阅读说明：本技术 抗her3抗体、其制法及其应用 (anti-HER 3 antibody, preparation method and application thereof ) 是由 瞿爱东 李翱翔 梁红远 祝婧烨 吴丽娜 黄海武 陆瑾 赵鑫 于 2014-08-14 设计创作，主要内容包括：本发明提供了一类抗HER3抗体、其制法及其应用。具体地,本发明提供了一种新型的抗HER3抗体,该抗体与HER3分子具有很强的亲和力,可特异性结合抗原分子,尤其是人鼠嵌合抗体有效降低了鼠抗的免疫原性。本发明的HER3抗体与西妥昔联用,能够显著增强西妥昔单抗的活性,并且能够降低西妥昔单抗的用量。(The invention provides an anti-HER 3 antibody, a preparation method and application thereof. Specifically, the invention provides a novel anti-HER 3 antibody, which has strong affinity with HER3 molecules and can be specifically combined with antigen molecules, particularly a human-mouse chimeric antibody, so that the immunogenicity of a mouse antibody is effectively reduced. The HER3 antibody is combined with cetuximab, so that the activity of the cetuximab can be obviously enhanced, and the dosage of the cetuximab can be reduced.)

1. An antibody heavy chain variable region comprising the following three Complementarity Determining Regions (CDRs):

(1) complementarity determining region CDR1

The complementarity determining region CDR1 is selected from: 17, 29 and 35 in SEQ ID No.:17, 29 and 35;

(2) complementarity determining region CDR2

The complementarity determining region CDR2 is selected from: 18, 30 and 36;

(3) complementarity determining region CDR3

The complementarity determining region CDR3 is selected from: 19, 31 and 37.

2. The heavy chain variable region of the antibody of claim 1, wherein the amino acid sequences of the three complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region are shown in SEQ ID NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19, respectively; or

The amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO. 29, SEQ ID NO. 30 and SEQ ID NO. 31; or

The amino acid sequences of three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 37;

preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 2, 10 or 14.

3. An antibody heavy chain having the heavy chain variable region of the antibody of claim 1; preferably, the antibody heavy chain has the amino acid sequence shown in SEQ ID No. 42, 43 or 45.

4. An antibody light chain variable region comprising the following three Complementarity Determining Regions (CDRs):

(1) complementarity determining region CDR1'

The complementarity determining region CDR1' is selected from: 20, 32 and 38;

(2) complementarity determining region CDR2'

The complementarity determining region CDR2' is selected from: 21, 33 and 39 of SEQ ID No.:21, 33;

(3) complementarity determining region CDR3'

The complementarity determining region CDR3' is selected from: 22, 34 and 40.

5. The antibody light chain variable region of claim 4, wherein the amino acid sequences of the three complementarity determining regions CDR1', CDR2', CDR3' of the light chain variable region are set forth in SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 22, respectively; or

The amino acid sequences of three complementarity determining regions CDR1', CDR2' and CDR3' of the light chain variable region are respectively shown in SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 34; or

The amino acid sequences of three complementarity determining regions CDR1', CDR2' and CDR3' of the light chain variable region are respectively shown in SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO. 40;

preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 4, 12 or 16.

6. A light chain of an antibody, wherein said light chain has the variable region of the light chain of the antibody of claim 4; preferably, the antibody light chain has an amino acid sequence as set forth in SEQ ID No. 46, 47 or 49.

7. A monoclonal antibody that specifically binds to HER3, said monoclonal antibody having the following properties:

(1) the affinity constant M of the compound with HER3 protein is more than or equal to 1 multiplied by 10^-10；

(2) Binds to the DI or DIII domain of the extracellular region of HER 3.

8. The monoclonal antibody of claim 7, wherein said monoclonal antibody has:

(1) the heavy chain variable region of claim 1, and/or

(2) The light chain variable region of claim 4; or

The monoclonal antibody has:

(1) the heavy chain of claim 3, and/or

(2) The light chain of claim 6.

9. A recombinant protein, said recombinant protein having:

(i) the heavy chain variable region of the antibody of claim 1, the antibody heavy chain of claim 3, the light chain variable region of the antibody of claim 4, the antibody light chain of claim 6, or the monoclonal antibody of claim 7; 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, said recombinant protein is specifically anti-HER 3.

In another preferred embodiment, the recombinant protein is selected from the group consisting of:

(a) a polypeptide having an amino acid sequence set forth in SEQ ID No. 42, 43, 45, 46, 47, or 49;

(b) a polypeptide which is derived from (a) and is specific against HER3, and which is formed by substituting, deleting or adding one or more (such as 1-20) amino acid residues to the amino acid sequence in (a).

10. An immunoconjugate, comprising:

(a) a carrier moiety comprising the heavy chain variable region of the antibody of claim 1, the antibody heavy chain of claim 3, the light chain variable region of the antibody of claim 4, the antibody light chain of claim 6, or the monoclonal antibody of claim 7 or the recombinant protein of claim 9; and

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

11. A polynucleotide encoding a protein selected from the group consisting of:

the variable region of the heavy chain of the antibody of claim 1, the heavy chain of the antibody of claim 3, the variable region of the light chain of the antibody of claim 4, the light chain of the antibody of claim 6, or the monoclonal antibody of claim 7 or the recombinant protein of claim 9.

12. A vector comprising the polynucleotide of claim 11.

13. A genetically engineered host cell comprising the vector or genome of claim 12 having the polynucleotide of claim 11 integrated therein.

14. A method for producing a recombinant polypeptide, the method comprising:

(a) culturing the host cell of claim 13 under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being the monoclonal antibody of claim 7 or the recombinant protein of claim 9.

15. A pharmaceutical composition comprising:

(i) the variable region of the heavy chain of the antibody of claim 1, the heavy chain of the antibody of claim 3, the variable region of the light chain of the antibody of claim 4, the light chain of the antibody of claim 6, or the monoclonal antibody of claim 7 or the recombinant protein of claim 9; and

(ii) a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 16, wherein the composition further comprises cetuximab.

17. The variable region of the heavy chain of the antibody of claim 1, the heavy chain of the antibody of claim 3, the variable region of the light chain of the antibody of claim 4, the light chain of the antibody of claim 6, or the monoclonal antibody of claim 7 or the recombinant protein of claim 9 for:

(a) separating, preparing, extracting and detecting cells; or

(b) Preparing products for separating, preparing, extracting and detecting cells.

18. A method for detecting the presence of HER3 protein in a sample, comprising the steps of:

(1) contacting a sample with the monoclonal antibody of claim 7;

(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.

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an anti-HER 3 antibody, and a preparation method and application thereof.

Background

HER3(ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine protein kinase erbB-3, SEQ ID NO: 41) is a member of the tyrosine kinase family of encoded Epidermal Growth Factor Receptors (EGFR), which also includes HER1 (also known as EGFR), HER2, and HER4, among others. The HER3 structure includes an extracellular region to which a ligand binds, a transmembrane region of an alpha helix, an intracellular region with a tyrosine kinase domain and a tyrosine-rich C-terminal phosphorylation site. The extracellular domain is further divided into I (L1), II (S1), III (L2) and IV (S2), domains I and III are ligand binding regions, and domains II and IV are dimerization domains exposed by conformational change after ligand binding. Heregulin (HRG) is a specific ligand for HER3, the expression and function of HER3 is influenced by the spatiotemporal expression of the ligand, and HRG stimulates intracellular signaling by promoting the formation of heterodimers of HER3 with other members of the HER family. Thus, it binds to this ligand, but is unable to transmit a signal into the cell via protein phosphorylation. However, it forms heterodimers with other HER family members having kinase activity. Heterodimerization results in activation of the receptor-mediated signaling pathway and transphosphorylation of its intracellular domain. Dimer formation among HER family members amplifies the signaling potential of HER3 and is a means for not only signal diversification, but also signal amplification. For example, HER2/HER3 heterodimer induces one of the most important mitogenic signals among HER family members via PI3K and the AKT pathway, and amplification of this gene and/or overexpression of its protein has been reported in many cancers, including prostate, bladder, and breast tumors. Alternative transcriptional splice variants encoding different isoforms have been characterized. One isoform lacks the intermembranous region and is secreted extracellularly. This form acts to modulate the activity of the membrane bound form. Other splice variants have also been reported, but they have not been fully characterized.

WO97/35885 relates to HER3 antibodies. WO2003/013602 relates to inhibitors of HER activity, including HER antibodies. WO2007/077028 and WO2008/100624 also relate to HER3 antibodies. However, there is still a lack in the art of a sensitive and specific antibody against HER 3.

Disclosure of Invention

The invention aims to provide a novel HER3 specific antibody or a fragment thereof.

Another object of the present invention is to provide a method for producing the above antibody or a fragment thereof and use thereof.

In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three Complementarity Determining Regions (CDRs):

(1) complementarity determining region CDR1

The complementarity determining region CDR1 is selected from: 17, 23, 29 and 35;

(2) complementarity determining region CDR2

The complementarity determining region CDR2 is selected from: 18, 24, 30 and 36;

(3) complementarity determining region CDR3

The complementarity determining region CDR3 is selected from: 19, 25, 31 and 37.

In another preferred embodiment, the amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19, respectively.

In another preferred embodiment, the amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO. 23, SEQ ID NO. 24 and SEQ ID NO. 25, respectively.

In another preferred embodiment, the amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO. 29, SEQ ID NO. 30 and SEQ ID NO. 31, respectively.

In another preferred embodiment, the amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO. 35, SEQ ID NO. 36 and SEQ ID NO. 37, respectively.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence as set forth in SEQ ID No. 2, 6, 10 or 12.

In a second aspect of the invention, there is provided an antibody heavy chain having the heavy chain variable region of the antibody of claim 1.

In another preferred embodiment, the antibody heavy chain has the amino acid sequence shown in SEQ ID No. 42, 43, 44 or 45.

In a third aspect of the present invention, there is provided an antibody light chain variable region, wherein the light chain variable region comprises the following three Complementarity Determining Regions (CDRs):

(1) complementarity determining region CDR1'

The complementarity determining region CDR1' is selected from: 20, 26, 32 and 38;

(2) complementarity determining region CDR2'

The complementarity determining region CDR2' is selected from: 21, 27, 33 and 39;

(3) complementarity determining region CDR3'

The complementarity determining region CDR3' is selected from: 22, 28, 34 and 40.

In another preferred embodiment, the amino acid sequences of the three complementarity determining regions CDR1', CDR2', CDR3' of the light chain variable region are shown in SEQ ID NO. 20, SEQ ID NO. 21 and SEQ ID NO. 22, respectively.

In another preferred embodiment, the amino acid sequences of the three complementarity determining regions CDR1', CDR2', CDR3' of the light chain variable region are shown in SEQ ID NO. 26, SEQ ID NO. 27 and SEQ ID NO. 28, respectively.

In another preferred embodiment, the amino acid sequences of the three complementarity determining regions CDR1', CDR2', CDR3' of the light chain variable region are shown in SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 34, respectively.

In another preferred embodiment, the amino acid sequences of the three complementarity determining regions CDR1', CDR2', CDR3' of the light chain variable region are shown in SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO. 40, respectively.

In another preferred embodiment, the light chain variable region has the amino acid sequence set forth in SEQ ID No. 4, 8, 12 or 16.

In a fourth aspect of the present invention, there is provided a light chain of an antibody, said light chain having the light chain variable region of the antibody of claim 3.

In another preferred embodiment, the antibody light chain has the amino acid sequence shown in SEQ ID No. 46, 47, 48 or 49.

In another preferred embodiment, the constant region of the heavy chain and/or the constant region of the light chain is human.

In a fifth aspect of the invention, there is provided a monoclonal antibody that specifically binds HER3, wherein the monoclonal antibody has the following properties:

(1) the affinity constant M of the compound with HER3 protein is more than or equal to 1 multiplied by 10^-10；

(2) Binds to the DI or DIII domain of the extracellular region of HER 3.

In another preferred embodiment, the monoclonal antibody inhibits the IC of HER3 binding to its ligand Heregulin₅₀The value is less than or equal to 3 nM.

In another preferred embodiment, the binding site of said monoclonal antibody to HER3 comprises one or more sites selected from the group consisting of: serine at position 125, aspartic acid at position 150, arginine at position 151 and histidine at position 467.

In another preferred embodiment, the monoclonal antibody has:

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

(2) The light chain variable region of claim 3.

In another preferred embodiment, the monoclonal antibody has:

(1) the heavy chain of claim 2; and/or

(2) The light chain of claim 4.

In another preferred embodiment, the monoclonal antibody comprises: single chain antibodies, diabodies, chimeric antibodies (e.g., human murine chimeric antibodies), murine antibodies, humanized antibodies, or combinations thereof.

In another preferred embodiment, the monoclonal antibody is an IgG (IgG1) type antibody.

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

(i) the variable region of the heavy chain of the antibody of claim 1, the heavy chain of the antibody of claim 2, the variable region of the light chain of the antibody of claim 3, the light chain of the antibody of claim 4, or the monoclonal antibody of claim 5; 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, said recombinant protein is specifically anti-HER 3.

In another preferred embodiment, the recombinant protein is selected from the group consisting of:

(a) a polypeptide having an amino acid sequence as set forth in SEQ ID No. 42-49;

(b) a polypeptide which is derived from (a) and is specific against HER3, and which is formed by substituting, deleting or adding one or more (such as 1-20) amino acid residues to the amino acid sequence in (a).

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

(a) a carrier moiety comprising the heavy chain variable region of the antibody of claim 1, the heavy chain of the antibody of claim 2, the light chain variable region of the antibody of claim 3, the light chain of the antibody of claim 4, or the monoclonal antibody of claim 5 or the recombinant protein of claim 7; and

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

In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.

In an eighth aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of:

the variable region of the heavy chain of an antibody according to the first aspect of the invention, the variable region of the heavy chain of an antibody according to the second aspect of the invention, the variable region of the light chain of an antibody according to the third aspect of the invention, the light chain of an antibody according to the fourth aspect of the invention, or a monoclonal antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

In another preferred embodiment, the polynucleotide has the DNA sequence shown in SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15 or 50-57.

According to a ninth aspect of the invention, there is provided a vector comprising a polynucleotide according to the eighth 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 tenth aspect of the invention, there is provided a genetically engineered host cell comprising a vector or genome according to the ninth aspect of the invention into which has been integrated a polynucleotide according to the eighth aspect of the invention.

In an eleventh 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 tenth aspect of the invention under conditions suitable for expression;

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

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

(i) the variable region of the heavy chain of the antibody of claim 1, the heavy chain of the antibody of claim 2, the variable region of the light chain of the antibody of claim 3, the light chain of the antibody of claim 4, or the monoclonal antibody of claim 5 or the recombinant protein of claim 7; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in the form of injection.

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating tumors selected from the group consisting of: non-small cell lung cancer, melanoma, head and neck tumors, gastric cancer, liver cancer, leukemia, kidney tumors, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, adrenal gland tumors, or bladder tumors.

In another preferred embodiment, the composition further comprises cetuximab.

In another preferred embodiment, the composition comprises the monoclonal antibody of the fifth aspect of the present invention and cetuximab, wherein the weight ratio of the monoclonal antibody to the cetuximab is 1-10: 10-1; preferably, the weight ratio of the monoclonal antibody to cetuximab is 1-5: 10-1; more preferably, the weight ratio of the monoclonal antibody to cetuximab is 1-5: 1

In a thirteenth aspect of the invention, there is provided a use of the heavy chain variable region of an antibody according to the first aspect of the invention, the heavy chain of an antibody according to the second aspect of the invention, the light chain variable region of an antibody according to the third aspect of the invention, the light chain of an antibody according to the fourth aspect of the invention, or the monoclonal antibody according to the fifth aspect of the invention or the recombinant protein according to the sixth aspect of the invention for:

(a) separating, preparing, extracting and detecting cells; or

(b) Preparing products for separating, preparing, extracting and detecting cells.

In another preferred embodiment, the cell is a cell expressing HER3 molecule; preferably a human cell expressing the HER3 molecule.

In another preferred embodiment, the product for separating, preparing, extracting and detecting cells comprises: a medium, a magnetic bead, a fluorescent-labeled antibody, a chemical-label-labeled antibody, a radioisotope-labeled antibody, a colloidal gold-labeled antibody, an enzyme-labeled antibody, or the like.

In another preferred embodiment, the product for separating, preparing, extracting and detecting cells comprises: devices, kits, and the like.

In a fourteenth aspect of the invention, there is provided a method for isolating in vitro a human cell expressing a HER3 molecule, comprising the steps of: the antibody of the fifth aspect of the invention or the recombinant protein of the sixth aspect of the invention (or a product for isolating, preparing, extracting and detecting cells) is co-incubated with or bound to a cell expressing HER3 molecule from a human, and the cell bound to the antibody is isolated (e.g. eluted or purified), thereby effecting isolation of the cell expressing HER3 molecule from the human.

In another preferred embodiment, the product for separating, preparing, extracting and detecting cells comprises: a medium containing the monoclonal antibody according to the fifth aspect of the present invention, magnetic beads, a fluorescent-labeled antibody, a chemical-label-labeled antibody, a radioisotope-labeled antibody, a colloidal gold-labeled antibody, an enzyme-labeled antibody, or the like.

In a fifteenth aspect of the invention, there is provided a method for detecting the presence of HER3 protein in a sample, said method comprising the steps of:

(1) contacting the sample with a monoclonal 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 another preferred example, in step (1) the sample is contacted with two antibodies against the HER3 protein, at least one of which is an antibody according to the fifth aspect of the invention, and detected by ELISA in step (2).

In another preferred embodiment, the "antigen-antibody complex" is a "first antibody-antigen-second antibody" ternary complex, wherein the first antibody is an antibody according to the fifth aspect of the invention and the binding epitope of the second antibody is different from the binding epitope of the first antibody.

In another preferred embodiment, the first antibody or the second antibody is detectably labeled.

In another preferred embodiment, the detectable label is a biotin label, a colloidal gold label, a horseradish peroxidase label, a radionuclide label, a fluorescein label, or a fluorescent protein label.

In another preferred example, the sample comprises: human or animal tissue samples, tumor resection samples, cultured human or animal cell samples.

In another preferred example, the detection is performed by ELISA method in step (2).

In another preferred embodiment, the method is used for non-diagnostic purposes.

In a sixteenth aspect of the invention, there is provided a method of treating a disorder associated with the HER3 molecule, comprising the steps of: administering to a subject in need of inhibition or treatment a monoclonal antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention or a pharmaceutical composition according to the twelfth aspect of the invention.

In another preferred embodiment, the HER3 molecule-related disease comprises a tumor, and an antagonistic organ transplant immune rejection.

In another preferred embodiment, the subject is a mammal (including a human).

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 detection results of the flow-type detector, in which fig. 1A shows the detection result of antibody 927, fig. 1B shows the detection result of 993, fig. 1C shows the detection result of antibody 1044, and fig. 1D shows the detection result of antibody 1050.

FIG. 2 shows IC of murine mAb inhibiting the binding of HER3 to ligand₅₀Figure 2A shows that HER3 antibody 927 inhibits binding of HER3 to ligand HRG, figure 2B shows that HER3 antibody 993 inhibits binding of HER3 to ligand HRG, figure 2C shows that HER3 antibody 1044 inhibits binding of HER3 to ligand HRG, and figure 2D shows that HER3 antibody 1050 inhibits binding of HER3 to ligand HRG.

FIG. 3 shows the in vivo tumor model experiment of human non-cell lung cancer A549, in which FIG. 3A is the detection result of tumor volume and FIG. 3B is the detection result of tumor weight.

Fig. 4 shows the synergistic effect of HER3 mab and cetuximab, fig. 4A shows the experiment for HER3 antibody 1044 inhibiting a431 growth synergistically with cetuximab, fig. 4B shows the experiment for HER3 antibody 927 inhibiting a431 growth synergistically with cetuximab, fig. 4C shows the experiment for HER3 antibody 993 inhibiting a431 growth synergistically with cetuximab, and fig. 4D shows the experiment for HER3 antibody 1050 inhibiting a431 growth synergistically with cetuximab. In the figure, the dosage of the cetuximab alone is 5 mu g/ml, and the dosage of the cetuximab of the synergistic group is 2.5 mu g/ml; the abscissa of each graph represents the number in μ g/ml (antibody amount).

Detailed Description

The inventor obtains a specific antibody of anti-HER 3 through extensive and intensive research, and experimental results show that the antibody can obviously inhibit the binding of HER3 and a ligand thereof. And the unexpected discovery that the antibody and cetuximab are used together to have a remarkable synergistic effect, so that the dosage of cetuximab can be obviously reduced. The present invention has been completed based on this finding.

HER3(ErbB-3, ERBB3, c-erbB-3, c-erbB3, receptor tyrosine protein kinase erbB-3, SEQ ID NO: 41) is a member of the tyrosine kinase family encoding Epidermal Growth Factor Receptor (EGFR), and in a preferred embodiment of the invention, the amino acid sequence of HER3 is as follows:

MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT(SEQ ID NO.:41)

as used herein, the term "heavy chain variableZone "and" V_H"may be used interchangeably.

As used herein, the terms "light chain variable region" and "V_L"may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to an antibody that specifically binds to HER3, such as a protein or polypeptide having a heavy chain (e.g. the amino acid sequence of SEQ ID No.:42, 43, 44, 45) and/or a light chain (e.g. the amino acid sequence of SEQ ID No.:46, 47, 48, 49). They may or may not contain the initial methionine.

The invention provides an antibody (monoclonal antibody) against HER3 or a fragment thereof.

Preferably, the heavy chain variable region of the antibody may have complementarity determining regions CDRs selected from the group consisting of: 17, 23, 29, 35, 18, 24, 30, 36, and 3 as shown in SEQ ID nos 19, 25, 31, 37.

More preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 2, 6, 10, 14.

Preferably, the light chain variable region of the antibody may have complementarity determining region CDRs selected from the group consisting of: the CDR1' shown in SEQ ID NO. 20, 26, 32, 38, the CDR2' shown in SEQ ID NO. 21, 27, 33, 39 and the CDR3' shown in SEQ ID NO. 22, 28, 34, 40.

More preferably, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 4, 8, 12, 16.

In another preferred embodiment, the antibody is an anti-HER 3 human murine chimeric monoclonal antibody, wherein the heavy chain constant region and/or the light chain constant region can be humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy or light chain constant region is that of human IgG1, IgG2, or the like.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains with variable regions, provided that the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy and light chains of the antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only complete monoclonal 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.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having human HER3 binding activity comprising the CDR regions described above. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted 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

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region as shown in SEQ ID Nos. 50-57 or may be a degenerate variant. As used herein, "degenerate variant" refers herein to a nucleic acid sequence that encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differs from the coding region sequence set forth in SEQ ID NO. 50-57.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, the polynucleotides that hybridize encode polypeptides having the same biological functions and activities as the mature polypeptides set forth in SEQ ID No. 10 and/or SEQ ID No. 15.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, 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. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

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. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

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 host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biological toxins (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and immunotherapy (Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research (Cancer Research)63, 3202); 4. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 5. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 6. liposomes (Mamot et al, 2005, cancer research 65, 11631); 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or active fragment thereof or fusion protein thereof, 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 pharmaceutical composition of the invention can be used directly for binding to the human HER3 molecule and thus can be used for the prevention and treatment of tumors. 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.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 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.

The main advantages of the invention are:

(1) a novel class of anti-HER 3 antibodies is provided, which have strong affinity to HER3 molecules and can specifically bind to antigen molecules, especially human-mouse chimeric antibodies, and the immunogenicity of mouse antibodies is effectively reduced.

(2) The anti-HER 3 antibody of the invention has significant activity in inhibiting tumor growth.

(3) The HER3 antibody is combined with cetuximab, so that the activity of the cetuximab can be obviously enhanced, and the dosage of the cetuximab can be reduced.

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, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: 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 by weight.

The experimental materials used in the examples of the present invention were obtained from commercial sources unless otherwise specified, wherein Balb/c mice were purchased from Shanghai Slek Inc.; CHO/dhfr^-Commodity number of cells purchased from American ATCC, ATCCCRL-9096^TM(ii) a Sp2/0-Ag14 mouse myeloma cells were purchased from ATCC, ATCC accession No.

CRL-1581^TM。