDR3, and the E-VL comprises 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, the E-HCDR1 is shown in SEQ ID NO: 6, the E-HCDR2 is shown in SEQ ID NO: 7, the E-HCDR3 is shown in SEQ ID NO: 8, the E-LCDR1 is shown in SEQ ID NO: 9, the E-LCDR2 is shown in SEQ ID NO: 10, and the E-LCDR3 is shown in 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 shown in SEQ ID NO: 30, the M-HCDR2 is shown in SEQ ID NO: 31, the M-HCDR3 is shown in SEQ ID NO: 32, the M-LCDR1 is shown in SEQ ID NO: 27, the M-LCDR2 is shown in SEQ ID NO: 33, and the M-LCDR3 is shown in SEQ ID NO: 29, or (ii) the M-HCDR1 comprises SEQ ID NO: 18, the M-HCDR2 comprises SEQ ID NO: 19, the M-HCDR3 comprises SEQ ID NO: 20, the M-LCDR1 comprises SEQ ID NO: 21, the M-LCDR2 comprises SEQ ID NO: 22, and the M-LCDR3 comprises SEQ ID NO: 23, or (iii) the M-HCDR1 is set forth in SEQ ID NO: 24, the M-HCDR2 is set forth in SEQ ID NO: 25, the M-HCDR3 is set forth in SEQ ID NO: 26, the M-LCDR1 is set forth in SEQ ID NO: 27, the M-LCDR2 is set forth in SEQ ID NO: 28, and the M-LCDR3 is set forth in 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 histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer.

2. The pharmaceutical composition according to claim 1, wherein 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.

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

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

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

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

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

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

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

10. The pharmaceutical composition according to claim 9, wherein the concentration of the excipient is 1 mM to 300 mM; preferably, the concentration of the excipient is 1 mM to 200 mM; more preferably, the concentration of the excipient is 1 mM to 20 mM.

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

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

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the bispecific antibody that specifically binds to HGFR and EGFR, (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 moieties 1, whose M-VH has: M-HCDR1 as shown in SEQ ID NO: 30, M-HCDR2 as shown in SEQ ID NO: 31, and M-HCDR3 as shown in SEQ ID NO: 32, and whose M-VL has: M-LCDR1 as shown in SEQ ID NO: 27, M-LCDR2 as shown in SEQ ID NO: 33, and M-LCDR3 as shown in SEQ ID NO: 29; another antigen binding moiety 1, whose M-VH has: M-HCDR1 as shown in SEQ ID NO: 18, M-HCDR2 as shown in SEQ ID NO: 19, and M-HCDR3 as shown in SEQ ID NO: 20, and whose M-VL has: M-LCDR1 as shown in SEQ ID NO: 21, M-LCDR2 as shown in SEQ ID NO: 22, and M-LCDR3 as shown in SEQ ID NO: 23; and The antigen binding moiety 2, whose E-VH has: E-HCDR1 as shown in SEQ ID NO: 6, E-HCDR2 as shown in SEQ ID NO: 7 and E-HCDR3 as shown in SEQ ID NO: 8, and its E-VL has: E-LCDR1 as shown in SEQ ID NO: 9, E-LCDR2 as shown in SEQ ID NO: 10 and E-LCDR3 as shown in SEQ ID NO: 11; or (ii) The bispecific antibody specifically binding to HGFR and EGFR comprises a HGFR antigen binding module 1 and an antigen binding module 2 that specifically binds to EGFR, The antigen binding moiety 1, whose M-VH has: M-HCDR1 as shown in SEQ ID NO: 18, M-HCDR2 as shown in SEQ ID NO: 19 and M-HCDR3 as shown in SEQ ID NO: 20, and its M-VL has: M-LCDR1 as shown in SEQ ID NO: 21, M-LCDR2 as shown in SEQ ID NO: 22 and M-LCDR3 as shown in SEQ ID NO: 23; and The antigen binding module 2, its E-VH has: E-HCDR1 as shown in SEQ ID NO: 6, E-HCDR2 as shown in SEQ ID NO: 7 and E-HCDR3 as shown in SEQ ID NO: 8, and its E-VL has: E-LCDR1 as shown in SEQ ID NO: 9, E-LCDR2 as shown in SEQ ID NO: 10 and E-LCDR3 as shown in SEQ ID NO:

11.

14. The pharmaceutical composition according to claim 13, wherein the bispecific antibody that specifically binds to HGFR and EGFR comprises: (i) the M-VH comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 16, and the M-VL comprises an amino acid sequence that has 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, wherein the M-VH of one antigen binding moiety 1 is represented by SEQ ID NO: 16, and the M-VL is represented by SEQ ID NO: 17; The M-VH of another antigen binding moiety 1 is shown in SEQ ID NO: 12, and the M-VL is shown in SEQ ID NO: 13; and The E-VH of the antigen binding moiety 2 is shown in SEQ ID NO: 3, and the E-VL is shown in SEQ ID NO: 5; or (ii) the bispecific antibody that specifically binds to HGFR and EGFR comprises an antigen-binding module 1 that specifically binds to HGFR and an antigen-binding module 2 that specifically binds to EGFR, The M-VH of the antigen binding module 1 is shown in SEQ ID NO: 12, and the M-VL is shown in SEQ ID NO: The antigen binding moiety 2 is shown as SEQ ID NO: 13, and the E-VH of the antigen binding moiety 2 is shown as SEQ ID NO: 3, and the E-VL is shown as SEQ ID NO:

5.

15. The pharmaceutical composition according to claim 13 or 14, wherein the bispecific antibody that specifically binds to HGFR and EGFR has a first chain as shown in SEQ ID NO: 34, a second chain as shown in SEQ ID NO: 35, a third chain as shown in SEQ ID NO: 36, and a fourth chain as shown in SEQ ID NO: 37; or It has a first strand as shown in SEQ ID NO:38, a second strand as shown in SEQ ID NO:39, a third strand as shown in SEQ ID NO:40, and a fourth strand as shown in SEQ ID NO:

41.

16. The pharmaceutical composition according to any one of claims 1 to 8, 13 to 15, comprising the following components: (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR, (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (c) 10 mg / mL to 100 mg / mL of sugar, and (d) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.5; Preferably, the pharmaceutical composition comprises the following components: (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR, (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80, (c) 20 mg / mL to 80 mg / mL of sucrose, and (d) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 4.8 to 6.2; More preferably, the pharmaceutical composition comprises the following components: (a) 80 mg / mL to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR, (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80, (c) 60 mg / mL to 80 mg / mL of sucrose, and (d) 5 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.

0.

17. The pharmaceutical composition according to any one of claims 1 to 15, comprising the following components: (a) 1 mg / mL to 250 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR, (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (c) 10 mg / mL to 100 mg / mL of sugar, (d) 1 mM to 300 mM excipients, (e) 10 U / mL to 3000 U / mL of enzyme, and (f) 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.5; Preferably, the pharmaceutical composition comprises the following components: (a) 1 mg / mL to 200 mg / mL of the bispecific antibody that specifically binds to HGFR and EGFR, (b) 0.1 mg / mL to 0.8 mg / mL of polysorbate 80, (c) 20 mg / mL to 80 mg / mL of sucrose, (d) 1 mM to 200 mM methionine, (e) 700 U / mL to 2400 U / mL of hyaluronidase, and (f) 5 mM to 50 mM histidine buffer; the pH of the pharmaceutical composition is 4.8 to 6.2; More preferably, the pharmaceutical composition comprises the following components: (a) 80 mg / mL to 180 mg / mL of a bispecific antibody that specifically binds to HGFR and EGFR, (b) 0.4 mg / mL to 0.8 mg / mL of polysorbate 80, (c) 60 mg / mL to 80 mg / mL of sucrose, (d) 1 mM to 20 mM methionine, (e) 1600 U / mL to 2400 U / mL of hyaluronidase, and (f) 5 mM to 30 mM histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer; the pH of the pharmaceutical composition is 5.0 to 6.

0.

18. A lyophilized preparation, which can form the pharmaceutical composition according to any one of claims 1 to 17 after reconstitution.

19. A method for preparing a lyophilized preparation, comprising the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 17.

20. A lyophilized preparation obtained by the method of claim 19.

21. A reconstituted solution, prepared by reconstituted the lyophilized preparation according to claim 18 or 20.

22. The pharmaceutical composition according to any one of claims 1 to 17 or the reconstituted solution according to claim 21, which is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation or an intramuscular injection preparation; preferably an intravenous injection preparation or a subcutaneous injection preparation.

23. A method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 17, or the lyophilized formulation of claim 18 or 20, or the reconstituted solution of 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.

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