CN114652824A

CN114652824A - anti-EGFR and PD-1 bispecific antibody formulations and methods of preservation

Info

Publication number: CN114652824A
Application number: CN202011543971.2A
Authority: CN
Inventors: 吴晓丽; 刘琴; 贾春媛; 涂晟; 韩建峰; 陈全民; 郭哲明; 周伟昌
Original assignee: Wuxi Biologics Shanghai Co Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2022-06-24

Abstract

The application discloses an anti-EGFR and PD-1 bispecific antibody preparation and a preservation method. The anti-EGFR and PD-1 bispecific antibody formulation comprises an anti-EGFR and PD-1 bispecific antibody; a buffer solution; a stabilizer; a surfactant; and an antioxidant.

Description

anti-EGFR and PD-1 bispecific antibody formulations and methods of preservation

Technical Field

The present application relates to recombinant anti-EGFR and PD-1 bispecific antibody formulations and methods of preservation. The anti-EGFR and PD-1 bispecific antibody formulations of the present application are excellent in stability, stable for at least 18 months at 2-8 ℃, stable for at least 6 months at room temperature, and stable after freezing and thawing.

Background

Epidermal Growth Factor Receptor (EGFR) is overexpressed in a variety of human cancers. EGFR can be activated by different ligands. Among these ligands, EGF is a high affinity ligand for EGFR. Binding of EGF to the extracellular region of EGFR induces receptor dimerization. EGFR may also pair with another member of the ErbB receptor, such as HER2, to form a heterodimer. EGFR dimerization stimulates its intrinsic kinase activity and subsequent phosphorylation of EGFR at several sites. This phosphorylation induces downstream activation and signaling and further initiates several signaling pathways, mainly the MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation. The entire EGF/EGFR pathway induces cell differentiation, migration, adhesion, and proliferation. EGFR is an important target for targeted therapy because it is overexpressed in a variety of human tumors.

Programmed death protein 1(PD-1, CD279) is a member of the CD28 family expressed on activated T cells and other immune cells. The involvement of PD-1 inhibits the function of these immune cells. There are two known ligands for PD-1, PD-L1(B7-H1, CD274) and PD-L2(B7-DC, CD273), both of which belong to the B7 family. PD-L1 expression is inducible in a variety of lymphoid and peripheral tissue cell types, whereas PD-L2 is more restricted to myeloid lineage cells including dendritic cells. The primary role of the PD-1 pathway is to down-regulate inflammatory immune responses in tissues and organs.

It was found that cancer cells are able to generate immune escape by upregulating the PD-1/PD-L1 pathway in the tumor microenvironment (Boussiositis 2016N Engl J Med). This mechanism is particularly seen in tumors with EGFR gene activating mutations. Modulation of the PD-1 pathway may be a typical mechanism of immune escape. As evidence, high PD-L1 expression was found in tumors of EGFR mutated patients (Azuma 2014Ann Oncol; Ramalingam 2016J Thorac Oncol). Therefore, in order to enhance the therapeutic effect, anti-EGFR and PD-1 bispecific antibodies targeting both EGFR and PD-1 have been developed in the prior art, which can increase the response rate of PD-L1 and EGFR double-positive tumors.

However, such bispecific antibodies are prone to aggregate and particle formation in solution, which has been a challenge in developing bispecific antibody formulations. Thus, the development of anti-EGFR and PD-1 bispecific antibody formulations generally requires that no significant changes in physical, chemical and biological stability are observed for at least 12 months at refrigeration temperatures (2-8 ℃). CN107198773A discloses a liquid preparation of a recombinant anti-PD-L1 fully human monoclonal antibody, which can be stably stored for at least 24 months at the temperature of 2-8 ℃, but the anti-EGFR and PD-1 bispecific antibody is different from the recombinant anti-PD-L1 fully human monoclonal antibody in terms of various properties, and whether the liquid preparation is suitable for the anti-EGFR and PD-1 bispecific antibody or not is still unknown. Also, CN110623921A discloses an injection preparation of anti-CD 3 and anti-CD 19 bispecific antibodies, which provides a good storage environment for the dual antibodies, effectively reduces the generation rate of antibody aggregates in the preparation, improves the physical stability of the antibodies, and reduces the potential safety risk, but the dual antibodies still differ from the anti-EGFR and PD-1 bispecific antibodies in that the formulation thereof for the anti-EGFR and PD-1 bispecific antibodies is not determined.

Clearly, there remains a need in the art for an antibody formulation specifically designed for anti-EGFR and PD-1 bispecific antibodies that can maintain long-term stability of the anti-EGFR and PD-1 bispecific antibody in the antibody formulation. At the same time, there remains a need in the art for a method of preserving anti-EGFR and PD-1 bispecific antibodies.

Disclosure of Invention

To solve the above technical problems, one aspect of the present application provides an anti-EGFR and PD-1 bispecific antibody formulation comprising: anti-EGFR and PD-1 bispecific antibodies; a buffer solution; a stabilizer; a surfactant; and an antioxidant.

Another aspect of the present application provides a method for preserving an anti-EGFR and PD-1 bispecific antibody, comprising: (1) formulating an anti-EGFR and PD-1 bispecific antibody formulation comprising: anti-EGFR and PD-1 bispecific antibodies; a buffer solution; a stabilizer; a surfactant; and an antioxidant; and (2) storing or freezing the preparation at 0-30 ℃.

In one embodiment of the application, the formulation comprises an anti-EGFR and PD-1 bispecific antibody comprising a first binding domain that binds EGFR and a second binding domain that binds PD-1, said first binding domain comprising heavy chain complementarity determining region 1(HCDR-1), HCDR-2, HCDR-3, light chain complementarity determining region 1(LCDR-1), LCDR-2 and LCDR-3, which comprise SEQ ID NOs: 6-11 and conservative modifications thereof, and the second binding domain comprises HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2 and LCDR-3, which comprise SEQ ID NOs: 12-17 or SEQ ID NO: 12-13, 18 and 15-17 and conservative modifications thereof. In another embodiment of the present application, the HCDR-1, HCDR-2, HCDR-3, LCDR-1, LCDR-2 and LCDR-3 sequences of the first and second binding domains of the anti-EGFR and PD-1 bispecific antibody are set forth in Table 1 below:

TABLE 1 CDR sequences of exemplary anti-EGFR and PD-1 bispecific antibodies

In one embodiment of the present application, the first binding domain comprises a variable region having an amino acid sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 2. 4 and 5 have at least 70%, 80%, 90%, 95% or 99% homology. In another embodiment of the present application, the second binding domain comprises a variable region having an amino acid sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 1 and 3 have at least 70%, 80%, 90%, 95% or 99% homology. In another embodiment of the present application, the variable region of the first binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 2. 4 and 5 and conservative modifications thereof. In another embodiment of the present application, the variable region of said second binding domain comprises a sequence selected from the group consisting of SEQ ID NO: 1 and 3 and conservative modifications thereof. In one embodiment of the present application, the variable region sequences of the first and second binding domains of the anti-EGFR and PD-1 bispecific antibody are shown in table 2 below:

TABLE 2 variable region sequences of the binding domains of exemplary anti-EGFR and PD-1 bispecific antibodies

In one embodiment of the application, the anti-EGFR and PD-1 bispecific antibody comprises a first single chain variable fragment (scFv) that binds EGFR and a second scFv that binds PD-1. In one embodiment of the present application, the first scFv has an amino acid sequence that differs from a sequence selected from the group consisting of SEQ ID NOs: 20. 22 and 23 have at least 70%, 80%, 90%, 95% or 99% homology. In one embodiment of the present application, the second scFv has an amino acid sequence that is identical to a sequence selected from the group consisting of SEQ ID NOs: 19 and 21 have at least 70%, 80%, 90%, 95% or 99% homology. In another embodiment of the present application, the first scFv comprises a sequence selected from the group consisting of SEQ ID NOs: 20. 22 and 23 and conservative modifications thereof. In another embodiment of the present application, the second scFv comprises a sequence selected from the group consisting of SEQ ID NOs: 19 and 21 and conservative modifications thereof. In another embodiment of the present application, the scFv sequences of the anti-EGFR and PD-1 bispecific antibody are shown in table 3 below:

TABLE 3 scFv sequences of exemplary anti-EGFR and PD-1 bispecific antibodies

In one embodiment of the application, the formulation comprises the anti-EGFR and PD-1 bispecific antibody in a concentration of 0.5 to 20mg/ml, preferably 1 to 5mg/ml, most preferably about 2 mg/ml.

In one embodiment of the present application, the buffer comprised by the formulation may be any suitable buffer known in the art. In one embodiment of the present application, the buffer comprises one or more selected from the group consisting of: acetic acid/sodium acetate buffer, histidine/hydrochloric acid buffer, and histidine/aspartic acid buffer. In a preferred embodiment of the present application, the buffer comprises a histidine/aspartate buffer. In another embodiment of the present application, the buffer is a histidine/aspartate buffer. In one embodiment of the present application, the buffer has a concentration of 5 to 50mM, preferably 10 to 30mM, more preferably 10 to 25mM, most preferably about 10 mM. In another embodiment of the present application, the buffer is a histidine/aspartate buffer and the concentration is about 10 mM.

In one embodiment of the present application, the formulation comprises a stabilizer that may be any suitable stabilizer known in the art. In one embodiment of the present application, the stabilizer comprises one or more selected from the group consisting of: sucrose, mannitol, arginine hydrochloride and glycine. In one embodiment of the present application, the stabilizing agent comprises sucrose. In one embodiment of the present application, the concentration of the stabilizer is from 2% to 10%, preferably about 8%. In another embodiment of the present application, the stabilizing agent is sucrose at a concentration of about 8%.

In one embodiment of the present application, the surfactant comprised by the formulation may be any suitable surfactant known in the art. In one embodiment of the present application, the surfactant comprises one or more selected from the group consisting of: poloxamer 188, polysorbate 20, and polysorbate 80. In one embodiment of the present application, the surfactant comprises polysorbate 80. In one embodiment of the present application, the concentration of the surfactant is from 0.005% to 0.02%, preferably about 0.01%. In one embodiment of the present application, the surfactant is polysorbate 80 at a concentration of about 0.01%. In another embodiment of the present application, the formulation comprises a surfactant and a chelating agent, preferably, the surfactant is polysorbate 80 and the chelating agent is disodium edetate.

In one embodiment herein, the formulation comprises an antioxidant which may be any suitable antioxidant known in the art. In one embodiment of the present application, the antioxidant comprises one or more selected from the group consisting of: methionine, cysteine and glutathione. In one embodiment of the present application, the antioxidant comprises methionine. In one embodiment of the present application, the concentration of the antioxidant is 0.5mM to 10mM, preferably about 5 mM. In one embodiment of the present application, the antioxidant is methionine at a concentration of about 5 mM.

In one embodiment of the present application, the formulation further comprises a chelating agent. The chelating agent comprised by the formulation may be any suitable chelating agent known in the art. In one embodiment of the present application, the chelating agent comprises one or more selected from the group consisting of: disodium edetate and diethylenetriaminepentaacetic acid (DTPA). In one embodiment of the present application, the concentration of the chelating agent is from 0.0001% to 0.01%, preferably about 0.002%. In one embodiment of the present application, the chelating agent comprises edetate disodium. In one embodiment of the present application, the chelating agent is edetate disodium at a concentration of about 0.002%.

In one embodiment of the present application, the pH of the formulation is between 4.0 and 5.5, preferably about 4.5.

In one embodiment of the present application, the formulation is in liquid form or lyophilized form. In one embodiment of the present application, the formulation is in liquid form. In one embodiment of the present application, the formulation is in a liquid form reconstituted from a lyophilized form.

In a preferred embodiment of the present application, the formulation comprises: 2mg/ml bispecific antibody against EGFR and PD-1; 10mM histidine/aspartate buffer; 8% of sucrose; 0.01% polysorbate 80; 5mM methionine; and 0.002% edetate disodium.

In one embodiment of the present application, the formulation is stored at 0-30 ℃. In one embodiment of the present application, the formulation is stored at 2-8 ℃ and 25 ± 2 ℃. In another embodiment of the present application, the formulation is stored at 2-8 ℃. In another embodiment of the present application, the formulation is stored at about 25 ± 2 ℃. In another embodiment of the present application, the formulation is stored at room temperature. In another embodiment of the present application, the formulation is stored at 25 ± 2 ℃. In another embodiment of the present application, the formulation is stored at about 25 ℃. In one embodiment of the present application, the formulation is stable for storage for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 6 months. In another embodiment of the present application, the formulation is stable for at least 18 months when stored at 2-8 ℃. In another embodiment of the present application, the formulation is stable for at least 6 months when stored at 25+2 ℃. In another embodiment of the present application, the formulation is stored frozen at-20 to-80 ℃. In another embodiment of the present application, the formulation is cryopreserved at about-80 ℃. In another embodiment of the present application, the formulation remains stable after freezing and thawing. In another embodiment of the present application, the formulation remains stable after 2-10 cycles, such as 5 cycles, of freeze-thawing. In another embodiment of the present application, the formulation remains stable after shaking.

Compared with the prior art, the anti-EGFR and PD-1 bispecific antibody preparation has excellent stability, can be stably stored for at least 18 months at 2-8 ℃, can be stably stored for at least 6 months at room temperature, and still keeps stable after freeze thawing and shaking.

Drawings

The present application is described in more detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a trend graph showing the SEC-HPLC main peak purity in different formulations for anti-EGFR and PD-1 bispecific antibodies of example 1;

FIG. 2 is a trend graph showing the SEC-HPLC main peak purity in different formulations for anti-EGFR and PD-1 bispecific antibodies of example 2;

FIG. 3 is a trend graph showing the SEC-HPLC main peak purity in different formulations for anti-EGFR and PD-1 bispecific antibodies of example 3;

FIG. 4 is a trend graph showing the SEC-HPLC main peak purity in different formulations for anti-EGFR and PD-1 bispecific antibodies of example 4;

FIG. 5 is a trend graph showing the SEC-HPLC main peak purity in different formulations for anti-EGFR and PD-1 bispecific antibodies of example 5;

FIG. 6 is a trend graph showing SEC-HPLC main peak purity in different formulations for anti-EGFR and PD-1 bispecific antibodies of example 6;

FIG. 7 is a trend graph showing the SEC-HPLC main peak purity in different formulations for anti-EGFR and PD-1 bispecific antibodies of example 7;

FIG. 8 is a trend graph showing SEC-HPLC main peak purity in different formulations of anti-EGFR and PD-1 bispecific antibodies of example 8 at 5 ℃;

FIG. 9 is a trend graph showing SEC-HPLC main peak purity in different formulations of anti-EGFR and PD-1 bispecific antibodies of example 8 at 25 ℃;

figure 10 is a trend graph showing the purity of the SEC-HPLC main peaks of the anti-EGFR and PD-1 bispecific antibodies in example 9 in different formulations.

Detailed Description

Definition of

In this application, percentages are weight/volume (w/v) percentages unless otherwise indicated.

In the present application, "main peak purity" refers to the percentage of the peak area of an antibody monomer in a spectrum measured by SEC-HPLC or the like in an antibody preparation to the total of all peak areas.

In this application, "stable storage" refers to an antibody formulation in which the antibody remains substantially stable, e.g., substantially free of degradation, substantially free of aggregation, or substantially retains biological activity, physically, chemically, and biologically during storage. In one embodiment of the present application, "stable preservation" means that the main peak purity is not less than 95% during preservation.

In the present application, "conservative modifications" refer to nucleotide and amino acid sequence modifications that do not significantly affect or alter the binding properties of an antibody encoded by or comprising the nucleotide sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into the sequence by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

As used herein, the term "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical preparation. Suitable buffers are well known in the art and can be found in the literature. For example, citrate, acetate, histidine, succinate, malate, phosphate or lactate salts, and/or their respective free acids or bases, as well as mixtures of various salts and/or their acids and bases, may be employed. Examples of pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, and phosphate buffer. For histidine buffers, i.e. buffers with histidine, typically L-histidine, as buffer, e.g. L-histidine/HCl buffer, comprising L-histidine or a mixture of L-histidine and L-histidine hydrochloride and pH adjustment with hydrochloric acid. Unless otherwise indicated, the term "L-histidine" as used herein to describe buffers refers to buffers containing L-histidine. The L-histidine/HCl buffer can be prepared by dissolving the appropriate or specified amounts of L-histidine and L-histidine hydrochloride in water, or by dissolving the appropriate or specified amounts of L-histidine in water and adjusting the pH to the desired value by adding hydrochloric acid. As the buffer used, the pH can be adjusted to the desired value with acids or bases known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid and citric acid, sodium hydroxide and potassium hydroxide. For histidine/aspartate buffers, they are prepared by dissolving the appropriate or specified amount of histidine in water and adjusting the pH to the desired value by adding an aqueous solution of aspartate.

As used herein, the term "surfactant" means a pharmaceutically acceptable, surface active agent. Examples of pharmaceutically acceptable surfactants include, but are not limited to, polyoxyethylene-sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and Sodium Dodecyl Sulfate (SDS). Examples of polyoxyethylene-sorbitan fatty acid esters include polysorbate 20 (polyoxyethylene sorbitan monolaurate, under the trademark Tween 20^TMSold) and polysorbate 80 (polyoxyethylene sorbitan monooleate, sold under the trademark Tween 80^TMSales). Examples of polyethylene-polypropylene copolymers are under the designation F68 or Poloxamer 188^TMThose that are sold. An example of a polyoxyethylene alkyl ether is under the trademark Brij^TMThose that are sold. Examples of alkylphenyl polyoxyethylene ethers are those sold under the trade name Triton X, e.g., p-tert-octylphenoxypolyethoxyethanol (under the trade name Triton X-100)^TMSold).

As used herein, the term "stabilizer" means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage, and use. Stabilizers include, but are not limited to, sugars, amino acids, polyols (e.g., mannitol, sorbitol, xylitol, dextran, glycerol, arabitol, propylene glycol, polyethylene glycol), cyclodextrins (e.g., hydroxypropyl- β -cyclodextrin, sulfobutylethyl- β -cyclodextrin, β -cyclodextrin), polyethylene glycols (e.g., PEG3000, PEG 3350, PEG 4000, PEG 6000), albumins (e.g., Human Serum Albumin (HSA), Bovine Serum Albumin (BSA)), salts (e.g., sodium chloride, magnesium chloride, calcium chloride), chelating agents (e.g., EDTA), as defined below. More than one stabilizer from the same or different groups may be present in the formulation.

As used herein, the term "antioxidant" means a pharmaceutically acceptable excipient that prevents oxidation of the active pharmaceutical ingredient. Examples of antioxidants include, but are not limited to, ascorbic acid, glutathione, cysteine, methionine, citric acid, and EDTA. As used herein, the term "chelating agent" refers to a compound containing a coordinating atom capable of forming a coordinate bond with a metal atom or ion. Examples of chelating agents include, but are not limited to, phosphates, alcoholamines, aminocarboxylates, hydroxycarboxylates, organophosphonates, and polyacrylics.

The technical solutions of the present application will be described in detail and fully with reference to the accompanying drawings, and it should be understood that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without any inventive step are within the scope of protection of the present application.

TABLE 4 Instrument

TABLE 5 reagents

TABLE 6 consumable

Example 1 buffer System screening

The formulation of the recombinant anti-EGFR and PD-1 bispecific antibody liquid formulations of this example is as follows:

formula 1: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM acetic acid/sodium acetate buffer system, pH 5.5; 336C-20170701.

And (2) formula: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM citric acid/sodium citrate buffer system, pH value is 5.5; 336C-20170702.

And (3) formula: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system, pH 5.5; 336C-20170706.

And (4) formula: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system, pH 6.0; 336C-20170707.

And (5) formula: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system, pH 6.5; 336C-20170708.

And (6) formula: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM disodium hydrogen phosphate/sodium dihydrogen phosphate buffer system, pH 6.5; 336C-20170709.

And (3) formula 7: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system, pH 7.0; 336C-20170710.

The preparation method of the recombinant anti-EGFR and PD-1 bispecific antibody liquid preparation with each formula comprises the following steps: recombinant anti-EGFR and PD-1 bispecific antibody (WBP336C ═ W336-T1U3.G10-4.uhIgG4.SP (dK), disclosed in CN109575139A) from Shanghai pharmacomycin biotechnology limited was ultrafiltered and transfused into 5 buffer system solutions in example 1, the protein concentration was adjusted to 5mg/ml to obtain a semi-finished product, and the semi-finished product was filtered through a 0.22 micron PVDF sterilization filter under laminar flow conditions and filled into penicillin bottles to obtain a sample to be tested.

The samples of the above formulations 1 to 5 were divided into two groups and subjected to the test under different conditions, namely, 60% RH (Relative Humidity) at 25 ℃ for 1 week and 2 weeks, and 60% RH at 5 ℃ for 1 week, 2 weeks and 4 weeks. The samples were subjected to size exclusion high performance liquid chromatography (SEC-HPLC) and the results are shown in Table 7 and FIG. 1.

TABLE 7 SEC-HPLC TEST RESULTS FOR EXAMPLE 1

Note: t0 represents the starting point sample, 05-1W represents the sample tested at 5 ℃ for 1 week, 05-2W represents the sample tested at 5 ℃ for 2 weeks, 05-4W represents the sample tested at 5 ℃ for 4 weeks, 25-1W represents the sample tested at 25 ℃ for 1 week, and 25-2W represents the sample tested at 25 ℃ for 2 weeks.

The results show that the main peak purity of the recombinant anti-EGFR and PD-1 bispecific antibody in formula 1, formula 3, formula 4 and formula 5 is higher than that of the other three formulas, indicating that formula 1, formula 3, formula 4 and formula 5 are more stable to the present antibody than the buffer system of the other three formulas.

Example 2 buffer System Screen 2

and (4) a formula 8: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM acetic acid/sodium acetate buffer system + 8% sucrose, pH 4.5; 336C-20171001.

And (3) formula 9: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system, pH 5.5; 336C-20171002.

The preparation method of the recombinant anti-EGFR and PD-1 bispecific antibody liquid preparation of each formula comprises the following steps: the recombinant anti-EGFR and PD-1 bispecific antibody (WBP336C ═ W336-T1U3.G10-4.uhIgG4.SP (dK), disclosed in CN109575139) from Shanghai pharmacomycin biotechnology limited is ultrafiltered to change the solution into the 2 kinds of buffer system solutions of example 2, and other auxiliary materials are added to adjust the protein concentration to the target concentration to obtain a semi-finished product, and the semi-finished product is filtered by a 0.22 micron PVDF sterilization filter under laminar flow condition and filled into a penicillin bottle to obtain a sample to be tested.

The samples of formulas 8 and 9 were divided into two groups and tested under different conditions, namely, 60% RH at 25 ℃ for 1 week and 2 weeks and 60% RH at 5 ℃ for 1 week and 2 weeks. Samples taken at each time point were subjected to size exclusion high performance liquid chromatography SEC-HPLC and the results are shown in Table 8 and FIG. 2.

TABLE 8 SEC-HPLC TEST RESULTS FOR EXAMPLE 2

Note: t0 represents the starting point sample, 05-1W represents the sample tested at 5 ℃ for 1 week, 05-2W represents the sample tested at 5 ℃ for 2 weeks, 25-1W represents the sample tested at 25 ℃ for 1 week, 25-2W represents the sample tested at 25 ℃ for 2 weeks.

The results show that the purity of the main peak of the recombinant anti-EGFR and PD-1 bispecific antibody in the formula 8 is higher than that in the formula 9, the phenomenon is similar to the situation that the polymer peak is reduced and the main peak is increased under the condition of pH 3.8, which is shown in the downstream development and purification process, and the results show that the SEC-HPLC data of the recombinant anti-EGFR and PD-1 bispecific antibody is more stable at low pH value.

Example 3 buffer System screening

formula 10: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM acetic acid/sodium acetate buffer system + 8% sucrose + 0.02% polysorbate 80+ 0.002% edetate disodium +5mM methionine, pH 4.5; 336C-20180203.

Formula 11: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +10mM histidine/aspartate buffer system + 8% sucrose + 0.02% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180205.

The preparation method of the liquid preparation of the recombinant anti-EGFR and PD-1 bispecific antibodies of each formula is the same as that of example 2.

The samples of the above formulations 10 and 11 were divided into two groups and subjected to the test under different conditions, namely, 60% RH at 25 ℃ for 1 month, 2 months and 3 months, and 60% RH at 5 ℃ for 1 month, 2 months and 3 months, respectively. Samples taken at each time point were subjected to size exclusion high performance liquid chromatography SEC-HPLC and the results are shown in Table 9 and FIG. 3.

TABLE 9 SEC-HPLC TEST RESULTS FOR EXAMPLE 3

Note: t0 represents the starting point sample, 05-1M represents the sample tested at 5 ℃ for 1 month, 05-2M represents the sample tested at 5 ℃ for 2 months, 05-3M represents the sample tested at 5 ℃ for 3 months, 25-1M represents the sample tested at 25 ℃ for 1 month, 25-2M represents the sample tested at 25 ℃ for 2 months, and 25-3M represents the sample tested at 25 ℃ for 3 months.

The results show that the main peak purity of the recombinant anti-EGFR and PD-1 bispecific antibody in formula 11 is higher than that in formula 10, and the results show that the SEC-HPLC data of the recombinant anti-EGFR and PD-1 bispecific antibody in a 10mM histidine/aspartic acid buffer system are more stable.

Example 4 buffer System screening four

the formulation is 12: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +10mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine, pH 4.5; 336C-20180301.

Formula 13: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180302.

The preparation method of the recombinant anti-EGFR and PD-1 bispecific antibody liquid preparation of each formula is the same as that of example 2.

The samples of the above formulations 12 and 13 were divided into two groups and subjected to the test under different conditions, namely, 60% RH at 25 ℃ for 1 week and 2 weeks and 60% RH at 5 ℃ for 1 week and 2 weeks, respectively. Samples taken at each time point were subjected to size exclusion high performance liquid chromatography SEC-HPLC and the results are shown in Table 10 and FIG. 4.

TABLE 10 SEC-HPLC test results for example 4

The results show that the main peak purity of the recombinant anti-EGFR and PD-1 bispecific antibody in formula 12 is higher than that in formula 13, and the SEC-HPLC results show that the recombinant anti-EGFR and PD-1 bispecific antibody is more stable in the 10mM histidine/aspartate buffer system than in the 20mM histidine/aspartate buffer system.

Example 5 stabilizer screening

The formulation of the recombinant anti-EGFR and PD-1 bispecific antibody liquid formulation of this example is shown below:

formula 14: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system + 8% sucrose + 0.01% polysorbate 80, pH 5.5; 336C-20171003.

Formula 15: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system + 2% sucrose + 4% mannitol + 0.01% polysorbate 80, pH 5.5; 336C-20171004.

Formula 16: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system + 1% arginine hydrochloride + 4% mannitol + 0.01% polysorbate 80, pH 5.5; 336C-20171007.

Formula 17: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system + 4% mannitol + 0.75% glycine + 0.01% polysorbate 80, pH 5.5; 336C-20171008.

The samples of the above formulations 14, 15, 16 and 17 were divided into two groups and subjected to the test under different conditions, namely, 25 ℃ 60% RH for 1 week and 2 weeks and 5 ℃ 60% RH for 1 week and 2 weeks, respectively. Samples taken at each time point were subjected to size exclusion high performance liquid chromatography SEC-HPLC and the results are shown in Table 11 and FIG. 5.

TABLE 11 SEC-HPLC TEST RESULTS OF EXAMPLE 5

The results show that the main peak purity of the recombinant anti-EGFR and PD-1 bispecific antibody in formula 14 is higher than the other three formulas, and the SEC-HPLC results show that the recombinant anti-EGFR and PD-1 bispecific antibody is more stable in the formula with 8% sucrose than the other formulas with mannitol, arginine hydrochloride and glycine.

Example 6 surfactant species screening

Formula 18: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/histidine hydrochloride buffer system + 2% sucrose + 4% mannitol + 0.2% poloxamer 188, pH 5.5; 336C-20171010.

The samples of formulations 15 and 18 above were divided into three groups and tested under different conditions, with 60% RH at 25 ℃ for 1 week and 2 weeks, 60% RH at 5 ℃ for 1 week and 2 weeks, and 5 cycles of freezing and thawing at-70 ℃ to room temperature. Samples taken at each time point were subjected to size exclusion high performance liquid chromatography SEC-HPLC and the results are shown in table 12 and fig. 6.

TABLE 12 SEC-HPLC TEST RESULTS FOR EXAMPLE 6

The results show that the main peak purity of the recombinant anti-EGFR and PD-1 bispecific antibody in formula 15 is higher than formula 18, and the results show that polysorbate 80 is significantly better than poloxamer 188 at freeze-thaw.

Example 7 antioxidant screening

formulation 19: 10mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium, pH 4.5; 336C-20180303.

The formulation 20: 10mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine, pH 4.5; 336C-20180307.

Formula 21: 10mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium, pH 5.0; 336C-20180306.

Formula 22: 10mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 5.0; 336C-20180304.

The samples of the above formulations 19, 20, 21 and 22 were divided into two groups and subjected to the test under different conditions, namely, 60% RH at 25 ℃ for 1 week and 2 weeks and 60% RH at 5 ℃ for 1 week and 2 weeks, respectively. Samples taken at each time point were subjected to size exclusion high performance liquid chromatography SEC-HPLC and the results are shown in Table 13 and FIG. 7.

TABLE 13 SEC-HPLC TEST RESULTS FOR EXAMPLE 7

The SEC-HPLC results showed that the main peak purity of the recombinant anti-EGFR and PD-1 bispecific antibody was slightly higher in formulas 20 and 22 than in formulas 19 and 21. Formulations 20 and 22 added more methionine than formulations 19 and 21, respectively, indicating that the addition of the antioxidant methionine was beneficial to maintain the stability of the samples.

Example 8 surfactant concentration and chelating agent screening

and (4) formula 8: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM acetic acid/sodium acetate buffer system + 8% sucrose, pH 4.5; 336C-20171101.

Formula 23: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM acetic acid/sodium acetate buffer system + 8% sucrose + 0.01% polysorbate 80, pH 4.5; 336C-20171102.

Formula 24: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM acetic acid/sodium acetate buffer system + 8% sucrose + 0.02% polysorbate 80, pH 4.5; 336C-20171103.

Formulation 25: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM acetic acid/sodium acetate buffer system + 8% sucrose + 0.05% polysorbate 80, pH 4.5; 336C-20171104.

Formulation 26: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM acetic acid/sodium acetate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.001% disodium edetate, pH 4.5; 336C-20171105.

The samples of formula 8, formula 23, formula 24, formula 25 and formula 26 were divided into four groups and tested under different conditions, respectively, 60% RH at 25 ℃ and 60% RH at 5 ℃ for 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks, shaking at 300rpm at 25 ℃ for 6 days, and freeze-thaw at-70 ℃ to room temperature for 5 cycles. The samples were subjected to size exclusion high performance liquid chromatography SEC-HPLC and appearance tests, the appearance test results are shown in Table 14, and the SEC-HPLC test results are shown in Table 15, FIG. 8 and FIG. 9.

TABLE 14 appearance test results of example 8

Note: a represents a colorless microemulsion light liquid; b represents a colorless, opalescent liquid with visible particles; t0 denotes the starting point sample, 05-1W denotes the sample tested at 5 ℃ for 1 week, 05-2W denotes the sample tested at 5 ℃ for 2 weeks, 05-4W denotes the sample tested at 5 ℃ for 4 weeks, 05-8W denotes the sample tested at 5 ℃ for 8 weeks, 05-12W denotes the sample tested at 5 ℃ for 12 weeks, 25-1W denotes the sample tested at 25 ℃ for 1 week, 25-2W denotes the sample tested at 25 ℃ for 2 weeks, 25-4W denotes the sample tested at 25 ℃ for 4 weeks, 25-8W denotes the sample tested at 25 ℃ for 8 weeks, 25-12W denotes the sample tested at 25 ℃ for 12 weeks; FT-5C represents samples tested for 5 cycles at-70 ℃ to room temperature freeze-thaw, and A-6D represents samples tested for 6 days at 25 ℃ under 300rpm shaking conditions.

The results show that the appearance of the sample of formula 8 is colorless microemulsion liquid with visible particles; the appearance of the samples of the formulas 23, 24, 25 and 26 is colorless microemulsion light liquid. The appearance result shows that the appearance of the sample can be effectively improved by adding the polysorbate 80, and the influence on the product quality caused by the appearance of visible particles in the sample is avoided.

TABLE 15 SEC-HPLC TEST RESULTS FOR EXAMPLE 8

Note: t0 denotes the starting point sample, 05-1W denotes the sample tested at 5 ℃ for 1 week, 05-2W denotes the sample tested at 5 ℃ for 2 weeks, 05-4W denotes the sample tested at 5 ℃ for 4 weeks, 05-8W denotes the sample tested at 5 ℃ for 8 weeks, 05-12W denotes the sample tested at 5 ℃ for 12 weeks, 25-1W denotes the sample tested at 25 ℃ for 1 week, 25-2W denotes the sample tested at 25 ℃ for 2 weeks, 25-4W denotes the sample tested at 25 ℃ for 4 weeks, 25-8W denotes the sample tested at 25 ℃ for 8 weeks, 25-12W denotes the sample tested at 25 ℃ for 12 weeks.

The SEC-HPLC results showed that the main peak purity of the recombinant anti-EGFR and PD-1 bispecific antibodies in formula 8 and formula 26 was higher than formula 23, formula 24, formula 25. Formulations 23, 24, and 25 contained polysorbate 80 at concentrations of 0.01%, 0.02%, and 0.05%, respectively, indicating that the decrease in purity of the main peak was greater after the sample was added with polysorbate 80, and the decrease increased with increasing polysorbate 80 concentration. Formulation 8 contained no polysorbate 80, but the sample had visible particles in appearance. Formulation 26 contained polysorbate 80 at a concentration of 0.01% and disodium edetate at a concentration of 0.002, and the SEC-HPLC main peak purity performance of the formulation 26 sample was consistent with that of the formulation 8 sample without polysorbate 80 added, with no visible particles in appearance. The addition of disodium edetate as a chelating agent was shown to inhibit protein aggregation caused by the addition of polysorbate 80.

Example 9 protein concentration screening

formulation 27: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180405.

Formula 28: 5mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180406.

Formulation 29: 6mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180407.

The formula is 30: 7mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180408.

Formulation 31: 8mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180409.

Formulation 32: 9mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180410.

Formulation 33: 10mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336C-20180411.

The samples of the above formula 27, formula 28, formula 29, formula 30, formula 31, formula 32 and formula 33 were divided into two groups and subjected to tests under different conditions, 60% RH at 25 ℃ and 60% RH at 5 ℃ for 3 days, 1 week and 2 weeks, respectively. The samples were subjected to size exclusion high performance liquid chromatography SEC-HPLC and the results of the SEC-HPLC tests are shown in Table 16 and FIG. 10.

TABLE 16 SEC-HPLC TEST RESULTS FOR EXAMPLE 9

Note: t0 represents the starting point sample, 05-3D represents the sample tested at 5 ℃ for 3 days, 05-1W represents the sample tested at 5 ℃ for 1 week, 05-2W represents the sample tested at 5 ℃ for 2 weeks, 25-3D represents the sample tested at 25 ℃ for 3 days, 25-1W represents the sample tested at 25 ℃ for 1 week, 25-2W represents the sample tested at 25 ℃ for 2 weeks.

The SEC-HPLC results showed that the main peak purity of the recombinant anti-EGFR and PD-1 bispecific antibody proteins tended to decrease with increasing protein concentration in the above formula 27, formula 28, formula 29, formula 30, formula 31, formula 32 and formula 33 in order to maintain the long term stability of the proteins in liquid formulations.

Example 10 stability study

formulation 27: 2mg/ml recombinant anti-EGFR and PD-1 bispecific antibody +20mM histidine/aspartate buffer system + 8% sucrose + 0.01% polysorbate 80+ 0.002% edetate disodium +5mM methionine at pH 4.5; 336S 20181201.

The preparation method of the recombinant anti-EGFR and PD-1 bispecific antibody liquid preparation with each formula comprises pilot scale production, purifying by each purification process step after cell culture, changing liquid into a final formula, and adjusting the formula to the final formula by adding auxiliary materials to prepare a stock solution. The stock solution is prepared into a finished product through the process steps of freezing and thawing, uniformly mixing, filtering and filling.

The finished products of the formula 27 are divided into two groups, and are respectively placed under different conditions for testing, and the accelerated stability test is carried out for 1 month, 3 months and 6 months under the condition of 60% RH at 25 ℃; long term stability test at 5 ℃ 60% RH for 3 months, 6 months, 9 months, 12 months, 18 months and 24 months. The assay items included protein concentration, color, clarity, visible foreign matter, insoluble microparticles, osmolarity, SEC-HPLC purity, charge heteroplasmon iCIEF, reduced CE-SDS purity, non-reduced CE-SDS purity, ELISA binding activity (PD1), and ELISA binding activity (EGFR). The test results are shown in tables 17 and 18.

TABLE 17 accelerated stability test results of example 10 at 25 deg.C 60% RH

Note: "accelerated 1M" means a sample tested at 25 ℃ for 1 month, "accelerated 3M" means a sample tested at 25 ℃ for 3 months, and "accelerated 6M" means a sample tested at 25 ℃ for 6 months.

TABLE 18 Long-term stability test results at 5 deg.C 60% RH for example 10

Note: "Long-term 3M" means a sample tested at 5 ℃ for 3 months, "Long-term 6M" means a sample tested at 5 ℃ for 6 months, "Long-term 9M" means a sample tested at 5 ℃ for 9 months, "Long-term 12M" means a sample tested at 5 ℃ for 12 months, and "Long-term 18M" means a sample tested at 5 ℃ for 18 months.

The data show that recombinant anti-EGFR and PD-1 bispecific antibody proteins exhibit more stable quality index under accelerated stability at 25 ℃ 60% RH and long-term stability test conditions at 5 ℃ in formulation 27.

The above are only examples of the present application, and do not limit the scope of the present application. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. It is not necessary to describe all embodiments in this context. The technical solutions like this formed by equivalent transformation or equivalent substitution fall within the protection scope of the present application.

Sequence listing

<110> Shanghai Yaoming Biotechnology Co., Ltd

<120> anti-EGFR and PD-1 bispecific antibody preparation and preservation method

<130> PCTF1707

<160> 23

<170> PatentIn version 3.3

<210> 1

<211> 242

<212> PRT

<213> Artificial sequence

<400> 1

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Gly Thr Tyr Leu Tyr Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Thr Leu Gly Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Leu

85 90 95

Thr His Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser

130 135 140

Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Thr Tyr Tyr

145 150 155 160

Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Leu Gly

165 170 175

Tyr Ile Asn Met Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys

180 185 190

Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met

195 200 205

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Ile Leu Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val

225 230 235 240

Ser Ser

<210> 2

<211> 241

<212> PRT

<213> Artificial sequence

<400> 2

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln

115 120 125

Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys

130 135 140

Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg

145 150 155 160

Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly

165 170 175

Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln

195 200 205

Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr

210 215 220

Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ala

<210> 3

<211> 242

<212> PRT

<213> Artificial sequence

<400> 3

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Gly Thr Tyr Leu Tyr Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Thr Leu Gly Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Leu

85 90 95

Thr His Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser

130 135 140

Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Thr Tyr Tyr

145 150 155 160

Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Leu Gly

165 170 175

Tyr Ile Asn Met Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys

180 185 190

Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met

195 200 205

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Ile Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val

225 230 235 240

Ser Ser

<210> 4

<211> 241

<212> PRT

<213> Artificial sequence

<400> 4

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln

115 120 125

Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys

130 135 140

Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg

145 150 155 160

Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly

165 170 175

Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln

195 200 205

Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr

210 215 220

Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ala

<210> 5

<211> 241

<212> PRT

<213> Artificial sequence

<400> 5

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln

115 120 125

Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys

130 135 140

Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg

145 150 155 160

Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly

165 170 175

Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr

210 215 220

Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ala

<210> 6

<211> 10

<212> PRT

<213> Artificial sequence

<400> 6

Gly Phe Ser Leu Thr Asn Tyr Gly Val His

1 5 10

<210> 7

<211> 16

<212> PRT

<213> Artificial sequence

<400> 7

Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser

1 5 10 15

<210> 8

<211> 11

<212> PRT

<213> Artificial sequence

<400> 8

Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr

1 5 10

<210> 9

<211> 11

<212> PRT

<213> Artificial sequence

<400> 9

Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His

1 5 10

<210> 10

<211> 7

<212> PRT

<213> Artificial sequence

<400> 10

Tyr Ala Ser Glu Ser Ile Ser

1 5

<210> 11

<211> 9

<212> PRT

<213> Artificial sequence

<400> 11

Gln Gln Asn Asn Asn Trp Pro Thr Thr

1 5

<210> 12

<211> 10

<212> PRT

<213> Artificial sequence

<400> 12

Gly Phe Thr Phe Thr Thr Tyr Tyr Ile Ser

1 5 10

<210> 13

<211> 17

<212> PRT

<213> Artificial sequence

<400> 13

Tyr Ile Asn Met Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 14

<211> 6

<212> PRT

<213> Artificial sequence

<400> 14

Leu Gly Tyr Phe Asp Tyr

1 5

<210> 15

<211> 16

<212> PRT

<213> Artificial sequence

<400> 15

Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Gly Thr Tyr Leu Tyr

1 5 10 15

<210> 16

<211> 7

<212> PRT

<213> Artificial sequence

<400> 16

Leu Val Ser Thr Leu Gly Ser

1 5

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence

<400> 17

Met Gln Leu Thr His Trp Pro Tyr Thr

1 5

<210> 18

<211> 6

<212> PRT

<213> Artificial sequence

<400> 18

Ile Gly Tyr Phe Asp Tyr

1 5

<210> 19

<211> 568

<212> PRT

<213> Artificial sequence

<400> 19

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Gly Thr Tyr Leu Tyr Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Thr Leu Gly Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Leu

85 90 95

Thr His Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser

130 135 140

Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Thr Tyr Tyr

145 150 155 160

Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Leu Gly

165 170 175

Tyr Ile Asn Met Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys

180 185 190

Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met

195 200 205

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Ile Leu Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val

225 230 235 240

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys

245 250 255

Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys

260 265 270

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

275 280 285

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

290 295 300

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

305 310 315 320

Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val

325 330 335

Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

340 345 350

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

355 360 365

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

370 375 380

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

385 390 395 400

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

405 410 415

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

420 425 430

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

435 440 445

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

450 455 460

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

465 470 475 480

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

485 490 495

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

500 505 510

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

515 520 525

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

530 535 540

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

545 550 555 560

Lys Ser Leu Ser Leu Ser Leu Gly

565

<210> 20

<211> 348

<212> PRT

<213> Artificial sequence

<400> 20

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln

115 120 125

Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys

130 135 140

Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg

145 150 155 160

Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly

165 170 175

Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln

195 200 205

Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr

210 215 220

Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ala Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

245 250 255

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

260 265 270

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

275 280 285

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

290 295 300

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

305 310 315 320

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

325 330 335

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

340 345

<210> 21

<211> 568

<212> PRT

<213> Artificial sequence

<400> 21

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Gly Thr Tyr Leu Tyr Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Thr Leu Gly Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Leu

85 90 95

Thr His Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser

130 135 140

Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Thr Tyr Tyr

145 150 155 160

Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr Leu Gly

165 170 175

Tyr Ile Asn Met Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys

180 185 190

Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met

195 200 205

Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

210 215 220

Ile Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val

225 230 235 240

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys

245 250 255

Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys

260 265 270

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

275 280 285

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

290 295 300

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

305 310 315 320

Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val

325 330 335

Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

340 345 350

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

355 360 365

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

370 375 380

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

385 390 395 400

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

405 410 415

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

420 425 430

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

435 440 445

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

450 455 460

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

465 470 475 480

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

485 490 495

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

500 505 510

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

515 520 525

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

530 535 540

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

545 550 555 560

Lys Ser Leu Ser Leu Ser Leu Gly

565

<210> 22

<211> 348

<212> PRT

<213> Artificial sequence

<400> 22

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln

115 120 125

Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys

130 135 140

Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg

145 150 155 160

Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly

165 170 175

Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln

195 200 205

Ser Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr

210 215 220

Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ala Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

245 250 255

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

260 265 270

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

275 280 285

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

290 295 300

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

305 310 315 320

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

325 330 335

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

340 345

<210> 23

<211> 348

<212> PRT

<213> Artificial sequence

<400> 23

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln

115 120 125

Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys

130 135 140

Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg

145 150 155 160

Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly

165 170 175

Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr

210 215 220

Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ala Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

245 250 255

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

260 265 270

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

275 280 285

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

290 295 300

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

305 310 315 320

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

325 330 335

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

340 345

Claims

1. An anti-EGFR and PD-1 bispecific antibody formulation comprising:

bispecific antibodies against EGFR and PD-1;

a buffer solution;

a stabilizer;

a surfactant; and

an antioxidant.

2. The formulation according to claim 1, wherein the concentration of the bispecific antibody is 0.5 to 20mg/ml, preferably 1 to 5mg/ml, most preferably 2 mg/ml.

3. The formulation of claim 1 or 2, wherein the buffer comprises one or more selected from the group consisting of: acetic acid/sodium acetate buffer, histidine/hydrochloric acid buffer and histidine/aspartic acid buffer, preferably including histidine/aspartic acid buffer, optionally, the buffer concentration is 5 to 50mM, preferably 10 to 30mM, more preferably 10 to 25mM, most preferably 10 mM.

4. The formulation of any one of claims 1 to 3, wherein the stabilizing agent comprises one or more selected from the group consisting of: sucrose, mannitol, arginine hydrochloride and glycine, the stabilizer preferably comprising sucrose, optionally at a concentration of 2% to 10%, preferably 8%.

5. The formulation of any one of claims 1 to 4, wherein the surfactant comprises one or more selected from the group consisting of: poloxamer 188, polysorbate 20 and polysorbate 80, the surfactant preferably including polysorbate 80, optionally at a concentration of 0.005% to 0.02%, preferably 0.01%.

6. The formulation of any one of claims 1 to 5, wherein the antioxidant comprises one or more selected from the group consisting of: methionine, cysteine or glutathione, the antioxidant preferably comprising methionine, optionally at a concentration of 0.5 to 10mM, preferably 5 mM.

7. The formulation according to any one of claims 1 to 6, characterized in that it further comprises a chelating agent comprising one or more selected from the group consisting of: edetate disodium and diethylenetriaminepentaacetic acid, the chelating agent preferably comprising edetate disodium, optionally at a concentration of 0.0001% to 0.01%, preferably 0.002%.

8. The formulation of any one of claims 1 to 7, wherein the formulation comprises:

2mg/ml bispecific antibody against EGFR and PD-1;

10mM histidine/aspartate buffer;

8% of sucrose;

0.01% polysorbate 80;

5mM methionine; and

0.002% of edetate disodium.

9. The formulation according to any one of claims 1 to 8, wherein the pH of the formulation is between 4.0 and 5.5, preferably 4.5, optionally the formulation is in liquid form or lyophilized form, preferably in liquid form reconstituted from lyophilized form.

10. A method of preserving an anti-EGFR and PD-1 bispecific antibody, comprising:

(1) formulating the formulation of any one of claims 1-9; and

(2) storing the preparation at 0-30 deg.C or freezing.