阅读说明：本技术 抗her3人源化单克隆抗体及其制剂 (anti-HER 3 humanized monoclonal antibody and preparation thereof ) 是由 瞿爱东 梁红远 徐帆洪 李翱翔 吴丽娜 祝婧烨 邱建华 陆瑾 张琳 赵鑫 宋效飞 于 2018-07-17 设计创作，主要内容包括：本发明提供了抗HER3人源化单克隆抗体及其制剂。具体地,本发明提供了一种新的抗HER3人源化抗体。本发明的抗体能够高特异性地结合HER3抗原,具有较高的亲和力和生物活性,以及低的免疫原性,结构稳定,成药性良好。并且本发明人源化抗体在本发明抗体制剂中的稳定性非常好,用于制备预防或治疗HER3相关的疾病的药物。(The invention provides an anti-HER 3 humanized monoclonal antibody and a preparation thereof. In particular, the invention provides a novel humanized antibody against HER 3. The antibody of the invention can be combined with HER3 antigen with high specificity, and has the advantages of high affinity, high bioactivity, low immunogenicity, stable structure and good drug-forming property. The humanized antibody has very good stability in the antibody preparation of the invention, and is used for preparing a medicament for preventing or treating HER3 related diseases.)

1. An antibody light chain variable region selected from the group consisting of:

(1) a light chain variable region having a sequence as set forth in SEQ ID No. 1; or

(2) A light chain variable region derived from the sequence of SEQ ID No. 1, having the function of the light chain variable region of (1), formed by substituting, deleting, modifying and/or adding at least one (e.g., 1-20, preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) amino acid residues in the amino acid sequence of SEQ ID No. 1.

2. A light chain of an antibody, wherein said light chain has the light chain variable region of claim 1.

3. A heavy chain variable region of an antibody, wherein said heavy chain variable region is selected from the group consisting of:

(i) a heavy chain variable region having a sequence as set forth in SEQ ID No. 2; or

(ii) A heavy chain variable region derived from the sequence of SEQ ID No. 2 having (i) the function of the heavy chain variable region, formed by substituting, deleting, modifying and/or adding at least one (e.g., 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, most preferably 1 or 2) amino acid residues in the amino acid sequence of SEQ ID No. 2.

4. An antibody heavy chain having the heavy chain variable region of claim 3.

5. An antibody, wherein said antibody has:

(1) the light chain variable region of claim 1; and/or

(2) The heavy chain variable region of claim 3;

alternatively, the antibody has: the light chain of claim 2; and/or the heavy chain of claim 4.

6. A recombinant protein, said recombinant protein having:

(i) the light chain variable region of claim 1, the light chain of claim 2, the heavy chain variable region of claim 3, the heavy chain of claim 4, or the antibody of claim 5; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

7. An antibody preparation, comprising:

(a) the antibody of claim 5; and

(b) a vector, said vector comprising: a buffer, sterile water, and optionally a surfactant.

8. A kit comprising the antibody preparation of claim 7 and a container holding said antibody preparation.

9. A CAR construct wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to HER3, and wherein the scFv has the light chain variable region of claim 1 and the heavy chain variable region of claim 3.

10. An antibody drug conjugate, comprising:

(a) an antibody moiety selected from the group consisting of: the light chain variable region of claim 1, the light chain of claim 2, the heavy chain variable region of claim 3, the heavy chain of claim 4, or the antibody of claim 5, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

Technical Field

The invention relates to the field of medicines, in particular to an anti-HER 3 humanized monoclonal antibody and a preparation thereof.

Background

An increasing number of researches have found that human epidermal growth factor 3 (HER 3) plays an important role in the development and progression of tumors. HER3 normally functions by forming heterodimers with EGFR or HER2 molecules, overexpression of EGFR and HER2 is often accompanied by overexpression of HER3 in cancer pathological tissues, and the fact that HER3 overexpression plays an important role in generating resistance to EGFR and HER2 targeted therapy is considered. After being combined with ligand Heregulin (HRG), HER3 undergoes conformational change, the binding sites with EGFR and HER2 are exposed, and then heterodimers are formed with EGFR and HER2 molecules, intracellular signals are activated, and tumor cell proliferation is promoted.

Clinical treatment of murine mabs is limited because they elicit human anti-mouse antibody responses (HAMA) in clinical treatment. The antibody humanization technology can greatly reduce the immunogenicity of the murine monoclonal antibody.

Thus, in view of the role and function of HER3 in various classes of related diseases, there remains a need in the art to develop humanized antibodies against HER3 that are suitable for treating patients.

Disclosure of Invention

The invention aims to provide a HER3 humanized antibody with high affinity and high biological activity and application thereof.

In a first aspect of the present invention, there is provided a light chain variable region of an antibody, said light chain variable region being selected from the group consisting of:

(1) a light chain variable region having a sequence as set forth in SEQ ID No. 1; or

(2) A light chain variable region derived from the sequence of SEQ ID No. 1, having the function of the light chain variable region of (1), formed by substituting, deleting, modifying and/or adding at least one (e.g., 1-20, preferably 1-15, more preferably 1-10, more preferably 1-8, more preferably 1-3, most preferably 1 or 2) amino acid residues in the amino acid sequence of SEQ ID No. 1.

In another preferred embodiment, the light chain variable region corresponds to the mutation at glycine 69 (G) in the sequence shown in SEQ ID No. 1.

In another preferred embodiment, the light chain variable region corresponds to the sequence shown in SEQ ID No. 1 in which glycine (G) at position 69 is mutated to serine (S) (i.e., G69S).

In another preferred embodiment, the light chain variable region sequence is as shown in SEQ ID No. 1 or 3.

In a second aspect of the invention, there is provided a light chain of an antibody, said light chain having a light chain variable region as described in the first aspect of the invention.

In another preferred embodiment, the light chain of the antibody further comprises a heavy chain constant region.

In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin, preferably of human origin.

In a third aspect of the present invention, there is provided a heavy chain variable region of an antibody, wherein the heavy chain variable region is selected from the group consisting of:

(i) a heavy chain variable region having a sequence as set forth in SEQ ID No. 2; or

(ii) A heavy chain variable region derived from the sequence of SEQ ID No. 2 having (i) the function of the heavy chain variable region, formed by substituting, deleting, modifying and/or adding at least one (e.g., 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 3, most preferably 1 or 2) amino acid residues in the amino acid sequence of SEQ ID No. 2.

In another preferred embodiment, the heavy chain variable region corresponds to the sequence shown in SEQ ID No. 2 with amino acid mutations selected from the group consisting of: arginine (R) at position 38, methionine (M) at position 48, valine (V) at position 68, or combinations thereof.

In another preferred embodiment, the mutation of the heavy chain variable region corresponding to the sequence shown in SEQ ID No. 2 is selected from the group consisting of: R38K, M48I, V68A, or a combination thereof; preferably, the mutation is R38K.

In another preferred embodiment, the heavy chain variable region sequence is as shown in SEQ ID No. 2 or 4.

In a fourth aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the third aspect of the invention.

In another preferred embodiment, the heavy chain of said antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin, preferably of human origin.

In a fifth aspect of the invention, there is provided an antibody having:

(1) a light chain variable region according to the first aspect of the invention; and/or

(2) A heavy chain variable region according to the third aspect of the invention;

alternatively, the antibody has: a light chain according to the second aspect of the invention; and/or a heavy chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody has a light chain variable region as set forth in SEQ ID No. 1 or 3; and/or a heavy chain variable region as set forth in SEQ ID No. 2 or 4.

In another preferred embodiment, the antibody has a mutation selected from the group consisting of:

(a) the variable region of the light chain of the antibody corresponds to the 69 th glycine (G) in the sequence shown in SEQ ID NO. 1; and/or

(b) The heavy chain variable region of the antibody corresponds to the sequence shown in SEQ ID NO. 2 with amino acid mutations selected from the group consisting of: arginine (R) at position 38, methionine (M) at position 48, valine (V) at position 68, or combinations thereof.

In another preferred embodiment, the variable region of the light chain of said antibody corresponds to the mutation of glycine 69 (G) in the sequence shown in SEQ ID No. 1.

In another preferred embodiment, the antibody further comprises the presence of an amino acid mutation corresponding to the heavy chain variable region sequence set forth in SEQ ID No. 2 selected from the group consisting of: arginine (R) at position 38, methionine (M) at position 48, valine (V) at position 68, or combinations thereof.

In another preferred example, the light chain variable region of the antibody corresponds to glycine (G) at position 69 mutated to serine (S) in the sequence shown in SEQ ID No.:1, and the heavy chain variable region of the antibody corresponds to arginine (R) at position 38 mutated to lysine (K) in the sequence shown in SEQ ID No.: 2.

In another preferred embodiment, the light chain variable region sequence of the antibody is as shown in SEQ ID No. 1 or 3; and/or the heavy chain variable region sequence of the antibody is shown in SEQ ID NO. 2 or 4.

In another preferred embodiment, the light chain variable region sequence of the antibody is as shown in SEQ ID No. 3; and the heavy chain variable region sequence of the antibody is shown as SEQ ID NO. 2 or 4.

In another preferred embodiment, the antibody has a light chain variable region having the sequence shown in SEQ ID No. 3; and the antibody has a heavy chain variable region with a sequence shown in SEQ ID NO. 4.

In another preferred embodiment, the antibody is a humanized antibody.

In another preferred embodiment, the antibody is specifically binding to HER 3.

In another preferred embodiment, the antibody has an affinity for human HER3 with a KD value (M) of 1.0E-8 to 2.0E-10.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody is a bispecific antibody.

In another preferred embodiment, the antibody is in the form of a drug conjugate.

In a sixth aspect of the present invention, there is provided a recombinant protein having:

(i) a light chain variable region according to the first aspect of the invention, a light chain according to the second aspect of the invention, a heavy chain variable region according to the third aspect of the invention, a heavy chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In a seventh aspect of the invention, there is provided an antibody preparation comprising:

(a) an antibody according to the fifth aspect of the invention; and

(b) a vector, said vector comprising: a buffer, sterile water, and optionally a surfactant.

In another preferred embodiment, in said formulation, said antibody is present at a concentration of 5-100 mg/mL; preferably 10-70mg/mL, more preferably 20-60 mg/mL.

In another preferred embodiment, the buffer is selected from the group consisting of: a citric acid buffer system, a histidine buffer system, or a combination thereof.

In another preferred embodiment, the buffer is a histidine buffer system.

In another preferred embodiment, the histidine buffer system is present in the formulation in a concentration of 0.5-20mM, preferably 1-10 mM.

In another preferred embodiment, the citric acid buffer system comprises histidine and histidine hydrochloride, preferably 1-10mM histidine and 1-10mM histidine hydrochloride, based on the total weight of the antibody formulation.

In another preferred embodiment, the buffer is a citric acid buffer system.

In another preferred embodiment, the concentration of the citric acid buffer system in the formulation is 5-100mM, preferably 10-50 mM.

In another preferred embodiment, the citric acid buffer system comprises sodium citrate and sodium chloride, preferably 10-50mM sodium citrate and 50-200mM sodium chloride, based on the total weight of the antibody formulation.

In another preferred embodiment, the surfactant is selected from the group consisting of: tween 80, tween 20, or a combination thereof.

In another preferred embodiment, the surfactant is present in the formulation in an amount of 0.005 to 0.2 wt.%, based on the total weight of the antibody formulation.

In another preferred embodiment, the surfactant is tween 80, and the concentration is 0.02 to 0.1 wt%.

In another preferred embodiment, the formulation has a pH in the range of 5.0 to 7.5, preferably 5.5 to 7.

In another preferred embodiment, the formulation is an injectable formulation.

In another preferred embodiment, the formulation comprises: an antibody according to the fifth aspect of the invention, a histidine buffer system, a surfactant, and the balance sterile water for injection.

In another preferred embodiment, the formulation comprises:

20-60mg/mL of the antibody of the fifth aspect of the invention;

histidine buffer system 1-10 mM;

tween 800.02-0.1 wt%;

and sterile water for injection as the balance, and the pH of the formulation is preferably in the range of 5.5 to 6.5.

In an eighth aspect of the invention, there is provided a kit comprising an antibody preparation according to the seventh aspect of the invention, and a container for holding the antibody preparation.

In a ninth aspect of the invention there is provided a CAR construct wherein the scFv segment of the antigen binding region of the CAR construct is a binding region that specifically binds to HER3 and has a light chain variable region according to the first aspect of the invention and a heavy chain variable region according to the third aspect of the invention.

In a tenth aspect of the invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the ninth aspect of the invention.

In another preferred embodiment, the immune cell is selected from the group consisting of: NK cells, T cells.

In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).

In an eleventh aspect of the present invention, there is provided an antibody drug conjugate comprising:

(a) an antibody moiety selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In another preferred embodiment, said antibody moiety is coupled to said coupling moiety by a chemical bond or a linker.

In a twelfth aspect of the invention, there is provided the use of an active ingredient selected from the group consisting of: the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, the immune cell according to the tenth aspect of the invention, the antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof, wherein the active ingredient is for use in the administration of a therapeutically effective amount of the active agent

(a) Preparing a detection reagent or a kit;

(b) preparing a medicament or preparation for preventing and/or treating HER3 related diseases; and/or

(c) Preparing a medicament or a preparation for preventing and/or treating cancer or tumor.

In another preferred embodiment, the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof.

In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, or a combination thereof.

In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.

In another preferred embodiment, the tumor is a tumor highly expressing HER 3.

In another preferred embodiment, the medicament or formulation is for the preparation of a medicament or formulation for the prevention and/or treatment of a disease associated with HER3 (positive expression).

In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).

In another preferred embodiment, the detection reagent or the kit is used for diagnosing HER3 related diseases.

In another preferred embodiment, the detection reagent or kit is for detecting HER3 protein in a sample.

In another preferred embodiment, the detection reagent is a detection sheet.

In a thirteenth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) an active ingredient selected from the group consisting of: a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, an immune cell according to the tenth aspect of the invention, an antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

In another preferred embodiment, the pharmaceutical composition is an injection.

In another preferred embodiment, the pharmaceutical composition is used for treating tumors.

In another preferred embodiment, the tumor is a tumor highly expressing HER 3.

In a fourteenth aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or

(2) A recombinant protein according to the sixth aspect of the invention;

(3) a CAR construct according to the ninth aspect of the invention.

In a fifteenth aspect of the invention, there is provided a vector comprising a polynucleotide according to the fourteenth aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In a sixteenth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the fifteenth aspect of the invention or having integrated into its genome a polynucleotide according to the fourteenth aspect of the invention.

In a seventeenth aspect of the invention, there is provided a method for the in vitro detection (including diagnostic or non-diagnostic) of HER3 protein in a sample, said method comprising the steps of:

(1) contacting said sample in vitro with an antibody according to the fifth aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of a complex is indicative of the presence of HER3 protein in the sample.

In an eighteenth aspect of the present invention, there is provided a detection board comprising: a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the invention or an antibody drug conjugate according to the eleventh aspect of the invention.

In a nineteenth aspect of the present invention, there is provided a kit comprising:

(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or

(2) A second container comprising a secondary antibody directed against the antibody according to the fifth aspect of the invention;

alternatively, the kit comprises a detection plate according to the eighteenth aspect of the invention.

In a twentieth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing a host cell according to the fourteenth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

In a twenty-first aspect of the invention, there is provided a method of treating HER 3-related disease, the method comprising: administering to a subject in need thereof an antibody according to the fifth aspect of the invention, an antibody-drug conjugate of said antibody, or a CAR-T cell expressing said antibody, or a combination thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the sequences of HER3 murine mAb and human Germline template variable regions. Wherein a is the light chain variable region and b is the heavy chain variable region. The upper row is a variable region sequence of the mouse monoclonal antibody, the lower row is a variable region sequence after CDR transplantation, the drawn part is a CDR region, and the gray mark part of amino acids are different amino acids in the framework regions of the mouse monoclonal antibody and the human Germline template.

FIG. 2 shows the results of solubility measurements by PEG precipitation, with the arrows indicating the pores where precipitation began to occur.

FIG. 3 shows the endogenous fluorescence detection profile of the humanized antibody of the present invention.

FIG. 4 shows the tumor suppression effect of the humanized antibody of the present invention in animals.

Detailed Description

The present inventors have unexpectedly obtained an anti-HER 3 humanized antibody having excellent affinity and good structural stability through extensive and intensive studies and extensive screening. Specifically, the humanized framework regions are selected to have similarity and human use frequency, the framework regions of IGKV3-20 x 01 and IGHV1-69 x 01 are selected for humanization, and mutation screening is carried out on the obtained humanized antibody. The humanized antibody, especially the mutated humanized antibody, has affinity similar to that of the chimeric antibody, and the solubility and endogenous fluorescence of the humanized antibody are preliminarily studied to verify that the humanized antibody has preliminary druggability, so that the humanized antibody can be further developed into humanized monoclonal antibody medicine for target treatment in the future. And the humanized antibody is subjected to primary druggability research, and the preparation formula of the anti-HER 3 humanized antibody is screened and determined. The present invention has been completed based on this finding.

Term(s) for

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three letter codes and the one letter codes for amino acids used in the present invention are as described in j. diol. chem,243, p3558 (1968).

As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; comprising contacting an anti-HER 3 antibody with a human or animal, a subject, a cell, a tissue, a physiological compartment or a physiological fluid.

As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, to a patient having one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect, comprising any of the anti-HER 3 antibodies of the invention and compositions thereof. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be 1, 2 or 3.

"sequence identity" as referred to herein means the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions. The sequence identity between a sequence described in the present invention and a sequence with which it is identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

HER3

Human epidermal growth factor 3 (HER 3) plays an important role in the development and progression of tumors. HER3 normally functions by forming heterodimers with EGFR or HER2 molecules, overexpression of EGFR and HER2 is often accompanied by overexpression of HER3 in cancer pathological tissues, and the fact that HER3 overexpression plays an important role in generating resistance to EGFR and HER2 targeted therapy is considered. After being combined with ligand Heregulin (HRG), HER3 undergoes conformational change, the binding sites with EGFR and HER2 are exposed, and then heterodimers are formed with EGFR and HER2 molecules, intracellular signals are activated, and tumor cell proliferation is promoted.

The anti-HER 3 murine monoclonal antibody 1044 (application number 2014104015453) can block the binding of HER3 molecules and HRG ligand thereof, thereby inhibiting the formation of heterodimer with other molecules of HER family, can bind with tumor surface HER3 molecules, inhibit the proliferation of human epidermal squamous cell carcinoma A431 cells, and has the potential of treating various HER3 over-expressed tumors.

1044 mouse monoclonal antibody light chain variable region (SEQ ID NO. 5)

DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPWTFGGGTKLEIKR

1044 mouse Single heavy chain variable region (SEQ ID NO. 6)

EVQLQQSGTELMKPGASVKISCKATGGTFSNYWIDWVKQRPGHGLEWIGEILPGSGGTDYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARDDYDVFAYWGQGTLVTVSA

Antibodies

As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of Ig can be divided into different subclasses according to the difference of amino acid composition of the heavy chain region and the number and position of the disulfide bonds of the heavy chain, for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda depending on the constant region. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The antibody light chain of the invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.

In the present invention, the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4, or variants thereof. The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminus to carboxy terminus. The 3 CDR regions of the light chain refer to LCDR1, LCDR2 and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3.

The term "murine antibody" is in the present invention a monoclonal antibody against HER3 prepared according to the knowledge and skill in the art. Preparation is performed by injecting a subject with HER3 antigen and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine HER3 antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or variant thereof.

The term "chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can reduce an immune response induced by the murine antibody.

The term "humanized antibody", also known as CDR-grafted antibody (CDR), refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. The humanized antibody can overcome the heterogenous reaction induced by the chimeric antibody carrying a great deal of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity.

The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., HER 3). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen binding fragment of an antibody" include

(i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains;

(ii)F(ab’)₂a fragment comprising a bivalent fragment of two Fab fragments connected by a disulfide bridge on the chain compare region;

(iii) an Fd fragment consisting of the VH and CH1 domains;

(iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding.

The term "CDR" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding. One of the most common definitions of the 6 CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interest NIH Publication 91-3242).

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on the HER3 molecule). Epitopes typically comprise at least 3,4,5,6,7,8,9,10,11,12,13,14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is administered at a rate of about less than 10^-7M, e.g. less than about 1O^-8M、1O^-9M or lO^-10M or less affinity (KD) binding.

The term "competes for binding" refers to an antibody that recognizes the same epitope (also referred to as an antigenic determinant) or a portion of the same epitope on the extracellular region of HER3 as the monoclonal antibody of the invention and binds to the antigen. An antibody that binds to the same epitope as a monoclonal antibody of the invention refers to an antibody that recognizes and binds to the amino acid sequence of HER3 recognized by a monoclonal antibody of the invention.

The term "KD" or "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the invention are present in an amount less than about 10^-7M, e.g. less than about 1O^-8M、1O^-9M or lO^-10M or less dissociation equilibrium constant (KD) binds to HER 3.

As used herein, the term "antigenic determinant" refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen-binding fragment of the invention.

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.

As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cell may be a eukaryotic, prokaryotic, or phage clonal cell line.

In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.

In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

anti-HER 3 humanized antibodies

The present invention provides an anti-HER 3 humanized antibody (hereinafter abbreviated as HER3 antibody). In particular, the present invention provides a highly specific and high affinity humanized antibody against HER3 comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.

In 1986, Jones et al firstly transplanted the heavy chain CDR of the murine monoclonal antibody to the framework region of the heavy chain of the human antibody, and then assembled with the light chain of the murine monoclonal antibody into a complete antibody and kept the affinity similar to that of the original murine monoclonal antibody, thereby providing a thought for the development of antibody humanization technology. Queen et al succeeded in constructing a humanized antibody against CD25 in 1989 by a CDR grafting method in which a human antibody Eu framework region was humanized and amino acids of a murine antibody were retained at partial sites of the framework region to maintain affinity. In 1992 Presta et al reported a successful humanization method by CDR grafting using human antibody subgroup consensus (consensus sequence) as a template. Pedersen et al, 1994, reported humanization of antibodies using surface remodeling (resurfacing). Hsiao et al, 1994, reported humanization methods for CDR grafting with human antibody Germine sequence framework regions. Jespers et al succeeded in constructing a humanization method by a method using a phage library (Shuffling library) in 1994.

The choice of human framework regions in antibody humanization is generally two, one is a known mature antibody and one is a human Germline sequence. Known mature antibody framework regions often contain somatic mutation sites that may confer potential immunogenicity. Compared with a mature antibody, the human Germline sequence framework region is theoretically lower in immunogenicity, more flexible in structure and strong in plasticity, and can easily accept different CDR regions. The human antibody Germline gene has certain bias in the use frequency of a human body, and the antibody obtained by selecting the Germline framework region with high use frequency for humanization has the advantages of low immunogenicity, high expression quantity, stable structure and the like, so that the Germline sequence with the highest similarity with a murine antibody is not selected during humanization, the similarity and the use frequency of the human body are considered, and the framework regions of IGKV3-20 x 01 and IGHV1-69 x 01 are selected for humanization through a large number of experimental screens. The invention selects the human antibody Germline framework region for CDR transplantation, so that the constructed humanized antibody has more stable structure, high expression quantity, low immunogenicity and higher druggability.

In particular, as described in the first to fifth aspects of the invention.

1044 light chain variable region after CDR-grafting (VL1, VL4 site retained murine monoclonal antibody amino acids) (SEQ ID NO: 1)

DIVMTQSPGTLSLSPGERATLSCRSSKSLLHSNGITYLYWYQQKPGQAPRLLIYQMSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCAQNLELPWTFGQGTKVEIKR

1044 heavy chain variable region following CDR grafting (VH74 site retaining murine monoclonal antibody amino acids) (SEQ ID NO: 2)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWIDWVRQAPGQGLEWMGEILPGSGGTDYNEKFKGRVTFTADTSTSTAYMELSSLRSEDTAVYYCARDDYDVFAYWGQGTLVTVSS

1044 humanized antibody light chain variable region (VL1, VL4, VL69 sites retain murine monoclonal antibody amino acids) (SEQ ID NO: 3)

DIVMTQSPGTLSLSPGERATLSCRSSKSLLHSNGITYLYWYQQKPGQAPRLLIYQMSNLASGIPDRFSSSGSGTDFTLTISRLEPEDFAVYYCAQNLELPWTFGQGTKVEIKR

1044 humanized antibody heavy chain variable region (VH38, VH74 site retained murine monoclonal antibody amino acids) (SEQ ID NO: 4)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWIDWVKQAPGQGLEWMGEILPGSGGTDYNEKFKGRVTFTADTSTSTAYMELSSLRSEDTAVYYCARDDYDVFAYWGQGTLVTVSS

In another preferred embodiment, the constant regions of the humanized antibody are identical to the constant regions of murine monoclonal antibody 1044 (application No. 2014104015453) to HER 3.

In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

The antibody of the present invention may be a double-chain or single-chain antibody, and may preferably be a fully humanized antibody.

The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab')₂Or other known antibody derivatives in the art, and any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.

The antibody of the invention may be a humanized antibody, CDR grafted and/or modified antibody targeting HER 3.

In the above-mentioned aspect of the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

The invention successfully carries out humanized transformation on the HER3 mouse monoclonal antibody, the humanized antibody reaches the affinity similar to that of a chimeric antibody, and the preliminary study on the solubility and endogenous fluorescence of the humanized antibody proves that the humanized antibody has preliminary druggability, so that the humanized monoclonal antibody can be further developed into a humanized monoclonal antibody medicament for targeted therapy in the future.

Preparation of antibodies

Any method suitable for producing monoclonal antibodies may be used to produce the HER3 antibody of the invention. For example, an animal may be immunized with a linked or naturally occurring HER3 protein or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Any suitable form of HER3 may be used as an immunogen (antigen) for the production of a non-human antibody specific for HER3, which antibody is screened for biological activity. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, baculovirus vectors, plasmids and non-viral vectors.

Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.

An exemplary method of humanizing the HER3 antibody of the invention is described in example 1.

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The term "nucleic acid molecule" refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The term "host cell" refers to a cell into which an expression vector has been introduced. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

The steps described in the present invention for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Antibody formulations

The antibody has different stability in different preparation buffers, and is represented by the change of charge heterogeneity, degradation, polymerization and the like of antibody molecules, and the change of the quality properties is related to the physicochemical properties of the antibody, so that the preparation buffers suitable for the antibody need to be screened according to the physicochemical properties of different antibodies in the development process of antibody drugs. The currently commonly used antibody preparation buffer systems include phosphate buffer, citric acid buffer, histidine buffer, and the like, and according to the antibody properties, saline ions with different concentrations or excipients such as sorbitol, trehalose, sucrose, and the like, and a proper amount of surfactants such as tween 20 or tween 80 and the like are added to maintain the stability of the antibody.

The antibody preparation of the invention is as described in the seventh aspect of the invention.

The antibody drug combination preparation can effectively inhibit side reactions such as aggregation precipitation, hydrolysis, oxidation, deamidation and the like of the humanized antibody, and can effectively improve the stability of the product under the conditions of pressurization (high temperature, strong light irradiation, freeze thawing and the like), acceleration and long-term refrigeration.

Pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or an active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

The pharmaceutical composition of the invention can be directly used for binding HER3 protein molecules, and thus can be used for preventing and treating HER3 related diseases. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

Where a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Detection use and kit

The antibodies of the invention are useful in detection applications, for example, for detecting a sample, thereby providing diagnostic information.

In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus, a biopsy as used in the present invention may comprise a tissue sample prepared, for example, by endoscopic methods or by needle or needle biopsy of an organ.

Samples for use in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

The main advantages of the invention

(a) The humanized antibody is a typical structure with better embedding of tryptophan and tyrosine, the screened human VH and VL framework regions have stable structures, and are better matched with the CDR regions of the mouse monoclonal antibody, the light chain variable region and the heavy chain variable region can be well paired together, and the humanized antibody has high affinity and stable structure.

(b) Compared with chimeric antibodies, the humanized antibody has excellent biological activity and specificity, lower immunogenicity and higher expression amount while retaining the affinity equivalent to HER 3.

(c) The humanized antibody has high solubility and good drug forming property.

(d) The humanized antibody of the invention has obvious characteristic of inhibiting tumor growth in an A549 tumor model, and the tumor growth inhibition activity is related to the treatment dosage, thus proving that the humanized anti-HER 3 monoclonal antibody still has good biological activity.

(e) Compared with the mature antibody, the humanized framework region is selected according to similarity and human use frequency, and the humanized antibody has lower immunogenicity, higher expression level (especially in mammalian cells such as CHO-K1 cells), more stable structure and better druggability.

(f) The stability of the humanized antibody of the present invention in the antibody preparation of the present invention is very good.

(g) The humanized antibody of the invention is constructed by stably expressing a cell strain in CHO-K1 cells, and the screened stable cell strain is cultured by Fed Batch in a shake flask, so that the expression quantity of the antibody reaches 4-5 g/L.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Experiments in which specific conditions are not specified in the examples or test examples of the present invention are usually performed under conventional conditions or under conditions recommended by the manufacturers of raw materials/goods; reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Materials and methods

1. Strains, cells, plasmids and test animals

Coli DH5 α competent cells were prepared by the present study room; FreeStyle 293-F cells and expression media were purchased from Gibco; the pSGHV0 eukaryotic expression plasmid was gifted by the biophysical system Dr.D.J.Leahy, university of John Hopkins medical school; babl/c nude mice were purchased from Shanghai Si Laike laboratory animals, Inc.

2. Primary reagent and apparatus

The site-directed mutagenesis kit is purchased from Beijing Saibaosheng Gene technology Co., Ltd; mabselect Sure affinity media was purchased from GE; rituximab (batch No. H0709), trastuzumab (batch No. N3674) were purchased from shanghai roche pharmaceutical limited; the recombinant protein of the HER3 extracellular region is expressed by CHO cells; goat anti-human antibodies were purchased from Jackson ImmunoResearch; polyethylene imine (PEI MW 25000) from Polysciences; polyethylene glycol 8000(PEG 8000) was purchased from sigma aldrich; SDS-MW Analysis Kit was purchased from Beckmann Kulter; OPM-CHO CD07, PFF05, CDF16 media were purchased from Orpummey Biotech, Inc., Shanghai; the 1044 mouse monoclonal antibody is antibody 1044 of 2014104015453.

3. Construction of humanized antibodies

After grafting CDR of 1044 mouse monoclonal antibody to human antibody framework region, humanized variable region gene sequence was synthesized by Shanghai Boshang biotechnology, and then the variable region genes of heavy chain and light chain were constructed respectively on pSGHV0 vector containing constant region of heavy chain and constant region of kappa chain of human IgG 1. The humanized antibody framework regions of VH38, VH48, VH68, VL69 and the like are subjected to back mutation by using a site-directed mutagenesis kit and confirmed by sequencing. The 1044 human murine chimeric antibody and each of the back-mutated humanized antibodies were transiently transfected with PEI in 293F cells and the affinity was determined using ProteOn XPR36 after centrifugation of the transiently transfected cell culture supernatants at 2000 rpm. Constructing stable CHO-K1 cell line with the humanized antibody sequence selected by Onapalmi Biotech limited, expressing the humanized antibody hu1044 with the stable cell line, FedBatch culturing the stable cell line in a shake flask at 5 × 10⁵The stable cell strain is inoculated into a culture flask at the cell density of/ml, the basic culture medium is OPM-CHOCD07, the PFF05 feed culture medium with the culture volume of 3 percent, 5 percent, 7 percent, 5 percent and 3 percent is supplemented on the 3 rd, 5 th, 7 th, 9 th and 11 th days of culture, the CDF16 feed culture medium with the culture volume of 0.3 percent is supplemented on the 5 th, 7 th, 9 th and 11 th days of culture, the glucose content is maintained at 2-6g/L, and the cell survival rate is keptThe culture is finished when the culture time is less than 60% or the culture time is 15 days, and the expression quantity of the antibody reaches 4-5 g/L. The humanized antibody was then purified using a MabselectSure Protein A affinity medium and concentrated to 60 mg/mL.

Determination of antibody affinity in ProteOn XPR36 protein interaction array System

The GLM chip transversely marks goat anti-human antibodies by 6 channels, the channels are rotated by 90 degrees to be changed into chimeric antibodies or humanized antibodies which are longitudinally captured and expressed by 293F cells, the channels are further transversely rotated, HER3 extracellular region proteins of 20nM, 10nM, 5nM, 2.5nM and 1.25nM are respectively injected into the channels 1-5, buffer solution is injected into the channel 6 to obtain 5 kinetic reaction curves which respectively represent the reaction results of antigens and capture antibodies with different concentrations, and the affinity constants (KD M) of the chimeric antibodies and the humanized antibody mutants are calculated by using a Langmuir mode.

Measurement of antibody solubility by PEG precipitation

FB1 solution (90g sodium chloride, 73.5g trisodium citrate, 7g polysorbate 80 dissolved in 1L water for injection, pH adjusted to 6.5) and FB2 buffer (0.3g histidine, 0.5g histidine hydrochloride, 0.08g polysorbate 20, 18.9g trehalose dissolved in 1L water for injection, pH adjusted to 6.0) were prepared. Two 40% PEG 8000 mother liquors were prepared with deionized water, the pH was adjusted to 6.5 and 6.0, respectively, and then the 40% PEG 8000 mother liquors were diluted to different concentrations with FB1 and FB2, respectively. 60mg/mL of the humanized antibody HER3 was ultrafiltered and replaced in FB1 and FB1 buffers, respectively, using ultrafiltration tubes at a final concentration of 20mg/mL in a volume of 100-fold. Then, rituximab, trastuzumab, HER3 humanized mab (FB1) and HER3 humanized mab (FB1) were diluted to 10mg/mL with respective buffers, 100 μ L of each antibody and 100 μ L of PEG 8000 (5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) at different concentrations were mixed in a 96-well plate, and the results were observed after 10-minute reaction.

6. Non-reducing capillary electrophoresis detection (CE-SDS)

Mu.g of the sample was taken, 70. mu.L of SDS sample buffer was added thereto to give a final volume of 95. mu.L, 5. mu.L of 250mM iodoacetamide was added thereto and mixed, and the mixture was centrifuged at 5000rpm for 1 minute, then the mixture was subjected to water bath at 65 ℃ for 4 minutes, cooled to room temperature, and then centrifuged at 5000rpm for 1 minute, and 100. mu.L of the mixture was taken and put into a capillary electrophoresis sampling flask for analysis. An uncoated-fused silica capillary (50 μm internal diameter) was used, with an effective length of 24.5 cm. And (3) electrically injecting a sample with 5kV reverse polarity for 20s, separating the sample at 15kV for 40min, and detecting the wavelength at 220 nm.

7. Ion exchange high performance liquid chromatography (IEX-HPLC)

Mobile phase A: 10mM sodium phosphate solution, pH 7.4; mobile phase B: 10mM sodium phosphate solution, 0.25M sodium chloride, pH 7.4; a chromatographic column: propac WCX-10 (4.0X 250mm) by Daian; a detector: an ultraviolet detector with the wavelength of 280 nm; column temperature: 35 ℃; sample introduction amount: 40 mu g of the mixture; the flow rate is 0.5 mL/min; separation gradient: increasing the content of mobile phase B from 0% to 30% within 27 min; collecting time: and (4) 41 min.

8. Size exclusion high performance liquid chromatography (SEC-HPLC)

Mobile phase: 0.2M sodium phosphate buffer, pH 6.8; a chromatographic column: SuperSW3000 (4.6X 300mm, 4 μm) from TOSOH Bioscience; a detector: an ultraviolet detector with the wavelength of 280 nm; flow rate: 0.35 mL/min; column temperature: 30 ℃; sample introduction amount: 30 mu g of the solution; collecting time: and 20 min.

9. Endogenous fluorescence detection

Respectively diluting the samples to 1.0mg/mL by using respective corresponding buffer solutions, adding the diluted samples into a cuvette, detecting on SpectraMax M5, wherein the excitation wavelength is 295nm, the emission wavelength is scanned from 310nm to 450nm, and the scanning step is 1 nm.

10. In vivo efficacy test of humanized antibody

Culturing human non-small cell lung cancer cell A549 to logarithmic growth phase, digesting with pancreatin, washing with serum-free medium, and culturing at a ratio of 1 × 10⁷The cell amount was inoculated subcutaneously on the back of 3-4-week-old Babl/c nude mice, and the nude mice were randomly grouped into 6 mice each when significant tumor outgrowth was observed two weeks later. The administration concentrations of the HER3 humanized monoclonal antibody are respectively as follows: 30mg/kg, 15mg/kg, 7.5mg/kg, treatment was administered i.p. with PBS as a control, twice weekly, and tumor volumes were measured twice weekly. Tumor volume size in nude mice: v is 0.5 × a × b²And a is the long diameter of the tumor entity, and b is the short diameter of the tumor entity.

11. Humanized antibody formulation screening

11.1 formulation screening I

The following three buffers were prepared: citrate buffer (containing 25mM sodium citrate, 140mM sodium chloride, 0.07% Tween-80, pH6.5), phosphate buffer (containing 50mM phosphate, 140mM sodium chloride, 0.07% Tween-80, pH8.0), histidine buffer (containing 2mM histidine, 2mM histidine hydrochloride, 1.5% trehalose, 0.07% Tween-80, pH6.0), purified humanized antibody were replaced with the above three buffers respectively, the final antibody concentration was 40mg/mL, and the change in charge heteroplasmon detection was carried out by IEX-HPLC method after leaving at-70 ℃, 2-8 ℃ and 37 ℃ for one week.

11.2 formulation screening II

The following two buffers were prepared: citric acid buffer (containing 25mM sodium citrate, 140mM sodium chloride, 0.07% Tween-80, pH6.5), histidine buffer (containing 2mM histidine, 2mM histidine hydrochloride, 0.07% Tween-80, pH6.0), adding 1.5% trehalose or 2% sorbitol to the two buffers, respectively, replacing the purified humanized antibody with the above buffers to give a final antibody concentration of 40mg/mL, standing at 37 deg.C for one week, and detecting the mass change by IEX-HPLC method or SEC-HPLC method.

11.3 formulation screening III

Histidine buffer (containing 2mM histidine, 2mM histidine hydrochloride, 0.07% Tween-80, pH6.0), 25, 50, 75, 125, 150mM sodium chloride were added, and then the humanized antibody was prepared in the above buffers of different salt ions at a final antibody concentration of 40mg/mL, and the influence of the salt ions on antibody turbidity was observed.

The humanized antibody was prepared to 40mg/mL using a histidine buffer (containing 2mM histidine, 2mM histidine hydrochloride, 0.07% Tween-80, pH6.0) and the mass change was measured by IEX-HPLC, SEC-HPLC and non-reducing CE-SDS after leaving at 37 ℃ for two weeks.