WO2016110267A1 - 具有共同轻链的双特异性抗体或抗体混合物 - Google Patents

具有共同轻链的双特异性抗体或抗体混合物 Download PDF

Info

Publication number
WO2016110267A1
WO2016110267A1 PCT/CN2016/070447 CN2016070447W WO2016110267A1 WO 2016110267 A1 WO2016110267 A1 WO 2016110267A1 CN 2016070447 W CN2016070447 W CN 2016070447W WO 2016110267 A1 WO2016110267 A1 WO 2016110267A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
light chain
antibody
binding
mixture
Prior art date
Application number
PCT/CN2016/070447
Other languages
English (en)
French (fr)
Inventor
徐霆
须涛
汪皛皛
李倩
逄敏洁
张慧敏
韩莉
张庆青
Original Assignee
苏州康宁杰瑞生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州康宁杰瑞生物科技有限公司 filed Critical 苏州康宁杰瑞生物科技有限公司
Priority to ES16734927T priority Critical patent/ES2811267T3/es
Priority to DK16734927.3T priority patent/DK3243840T3/da
Priority to CN201680005167.4A priority patent/CN107108747B/zh
Priority to US15/541,921 priority patent/US10808043B2/en
Priority to JP2017536858A priority patent/JP7041516B2/ja
Priority to EP16734927.3A priority patent/EP3243840B8/en
Priority to CN202111616341.8A priority patent/CN114276458A/zh
Publication of WO2016110267A1 publication Critical patent/WO2016110267A1/zh
Priority to US17/022,545 priority patent/US11396557B2/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to bispecific antibodies or antibody mixtures, and to methods of making such bispecific antibodies or antibody mixtures.
  • the invention also relates to nucleic acid molecules encoding the bispecific antibodies or antibody mixtures, recombinant vectors and recombinant cells comprising the nucleic acid molecules, and methods of detecting and quantifying the bispecific antibodies or antibody mixtures.
  • monoclonal antibody drugs has grown rapidly in the past 15 years and has become a growth point for the pharmaceutical industry. Since 1996, a total of 30 or so monoclonal antibodies have been approved for marketing. Among them, nine monoclonal antibodies have annual sales of more than one billion US dollars. The total sales of monoclonal antibodies in 2010 exceeded $30 billion and the annual growth rate exceeded 10%. Because monoclonal antibodies have strong target specificity, they can only inhibit single targets, and in many diseases, including tumors and autoimmune diseases, it is necessary to inhibit multiple signaling pathways to avoid compensatory effects. For viral infections, due to the high mutation rate of the virus, it is often necessary to inhibit multiple antigenic sites to prevent escape. Therefore, there are several alternatives that can solve such problems.
  • An alternative is to use a polyclonal antibody, or to obtain a heterodimer such as a bispecific antibody by engineering the Fc portion of the antibody, at least for two different antigens or different binding sites for the same antigen.
  • Yet another approach is to treat with a mixture of antibodies, which may comprise two or more antibodies directed against different epitopes on the same target, or a mixture of antibodies directed against different targets.
  • Bispecific antibodies are immunoglobulin molecules containing two different ligand binding sites. It replaces the same sequence of the classical antibody Fab, but uses two different Fab sequences, so the Y-arms can bind to different epitopes.
  • the use of bispecific antibodies in the treatment of cancer has been reviewed in several literatures (Carter 2001; Chames and Baty 2009; Chames and Baty 2009).
  • One arm of BsAbs can connect to the relevant antigen on the surface of tumor cells, while the other arm can trigger immune effector cells to further kill cells and kill cancer tumor cells through the immune system.
  • Carter et al. used the "knob into hole” model to engineer a partial amino acid of the heavy chain of the antibody as early as the 1990s, and successfully achieved the preparation of bispecific antibodies (Ridgway, Presta et al. 1996; Carter 2001).
  • the “hole-hole” model is still insufficient for hindering the formation of homodimers, and there are still about 5% homodimers left.
  • the team then further increased the heterodimer content by random mutation-phage display, but did not solve the underlying problem.
  • the inventors of the present invention modify the CH3-related amino acids of Fc by a charge amino acid interaction network to attenuate the region's own interactions (favoring to form homodimers) and enhance the interaction between the regions (favoring the formation of heterodimers), Successfully solved the residue of 5% homodimer in the "handle-hole" model, and the related method has been published (publication number: CN102558355A).
  • the Applicant has invented a method for producing a mixture comprising two or more homodimeric proteins or antibodies in a single recombinant cell by mutating the Fc portion to alter the direct interaction of the Fc. This approach avoids the potential for process control and amplification due to mixed cell culture, providing a more cost-effective way to prepare and produce antibody mixtures.
  • This program has also been published (publication number: CN 103388013A).
  • Herceptin also known as trastuzumab, Trastuzumab
  • trastuzumab a monoclonal antibody against human human epidermal growth factor receptor 2 (HER2).
  • the antibody which acts on the HER2-Neu surface protein of breast cancer cells, interferes with the biological processes of cancer cells and ultimately causes death.
  • the main suitable population of Herceptin is breast cancer patients with HER2 overexpression (immunohistochemistry 3+ or fluorescence in situ hybridization FISH positive), which accounts for 20-30% of all breast cancer patients.
  • Pertuzumab is a recombinant monoclonal antibody that binds to the extracellular domain II region of the HER-2 receptor, inhibits the formation of dimers, and inhibits receptor-mediated signal transduction pathways (Agus). DB, Gordon MS, Taylor C, et al. 2005). This may partly explain Patuxu Monoclonal antibody inhibits the growth of HER-2 low-expression tumors, while trastuzumab binds to the extracellular IV region of the HER-2 receptor, and the formation of dimers does not involve the IV region, so trastuzumab only HER-2 overexpressing breast cancer patients are effective. Phase II clinical studies of pertuzumab in the treatment of HER-2 low-expression advanced breast cancer are currently underway.
  • the present invention by taking pertuzumab and trastuzumab as an example, prepares a bispecific antibody and antibody mixture having both the function of pertuzumab and trastuzumab, and finds on the basis of this A new method for preparing bispecific antibodies or antibody mixtures in which light and heavy chains can be correctly combined.
  • bispecific antibodies or antibody mixtures with a common light chain enable the correct combination of light and heavy chains and have good binding properties, biological activity and stability, even biological activity compared to the two original antibodies Better than the original antibody.
  • a first aspect of the invention relates to a bispecific antibody or antigen binding portion thereof, characterized in that said bispecific antibody or antigen binding portion thereof has a common light chain, said common light chain means that two light chains have the same sequence .
  • the heavy chain thereof is capable of correctly binding to the light chain under physiological conditions or in vitro protein expression states, respectively.
  • the bispecific antibody or antigen binding portion thereof The common light chain was engineered from two original monoclonal antibodies (known monoclonal antibodies) that differed from at least the light chain sequence of one of the two original monoclonal antibodies.
  • the common light chain is identical to or modified on the basis of one of the two original monoclonal antibodies (eg, amino acid sequence modification), and the purpose of the modification is to Maintain affinity with the respective antigen or epitope.
  • the amino acid sequence modification comprises a mutation, deletion or addition of an amino acid, such as a mutation, deletion or addition of no more than 3 amino acids, preferably no more than 2 amino acids, more preferably no more than 1 amino acid.
  • the heavy chain Fc segment of the bispecific antibody or antigen binding portion thereof is engineered to be more advantageous for the formation of a heterodimeric protein.
  • the two original monoclonal antibodies are Patuxa antibody and Trastaff antibody.
  • the common light chain is capable of binding to the heavy chain of pertuzumab and trastuzumab, respectively.
  • the common light chain is selected from the light chain of pertuzumab or trastuzumab or a mutant thereof.
  • the heavy chain (including the variable region and the constant region) of the bispecific antibody or antigen binding portion thereof may be identical to the two original monoclonal antibodies or may be modified to facilitate formation. Heterodimeric protein; the modification is, for example, engineering the heavy chain Fc segment to facilitate formation of a heterodimeric protein.
  • the sequence of the variable region of the common light chain comprises a sequence selected from amino acids 1 to 107 of SEQ ID NO: 1 to SEQ ID NO: 6.
  • the sequence of the light chain constant region comprises the sequence of amino acids 108 to 214 of SEQ ID NO: 1.
  • the heavy chain variable regions are the heavy chain variable regions of pertuzumab and trastuzumab, respectively.
  • sequences of the two heavy chain variable regions comprise the sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
  • sequences of the two heavy chain Fc segments thereof comprise the sequences set forth in SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
  • sequences of the two heavy chains comprise the sequences set forth in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.
  • a second aspect of the invention relates to a mixture of antibodies or antigen binding portions thereof which are capable of being correctly produced in a cell, said mixture comprising at least two antibodies or antigen binding portions thereof,
  • the antibody or antigen binding portion thereof has a common light chain, which means that the variable regions of the two light chains have the same sequence.
  • the heavy chain of the antibody or antigen binding portion thereof is capable of correctly binding to the light chain under physiological conditions or protein expression states in vitro, respectively.
  • the common light chain of the bispecific antibody or antigen binding portion thereof is engineered from two original monoclonal antibodies (known monoclonal antibodies), the common light chain being at least two The light chain sequence of one of the original monoclonal antibodies was different.
  • the common light chain is identical to or modified on the basis of one of the two original monoclonal antibodies (eg, amino acid sequence modification), and the purpose of the modification is to Maintain affinity with the respective antigen or epitope.
  • the amino acid sequence modification comprises a mutation, deletion or addition of an amino acid, such as a mutation, deletion or addition of no more than 3 amino acids, preferably no more than 2 amino acids, more preferably no more than 1 amino acid.
  • the heavy chain of the bispecific antibody or antigen binding portion thereof is derived from two original monoclonal antibodies, the heavy chain variable region sequence of the bispecific antibody or antigen binding portion thereof The sequence of the and/or CH1 domain is identical to the original monoclonal antibody.
  • the heavy chain (including the variable region and the constant region) of the bispecific antibody or antigen binding portion thereof may be identical to the two original monoclonal antibodies or modified to be more advantageous
  • the homodimeric protein is formed; the modification is, for example, engineering the heavy chain Fc segment to facilitate formation of the homodimeric protein.
  • the two original monoclonal antibodies are a Patuxa antibody and a Herceptin antibody.
  • the common light chain is capable of binding to the heavy chain of pertuzumab and trastuzumab, respectively.
  • the common light chain is selected from the light chain of pertuzumab or trastuzumab or a mutant thereof.
  • the sequence of the variable region of the common light chain comprises a sequence selected from amino acids 1 to 107 of SEQ ID NO: 1 to SEQ ID NO: 6.
  • the sequence of the light chain constant region comprises the sequence of amino acids 108 to 214 of SEQ ID NO: 1.
  • the heavy chain variable region of the antibody or antigen binding portion thereof is a heavy chain variable region of pertuzumab and trastuzumab, respectively.
  • sequences of the two heavy chain variable regions comprise the sequences set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
  • sequences of the two heavy chain Fc segments thereof comprise the sequences set forth in SEQ ID NO:27 and SEQ ID NO:28, respectively.
  • the heavy chain sequence of the antibody or antigen binding portion thereof comprises the sequences set forth in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.
  • a third aspect of the invention relates to a variant protein of the extracellular region of the HER2 protein which has a mutation selected from the group consisting of the sequence of the extracellular region of the wild type HER2 protein:
  • the 558th glutamic acid is mutated to alanine.
  • the 573th phenylalanine is mutated to alanine.
  • the serine at position 288 is mutated to alanine.
  • the 296th histidine is mutated to alanine.
  • the HER2 variant protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
  • sequence of the extracellular region of the wild type HER2 protein is set forth in SEQ ID NO: 18.
  • a fourth aspect of the invention relates to a nucleic acid molecule encoding the bispecific antibody or antigen binding portion thereof according to any one of the first aspects of the invention, or the antibody or antigen binding portion thereof, or A partial sequence (eg, a light chain and/or a heavy chain) of the antibody or antigen binding portion thereof, or a HER2 variant protein of any of the third aspects.
  • a fifth aspect of the invention relates to a recombinant vector comprising the nucleic acid molecule of any of the fourth aspects of the invention.
  • a sixth aspect of the invention relates to a recombinant cell comprising the recombinant vector of any of the fifth aspects of the invention or the nucleic acid molecule of any of the fourth aspect.
  • a seventh aspect of the invention relates to a method for preparing a bispecific antibody or antigen-binding portion thereof according to two monoclonal antibodies or antigen-binding portions thereof directed against different antigenic epitopes, comprising the steps of:
  • a common light chain sequence capable of binding to the two monoclonal antibodies heavy chain, respectively is obtained, wherein the common light chain means that the two light chains have the same sequence, preferably, The common light chain is a light chain of one of the monoclonal antibodies or a mutant of one of the monoclonal antibodies.
  • the heavy chain sequence and the common light chain sequence of the two monoclonal antibodies are respectively constructed in an expression vector to obtain two recombinant expression vectors; preferably, the heavy chain sequence, particularly the Fc fragment, is mutated to be more advantageous for having different heavy chains. Binding of the Fc segments of the two monoclonal antibodies;
  • the two recombinant expression vectors are transferred into the same host cell, and expression is induced to obtain a bispecific antibody or antigen-binding portion thereof.
  • variable region of the common light chain is obtained by first determining an interface amino acid in contact between a light chain variable region of two monoclonal antibodies and a respective antigen or antigen epitope, Then, when the variable region of the light chain variable of any one of the monoclonal antibodies is selected as the candidate common light chain, the interface light amino acid of the common light chain and the antigen or antigen epitope of the monoclonal antibody is contacted with another The difference amino acid when comparing the amino acids of the light chain variable region of the monoclonal antibody, the light chain variable region having a small number of differential amino acids is the variable region of the common light chain; preferably, the common light chain variable region is further Mutations to obtain variable regions of a common light chain that have better affinity for antigen or antigenic epitopes.
  • the constant region of the common light chain is obtained by using the light chain constant region of the monoclonal antibody of the monoclonal antibody that has been determined to provide the common light chain as a common light
  • the constant region of the strand, or the light chain constant region is further mutated to obtain a constant region of the common light chain.
  • the mutation refers to mutation of an interfacial amino acid therein.
  • said affinity to an antigen or antigenic epitope is greater than the affinity of two antigens or antigenic epitopes corresponding to the common light chain to the bispecific antibody or antigen binding portion thereof A balance is reached to give the bispecific antigen or antigen binding portion thereof better biological activity and physicochemical properties (e.g., stability).
  • An eighth aspect of the invention relates to a method of preparing a mixture comprising at least two monoclonal antibodies or antigen-binding portions thereof, the method comprising the steps of:
  • the heavy chain sequence and the common light chain sequence of the two monoclonal antibodies are respectively constructed in an expression vector to obtain two recombinant expression vectors; preferably, the heavy chain sequence, particularly the Fc fragment, is mutated to be more advantageous for having the same heavy chain.
  • the two recombinant expression vectors are transferred into the same host cell, and expression is induced to obtain a mixture of antibodies or antigen-binding portions thereof.
  • variable region of the common light chain is obtained by first determining an interface amino acid in contact between a light chain variable region of two monoclonal antibodies and a respective antigen or antigen epitope, Then, when the variable region of the light chain variable of any one of the monoclonal antibodies is selected as the candidate common light chain, the interface light amino acid of the common light chain and the antigen or antigen epitope of the monoclonal antibody is contacted with another The difference amino acid when comparing the amino acids of the light chain variable region of the monoclonal antibody, the light chain variable region having a small number of differential amino acids is the variable region of the common light chain; preferably, the common light chain variable region is further Mutations to obtain variable regions of a common light chain that have better affinity for antigen or antigenic epitopes.
  • the constant region of the common light chain is obtained by using the light chain constant region of the monoclonal antibody of the monoclonal antibody that has been determined to provide the common light chain as a common light
  • the constant region of the strand, or the light chain constant region is further mutated to obtain a constant region of the common light chain.
  • the mutation refers to mutation of an interfacial amino acid therein.
  • the affinity for the antigen or antigen epitope is better to mean that between the common light chain and the affinity of the two antigens or epitopes corresponding to the two monoclonal antibodies in the mixture Equilibrium gives the mixture better biological activity and physicochemical properties (eg stability).
  • the invention also relates to a method of detecting whether and/or quantifying an antibody or antigen-binding portion thereof, or a method thereof, comprising the following steps (see the schematic of Figure 25):
  • the antibody or antigen binding portion thereof is judged to be a bispecific antibody or antigen binding portion thereof; optionally, the bispecific antibody is further further based on the obtained positive value Or the antigen binding portion thereof is quantified.
  • the antigen-binding portions 1 and 2 refer to a portion of a bispecific antibody or an antigen-binding portion thereof, respectively, which binds to a different antigen or antigen epitope; in an embodiment of the present invention, The antigen-binding portions 1 and 2 were respectively engineered on the basis of two original antibodies, and the antigen-binding portions 1 and 2 were identical to the antigen or antigen epitope bound to the two original antibodies, respectively.
  • the specific antigen 1 and specific antigen 2 are HERm1 and HERm2.
  • the labeled specific antigen is a specific antigen labeled with biotin.
  • the detection molecule refers to a substrate molecule that can be used for detection, such as HRP-labeled streptavidin.
  • the present invention also relates to a method of detecting whether a mixture of an antibody or an antigen-binding portion thereof is a homodimeric protein, the mixture comprising two antibodies (antibody 1 and antibody 2) or an antigen-binding portion thereof, the method comprising the following Step (see the schematic of Figure 26):
  • step 2) When step 2) is positive, and the reaction is concentration dependent, and step 3) is negative, it is judged to be homodimeric protein in the mixture and does not contain heterodimeric protein; when step 2) is positive At the same time, when step 3) is positive, it is judged that the mixture contains both homodimeric protein and heterodimeric protein.
  • the specific antigen 1 and specific antigen 2 are HERm1 and Herm2.
  • the labeled specific antigen is an antigen labeled with biotin.
  • the detection molecule refers to a substrate molecule that can be used for detection, such as HRP-labeled streptavidin.
  • the invention further relates to a composition (e.g., a pharmaceutical composition) comprising the bispecific antibody or antigen binding portion thereof of any of the first aspects of the invention, and optionally a pharmaceutically acceptable carrier or excipient.
  • a composition e.g., a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody or antigen binding portion thereof of any of the first aspects of the invention, and optionally a pharmaceutically acceptable carrier or excipient.
  • the invention further relates to a composition (e.g., a pharmaceutical composition) comprising a mixture of any of the second aspects of the invention, and optionally a pharmaceutically acceptable carrier or excipient.
  • a composition e.g., a pharmaceutical composition
  • a pharmaceutical composition comprising a mixture of any of the second aspects of the invention, and optionally a pharmaceutically acceptable carrier or excipient.
  • the invention further relates to a kit comprising the bispecific antibody or antigen binding portion thereof of any of the first aspects of the invention, and optionally a buffer and/or instructions.
  • the kit is for use in diagnosing a HER2-positive tumor (eg, breast cancer, gastric cancer).
  • a HER2-positive tumor eg, breast cancer, gastric cancer.
  • the invention further relates to a kit comprising the mixture of any of the second aspects of the invention, and optionally a buffer or instructions.
  • the kit is for use in diagnosing a HER2-positive tumor (eg, breast cancer, gastric cancer).
  • a HER2-positive tumor eg, breast cancer, gastric cancer.
  • the invention further relates to the use of a bispecific antibody or antigen binding portion thereof according to any of the first aspects of the invention for the manufacture of a medicament for the prevention and/or treatment of a HER2-positive tumor, such as breast cancer, gastric cancer.
  • the invention further relates to the use of a mixture according to any of the second aspects of the invention for the manufacture of a medicament for the prevention and/or treatment of HER2-positive tumors, such as breast cancer, gastric cancer.
  • the invention further relates to the use of a bispecific antibody or antigen binding portion thereof according to any of the first aspects of the invention for the preparation of a reagent or kit for diagnosing a HER2-positive tumor, such as breast cancer, gastric cancer.
  • the invention further relates to the use of a mixture according to any of the second aspects of the invention for the preparation of a reagent or kit for diagnosing a HER2-positive tumor, such as breast cancer, gastric cancer.
  • the present invention also relates to a variant protein of the extracellular region of the HER2 protein according to any one of the third aspects of the present invention, which is for detecting the bispecific antibody or antigen-binding portion thereof according to any one of the first aspect or for detecting any of the second aspect The use of the mixture of items.
  • the invention further relates to a method of preventing and/or treating a HER2-positive tumor, such as breast cancer, gastric cancer, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the bispecific of any of the first aspects of the invention The step of a sex antibody or antigen binding portion thereof.
  • the invention further relates to a method of preventing and/or treating a HER2-positive tumor, such as breast cancer, gastric cancer, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a mixture of any of the second aspects of the invention. step.
  • the invention further relates to a method of diagnosing a HER2-positive tumor (e.g., breast cancer, gastric cancer), the method comprising the step of using the bispecific antibody or antigen-binding portion thereof of any of the first aspects of the invention.
  • a HER2-positive tumor e.g., breast cancer, gastric cancer
  • the invention further relates to a method of diagnosing a HER2-positive tumor, such as breast cancer, gastric cancer, comprising the step of using a mixture of any of the second aspects of the invention.
  • the invention further relates to a method of detecting a bispecific antibody or antigen binding portion thereof according to any of the first aspects of the invention or a method of detecting a mixture according to any of the second aspects of the invention, the method comprising using any of the third aspect of the invention A step of a variant protein of the extracellular region of the HER2 protein.
  • the present invention also relates to the bispecific antibody or antigen-binding portion thereof according to any one of the first aspects of the present invention for use in the prevention and/or treatment of a HER2-positive tumor (e.g., breast cancer, gastric cancer).
  • a HER2-positive tumor e.g., breast cancer, gastric cancer.
  • the invention further relates to a mixture according to any of the second aspects of the invention for use in the prevention and/or treatment of HER2-positive tumors (e.g. breast cancer, gastric cancer).
  • HER2-positive tumors e.g. breast cancer, gastric cancer.
  • the term "antibody” refers to an immunoglobulin molecule usually composed of two identical pairs of polypeptide chains each having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • variable regions and constant The region is joined by a "J" region of about 12 or more amino acids, which also contains a "D” region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain CL.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with high denaturation (referred to as complementarity determining regions (CDRs)) interspersed with more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy/light chain pair form an antibody binding site, respectively.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342: 878-883.
  • the term "antibody” is not limited by any particular method of producing antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3 or IgG4 subtype
  • IgA1, IgA2, IgD, IgE or IgM antibodies for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding portion" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (eg, HER2) to which the antibody binds, competing with the intact antibody for the antigen Specific binding.
  • antigen eg, HER2
  • the term "antigen-binding portion" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (eg, HER2) to which the antibody binds, competing with the intact antibody for the antigen Specific binding.
  • the antigen binding portion comprises a Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining region (CDR) fragment, Single-chain antibodies (eg, scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • CDR complementarity determining region
  • an antibody eg, the antibody fragment described above
  • a given antibody eg, monoclonal antibody 2E12
  • the method is to specifically screen the antigen-binding portion of the antibody.
  • Fv fragment means a single arm of V H and V L domains of an antibody thereof; in the present invention, the term “Fd fragment” means an antibody fragment consisting of V H and C H 1 domains of a ; term “dAb fragment” means antibody fragment (Ward et al., Nature341: 544-546 (1989)) by the V H domains; the term “Fab fragment” means a L V L, V H, C, and C An antibody fragment consisting of an H 1 domain; the term “F(ab') 2 fragment” means an antibody fragment comprising two Fab fragments joined by a disulfide bridge on the hinge region.
  • antibody Fc fragment is a term well known to the skilled person and is defined based on papain cleavage of an antibody, and refers to a human immunoglobulin chain constant region, particularly an immunoglobulin heavy chain constant region. a carboxy terminus or a portion thereof.
  • an immunoglobulin Fc region may comprise a combination of two or more domains of heavy chain CH2, CH3, CH4 and an immunoglobulin hinge region.
  • Immunoglobulins can be divided into different classes, mainly five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG-1, IgG-2. , IgG-3, IgG-4, IgA-1, and IgA-2. Selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is well within the purview of those skilled in the art.
  • the antibody Fc fragment used in the invention comprises at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain, such as human IgGl Fc.
  • the term "bispecific antibody” is capable of binding to two antigens or antigenic epitopes, respectively, including light and heavy chains of an antibody capable of specifically binding to a first antigen or antigenic epitope, and capable of specificity The light and heavy chains of an antibody that binds to a second antigen or antigenic epitope.
  • the light chain of the antibody capable of specifically binding to the first antigen or antigen epitope in the bispecific antibody and the light chain of the antibody capable of specifically binding the second antigen or antigen epitope have The same sequence.
  • the heavy chain of the antibody capable of specifically binding to the first antigen or antigen epitope in the bispecific antibody and the heavy chain of the antibody capable of specifically binding the second antigen or antigen epitope have Different sequences.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or an antibody.
  • epitope is also referred to in the art as an "antigenic determinant.”
  • An epitope or antigenic determinant typically consists of a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and typically has specific three dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "or” conformational.
  • the light chain sequences (especially the variable region sequences) of two monoclonal antibodies (ie, the original antibodies) directed against different antigens or antigenic epitopes are analyzed and verified to obtain two monoclonal antibodies.
  • the common light chain can be used to express a bispecific antibody, and can also be used to express a mixture containing two antibodies; when expressing a bispecific antibody, the antibody contains a light capable of binding to the first antigen. a chain and a heavy chain, and a light chain and a heavy chain capable of binding to a second antigen, wherein the two light chain sequences are identical, that is, the common light chain; when expressing the antibody mixture, each antibody contains two The light chain and the heavy chain, wherein the light chain sequences are identical, that is, the common light chain.
  • the light chain constant region of the two original antibodies may be a kappa type or a lambda type;
  • the kappa type light chain constant region includes various isotypes such as Km1, Km1, 2, Km3;
  • the lambda light chain constant region includes various isoforms such as CL1, CL2, CL3, CL6, and CL7.
  • variable regions are critical for the specific binding of antigens and antibodies, and therefore the selection and modification of variable region sequences is particularly critical when engineering or obtaining antibodies. Therefore, in the present invention, in order to obtain a bispecific antibody or antibody mixture having a common light chain of the present invention, it is first necessary to obtain a variable region of the common light chain. After selecting the light chain variable region of one of the original monoclonal antibodies or a mutant sequence thereof as the variable region of the common light chain according to the above method of the present invention, the constant region of the common light chain is determined.
  • the light chain constant region of this monoclonal antibody that has been identified as the variable region of the common light chain is selected as the constant region of the common light chain; of course, in some cases, another The light chain constant region of a monoclonal antibody acts as a constant region of the common light chain.
  • the original light chain constant region can be engineered (e.g., addition, deletion or mutation of amino acids, etc.) according to techniques well known in the art to obtain a more constant region of the common light chain, for example by modification to make it better. ADCC, CDC, endocytosis, stability, immunogenicity or half-life.
  • the heavy chain types of the two original antibodies may be the same or different, preferably of the same type.
  • the bispecific antibody and antibody mixture are prepared such that the variable region of the heavy chain sequence and the sequence of the CH1 domain are unchanged compared to the original antibody.
  • both arms of the bispecific antibody or antibodies containing both antibody mixtures are derived from two original monoclonal antibodies.
  • the sequence of the light chain variable region needs to be altered to obtain a common light chain without the need to alter the sequence of the heavy chain variable region. That is, in the constructed bispecific antibody and antibody mixture, the heavy chain variable region sequence of the antibody may be identical to the original antibody, but at least one of the light chain variable region sequences is different from the original antibody.
  • two original monoclonal antibodies can be selected according to different needs or purposes.
  • two monoclonal antibodies against different antigenic epitopes of the same antigen can be selected, or one of the antibodies can be linked to the relevant antigen on the surface of the tumor cell.
  • Another antibody can trigger immune effector cells to further kill the cells.
  • heavy chains such as the Fc segment
  • Fc segment in the preparation of a bispecific antibody, can be engineered using prior art techniques to facilitate the formation of heterodimeric proteins when the antibody is expressed.
  • heavy chains such as the Fc segment
  • Fc segment can be engineered using prior art techniques to facilitate the formation of homodimeric proteins when the antibody is expressed.
  • techniques for fusing polypeptides having different antigen recognition epitopes include, but are not limited to, heterodimeric Fc fusion techniques as in the specific embodiments, and may also be "Fab" techniques, see Figure 1. .
  • the heterodimeric Fc fusion technique used in the present invention may be a "handle"-"hole” model or a “charge repulsion” model, but is not limited only to the two models. .
  • the antibody mixture platform used in the present invention to produce in a single recombinant cell production can be a "charge repulsion" model, but is not limited only to this model.
  • the nucleic acid molecule when used to prepare a bispecific antibody or antibody mixture, encodes a light chain and/or heavy chain of an antibody against a first antigen, or an antibody encoding a second antigen Light chain and / or heavy chain.
  • the light chain is a common light chain; in an embodiment of the invention, the Fc segment of the heavy chain is engineered.
  • the vector may be a cloning vector or an expression vector.
  • the cloning vector is used to clone a related fragment of an antibody; the expression vector is for expression of a bispecific antibody or antibody mixture.
  • a vector suitable for antibody expression can be selected according to common knowledge in the art.
  • the expression vector is pcDNA4m, which is a vector obtained by engineering on the basis of the vector pcDNA4/myc-HisA.
  • the expression vector comprises a nucleic acid molecule encoding a light chain and/or a heavy chain of an antibody against a first antigen, or a light chain and/or heavy comprising an antibody encoding a second antigen Chain of nucleic acid molecules.
  • the host cell is a host cell suitable for antibody expression, such as a prokaryotic cell (eg, E. coli) or a eukaryotic cell; the eukaryotic cell is, for example, a yeast cell, a plant cell, or a mammal.
  • Animal cells such as CHO cells, HEK293 Cells or myeloma cells, etc.
  • the host cell contains both an expression vector for the light chain and/or heavy chain of the antibody against the first antigen and a light chain and/or heavy chain for the antibody against the second antigen.
  • An expression vector in a particular embodiment of the invention, the light chain is a common light chain; in an embodiment of the invention, the Fc portion of the heavy chain is engineered.
  • Bispecific antibodies or antibody mixtures can be purified from host cells using standard experimental means. Purification methods include, but are not limited to, chromatographic techniques such as size exclusion, ion exchange, affinity chromatography, and ultrafiltration. In an embodiment of the invention, the bispecific antibody and antibody mixture are purified by ProteinA affinity chromatography.
  • the bispecific antibody or antigen-binding portion thereof of the present invention or a mixture thereof may also be used in combination with a chemotherapeutic drug and/or other antibodies, and thus the composition of the present invention may further contain a chemotherapeutic drug and/or other antibody. .
  • the chemotherapeutic drugs include, but are not limited to, Adriamycin, cyclophosphamide, and taxanes [Taxol and Taxotere], capecitabine (Xeloda). ), Gemzar, Navelbine, Tamoxifen, Aromatase Inhibitors (Rining, Furlong, Arnold New), 5-FU Glycerate, Campitosar, Oxaliplatin, cisplatin, carboplatin, estramustine, novantrone, prednisone, vincristine (Oncovin), etc., or a combination thereof.
  • a HER2 protein mutant capable of specifically binding only to one of pertuzumab and Herceptin monoclonal antibody is prepared by mutation of the HER2 protein.
  • the bispecific antibodies and antibody mixtures are identified using these mutants.
  • the double antigen sandwich ELISA also called bridge ELISA
  • the mutated HER2 protein is combined with the mutated HER2 protein to identify whether the antibody is a bispecific antibody or whether the antibody mixture contains a homodimeric protein, and further to the bispecific antibody. Or homodimeric proteins in the antibody mixture are quantified.
  • the double antigen sandwich ELISA is known in the art, and its working principle is to utilize the antigen and the enzyme target antigen attached to the solid phase carrier to respectively bind the two antigens on the detected antibody molecule in the sample. Binding to form a solid phase antigen-antibody-enzyme-labeled antigen immune complex.
  • the detection step of the method includes, for example: (1) coating a specific antigen with a solid phase carrier. Incubate for a period of time to form a solid phase antigen and wash to remove unbound antigen and impurities. (2) Add the sample to be inspected, incubate, and fully react the antibody in the sample with the antigen on the solid phase carrier to form a solid phase antigen-antibody complex.
  • washing remove other unbound material. (3) Add the enzyme-labeled antigen and incubate to form a solid phase antigen-test antibody-enzyme-labeled antigen sandwich complex. Washing removes unbound enzyme-labeled antigen. (4) Adding substrate color. The enzyme-catalyzed substrate on the solid phase produces a colored product, and the amount of antibody in the sample is measured by colorimetry.
  • the HER2-positive tumor includes both tumors overexpressing HER2 protein (for example, breast cancer, gastric cancer, esophageal cancer, ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer). Also included are tumors with low expression of the HER2 protein (e.g., HER2 low expression breast cancer, gastric cancer, lung cancer, etc.).
  • tumors overexpressing HER2 protein for example, breast cancer, gastric cancer, esophageal cancer, ovarian cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer.
  • tumors with low expression of the HER2 protein e.g., HER2 low expression breast cancer, gastric cancer, lung cancer, etc.
  • the present invention obtains a common light chain capable of binding to two monoclonal antibodies heavy chains by analyzing the light chain sequences of two different monoclonal antibodies, and on the basis of this, prepares a bispecific antibody and antibody mixture having a common light chain.
  • the bispecific antibody and antibody mixture prepared by this method have good binding properties, biological activity and stability, and may be superior to the original antibody in biological activity.
  • the common light chain technology is simple and controllable, and effectively solves the problem of heavy and light chain mismatch in bispecific antibodies without affecting the stability, activity and purity of the antibody; for the antibody mixture, it is Expression in the same host cell can avoid the difficulty of culturing mixed cell populations, and is more conducive to scale-up production.
  • Figure 1 Schematic representation of heterodimeric protein fusion.
  • Figure a shows the heterodimeric Fc fusion technique and
  • Figure b shows the "Fab" technique.
  • FIG. 1 Recognition of light chain hypervariable regions of pertuzumab and trastuzumab, where A is the recognition result of the light chain hypervariable region of pertuzumab, and B is the recognition of the light chain hypervariable region of trastuzumab
  • A is the recognition result of the light chain hypervariable region of pertuzumab
  • B is the recognition of the light chain hypervariable region of trastuzumab
  • C is the result of a comprehensive analysis of the light chain sequence alignment of the pertuzumab and the trastuzumab and the antigen interface.
  • FIG. 1 Schematic diagram of trastuzumab Fab fragment and Her2 extracellular domain (ECD).
  • TmabCLC1 to 6 TmabCLC1 to 6; 7 to 12: PmabCLC1 to 6; M: protein amount standard.
  • Figure 8 Affinity of Pertuzumab with a common light chain for its specific antigen HER2m2
  • KN026 transient expression cell culture supernatant 1: KN026 transient expression cell culture supernatant; 2: KN026 affinity chromatography flow through; 3: KN026 one-step affinity chromatography after purification of protein sample (reduction); 4: KN026 one-step affinity chromatography after purification of protein samples (non Reduction)
  • M Protein amount standard.
  • Figure 25 is a schematic representation of a method of detecting whether an antibody is a bispecific antibody and a method of quantification.
  • Figure 26 is a schematic representation of a method of detecting whether an antibody mixture is a homodimeric protein.
  • Figure 27 Dose dependence of PTmab bispecific antibody on a transplanted tumor model of HER2 low expression tumor strain human non-small cell lung cancer NCI-H522 mouse.
  • Figure 28 PTmab bispecific antibody to HER2 low expression tumor strain human non-small cell lung cancer
  • the pharmacodynamic effect of the NCI-H522 mouse xenograft model was comparable to that of the equimolar Trastuzumab standard plus the equimolar Pertuzumab standard.
  • the complex crystal structure of trastuzumab and pertuzumab was obtained from the protein database (PDB, www.pdb.org).
  • the trastuzumab PDB number was 1N8Z and the pertuzumab PDB number was 1S78. .
  • Two screening strategies are available to identify amino acid contacts between CH3-CH3: (i) distance of amino acid action (ii) solvent accessible area analysis. Here, screening is based on the distance of amino acid action.
  • an interfacial amino acid refers to those amino acids whose distance between the heavy chain of the side chain and the heavy atom of any one of the other amino acids is less than a threshold.
  • the threshold is selected as Also available in some literature (Bahar and Jernigan 1997).
  • Table 1 is a list of amino acids interacting with the trastuzumab light chain and the antigen HER2. Listed in Table 1 are the 12 interface amino acids of trastuzumab screened by the amino acid contact screening rules.
  • Table 2 is a list of amino acids interacting with the pertuzumab light chain and the antigen HER2. Listed in Table 2 are the eight interface amino acids of pertuzumab screened by the amino acid contact screening rules.
  • the pertuzumab and trastuzumab light chains were identified by the hypervariable region recognition system, kabat numbering, and the recognition software was http://www.bioinf.org.uk/abs/abnum/.
  • the recognition results of the light chain hypervariable region of the pertuzumab are shown in Figure 2-A, and the light chain recognition results of the homozygous monoclonal antibody in trastuzumab are shown in Figure 2-B.
  • Pactuzumab and trastuzumab light chain sequence alignment and antigen interface amino acid comprehensive analysis results are shown in Figure 2-C, pertuzumab (P-mab) and trastuzumab (T-mab)
  • the light chain is in contact with the antigen and the amino acid is shown in background color black.
  • the trastuzumab light chain is a common light chain
  • the difference amino acid obtained by comparing the interface amino acid in contact with the antigen on the common light chain with the interface amino acid on the light chain of the pertuzumab (P-mab) table 3.
  • the difference amino acid obtained by comparing the interface amino acid in contact with the antigen on the common light chain with the interface amino acid on the trastuzumab (T-mab) light chain is as follows. Table 4.
  • the light chain of trastuzumab (T-mab) was selected as the framework.
  • the T31I or / and T94Y mutations were introduced to obtain the common light chain sequence CLC1 to CLC4 of the paclitaxel and trastuzab bispecific antibodies; the light chain of the pertuzumab (P-mab) was selected as the framework, and T31I or / was introduced.
  • the Y94T mutation the common light chain sequence CLC5-CLC6 of the paclitaxel and trastuzin bispecific antibody was obtained.
  • the amino acid sequences of each common light chain are as follows:
  • H91A, R50A, W95A, Y100aA have a major impact on the binding of trastuzumab to the extracellular domain of HER2.
  • the Hyun-Soo Cho team crystallized the complex of the trastuzumab Fab fragment and the HER2 extracellular domain (ECD) in 2003 (PDB number: 1N8Z), and the results were published in Nature.
  • Combination 1 P571, P572 itself and the Fab several key amino acids have a force formation. Consider that these two amino acids are at the loop corner, such as mutations will affect their structural stability.
  • Combination 2 GLU558 forms an ionic bond with the trastose Fab heavy chain ARG50, and forms a van der Waals force with multiple amino acids of the Fab heavy chain.
  • the disruption can block the action of the trastuzole Fab and HER2, so GLU558 ⁇ ALA558; PHE573 and Fab are selected.
  • Multiple amino acids in the heavy chain form van der Waals forces, including key amino acids ARG508, TRP99, and TYR105. Destruction can block the action of Fab and HER2, so PHE573 ⁇ ALA573 will be selected.
  • the HER2 variant protein recognized only by Trastuzumab was named HER2m1
  • the HER2 variant proteins recognized only by Pertuzumab were named HER2m2 and HER2m3.
  • the amino acid sequences of the HER2 variant proteins are:
  • the commercial vector pcDNA4/myc-HisA contains two PvuII cleavage sites at positions of approximately 1411 bp and 3160 bp, respectively. Site-directed mutagenesis of the plasmid, mutation of base C at position 3160 bp to G, removal of the PvuII restriction site at this position, leaving only one restriction site at about 1411 bp, the new vector was named pcDNA4m.
  • Primers were designed based on the DNA sequence (AY623427) of the crystallizable fragment (Fc) of human immunoglobulin gamma1 (IgG1) on NCBI, as follows
  • the upstream primers were supplemented with HindIII and BamHI cleavage sites for subsequent cloning, and EcoRI cleavage sites were added to the downstream primers.
  • the gene of Fc fragment was amplified by using the whole cDNA of PBMC as a template, and then cloned into the transformed vector pcDNA4m by double-digestion with HindIII and EcoRI of Takara. The accuracy of the plasmid was verified by sequencing, and the recombinant plasmid pcDNA4m-Fc was obtained.
  • Primers were designed based on the DNA sequence information of the HER2 protein on NCBI (NM_004448.2), and the extracellular domain (amino acid residues 1 to 652) of the wild type HER2 protein was cloned using the following primers:
  • the primer contains an upstream HindIII recognition site and a downstream BamHI recognition site.
  • a 1.9 kb DNA fragment encoding the extracellular domain of HER2wt was amplified and cloned into a commercial T vector (pMD19-T Simple Vector; From the Takara company, the T-Her2 ECD plasmid was obtained and sequenced to confirm the correctness of the sequence.
  • HER2 variant protein HER2m1 recognized only by Trastuzumab and the HER2 variant protein HER2m2 and HER2m3 recognized only by Pertuzumab
  • corresponding mutation primers were designed according to the mutation site:
  • the T-Her2 ECD plasmid was used as a template, and the above primers were used for site-directed mutagenesis to obtain three HER2 extracellular domain variant proteins (HER2m1, HER2m2, HER2m3). Then, it was cloned into the vector pcDNA4m-Fc by HindIII and BamHI of Takara, and the three genes of HER2m1, HER2m2 and HER2m3 were respectively fused to the 5' end of the Fc gene to obtain three new vectors, which were named: pcDNA4m- Her2m1-Fc, pcDNA4m-Her2m2-Fc, pcDNA4m-Her2m3-Fc. These three vectors can be used to express the fusion proteins HER2m1-Fc, HER2m2-Fc, HER2m3-Fc in mammalian cells.
  • transfected cell suspension was counted to have a cell density of 3.5 to 4 ⁇ 10 6 cells/mL, and the cell suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.
  • the cells were resuspended in 40 mL x 3 fresh Freestyle 293 medium, centrifuged again at 1000 rpm for 5 min, and the supernatant was discarded.
  • 293 cells were resuspended in 200 mL x 3 Freestyle 293 medium.
  • the expression vectors of the three HER2 variant proteins obtained in Example 2-4 were each taken at 200 ⁇ g, and diluted with 2 mL of Freestyle 293 medium, respectively.
  • 1.5 mL of Polyethylenimine was diluted with 5 mL of Freestyle 293 medium, and the desired PEI solution was converted.
  • 2 mL of the PEI solution was added to the diluted 2 mL expression plasmid and mixed, and allowed to stand at room temperature for 5 minutes.
  • Three plasmid/PEI mixtures were separately added to three 200 mL cell suspensions, placed at 37 ° C, 10% CO 2 , 90 rpm; and 50 ⁇ g/L IGF-1 was added.
  • 200 mL of EX293 medium, 2 mM Glutamine and 50 ug/L IGF-1, 135 rpm were added to each of the transformed samples.
  • HER2 variant protein cell transient expression culture supernatant After 5-6 days of culture, 3 parts of 400 mL of HER2 variant protein cell transient expression culture supernatant were collected, and the HER2 variant protein sample was preliminarily purified by ProteinA affinity chromatography.
  • the expression level of HER2m3 was very low, and the titer of the template protein in the cell culture supernatant was less than 0.5 mg/L, which was presumably mainly due to the instability of the protein variant, so the protein was not further purified.
  • the resulting expression levels of the HER2ml and HER2m2 variant proteins were approximately 20 mg/L by purification.
  • the obtained protein sample is initially detected by SDS-PAGE, which can be clearly See the target strip (see Figure 4).
  • the plates were coated with Trastuzumab monoclonal antibody or Pertuzumab mAb, overnight at 4 °C. Thereafter, a 3% BSA solution was added and blocked at room temperature for 2 hours.
  • the sample to be tested (HER2m1 or HER2m2 protein) was pre-labeled with biotin, and then the biotinylated proteins HER2m1-Biotin and HER2m2-Biotin were diluted 1:4 from 16 ⁇ g/mL to 0.224ng/ ⁇ L for a total of 9 gradient.
  • a gradient dilution of the biotinylated HER2 variant protein sample was added to the plate and reacted for 2 hours at room temperature.
  • HRP-labeled streptavidin was then added and allowed to stand at room temperature for 1.5 hours to finally catalyze the color reading of the substrate.
  • the obtained data was fitted by a four-parameter method to obtain an affinity curve.
  • the apparent affinity of the HER2m1 protein for Pertuzumab was reduced by a factor of 20 compared to its apparent affinity for Trastuzumab, which is considered to be a Trastuzumab-specific antigenic protein.
  • the apparent affinity of the HER2m2 protein for Trastuzumab was reduced by >2 orders of magnitude compared to the apparent affinity for Trastuzumab, which was expressed as a Pertuzumab-specific antigen.
  • Example 4 Replaces the Tmab and Pmab original light chains with a common light chain and verifies the effect of the common light chain
  • the corresponding DNA sequence was designed using the DNAworks online tool (http://helixweb.nih.gov/dnaworks/).
  • the heavy chain gene of Trastuzumab (SEQ ID NO: 16) and the heavy chain gene of Pertuzumab (SEQ ID NO: 17) were obtained by artificial synthesis.
  • the amino acid sequences of a group of common light chains obtained in Example 1 SEQ ID NOS: 1-6
  • the corresponding coding DNA sequence was designed using the DNAworks online tool (http://helixweb.nih.gov/dnaworks/).
  • the common light chain genes CLC1 (SEQ ID NO: 7) and CLC5 (SEQ NO: 11) of the Pmab-Tmab bispecific antibody were obtained by artificial synthesis.
  • mutant primers were designed according to the sequence of CLC2 to CLC6, and the sequences are as follows:
  • the CLC1 gene was used as a template, and the gene sequences of CLC2 ⁇ CLC4 were obtained by site-directed mutagenesis of T31I and T94Y primers (SEQ ID NO:8 to SEQ ID NO:10).
  • the CLC6 gene was obtained by site-directed mutagenesis using Y94T primers. Sequence (SEQ ID NO: 12).
  • the heavy chain gene of the synthesized Trastuzumab, the heavy chain gene of Pertuzumab, and the common light chain gene (CLC1 to CLC6) were subcloned into the engineered vector pcDNA4m by double digestion with HindIII and EcoRI of Takara, respectively.
  • recombinant plasmid DNA was obtained: pcDNA4m-TmabHC, pcDNA4m-PmabHC, and the common light chain related vector pcDNA4m-CLC1 to pcDNA4m-CLC6.
  • the above-described well-established common light chain gene expression vector pcDNA4m-CLC1 to 6 was digested with Bgl II, Pvu II of Takara. The digested product was separated and purified by 0.8% agarose electrophoresis, and the DNA fragment containing the common gene was recovered by about 2 kb.
  • the pcDNA4m-TmabHC was digested with BglII and NruI to recover a DNA fragment containing about 6 kb of TmabHC gene; pcDNA4m-PmabHC A 6 kb DNA fragment containing the PmabHC gene was double-digested with BglII and NruI.
  • the digested DNA fragments were ligated, and the expression elements of TmabHC or PmabHC were integrated with the common light chain expression elements of different sequences to obtain recombinant plasmids pcDNA4m-Tmab-CLC1, pcDNA4m-Tmab-CLC2, pcDNA4m-Tmab.
  • transfected cell suspension was counted to have a cell density of 3.5 to 4 ⁇ 10 6 cells/mL, and the cell suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.
  • the cells were resuspended in 10 mL x 12 fresh Freestyle 293 medium, centrifuged again at 1000 rpm for 5 min, and the supernatant was discarded.
  • 293 cells were resuspended in 50 mL x 12 Freestyle 293 medium.
  • the expression vectors of the 12 common light chain monoclonal antibodies obtained in Example 4-1 were each taken at 50 ⁇ g, and diluted with 0.5 mL of Freestyle 293 medium, respectively.
  • 1.5 mL of Polyethylenimine was diluted with 5 mL of Freestyle 293 medium, and the desired PEI solution was converted.
  • 0.5 mL of the PEI solution was added to the diluted 0.5 mL expression plasmid and mixed, and allowed to stand at room temperature for 5 minutes.
  • the protein samples purified by one-step affinity chromatography were subjected to preliminary detection by non-reducing SDS-PAGE. It can be seen that, as shown in Figure 6, all of the common light chain monoclonal antibody proteins can clearly see two bands on the reducing gel, which are light chain bands between 25 kDa and 35 kDa and 85 kDa and 50 kDa. Heavy chain strip between.
  • the purity of the protein samples was determined by SE-HPLC and the results are shown in Table 6.
  • Point mutation of the Fc fragment of TmabHC in pcDNA4m-Tmab-CLC1, making TmabHC a TmabHC-knob (the heavy chain sequence is shown as SEQ ID NO: 19; the mutated residue is: S354C, T366W,), and further constructed pcDNA4m-Tmabknob-CLC1; the amino acid residue in PmabHC in pcDNA4m-Pmab-CLC1 was mutated to PmabHC-hole by the site-directed mutagenesis (the heavy chain sequence is represented by SEQ ID NO: 20, the residue of the mutation)
  • the base is: Y349C, T366S, L368A, Y407V), and further construct pcDNA4m-Pmabhole-CLC1.
  • the specific mutation scheme is referred to CN102558355A.
  • the two newly constructed plasmids will be used to construct a Pmab-
  • KN026 Protein A affinity chromatography and ion exchange chromatography and molecular sieve chromatography and designated as KN026.
  • the instantaneous expression level of KN026 can be up to 80 mg/L according to OD280.
  • the purified protein samples were initially tested by SDS-PAGE. It can be seen that, as shown in Figure 9, the KN026 bispecific antibody protein can clearly see two bands on the reducing gel, which are light chain bands between 25 kDa and 35 kDa and weights between 85 kDa and 50 kDa. Chain belt. At the same time, under non-reducing conditions, KN026 is a strip. The purity of the protein sample was determined by SE-HPLC and the results are shown in Figure 10 with a purity of about 95%.
  • the plate was coated with the Trastuzumab specific antigen protein HER2m1 and incubated overnight at 4 °C. Thereafter, a 3% BSA solution was added and blocked at room temperature for 2 hours.
  • the samples to be tested were diluted 1:3 from 5 ⁇ g/mL up to 1.06 ng/uL for a total of eight gradients.
  • a gradient-diluted sample to be tested was added to the plate, and reacted at room temperature for 2 hours.
  • the biotinylated Pertuzumab-specific antigen protein HER2m2-Biotin was then added to the ELISA plate and reacted with the sample to be tested for 2 hours at room temperature.
  • HRP-labeled streptavidin was then added and allowed to react with HER2m2-Biotin for 1.5 hours at room temperature to finally catalyze the substrate color reading.
  • the obtained data was fitted by a four-parameter method to obtain an affinity curve.
  • TmabHC was subjected to point mutation, and TmabHC was made into TmabHC-mix1 (wherein the heavy chain sequence is shown as SEQ ID NO: 21), and pcDNA4m-Tmabmix1-CLC1 was further constructed.
  • Patent CN 103388013A Example 1 These two newly constructed plasmids will be constructed on a "charge-rejection" mixture model using a common light chain model to construct a mixture of Pmab, Tmab antibodies that can be expressed in a single recombinant cell line.
  • the plasmid/PEI mixture was added to a 600 mL cell suspension, placed at 37 ° C, 10% CO 2 , 90 rpm; and 50 ⁇ g/L IGF-1 was added. Four hours later, 600 mL of EX293 medium, 2 mM Glutamine and 50 ⁇ g/L IGF-1, respectively, were incubated at 135 rpm in the transformed samples. Add 3.8 mM VPA after 24 hours.
  • KN010 a Pmab and Tmab antibody mixture protein sample was initially purified by ProteinA affinity chromatography and designated as KN010. According to OD280, the instantaneous expression level of KN010 can reach 100 mg/L.
  • the protein samples purified by one-step affinity chromatography were initially detected by SDS-PAGE. It can be seen that, as shown in Fig. 12, the common light chain mixed monoclonal antibody protein product can clearly see two bands on the reducing gel, which are light chain bands between 25 kDa and 35 kDa and between 85 kDa and 50 kDa. Heavy chain strips. At the same time, under non-reducing conditions, KN010 is a strip. The purity of the protein sample was determined by SE-HPLC and the results are shown in Figure 13, with a purity of about 95%.
  • the same antigen Bridging ELISA was used to verify that the KN010 antibody protein can recognize the specific antigens of Trastuzumab and Pertuzumab, respectively. It was verified by the hetero-antigen Bridging ELISA that KN010 could not recognize this pair of antigens at the same time.
  • the plate was coated with the Trastuzumab specific antigen protein HER2m1 and incubated overnight at 4 °C. Thereafter, a 3% BSA solution was added and blocked at room temperature for 2 hours.
  • the sample to be tested was diluted 1:4 from 2.5 ⁇ g/mL up to 0.61 ng/ ⁇ L for a total of seven gradients. A gradient-diluted sample to be tested was added to the plate, and reacted at room temperature for 2 hours. Thereafter, the biotinylated Pertuzumab specific antigen protein HER2m2-Biotin, or biotinylated HER2m1-Biotin was added to the plate, and the sample to be tested was reacted at room temperature for 2 hours.
  • HRP-labeled streptavidin was then added and allowed to interact with HER2m2-Biotin or HER2m1-Biotin for 1.5 hours at room temperature to finally catalyze the substrate color reading.
  • the obtained data was fitted by a four-parameter method to obtain an affinity curve.
  • the plate was coated with Perstuzumab specific antigen protein HER2m2 and left overnight at 4 °C. Thereafter, a 3% BSA solution was added and blocked at room temperature for 2 hours.
  • the sample to be tested was diluted 1:4 from 2.5 ⁇ g/mL up to 0.61 ng/ ⁇ L for a total of seven gradients.
  • a gradient-diluted sample to be tested was added to the plate, and reacted at room temperature for 2 hours.
  • the biotinylated Pertuzumab-specific antigen protein HER2m2-Biotin was added to the plate, and the sample to be tested was reacted at room temperature for 2 hours.
  • HRP The labeled streptavidin was reacted with HER2m2-Biotin for 1.5 hours at room temperature to finally catalyze the substrate color reading.
  • the obtained data was fitted by a four-parameter method to obtain an affinity curve.
  • both arms of the KN010 protein were shown to recognize both the antigen of Trastuzumab or the antigen of Pertuzumab by the same antigen bridge ELISA.
  • the KN010 protein includes at least two antibodies that recognize different antigen targets.
  • the hetero-antigen bridge ELISA did not develop color, which proved that the arms of KN010 could not recognize both antigens at the same time, that is, the components of KN010 that did not contain Ptmab heterodimer.
  • Flow cytometry was used to observe the binding of HER2 high-expression breast cancer cell line BT474 to Ptmab bispecific antibody, Pertuzumab, Trastuzumab and other HER2 antibodies, and to investigate the concentration dependence of its action.
  • BT474 cells were taken, resuspended in 5% BSA/PBS after digestion, and 3 ⁇ 10 5 cells/tube were added to each 1.5 mL centrifuge tube; the sample to be tested was diluted 3-fold from 100 ⁇ g/mL to 0.001694 ⁇ g/ mL, a total of 11 concentrations.
  • the sample was reacted with the cells, and then the FITC-rabbit anti-human IgG assay was added to bind the cells to the test antibody, and the mean fluorescence value (MFI) was read by flow cytometry.
  • MFI mean fluorescence value
  • Flow cytometry was used to observe the binding of HER2-highly expressed gastric cancer cell line N-87 to Ptmab bispecific antibody KN026, Pertuzumab, Trastuzumab and other HER2 antibodies, and to investigate the concentration dependence of its action.
  • N-87 cells were taken, resuspended in 5% BSA/PBS after digestion, and 3 ⁇ 10 5 cells/tube were added to each 1.5 mL centrifuge tube; the sample to be tested was diluted from 40 ⁇ g/mL to 29766. Ug/mL, a total of 13 concentrations.
  • the sample was reacted with the cells, and then the FITC-rabbit anti-human IgG assay was added to bind the cells to the test antibody, and the mean fluorescence value (MFI) was read by flow cytometry.
  • MFI mean fluorescence value
  • KN026 the Ptmab bispecific antibody
  • Pertuzumab and Trastuzumab all have significant binding to N-87, and this effect is dependent on the concentration. It can be seen from the EC50 of the binding curve that KN026 has a slightly lower affinity for the HER2 protein on the surface of N-87 cells than Trastuzumab and Pertuzumab.
  • Flow cytometry was used to observe the binding of HER2 high-expression breast cancer cell line BT474 to Pmab, Tmab antibody mixture, Pertuzumab, Trastuzumab and other HER2 antibodies, and to investigate the concentration dependence of its action.
  • BT474 cells were taken, resuspended in 5% BSA/PBS after digestion, and 15 ⁇ 10 6 cells/tube were added to each 1.5 mL centrifuge tube; the sample to be tested was diluted 3-fold from 1000 ⁇ g/mL to 0.01694 ⁇ g/ mL, a total of 11 concentrations.
  • the sample was reacted with the cells, and then the FITC-rabbit anti-human IgG assay was added to bind the cells to the test antibody, and the mean fluorescence value (MFI) was read by flow cytometry.
  • MFI mean fluorescence value
  • the Pmab, Tmab antibody mixture (KN010) and Pertuzumab and Trastuzumab all have significant binding to BT474, and this effect has a concentration dependency. It can be seen from the EC50 of the binding curve that the affinity of KN010 for the HER2 protein on the surface of BT474 cells is between Trastuzumab and Pertuzumab.
  • the CKK-8 method was used to observe the proliferation of HER2 high-expression breast cancer cell line BT474 in the presence of Ptmab bispecific antibody, Pertuzumab, Trastuzumab and other HER2 antibodies, so as to compare and evaluate the proliferation of BT474 cancer cells by Ptmab bispecific antibody.
  • the inhibitory effect of the action was used to observe the proliferation of HER2 high-expression breast cancer cell line BT474 in the presence of Ptmab bispecific antibody, Pertuzumab, Trastuzumab and other HER2 antibodies.
  • BT474 cells were used in 96-well plates at a density of 10,000 cells/well and cultured at 37 °C for 16 h.
  • Different concentrations of samples were prepared using assay medium (DMEM medium supplemented with 1% fetal bovine serum): up to 10 ⁇ g/ml to 0.0015 ⁇ g/ml, 3 fold dilutions, for a total of 9 concentrations.
  • 150 ⁇ l of sample was added to each well, and cell viability was determined by CCK-8 kit (DOJINDO) 72 h later.
  • the obtained cell viability value was plotted against the logarithm of the sample concentration, and fitted by a four-parameter method to obtain a sample to be tested (Ptmab bispecific antibody KN026) and a reference product (Trastuzumab and Cell killing curve of Trastuzumab + Pertuzumab in combination).
  • MTT assay was used to observe the proliferation of N-87 cells with high expression of HER2 in the presence of Ptmab bispecific antibody, Pertuzumab, Trastuzumab and other HER2 antibodies, so as to compare and evaluate Ptmab bispecific antibody against N-87 cancer cells.
  • the inhibitory effect of proliferation was used to observe the proliferation of N-87 cells with high expression of HER2 in the presence of Ptmab bispecific antibody, Pertuzumab, Trastuzumab and other HER2 antibodies, so as to compare and evaluate Ptmab bispecific antibody against N-87 cancer cells. The inhibitory effect of proliferation.
  • N-87 cells were used in 96-well plates at a density of 10,000 cells/well and cultured at 37 °C for 16 h.
  • Different concentrations of samples were prepared using assay medium (RPMI-1640 medium supplemented with 1% fetal bovine serum): up to 10 ⁇ g/ml to 0.0015 ⁇ g/ml, 3 fold dilutions, for a total of 9 concentrations.
  • 150 ⁇ l of sample was added to each well, and cell viability was determined by CCK-8 kit (DOJINDO) 72 h later.
  • the obtained cell viability value is plotted against the logarithm of the sample concentration, and is fitted by a four-parameter method to obtain cell killing of the test sample (Ptmab bispecific antibody) and the reference product (Trastuzumabd and Trastuzumab + Pertuzumab combined administration). curve.
  • KN026 has a significant killing effect on the combination of Trastuzumab and Trastuzumab+Pertuzumab, and this effect is concentration dependent.
  • the bispecific antibody has a significantly better inhibitory effect on N-87 than Trastuzumab alone or in combination with Pertuzumab.
  • the Tm value of the Ptmab bispecific antibody KN026 and the reference antibody was determined by DSC (differential scanning calorimeter), and the thermal stability of the Ptmab bispecific antibody was initially determined based on this.
  • the sample protein was prepared in a concentration of 2 mg/mL in 1 ⁇ PBS buffer (pH 7.4).
  • the specific heat capacity (Cp) of the sample or blank buffer was scanned at a rate of 60 ° C/hr starting at 10 °C.
  • the results of the sample scanning are respectively subtracted from the results of the corresponding buffers, and the obtained Cp value is plotted against the temperature, wherein the temperature corresponding to the peak value at which the Cp value is significantly increased is the Tm value of the sample.
  • the Ptmab bispecific antibody KN026 and the Trastuzumab reference sample showed two distinct Tm values, including a CH2 dissolution temperature of about 60 ° C and a dissolution temperature of CH 3 at about 80 °C.
  • Tm value around 80 °C, bispecific antibody was slightly lower, but still higher than 80 ° C, compared with the reference product is not obvious, not It is believed to have an effect on the thermal stability of the antibody.
  • ICR mice were randomly divided into two groups.
  • the experimental group received a single intraperitoneal injection of Ptmab bispecific antibody KN02610mg/kg, and the reference group received a single intraperitoneal injection of Trastuzumab standard 10mg/kg.
  • Each group of animals was divided into three echelons, and four animals in each echelon took blood at time points.
  • the non-endpoint blood sampling 5 min–96 h
  • the blood was collected from the orbital venous plexus by about 0.2 ml.
  • the inferior vena cava was euthanized after inhalation of isoflurane.
  • the serum was separated and stored in a refrigerator at -80 °C.
  • Serum samples were tested for blood concentration using Tmab and Ptmab-specific ELISA, and the serum antibody content in the serum was plotted against the time of blood collection to obtain the pharmacokinetic profile of the bispecific antibody (KN026) and the reference antibody (Herceptin) (see Figure 21) and further calculation of the corresponding pharmacokinetic parameters (Table 7). It can be seen that the half-life of Ptmab bispecific antibody (KN026) in mice is slightly lower than that of Trastuzumab, but still greater than 10 days, similar to the half-life of most monoclonal antibodies in mice, Ptmab can be considered in mice. The stability is similar to conventional mAb drugs.
  • mice were subcutaneously inoculated with human ovarian cancer SKOV3 cells at a dose of 5 ⁇ 10 6 cells+50% matrigel/only.
  • Tumor-forming mice were randomly divided into groups of 6 (male and female). When the tumor size is about 100-150 mm 3 in diameter, the injection of the anti-tumor drug is started. Administration intraperitoneally, twice a week; continuous administration for 2 weeks. The size of the tumor was measured twice a week.
  • the experimental group was administered with Ptmab bispecific antibody KN02620 mg/kg each time, the reference group was administered Trastuzumab standard, or Pertuzumab standard 20 mg/kg, and the blank control group was administered the same volume of PBS buffer each time.
  • the experimental group and the reference group were compared with the blank control group, and the nude mice of SKOV3 were moved.
  • the tumor model showed a certain tumor inhibition effect.
  • the PTmab bispecific antibody showed stronger antitumor effect than the parental reference product Trastuzumab standard or the Pertuzumab standard alone.
  • Balb/c nude mice were subcutaneously inoculated with human gastric cancer N-87 cells at a dose of 4 ⁇ 10 6 cells/only. Tumor-forming mice were randomly divided into groups of 6 (male and female). When the tumor size was about 100-130 mm 3 in diameter, the injection of the anti-tumor drug was started. IP administration, twice a week; continuous administration for 4-5 weeks. The size of the tumor was measured twice a week.
  • the experimental group was administered with Ptmab bispecific antibody KN0265 mg/kg each time, the reference group was administered Trastuzumab standard, or Pertuzumab standard 5 mg/kg, and the blank control group was administered the same volume of PBS buffer each time.
  • the experimental group and the reference group were compared with the blank control group, and showed a certain tumor suppressing effect on the N-87 nude mouse xenograft model.
  • the Ptmab bispecific antibody showed significantly better antitumor effect than the reference product Trastuzumab standard or the Pertuzumab standard alone.
  • Balb/c nude mice were subcutaneously inoculated with human gastric cancer N-87 cells at a dose of 4 ⁇ 10 6 cells/only. Tumor-forming mice were randomly divided into groups of 6 (male and female). When the tumor size is about 100-120 mm 3 in diameter, the injection of anti-tumor drugs is started for the experiment. IP administration, twice a week, for 3 weeks. The size of the tumor was measured twice a week.
  • the experimental group was administered with the PTmab bispecific antibody KN0262.5 mg/kg, and the reference group was administered with the Trastuzumab standard in combination with the Pertuzumab standard, 2.5 mg/kg, and the blank control group was administered the same volume of PBS buffer each time.
  • the experimental group and the reference group were compared with the blank control group, and showed a certain tumor suppressing effect on the N-87 nude mouse xenograft model.
  • the PTmab bispecific antibody showed a stronger antitumor effect than the equivalent molar amount of the reference product Trastuzumab standard plus the Pertuzumab standard.
  • Example 15 Dose-dependent expression of PTmab bispecific antibody in a transplanted tumor model of HER2 low expression tumor strain human non-small cell lung cancer NCI-H522 mouse
  • NOD/SCID immunodeficient mice were subcutaneously inoculated with non-small cell lung cancer NCI-H522 cells and matrigel mixture (cell: matrigel ratio of 1:1) to establish a model, each inoculation dose of 5 ⁇ 10 6 cells / only, will be small tumor formation Rats were randomized into groups of 6 (half male and female). When the tumor size was as long as about 100 mm 3 , the injection of the anti-tumor drug was started for the experiment. The day of the first IP (abdominal) administration was marked as day 0. After IP administration once a week, the concentration was halved for the first dose; continuous administration for 7 weeks. The size of the tumor was measured twice a week.
  • the experimental group was divided into three groups, and the PTmab bispecific antibody KN026 was administered according to the following doses: 30 mg/kg for the first time, then 15 mg/kg for each week after the first dose; 10 mg/kg for the first time, and then given once a week.
  • the drug was 5 mg/kg; the first dose was 3 mg/kg, and then 1.5 mg/kg was administered once a week.
  • the blank control group was administered the same volume of PBS buffer each time.
  • the three experimental groups showed significant tumor suppressing effects on the NCI-H522 mouse xenograft model relative to the blank control group, and the effect was dose-dependent.
  • Example 16 Pharmacological effect of PTmab bispecific antibody on transplanted tumor model of HER2 low expression tumor strain human non-small cell lung cancer NCI-H522 mouse
  • NOD/SCID immunodeficient mice were subcutaneously inoculated with non-small cell lung cancer NCI-H522 cells and matrigel mixture (cell: matrigel ratio of 1:1) to establish a model, each inoculation dose of 5 ⁇ 10 6 cells / only, will be small tumor formation Rats were randomized into groups of 6 (half male and female). When the tumor size was as long as about 100 mm 3 , the injection of the anti-tumor drug was started for the experiment. The first IP (abdominal) administration was marked on day 0 and the dose was 5 mg/kg. After IP administration once a week, the concentration was halved for the first dose, ie 2.5 mg/kg; continuous administration for 7 weeks. The size of the tumor was measured twice a week.
  • the experimental group was administered with the PTmab bispecific antibody KN026, and the reference group was administered with the Trastuzumab standard in combination with the Pertuzumab standard.
  • Each drug was administered in the aforementioned dose, ie, 5 mg P+5 mg T/kg for the first time, and then every week.
  • the second dose was 2.5 mg P + 2.5 mg T / kg, and the blank control group was administered the same volume of PBS buffer each time.
  • the experimental group and the reference group were compared with the blank control group, and showed a significant tumor suppressing effect on the NCI-H522 mouse xenograft model.
  • the PTmab bispecific antibody alone has a comparable antitumor effect when used in combination with the same molar amount of the reference product Trastuzumab standard plus the Pertuzumab standard.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Veterinary Medicine (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明提供了具有共同轻链的双特异性抗体或抗体混合物及其制备方法。本发明还提供了编码所述抗体或混合物的核酸分子、含有该核酸分子的重组载体和重组细胞，以及所述抗体或混合物的检测和定量方法。

Description

具有共同轻链的双特异性抗体或抗体混合物 技术领域
本发明涉及双特异性抗体或抗体混合物，以及所述双特异性抗体或抗体混合物的制备方法。本发明还涉及编码所述双特异性抗体或抗体混合物的核酸分子、含有该核酸分子的重组载体和重组细胞，以及所述双特异性抗体或抗体混合物的检测和定量方法。
背景技术
单克隆抗体药物的上市在近十五年内增长迅速，成为制药行业的成长点。自1996年起，一共有30个左右单抗药物被批准上市。其中有九个单抗药物年销售超过十亿美元。2010年单抗药物总销售超过300亿美元，并且年增长率超过10％。由于单克隆抗体的靶标特异性强，只能抑制单一靶点，而在多种疾病中，包括肿瘤和自体免疫性疾病等，需要抑制多重信号通路来避免代偿效应。对于病毒感染疾病，由于病毒的高突变率，往往需要抑制多抗原位点来防止逃逸。因此，有以下几种备选方案可以解决此类问题。一种备选方案是使用多克隆抗体，或通过改造抗体Fc段获得异二聚体如双特异性抗体，至少可以对两个不同抗原或者同一个抗原的不同结合位点具有活性。还有一种方案是使用抗体混合物来治疗，抗体混合物可包含两种或更多种针对同一靶物上不同表位的抗体，或针对不同靶物的抗体的混合物。
双特异性抗体(Bispecific antibody,BsAbs)是含有两个不同配体结合位点的免疫球蛋白分子。它取代了经典的抗体Fab两臂相同的序列，而是用两个不同的Fab序列，因此Y型两臂可以结合不同的抗原表位。双特异性抗体在癌症治疗中的应用已经被多篇文献所综述(Carter 2001；Chames and Baty 2009；Chames and Baty 2009)。BsAbs的一臂可以连接肿瘤细胞表面的相关抗原而另外一臂则可以触发免疫效应细胞进一步杀伤细胞，通过免疫体系来杀死癌症肿瘤细胞。
对于双特异性抗体的制备，早在90年代，Carter等人用“把手-孔洞”(knob into hole)模型改造抗体重链的部分氨基酸，比较成功的实现了双特异性抗体的制备(Ridgway,Presta et al.1996；Carter 2001)。然而在他们的研究结果中，“孔洞-孔洞”模型对阻碍同二聚体的形成的能力仍然不够，依旧遗留了大概有5％的同二聚体。之后该研究组通过随机突变-噬菌体展示等方法进一步提高异二聚体的含量，但也没有解决根本问题。
本发明的发明人通过电荷氨基酸相互作用网络修改Fc的CH3相关氨基酸来减弱区域自身相互作用(有利于形成同二聚体)并增强区域之间的相互作用(有利于形成异二聚体)，成功的解决了“把手-孔洞”模型中5％同二聚体的残留，相关方法已经发表专利(公开号：CN102558355A)。
相对异二聚体平台技术，混合抗体生产平台的发展相对比较早期。其中最受关注的是丹麦的Symphogen A/S公司的抗体混合物技术。该技术首先通过抗体筛选平台的筛选获得多个针对同一靶标的抗体，随后针对每个抗体分别进行细胞株构建。之后将不同细胞摇瓶培养的种子液进行混合，最后进行混合物逐步扩大培养放大，并进行纯化工艺优化获得最终的产品。尽管使用这一方法通过培养多个细胞的混合群可以直接从一个重组生产过程中获得多个抗体，但是由于对混合细胞群培养的控制的难度，以及因此带来的放大生产的复杂性，使得该方案依然有一些潜在的问题。
本发明申请人通过对Fc部分进行突变，改变Fc直接相互作用，发明了一种用于在单一重组细胞中生成包含两种或多种同二聚体蛋白或抗体混合物的方法。该方案避免了混合细胞培养带来的工艺控制及放大的潜在困难，提供了一种更为经济有效的抗体混合物制备与生产方式。此方案也已发表专利(公开号：CN 103388013A)。
但不论上述哪种方法，在利用全抗体框架制备双特异性抗体或抗体混合物时，都可能出现轻链和重链之间错配的现象，进而影响抗体的活性，目前本领域比较成熟的方法是Roche(Genentech)开发的Crossmab，即通过对其中一组Fab进行轻链-重链序列的互相替换来防止该轻链序列与另一组轻链-重链之间的错配(专利号US20090162359,US20120164726)。此方法虽然能解决大部分的重链-轻链错配问题，但是又会因为对重轻链序列进行人为改造而带来新的问题，如轻链的解离，聚体含量增加，以及对有些Fab序列，会对抗原表位的识别造成一些影响。
赫赛汀(Herceptin，也叫曲妥珠单抗，Trastuzumab)作为第一个在乳腺癌中显示有切实疗效的治疗性单抗，为人类的抗人类表皮生长因子受体2(HER2)单克隆抗体，它作用于乳腺癌细胞的HER2-Neu表面蛋白，干扰癌细胞的生物学进程，最终致其死亡。赫赛汀(Herceptin)的主要适宜人群是HER2过度表达(免疫组化3+或者荧光原位杂交FISH阳性)的乳腺癌患者，而该人群约占所有乳腺癌患者的20～30％。
帕妥珠单抗(pertuzumab)是一种重组的单克隆抗体，与HER-2受体胞外结构域Ⅱ区结合，抑制二聚体的形成，抑制受体介导的信号转导通路(Agus DB，Gordon MS，Taylor C，et al.2005)。这可能部分解释帕妥珠 单抗抑制HER-2低表达肿瘤生长的原因，而曲妥珠单抗与HER-2受体的细胞外Ⅳ区结合，二聚体的形成不涉及Ⅳ区，因此曲妥珠单抗只对HER-2过表达的乳腺癌患者有效。目前正在进行帕妥珠单抗治疗HER-2低表达晚期乳腺癌的Ⅱ期临床研究。Baselga(Baselga J，et al.2007)等的研究显示帕妥珠单抗联合赫赛汀(曲妥珠单抗)对难治HER-2阳性乳腺癌患者具有确凿的抗肿瘤活性。该研究显示1/5的患者对帕妥珠单抗治疗有效(肿瘤缩小或消失)，另外1/5的患者病情保持稳定达6个月以上。帕妥珠单抗治疗乳腺癌的III期临床试验结果显示，该药能极长延长ERBB2阳性转移性乳腺癌患者的无进展生存期。
日前，罗氏公司公布了一项最新实验结果，该试验是一项评估采用帕妥珠单抗和赫赛汀(曲妥珠单抗)联合化疗(多西他赛)，来治疗早期原癌基因人类表皮生长因子受体2(HER2)阳性乳腺癌女性患者的疗效的II期新辅助治疗研究。美国癌症研究协会(CTRC-AACR)在圣安东尼奥乳腺癌研讨会(SABCS)上发布的数据显示，在术前新辅助治疗中给予两种抗体联合多西他赛治疗的乳腺肿瘤完全消失率(45.8％的病例完全缓解率)较赫赛汀联合多西他赛(29.0％的病例完全缓解率)显著提高50％以上。和赫赛汀及化疗相比，帕妥珠单抗和帕妥珠单抗联合多西他赛不会导致副作用或心脏风险显著增加。
本发明即以帕妥珠单抗和曲妥珠单抗为例，制备得到了同时具备帕妥珠单抗和曲妥珠单抗功能的双特异性抗体和抗体混合物，并在此基础上找到了一种制备轻链和重链能够正确组合的双特异性抗体或抗体混合物的新方法。
发明内容
本发明的发明人通过反复实验，令人惊奇地发现，可以将两个原始抗体或抗体混合物中的轻链替换为共同轻链，以得到具有共同轻链的双特异性抗体或抗体混合物，该具有共同轻链的双特异性抗体或抗体混合物能够实现轻链和重链的正确组合，并且与两个原始抗体相比，具有良好的结合特性、生物学活性和稳定性，甚至在生物学活性上优于原始抗体。
本发明第一方面涉及双特异性抗体或其抗原结合部分，其特征在于所述双特异性抗体或其抗原结合部分具有共同轻链，所述共同轻链是指两条轻链具有相同的序列。
在本发明的一个实施方案中，其重链能够分别与所述轻链在生理条件或体外的蛋白表达状态下正确结合。
在本发明的一个实施方案中，所述双特异性抗体或其抗原结合部分的 共同轻链从两株原始单克隆抗体(已知单克隆抗体)改造获得，所述共同轻链至少与两株原始单克隆抗体中一株的轻链序列不同。在本发明的一个实施方案中，所述共同轻链与两株原始单克隆抗体中一株的轻链相同，或者在其基础上经过改造(例如氨基酸序列改造)获得，改造的目的是尽可能保持与各自的抗原或抗原表位的亲和力。在本发明的一个实施方案中，所述氨基酸序列改造包括氨基酸的突变、缺失或添加，例如突变、缺失或添加不超过3个氨基酸，优选不超过2个氨基酸，更优选不超过1个氨基酸。
在本发明的一个实施方案中，所述双特异性抗体或其抗原结合部分的重链Fc段经过改造以更有利于形成异二聚体蛋白。
在本发明的一个实施方案中，两株原始单克隆抗体为帕妥珠抗体和曲妥珠抗体。
在本发明的一个实施方案中，其中所述的共同轻链能够分别与帕妥珠单抗和曲妥株单抗的重链结合。
在本发明的一个实施方案中，其中所述的共同轻链选自帕妥珠单抗或曲妥株单抗的轻链或者它们的突变体。在本发明的一个实施方案中，所述双特异性抗体或其抗原结合部分的重链(包括可变区和恒定区)可以与两株原始单克隆抗体相同，或者经过改造以更有利于形成异二聚体蛋白；所述改造例如为对重链Fc段进行改造以更有利于形成异二聚体蛋白。
在本发明的一个实施方案中，其中所述共同轻链的可变区的序列包含选自如SEQ ID NO：1～SEQ ID NO：6中第1～107位氨基酸所示的序列。
在本发明的一个实施方案中，其中所述轻链恒定区的序列包含SEQ ID NO：1中第108～214位氨基酸所示的序列。
在本发明的一个实施方案中，其重链可变区分别为帕妥珠单抗和曲妥株单抗的重链可变区。
在本发明的一个实施方案中，其两条重链可变区的序列分别包含如SEQ ID NO：23和SEQ ID NO：24所示的序列。
在本发明的一个实施方案中，其两条重链Fc段的序列分别包含如SEQ ID NO：25和SEQ ID NO：26所示的序列。
在本发明的一个实施方案中，其两条重链的序列分别包含如SEQ ID NO：19和SEQ ID NO：20所示的序列。
本发明第二方面涉及能够在一个细胞中正确产生的抗体或其抗原结合部分的混合物，所述混合物包括至少两种抗体或其抗原结合部分，所述 抗体或其抗原结合部分具有共同轻链，所述共同轻链是指两条轻链的可变区具有相同的序列。
在本发明的一个实施方案中，其中所述抗体或其抗原结合部分的重链能够分别与所述轻链在生理条件或体外的蛋白表达状态下正确结合。
在本发明的一个实施方案中，所述双特异性抗体或其抗原结合部分的共同轻链从两株原始单克隆抗体(已知单克隆抗体)改造获得，所述共同轻链至少与两株原始单克隆抗体中一株的轻链序列不同。在本发明的一个实施方案中，所述共同轻链与两株原始单克隆抗体中一株的轻链相同，或者在其基础上经过改造(例如氨基酸序列改造)获得，改造的目的是尽可能保持与各自的抗原或抗原表位的亲和力。在本发明的一个实施方案中，所述氨基酸序列改造包括氨基酸的突变、缺失或添加，例如突变、缺失或添加不超过3个氨基酸，优选不超过2个氨基酸，更优选不超过1个氨基酸。
在本发明的一个实施方案中，所述双特异性抗体或其抗原结合部分的重链来源于两株原始单克隆抗体，所述双特异性抗体或其抗原结合部分的重链可变区序列和/或CH1结构域序列与原始单克隆抗体相同。
在本发明的一个实施方案中，所述双特异性抗体或其抗原结合部分的的重链(包括可变区和恒定区)可以与两株原始单克隆抗体相同，或者经过改造以更有利于形成同二聚体蛋白；所述改造例如为对重链Fc段进行改造以更有利于形成同二聚体蛋白。
在本发明的一个实施方案中，两株原始单克隆抗体为帕妥珠抗体和赫赛汀抗体。
在本发明的一个实施方案中，其中所述的共同轻链能够分别与帕妥珠单抗和曲妥株单抗的重链结合。
在本发明的一个实施方案中，其中所述的共同轻链选自帕妥珠单抗或曲妥株单抗的轻链或者它们的突变体。
在本发明的一个实施方案中，其中所述共同轻链的可变区的序列包含选自如SEQ ID NO：1～SEQ ID NO：6中第1～107位氨基酸所示的序列。
在本发明的一个实施方案中，其中所述轻链恒定区的序列包含SEQ ID NO：1中第108～214位氨基酸所示的序列。
在本发明的一个实施方案中，其中所述抗体或其抗原结合部分的重链可变区分别为帕妥珠单抗和曲妥株单抗的重链可变区。
在本发明的一个实施方案中，其两条重链可变区的序列分别包含如SEQ ID NO：23和SEQ ID NO：24所示的序列。
在本发明的一个实施方案中，其两条重链Fc段的序列分别包含如SEQ ID NO：27和SEQ ID NO：28所示的序列。
在本发明的一个实施方案中，其中所述抗体或其抗原结合部分的重链序列分别包含如SEQ ID NO：21和SEQ ID NO：22所示的序列。
本发明第三方面涉及HER2蛋白胞外区域的变体蛋白，其与野生型HER2蛋白胞外区域的序列相比，具有选自如下一组的突变：
1)第558位谷氨酸的突变和第573位苯丙氨酸的突变；
2)第288位丝氨酸的突变和第296位组氨酸的突变。
在本发明的一个实施方案中，将第558位谷氨酸突变为丙氨酸。
在本发明的一个实施方案中，将第573位苯丙氨酸突变为丙氨酸。
在本发明的一个实施方案中，将第288位丝氨酸突变为丙氨酸。
在本发明的一个实施方案中，将第296位组氨酸突变为丙氨酸。
在本发明的一个实施方案中，所述HER2变体蛋白包含选自如SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的氨基酸序列。
在本发明的一个实施方案中，野生型HER2蛋白胞外区域的序列如SEQ ID NO：18所示。
本发明第四方面涉及核酸分子，其编码本发明第一方面任一项的双特异性抗体或其抗原结合部分或者第二方面任一项的混合物中所述的抗体或其抗原结合部分、或者所述抗体或其抗原结合部分的部分序列(例如轻链和/或重链)，或者编码第三方面任一项的HER2变体蛋白。
本发明第五方面涉及重组载体，其含有本发明第四方面任一项的核酸分子。
本发明第六方面涉及重组细胞，其含有本发明第五方面任一项的重组载体或第四方面任一项的核酸分子。
本发明第七方面涉及一种根据两株针对不同抗原表位的单克隆抗体或其抗原结合部分制备双特异性抗体或其抗原结合部分的方法，其包括以下步骤：
根据两株单克隆抗体的轻链序列得到能够分别与两株单抗重链结合的共同轻链序列，所述共同轻链是指两条轻链具有相同的序列，优选地， 该共同轻链为其中一株单抗的轻链或者为其中一株单抗轻链的突变体。
在本发明的一个实施方案中，其还进一步包括以下步骤：
分别将两株单抗的重链序列和共同轻链序列构建于表达载体中，得到两个重组表达载体；优选地，对重链序列特别是Fc段进行突变，以更有利于具有不同重链的两株单抗的Fc段的结合；
将两个重组表达载体转入同一宿主细胞，诱导表达，得到双特异性抗体或其抗原结合部分。
在本发明的一个实施方案中，其中所述共同轻链的可变区的获取方法为，首先确定两株单抗的轻链可变区与各自抗原或抗原表位之间接触的界面氨基酸，然后确定以任意其中一株单抗的轻链可变区为候选共同轻链的可变区时，该共同轻链与该株单抗的抗原或抗原表位之间接触的界面氨基酸与另一株单抗轻链可变区的氨基酸相比较时的差异氨基酸，选取差异氨基酸数量较少的轻链可变区为共同轻链的可变区；优选地，对该共同轻链可变区进一步突变以获得与抗原或抗原表位亲合力更好的共同轻链的可变区。在本发明的一个实施方案中，其中所述共同轻链的恒定区的获取方法为，将已确定为提供共同轻链的可变区的这一株单克隆抗体的轻链恒定区作为共同轻链的恒定区，或对该轻链恒定区进一步突变以获得共同轻链的恒定区。
在本发明的一个实施方案中，所述突变是指对其中的界面氨基酸进行突变。
在本发明的一个实施方案中，所述与抗原或抗原表位亲和力更好是指在该共同轻链与该双特异性抗体或其抗原结合部分对应的两个抗原或抗原表位的亲和力之间达到一个平衡，使该双特异性抗原或其抗原结合部分具有更好的生物学活性和理化特性(例如稳定性)。
本发明第八方面涉及一种制备包括至少两种单克隆抗体或其抗原结合部分的混合物的方法，所述方法包括以下步骤：
根据两株单克隆抗体的轻链序列得到能够分别与两株单抗重链结合的共同轻链序列，所述共同轻链是指两条轻链具有相同的序列，优选地，该共同轻链为其中一株单抗的轻链或者为其中一株单抗轻链的突变体。
在本发明的一个实施方案中，其进一步包括以下步骤：
分别将两株单抗的重链序列和共同轻链序列构建于表达载体中，得到两个重组表达载体；优选地，对重链序列特别是Fc段进行突变，以更有利于具有相同重链的单抗的Fc段的结合；
将两个重组表达载体转入同一宿主细胞，诱导表达，得到抗体或其抗原结合部分的混合物。
在本发明的一个实施方案中，其中所述共同轻链的可变区的获取方法为，首先确定两株单抗的轻链可变区与各自抗原或抗原表位之间接触的界面氨基酸，然后确定以任意其中一株单抗的轻链可变区为候选共同轻链的可变区时，该共同轻链与该株单抗的抗原或抗原表位之间接触的界面氨基酸与另一株单抗轻链可变区的氨基酸相比较时的差异氨基酸，选取差异氨基酸数量较少的轻链可变区为共同轻链的可变区；优选地，对该共同轻链可变区进一步突变以获得与抗原或抗原表位亲合力更好的共同轻链的可变区。在本发明的一个实施方案中，其中所述共同轻链的恒定区的获取方法为，将已确定为提供共同轻链的可变区的这一株单克隆抗体的轻链恒定区作为共同轻链的恒定区，或对该轻链恒定区进一步突变以获得共同轻链的恒定区。
在本发明的一个实施方案中，所述突变是指对其中的界面氨基酸进行突变。
在本发明的一个实施方案中，所述与抗原或抗原表位亲和力更好是指在该共同轻链与该混合物中两株单抗对应的两个抗原或抗原表位的亲和力之间达到一个平衡，使该混合物具有更好的生物学活性和理化特性(例如稳定性)。
本发明还涉及一种检测抗体或其抗原结合部分是否为双特异性抗体或其抗原结合部分和/或对其定量的方法，所述方法包括以下步骤(参见图25的示意图)：
1)分别制备能够和双特异性抗体或其抗原结合部分中的抗原结合部分1结合而不和抗原结合部分2结合的特异性抗原1，以及能够和抗原结合部分2结合而不和抗原结合部分1结合的特异性抗原2；
2)取特异性抗原1(或者特异性抗原2)包被酶标板，加入待检抗体，反应一段时间，再加入标记的特异性抗原2(或者特异性抗原1)，反应一段时间，最后加入能够与前述标记分子结合的检测分子，反应一段时间，所述检测分子带有可检测的标记，根据检测原理读数，判断为反应阳性或阴性；
3)当反应为阳性、并且该反应具有浓度依赖性时，则判断该抗体或其抗原结合部分为双特异性抗体或其抗原结合部分；任选地，根据所得阳性数值进一步对双特异性抗体或其抗原结合部分进行定量。
在本发明中，所述抗原结合部分1和2分别是指双特异性抗体或其抗原结合部分中的两个分别与不同抗原或抗原表位结合的部分；在本发明的实施方案中，所述抗原结合部分1和2分别在两株原始抗体的基础上改造获得，并且抗原结合部分1和2分别和两株原始抗体结合的抗原或抗原表位相同。
在本发明的一个实施方案中，所述特异性抗原1和特异性抗原2是HERm1和HERm2。
在本发明的一个实施方案中，所述标记的特异性抗原是用生物素标记的特异性抗原。
在本发明的一个实施方案中，所述检测分子是指可用于检测的底物分子，例如为HRP标记的链霉亲和素。
本发明还涉及一种检测抗体或其抗原结合部分的混合物是否为同二聚体蛋白的方法，所述混合物包括两种抗体(抗体1和抗体2)或其抗原结合部分，所述方法包括以下步骤(参见图26的示意图)：
1)分别制备能够和抗体1结合而不和抗体2结合的特异性抗原1，以及能够和抗体2结合而不和抗体1结合的特异性抗原2；
2)取特异性抗原1(或者特异性抗原2)包被酶标板，加入待检混合物，反应一段时间，再加入标记的特异性抗原1(或者特异性抗原2)，反应一段时间，最后加入能够与前述标记分子结合的检测分子，反应一段时间，所述检测分子带有可检测的标记，根据检测原理读数，判断为反应阳性或阴性；
3)另取特异性抗原1(或者特异性抗原2)包被酶标板，加入待检混合物，反应一段时间，再加入标记的特异性抗原2(或者特异性抗原1)，反应一段时间，最后加入能够与前述标记分子结合的检测分子，反应一段时间，所述检测分子带有可检测的标记，根据检测原理读数，判断为反应阳性或阴性；
4)当步骤2)反应阳性、并且该反应具有浓度依赖性，同时步骤3)反应阴性时，则判断混合物中为同二聚体蛋白并且不含异二聚体蛋白；当步骤2)反应阳性同时步骤3)反应阳性时，则判断混合物中既含有同二聚体蛋白也含有异二聚体蛋白。
在本发明的一个实施方案中，所述特异性抗原1和特异性抗原2是HERm1和Herm2。
在本发明的一个实施方案中，所述标记的特异性抗原是用生物素标记的抗原。
在本发明的一个实施方案中，所述检测分子是指可用于检测的底物分子，例如为HRP标记的链霉亲和素。
本发明还涉及组合物(例如药物组合物)，其含有本发明第一方面任一项的双特异性抗体或其抗原结合部分，以及任选的药学上可接受的载体或赋形剂。
本发明还涉及组合物(例如药物组合物)，其含有本发明第二方面任一项的混合物，以及任选的药学上可接受的载体或赋形剂。
本发明还涉及试剂盒，其含有本发明第一方面任一项的双特异性抗体或其抗原结合部分，以及任选的缓冲液和/或说明书。
在本发明的一个实施方案中，所述试剂盒用于诊断HER2阳性肿瘤(例如乳腺癌、胃癌)。
本发明还涉及试剂盒，其含有本发明第二方面任一项的混合物，以及任选的缓冲液或说明书。
在本发明的一个实施方案中，所述试剂盒用于诊断HER2阳性肿瘤(例如乳腺癌、胃癌)。
本发明还涉及本发明第一方面任一项的双特异性抗体或其抗原结合部分在制备预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)的药物中的用途。
本发明还涉及本发明第二方面任一项的混合物在制备预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)的药物中的用途。
本发明还涉及本发明第一方面任一项的双特异性抗体或其抗原结合部分在制备诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的试剂或试剂盒中的用途。
本发明还涉及本发明第二方面任一项的混合物在制备诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的试剂或试剂盒中的用途。
本发明还涉及本发明第三方面任一项的HER2蛋白胞外区域的变体蛋白用于检测第一方面任一项的双特异性抗体或其抗原结合部分或者用于检测第二方面任一项的混合物的用途。
本发明还涉及预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)的方法，所述方法包括给有需要的受试者预防或治疗有效量的本发明第一方面任一项的双特异性抗体或其抗原结合部分的步骤。
本发明还涉及预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)的方法，所述方法包括给有需要的受试者预防或治疗有效量的本发明第二方面任一项的混合物的步骤。
本发明还涉及诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的方法，所述方法包括使用本发明第一方面任一项的双特异性抗体或其抗原结合部分的步骤。
本发明还涉及诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的方法，所述方法包括使用本发明第二方面任一项的混合物的步骤。
本发明还涉及检测本发明第一方面任一项的双特异性抗体或其抗原结合部分或者检测本发明第二方面任一项的混合物的的方法，所述方法包括使用本发明第三方面任一项的HER2蛋白胞外区域的变体蛋白的步骤。
本发明还涉及本发明第一方面任一项的双特异性抗体或其抗原结合部分，其用于预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)。
本发明还涉及本发明第二方面任一项的混合物，其用于预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)。
以下对本发明做进一步描述。
在本发明中，术语“抗体”是指通常由两对相同的多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒 定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
在本发明中，术语抗体的“抗原结合部分”是指全长抗体的一个或多个部分，所述部分保持结合抗体所结合的相同抗原(例如，HER2)的能力，与完整抗体竞争对抗原的特异性结合。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗原结合部分。在一些情况下，抗原结合部分包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如单克隆抗体2E12)获得抗体的抗原结合部分(例如，上述抗体片段)，并且以与对于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合部分。
在本发明中，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature341:544-546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域 组成的抗体片段；术语“F(ab')2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在本发明中，术语“抗体Fc段””是熟练的技术人员公知的术语并基于抗体的木瓜蛋白酶裂解而定义，指的是人免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧基端或其中的一部分。例如，免疫球蛋白Fc区可包括重链CH2、CH3、CH4的两个或更多结构域与免疫球蛋白铰链区的组合。根据重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类，主要有5类免疫球蛋白：IgA，IgD，IgE，IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG-l，IgG-2，IgG-3，IgG-4，IgA-l和IgA-2。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。
在本发明一个的实施方案中，本发明所用的抗体Fc段包括至少一个免疫球蛋白绞链区，一个CH2结构域和一个CH3结构域，例如为人IgG1Fc。
在本发明中，术语“双特异性抗体”能够分别和两种抗原或抗原表位结合，其包括能够特异性结合第一抗原或抗原表位的抗体的轻链和重链，以及能够特异性结合第二抗原或抗原表位的抗体的轻链和重链。在本发明的一个实施方案中，所述双特异性抗体中能够特异性结合第一抗原或抗原表位的抗体的轻链和能够特异性结合第二抗原或抗原表位的抗体的轻链具有相同的序列。在本发明的一个实施方案中，所述双特异性抗体中能够特异性结合第一抗原或抗原表位的抗体的重链和能够特异性结合第二抗原或抗原表位的抗体的重链具有不同的序列。
在本发明中，术语“表位”或“抗原表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
在本发明中，20种常规氨基酸和其缩写遵从常规用法。参见Immunology-A Synthesis(第2版,E.S.Golub和D.R.Gren,Eds.,Sinauer  Associates,Sunderland,Mass.(1991)),其通过引用合并入本文。
在本发明中，对两株针对不同抗原或抗原表位的单克隆抗体(即原始抗体)的轻链序列(特别是可变区序列)进行分析和验证，获得能够和两株单克隆抗体的重链结合的共同轻链。该共同轻链和重链结合后，仍能特异性结合原单抗的抗原或抗原表位。
在本发明中，该共同轻链可以用于表达双特异性抗体，也可以用于表达含有两种抗体的混合物；在表达双特异性抗体时，该抗体含有能够和第一种抗原结合的轻链和重链，以及能够和第二种抗原结合的轻链和重链，其中的两条轻链序列完全相同，即为该共同轻链；在表达抗体混合物时，每种抗体各含有两条轻链和重链，其中的轻链序列完全相同，即为该共同轻链。
在本发明中，所述两株原始抗体的轻链恒定区可以为κ型或λ型；所述κ型轻链恒定区包括各种同种异型，如Km1、Km1,2、Km3；所述λ型轻链恒定区包括各种同种异型，如CL1、CL2、CL3、CL6和CL7。
本领域公知，可变区对抗原和抗体的特异性结合至关重要，因此在改造或获得抗体时，对可变区序列的选择和改造尤为关键。因此，在本发明中，为了获得本发明的具有共同轻链的双特异性抗体或抗体混合物时，首先需要获得该共同轻链的可变区。在根据本发明上述的方法选择其中一株原始单克隆抗体的轻链可变区或其突变序列作为该共同轻链的可变区后，再确定该共同轻链的恒定区。通常情况下，会选择已确定为该共同轻链的可变区的这一株单克隆抗体的轻链恒定区作为该共同轻链的恒定区；当然，在某些情况下，也可以选择另一株单克隆抗体的轻链恒定区作为共同轻链的恒定区。如果有必要，可以根据本领域的公知技术对原轻链恒定区进行改造(例如氨基酸的添加、缺失或突变等)以获得更适宜的共同轻链的恒定区，例如通过改造使其具有更好的ADCC、CDC、内吞、稳定性、免疫原性或半衰期等。
在本发明中，两株原始抗体的重链类型可以相同或不同，优选为类型相同。在本发明的一个实施方案中，在制备双特异性抗体和抗体混合物时，与原始抗体相比，其重链序列的可变区和CH1结构域的序列不变。
在本发明中，所述双特异性抗体中的两个臂或含有两种抗体混合物中的抗体都来源于两株原始单克隆抗体。在构建双特异性抗体或抗体混合物时，仅需要改变轻链可变区的序列以获得共同轻链，而不需要改变重链可变区的序列。也就是说，在构建的双特异性抗体和抗体混合物中，抗体的重链可变区序列与原始抗体可以是相同的，但至少其中一个轻链可变区序列与原始抗体不同。
在本发明中，可以根据不同的需要或目的选择两种原始单克隆抗体，例如可以选择针对同一抗原不同抗原表位的两株单抗，也可以其中一种抗体连接肿瘤细胞表面的相关抗原，而另一种抗体可以触发免疫效应细胞以进一步杀伤细胞。
在本发明的实施方案中，在制备双特异抗体时，可以利用现有技术对重链例如Fc段进行改造，以在抗体表达时，更有利于异二聚体蛋白的形成。
在本发明的实施方案中，在制备抗体混合物时，可以利用现有技术对重链例如Fc段进行改造，以在抗体表达时，更有利于同二聚体蛋白的形成。
在本发明中，通过改造抗体重链的Fc段以更有利于同二聚体或异二聚体蛋白的技术为本领域所公知，例如可参考Ridgway,Presta et al.1996；Carter 2001，专利CN 102558355A，专利CN 103388013A。
在本发明的实施方案中，用到融合具有不同抗原识别表位的多肽的技术包括但不仅限于如具体实施例中的异二聚体Fc融合技术，也可以是“Fab”技术,见图1。
在本发明的实施方案中，本发明所用到的异二聚体Fc融合技术，可以为“把手”-“孔洞”模型，也可以是“电荷排斥”模型，但不仅仅限制于这两种模型。
在本发明的实施方案中，本发明所用到的能够在单一重组细胞生产制备的抗体混合物平台可以是“电荷排斥”模型，但不仅仅限制于这一模型。
在本发明的一些实施方案中，当用于制备双特异抗体或抗体混合物时，所述核酸分子编码针对第一抗原的抗体的轻链和/或重链，或者编码针对第二抗原的抗体的轻链和/或重链。在本发明的实施方案中，所述轻链为共同轻链；在本发明的实施方案中，所述重链的Fc段进行了改造。
在本发明的一些实施方案中，所述载体可以为克隆载体或表达载体。所述克隆载体用于克隆抗体的相关片段；所述表达载体用于表达双特异抗体或抗体混合物。可以根据本领域的公知常识选择适合抗体表达的载体。在本发明的具体实施方案中，所述表达载体为pcDNA4m，其是在载体pcDNA4/myc-HisA的基础上进行改造后得到的载体。
在本发明的一些实施方案中，所述表达载体中含有编码针对第一抗原的抗体的轻链和/或重链的核酸分子，或者含有编码针对第二抗原的抗体的轻链和/或重链的核酸分子。
在本发明的一些实施方案中，所述宿主细胞为适合抗体表达的宿主细胞，例如为原核细胞(例如E.Coli)或真核细胞；所述真核细胞例如为酵母细胞、植物细胞或哺乳动物细胞，所述哺乳动物细胞例如为CHO细胞、HEK293 细胞或骨髓瘤细胞等。
在本发明的一些实施方案中，所述宿主细胞中同时含有表达针对第一抗原的抗体的轻链和/或重链的表达载体以及表达针对第二抗原的抗体的轻链和/或重链的表达载体；在本发明的具体实施方案中，所述轻链为共同轻链；在本发明的实施方案中，所述重链的Fc段进行了改造。当用于表达双特异抗体时，通过Fc段的改造，使针对不同抗原的抗体的轻链和重链更容易结合，以形成双特异抗体；当用于表达抗体混合物时，通过Fc段的改造，使针对同一抗原的抗体的轻链和重链更容易结合，以形成抗体混合物。
双特异性抗体或抗体混合物可以用标准的实验手段从宿主细胞中纯化。纯化方法包括但不限于色谱技术如体积排阻、离子交换、亲和色谱法及超滤法。在本发明的实施方案中，通过ProteinA亲和层析法对双特异性抗体和抗体混合物进行纯化。
在本发明中，本发明的双特异性抗体或其抗原结合部分或其混合物还可与化疗药物和/或其它抗体联用，因此本发明的组合物中还可含有化疗药物和/或其它抗体。
在本发明中，所述化疗药物包括但不限于：阿霉素(Adriamycin)、环磷酰胺和紫杉烷类[紫杉醇(Taxol)和多西他赛(Taxotere)]、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)等，或它们的组合。
在本发明中，通过对HER2蛋白突变，制备得到了仅能够和帕妥珠单抗和赫赛汀单抗中的一种特异性结合的HER2蛋白突变体。在本发明的实施方案中，利用这几种突变体对双特异抗体和抗体混合物进行鉴定。
在本发明中，通过双抗原夹心ELISA(也叫桥式ELISA)方法结合突变的HER2蛋白鉴定抗体是否为双特异性抗体或者抗体混合物中是否含有同二聚体蛋白，并进一步对双特异性抗体或抗体混合物中的同二聚体蛋白进行定量。
在本发明中，所述双抗原夹心法ELISA为本领域所公知，其工作原理为利用连接于固相载体上的抗原和酶标抗原分别与样品中被检测抗体分子上两个抗原结合位点结合，形成固相抗原-抗体-酶标抗原免疫复合物。该方法的检测步骤例如包括：⑴将特异性抗原包被固相载体。孵育一定时间，使形成固相抗原，洗涤除去未结合的抗原和杂质。⑵加待检标本，孵育，使标本中的抗体与固相载体上的抗原充分反应，形成固相抗原抗体复合物。洗涤 除去其他未结合物质。⑶加酶标抗原，孵育，使形成固相抗原-待测抗体-酶标抗原夹心复合物。洗涤除去未结合酶标抗原。⑷加底物显色。固相上的酶催化底物产生有色产物,通过比色,测标本中抗体的量。
在本发明中，所述HER2阳性肿瘤既包括HER2蛋白过表达的肿瘤(例如乳腺癌、胃癌、食管癌、卵巢癌、子宫内膜癌、膀胱癌、肺癌、结肠癌和头颈部肿瘤)，也包括HER2蛋白低表达的肿瘤(例如HER2低表达的乳腺癌、胃癌、肺癌等)。
本发明通过分析两株不同单抗的轻链序列，获得能够分别与两株单抗重链结合的共同轻链，并在此基础上制备了具有共同轻链的双特异性抗体和抗体混合物，实验证明利用该方法制备得到的双特异性抗体和抗体混合物具有良好的结合特性、生物学活性和稳定性，并且在生物学活性方面可能优于原始抗体。
该共同轻链技术简单、可控，在不影响抗体稳定性、活性及纯度的情况下，有效解决了双特异性抗体中重轻链错配的难题；对于抗体混合物，更是使其可以在同一宿主细胞中表达，能避免混合细胞群培养的难度，更有利于放大生产。
附图说明
图1.异二聚体蛋白融合示意图。a图表示异二聚体Fc融合技术,b图表示“Fab”技术。
图2帕妥珠单抗和曲妥珠单抗轻链高变区的识别，其中A为帕妥珠单抗轻链高变区识别结果，B为曲妥珠单抗轻链高变区识别结果，C为帕妥珠单抗和曲妥珠单抗轻链序列比对及抗原界面氨基酸综合分析结果。
图3.曲妥珠单抗Fab片段和Her2胞外区域(ECD)结构图。
图4.Her2m1和Her2m2变体蛋白SDS-PAGE电泳分析结果(18％SDS-PAGE非还原条件)。
1：HER2m1；2：HER2m2；M：蛋白质量标准。
图5.ELISA法检测HER2变体蛋白对Trastuzumab或Pertuzumab的特异性结合
图6：一步亲和层析纯化得到的共同轻链单抗蛋白样品利用非还原的SDS-PAGE进行初步的检测(12％SDS-PAGE还原条件)
1～6：TmabCLC1～6；7～12：PmabCLC1～6；M：蛋白质量标准。
图7：带有共同轻链的Trastuzumab对其特异性抗原HER2m1的亲和力
图8：带有共同轻链的Pertuzumab对其特异性抗原HER2m2的亲和力
图9一步亲和层析纯化得到的KN026抗体蛋白样品利用SDS-PAGE进行初步的检测(12％SDS-PAGE还原条件)
1：KN026瞬时表达细胞培养上清；2：KN026亲和层析流穿；3：KN026一步亲和层析后纯化蛋白样品(还原)；4：KN026一步亲和层析后纯化蛋白样品(非还原)M：蛋白质量标准。
图10KN026抗体蛋白纯度的SE-HPLC检测结果
图11双特异性抗体KN026识别两种抗原的亲和曲线
图12一步亲和层析纯化得到的KN010抗体蛋白样品利用SDS-PAGE进行初步的检测(12％SDS-PAGE还原条件)
图13混合抗体蛋白KN026纯度的SE-HPLC检测结果
图14混合抗体蛋白KN026识别两种抗原的亲和曲线
图15 Ptmab双特异性抗体(KN026)以及Pertuzumab、Trastuzumab与BT474细胞结合的浓度依存性曲线
图16 Ptmab双特异性抗体(KN026)以及Pertuzumab、Trastuzumab与N-87细胞结合的浓度依存性曲线
图17 Pmab、Tmab抗体混合物(KN010)以及Pertuzumab、Trastuzumab与BT474细胞结合的浓度依存性曲线
图18 KN026、Trastuzumab及Trastuzumab+Pertuzumab联合用药对人乳腺癌BT474细胞增殖的抑制作用
图19 KN026、Trastuzumab及Trastuzumab+Pertuzumab联合用药对人胃癌N-87细胞增殖的抑制作用
图20 Ptmab双特异性抗体KN026(浅色曲线)以及Trastuzumab参比样品(深色曲线)的热稳定性(Tm值)检测
图21 KN026和Trastuzumab的药代曲线
图22 Ptmab双特异性抗体对人卵巢癌SKOV3裸鼠移植瘤的肿瘤体积的影响
图23 Ptmab双特异性抗体对人胃癌N-87裸鼠移植瘤的肿瘤体积的影响
图24PTmab双特异性抗体对人胃癌N-87裸鼠移植瘤的肿瘤体积的影响
图25检测抗体是否为双特异性抗体的方法以及定量方法的示意图。
图26检测抗体混合物是否为同二聚体蛋白的方法的示意图。
图27 PTmab双特异性抗体对HER2低表达肿瘤株人非小细胞肺癌NCI-H522小鼠移植瘤模型的剂量依赖性。
图28 PTmab双特异性抗体对HER2低表达肿瘤株人非小细胞肺癌 NCI-H522小鼠移植瘤模型的药效作用与等摩尔数Trastuzumab标准品加等摩尔数Pertuzumab标准品联合使用时相当。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1共同轻链的获取
1.序列及结构获得
从蛋白质数据库(PDB,www.pdb.org)中获得曲妥珠单抗和帕妥珠单抗的复合体晶体结构，曲妥珠单抗PDB编号为1N8Z,帕妥珠单抗PDB编号为1S78。有两个筛选策略可用来识别CH3-CH3之间的氨基酸接触：(i)氨基酸作用的距离(ii)溶剂可及区域分析。这里根据氨基酸作用距离进行筛选。
2.单抗轻链及抗原HER2界面氨基酸获取
根据氨基酸接触规则，界面氨基酸指侧链重原子与另外一条链的任何一个氨基酸的重原子之间的距离小于一个阈值的那些氨基酸。在这里阈值选择为
Figure PCTCN2016070447-appb-000001
在某些文献中也可以选择
Figure PCTCN2016070447-appb-000002
(Bahar和Jernigan 1997)。表1为曲妥珠单抗轻链及抗原HER2相互作用的氨基酸列表。表1所列的为通过氨基酸接触筛选规则所筛选出的曲妥珠单抗的12个界面氨基酸。
表1曲妥珠单抗轻链-抗原HER2界面氨基酸列表
Figure PCTCN2016070447-appb-000003
Figure PCTCN2016070447-appb-000004
表2为帕妥珠单抗轻链及抗原HER2相互作用的氨基酸列表。表2所列的为通过氨基酸接触筛选规则所筛选出的帕妥珠单抗的8个界面氨基酸。
表2帕妥珠单抗轻链-抗原HER2界面氨基酸列表
Figure PCTCN2016070447-appb-000005
3.帕妥珠单抗和曲妥珠单抗轻链高变区(CDRL1,CDRL2,CDRL3)的识别
将帕妥珠单抗和曲妥珠单抗轻链通过高变区识别系统–kabat编号进行识别，识别软件为http://www.bioinf.org.uk/abs/abnum/。帕妥珠单抗轻链高变区识别结果见图2-A，曲妥珠单抗高变区轻链识别结果见图2-B。
4.帕妥珠单抗和曲妥珠单抗轻链序列比对及轻链与抗原界面氨基酸综合分析
帕妥珠单抗和曲妥珠单抗轻链序列比对及抗原界面氨基酸综合分析结果见图2-C，帕妥珠单抗(P-mab)和曲妥珠单抗(T-mab)轻链与抗原接触氨基酸用背景颜色黑色显示。如以曲妥珠单抗轻链为共同轻链，则该共同轻链上与抗原接触的界面氨基酸与帕妥珠单抗(P-mab)轻链上的界面氨基酸相比较得到的差异氨基酸见表3。
表3帕妥珠单抗(P-mab)和曲妥珠单抗(T-mab)轻链与抗原接触的差异氨基酸
Figure PCTCN2016070447-appb-000006
如以帕妥珠单抗轻链为共同轻链，则该共同轻链上与抗原接触的界面氨基酸与曲妥珠单抗(T-mab)轻链上的界面氨基酸相比较得到的差异氨基酸见表4。
表4帕妥珠单抗(P-mab)和曲妥珠单抗(T-mab)轻链与抗原接触的差异氨基酸
Figure PCTCN2016070447-appb-000007
从帕妥珠单抗(P-mab)和曲妥珠单抗(T-mab)轻链与抗原接触的差异氨基酸分析，分别选择曲妥珠单抗(T-mab)的轻链为框架，引入T31I或/及T94Y突变，得到帕妥珠、曲妥珠双特异性抗体的共同轻链序列CLC1～CLC4；选择帕妥珠单抗(P-mab)的轻链为框架，引入T31I或/及Y94T突变，得到帕妥珠、曲妥珠双特异性抗体的共同轻链序列CLC5～CLC6。各共同轻链的氨基酸序列如下：
CLC1
Figure PCTCN2016070447-appb-000008
CLC2
Figure PCTCN2016070447-appb-000009
CLC3
Figure PCTCN2016070447-appb-000010
CLC4
Figure PCTCN2016070447-appb-000011
CLC5
Figure PCTCN2016070447-appb-000012
CLC6
Figure PCTCN2016070447-appb-000013
实施例2抗原蛋白HER2变体蛋白的制备及功能验证
1.只结合帕妥珠单抗HER2变体蛋白的设计
Robert F.Kelley’and Mark P.O’Connell在1993年公布了曲妥珠单抗及相应的突变和HER2胞外区域(ECD)结合的动力学参数。其中H91A，R50A, W95A，Y100aA对曲妥珠单抗与HER2胞外域的结合造成了重大影响。Hyun-Soo Cho团队在2003年对曲妥珠单抗Fab片段和HER2胞外区域(ECD)的复合物进行了结晶(PDB编号：1N8Z)，该项结果发表于Nature上。通过分析曲妥珠单抗Fab片段和HER2胞外区域(ECD)(其序列为SEQ ID NO：18所示)的复合物的结构，我们获得了曲妥珠单抗Fab片段和HER2胞外区域(ECD)的界面接触氨基酸，如表5。
着重对H91L，R50H,W95H，Y100aH接触的氨基酸进行分析。发现有两个组合可能对这几个氨基酸结合具有重要作用。组合1：ASP570，PRO571和PRO572，组合2：GLU558和PHE573。
表5曲妥珠单抗Fab片段和HER2胞外区域(ECD)的界面接触氨基酸
Figure PCTCN2016070447-appb-000014
Figure PCTCN2016070447-appb-000015
从以上结果可以看出
组合1：P571,P572本身与Fab几个关键氨基酸都有作用力的形成。考虑这两个氨基酸处于loop转角处，如突变将影响本身结构稳定性。
组合2：GLU558与曲妥珠Fab重链ARG50形成离子键，同Fab重链多个氨基酸形成范德华力，破坏可以阻断曲妥珠Fab与HER2的作用，因此选择将GLU558→ALA558；PHE573与Fab重链多个氨基酸形成范德华力，包括关键氨基酸ARG508,TRP99,TYR105，破坏可以阻断Fab与HER2的作用，因此选择将PHE573→ALA573。
以上分析结果可以见图3。
2.只结合曲妥珠单抗的HER2变体蛋白的设计
Matthew C.Franklin在Cancer cell上发表了帕托珠单抗Fab与HER2胞外结构的复合物结构。该团队同时用丙氨酸扫描的方法研究了HER2哪些关键氨基酸会影响与帕托珠单抗Fab的结合。其研究表明，HER2蛋白表面H296,S288,L295等氨基酸具有明显的作用，本发明选择了S288A/H296A双突变用于获得只结合曲妥珠单抗的HER2抗原。
其中只被Trastuzumab识别的HER2变体蛋白命名为HER2m1，只被Pertuzumab识别的HER2变体蛋白命名为HER2m2和HER2m3。HER2变体蛋白的氨基酸序列分别为：
HER2m1：
Figure PCTCN2016070447-appb-000016
Figure PCTCN2016070447-appb-000017
HER2m2：
Figure PCTCN2016070447-appb-000018
HER2m3：
Figure PCTCN2016070447-appb-000019
3.商业化哺乳动物细胞表达载体pcDNA4/myc-HisA的改造
商业化载体pcDNA4/myc-HisA(Invitrogen，V863-20)含有两个PvuII酶切位点，分别在大约1411bp和3160bp的位置。对质粒定点突变，将3160bp位置的碱基C突变为G，去除在该位置的PvuII酶切位点，只保留在大约1411bp处的一个酶切位点，新的载体命名为pcDNA4m。
根据NCBI上人免疫球蛋白gamma1(IgG1)的可结晶片段(Fc)的DNA序列(AY623427)设计引物，如下
Figure PCTCN2016070447-appb-000020
其中上游引物中添加了HindIII以及BamHI的酶切位点，用于后续克隆，下游引物中添加了EcoRI的酶切位点。
以PBMC的全cDNA为模板，扩增得到Fc片段的基因，之后经Takara公司的HindIII与EcoRI双酶切克隆到改造的载体pcDNA4m中，测序验证构建质粒的准确性，获得重组质粒pcDNA4m-Fc。
4.HER2变体蛋白真核表达载体的构建
根据NCBI上HER2蛋白的DNA序列信息(NM_004448.2)设计引物，将野生型HER2蛋白的胞外结构域(第1至第652位氨基酸残基)克隆出来，所用引物如下：
Figure PCTCN2016070447-appb-000021
引物中包含了上游的HindIII识别位点以及下游的BamHI识别位点。利用BT474细胞(购自中科院上海细胞库)的cDNA作为模板，扩增得到编码HER2wt的胞外结构域的1.9kb长的DNA片段并克隆进商业化的T载体中(pMD19-T Simple Vector；购自Takara公司)，得到T-Her2ECD质粒，测序确认序列的正确性。
根据前述得到的只被Trastuzumab识别的HER2变体蛋白HER2m1以及只被Pertuzumab识别的HER2变体蛋白HER2m2以及HER2m3的氨基酸序列，根据突变位点设计相应的突变引物：
Figure PCTCN2016070447-appb-000022
Figure PCTCN2016070447-appb-000023
以T-Her2ECD质粒为模板，利用上述引物进行定点突变，得到三个HER2胞外结构域的变体蛋白(HER2m1，HER2m2，HER2m3)的基因。之后，经Takara公司的HindIII与BamHI双酶切克隆到载体pcDNA4m-Fc上，将HER2m1，HER2m2，HER2m3三个基因分别融合于Fc基因的5’端，得到三个新载体，命名为：pcDNA4m-Her2m1-Fc，pcDNA4m-Her2m2-Fc，pcDNA4m-Her2m3-Fc。这三个载体可用于在哺乳动物细胞中表达融合蛋白HER2m1-Fc，HER2m2-Fc，HER2m3-Fc。
5.HER2变体蛋白的瞬时表达及纯化
转染前两天，准备200mL×3经悬浮驯化的HEK293(ATCC，CRL-1573TM)细胞用于瞬时转染，接种密度为0.8×106cells/mL。两天后对待转染细胞悬液进行计数细胞密度为3.5～4×106cells/mL，取细胞悬液1000rpm离心5min，弃上清液。用40mL×3的新鲜的Freestyle293培养基重新悬浮细胞，再次1000rpm离心5min，弃上清液。用200mL×3Freestyle293培养基重新悬浮293细胞。将实施例2-4中所得的3个HER2变体蛋白的表达载体各取200μg，分别用2mL Freestyle293培养基稀释。随后用5mL Freestyle293培养基稀释1.5mL Polyethylenimine，配置转化所需PEI溶液。分别在稀释好的2mL的表达用质粒中加入2mL的PEI溶液并混匀，室温静置5分钟。将3份质粒/PEI混合物分别加入3份200mL细胞悬液中，放置在37℃，10％CO2，90rpm中培养；同时补加50μg/L IGF-1。四小时后于每份转化样品中再分别补加200mL EX293培养基，2mM Glutamine和50ug/L IGF-1，135rpm培养。二十四小时后加3.8mM VPA。
培养5～6天后，分别收集3份400mL的HER2变体蛋白细胞瞬时表达培养上清液，通过ProteinA亲和层析法，初步纯化得到HER2变体蛋白样品。其中HER2m3表达水平非常低，其细胞培养上清中模板蛋白的滴度小于0.5mg/L，推测主要是由于蛋白变体不稳定造成的，因此对该蛋白没有进一步纯化。得到的HER2m1和HER2m2变体蛋白通过纯化计算的表达水平约20mg/L。得到的蛋白样品利用SDS-PAGE进行初步的检测，可以清晰 的看到目的条带(见图4)。
6.用ELISA法检测HER2变体蛋白对Trastuzumab或Pertuzumab的特异性结合
用Trastuzumab单抗蛋白或Pertuzumab单抗包被酶标板，4℃过夜。之后加入3％BSA溶液，室温封闭2小时。待检样品(HER2m1或是HER2m2蛋白)预先用生物素标记，之后生物素化的蛋白HER2m1-Biotin以及HER2m2-Biotin自16μg/mL起，1:4梯度稀释，直至0.224ng/μL，共9个梯度。酶标板中加入梯度稀释的生物素化的HER2变体蛋白样品，室温反应2小时。之后加入HRP标记的链霉亲和素，室温作用1.5小时，最后催化底物显色读数。得到的数据通过四参数法拟合，得到亲和曲线。
如图5所示，HER2m1蛋白对Pertuzumab的表观亲和力与其对Trastuzumab的表观亲和力相比，降低了20倍，可以认为该变体蛋白为Trastuzumab特异性抗原蛋白。而HER2m2蛋白对Trastuzumab的表观亲和力比起对Trastuzumab的表观亲和力降低了>2个数量级，表现为Pertuzumab特异性抗原。
实施例4用共同轻链替换Tmab及Pmab原始轻链，并验证共同轻链的效果
1.携带共同轻链的Tmab及Pmab单抗真核表达载体的构建
根据专利US2009/0285837A1搜索到的Trastuzumab和Pertuzumab全抗体的氨基酸序列(专利中图2及图16)，利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列，并通过人工合成方法获得Trastuzumab的重链基因(SEQ ID NO:16)、Pertuzumab的重链基因(SEQ ID NO:17)。根据实施例1中得到的一组共同轻链的氨基酸序列(SEQ ID NO:1～6)，利用DNAworks在线工具(http://helixweb.nih.gov/dnaworks/)设计对应的编码DNA序列，并通过人工合成方法获得Pmab-Tmab双特异性抗体的共同轻链基因CLC1(SEQ ID NO：7)以及CLC5(SEQ NO：11)。
之后根据CLC2～CLC6的序列设计突变引物，序列如下：
Figure PCTCN2016070447-appb-000024
Figure PCTCN2016070447-appb-000025
以CLC1基因为模板，利用T31I及T94Y两对引物定点突变得到CLC2～CLC4的基因序列(SEQ ID NO：8～SEQ ID NO：10)；以CLC5为模板，利用Y94T引物对定点突变得到CLC6基因序列(SEQ ID NO：12)。
合成好的Trastuzumab的重链基因、Pertuzumab的重链基因、以及共同轻链基因(CLC1～CLC6)分别经Takara公司的HindIII与EcoRI双酶切亚克隆到改造的载体pcDNA4m中，测序验证构建质粒的准确性，获得重组质粒DNA即：pcDNA4m-TmabHC，pcDNA4m-PmabHC以及共同轻链相关载体pcDNA4m-CLC1～pcDNA4m-CLC6。
取上述构建成功的共同轻链基因表达载体pcDNA4m-CLC1～6用Takara公司的Bgl II，Pvu II双酶切。酶切产物通过0.8％的琼脂糖电泳分离纯化，并分别回收约2kb大小含有共同基因的DNA片段；pcDNA4m-TmabHC经BglII，NruI双酶切回收约6kb大小含有TmabHC基因的DNA片段；pcDNA4m-PmabHC经BglII，NruI双酶切回收约6kb大小含有PmabHC基因的DNA片段。随后将酶切处理过的DNA片段进行连接，将TmabHC或PmabHC的表达原件与不同序列的共同轻链表达原件整合在一起，获得重组质粒pcDNA4m-Tmab-CLC1、pcDNA4m-Tmab-CLC2、pcDNA4m-Tmab-CLC3、pcDNA4m-Tmab-CLC4、pcDNA4m-Tmab-CLC5、pcDNA4m-Tmab-CLC6、pcDNA4m-Pmab-CLC1、pcDNA4m-Pmab-CLC2、pcDNA4m-Pmab-CLC3、pcDNA4m-Pmab-CLC4、pcDNA4m-Pmab-CLC5、pcDNA4m-Pmab-CLC6。
2.携带共同轻链的Tmab及Pmab单抗的瞬时表达及纯化
转染前两天，准备50mL×12经悬浮驯化的HEK293(ATCC，CRL-1573TM)细胞用于瞬时转染，接种密度为0.8×106cells/mL。两天后对待转染细胞悬液进行计数细胞密度为3.5～4×106cells/mL，取细胞悬液1000rpm离心5min，弃上清液。用10mL×12的新鲜的Freestyle293培养基重新悬浮细胞，再次1000rpm离心5min，弃上清液。用50mL×12Freestyle293培养基重新悬浮293细胞。将实施例4-1中所得的12个共同轻链单抗相关的表达载体各取50μg，分别用0.5mL Freestyle293培养基稀释。随后用5mL Freestyle293培养基稀释1.5mL Polyethylenimine，配置转化所需PEI溶液。分别在稀释好的0.5mL的表达用质粒中加入0.5mL的PEI溶 液并混匀，室温静置5分钟。将12份质粒/PEI混合物分别加入12份50mL细胞悬液中，放置在37℃，10％CO2，90rpm中培养；同时补加50μg/L IGF-1。四小时后于每份转化样品中再分别补加50mL EX293培养基，2mM Glutamine和50μg/L IGF-1，135rpm培养。二十四小时后加3.8mM VPA。
培养5～6天后，分别收集12份100mL的共同轻链单抗细胞瞬时表达培养上清液，通过ProteinA亲和层析法，初步纯化得到12个共同轻链单抗蛋白样品：Tmab-CLC1～6以及Pmab-CLC1～6；每个单抗通过纯化计算的表达水平见表6。
一步亲和层析纯化得到的蛋白样品利用非还原的SDS-PAGE进行初步的检测。可以看到，如图6所示，所有的共同轻链单抗蛋白在还原胶上都能看到清晰的两条带，分别为位于25kDa与35kDa之间的轻链条带及位于85kDa和50kDa之间的重链条带。蛋白样品的纯度通过SE-HPLC进行检测，结果见表6。
表6共同轻链单抗细胞瞬时表达水平及一步纯化后后样品纯度
Figure PCTCN2016070447-appb-000026
3.用ELISA法对携带共同轻链的Tmab及Pmab单抗进行亲和力分析
检测带有共同轻链的Tratuzumab及Pertuzumab对其各自的特异性抗原的亲和力变化，所用的间接ELISA的方法与实施例2-6中所述相似。其中，检测带有共同轻链的Tratuzumab的亲和力变化时，使用其特异性抗原蛋白HER2m1进行检测；检测带有共同轻链的Pertuzumab的亲和力变化时，使用其特异性抗原蛋白HER2m2进行检测。得到的亲和力曲线见图7～8， 根据EC50判断，当使用由原始Trastuzumab的轻链改造而来的共同轻链(CLC1～CLC4)时，Trastuzumab对其特异性抗原HER2m1的亲和力没有明显改变；而Pertuzumab对其特异性抗原HER2m2的亲和力略有降低，但是这种变化在可接受范围内。而当使用由原始Pertuzumab的轻链改造得到的共同轻链(CLC5和CLC6)时，Pertuzumab对其特异性抗原HER2m2的亲和力没有明显改变，但是Trastuzumab对其特异性抗原HER2m1的亲和力降低了近一倍。
实施例5 Ptmab双特异性抗体的制备和鉴定
1.Ptmab双特异性抗体的瞬时表达及纯化
对pcDNA4m-Tmab-CLC1中的TmabHC的Fc片段进行点突变，使TmabHC成为TmabHC-knob(重链序列为SEQ ID NO：19所示；突变的残基为：S354C,T366W,)，并进一步构建pcDNA4m-Tmabknob-CLC1；同时利用定点突变，将pcDNA4m-Pmab-CLC1中的PmabHC中的氨基酸残基参照专利CN102558355A，突变为PmabHC-hole(重链序列为SEQ ID NO：20所示，突变的残基为：Y349C,T366S,L368A,Y407V)，并进一步构建pcDNA4m-Pmabhole-CLC1。具体突变方案参照CN102558355A。这两个新构建的质粒将在“把手-孔洞”模型上，利用共同轻链模型构建Pmab-Tmab双特异性抗体。
2.Ptmab双特异性抗体的瞬时表达及纯化
转染前两天，准备600mL经悬浮驯化的HEK293(ATCC，CRL-1573TM)细胞用于瞬时转染，接种密度为0.8×106cells/mL。两天后对待转染细胞悬液进行计数细胞密度为3.5～4×106cells/mL，取细胞悬液1000rpm离心5min，弃上清液。用100mL的新鲜的Freestyle293培养基重新悬浮细胞，再次1000rpm离心5min，弃上清液。用600mL Freestyle293培养基重新悬浮293细胞。取pcDNA4m-Tmabknob-CLC1及pcDNA4m-Pmabhole-CLC1各300μg混匀后用3mL Freestyle293培养基稀释。随后用5mL Freestyle293培养基稀释1.5mL Polyethylenimine，配制转化所需PEI溶液，并在稀释好的3mL混合质粒中加入3mL的PEI溶液并混匀，室温静置5分钟。将质粒/PEI混合物加入600mL的细胞悬液中，放置在37℃，10％CO2，90rpm中培养；同时补加50μg/L IGF-1。四小时后于转化样品中再分别补加600mL EX293培养基，2mM Glutamine和50ug/L IGF-1，135rpm培养。二十四小时后加3.8mM VPA。
培养6～7天后，收集1200mL的Ptmab双特异性抗体细胞瞬时表达培 养上清液，通过ProteinA亲和层析法以及离子交换层析法和分子筛层析法初步纯化得到Ptmab双特异性蛋白样品，命名为KN026。根据OD280计算得到KN026瞬时表达水平可达到80mg/L。
纯化得到的蛋白样品利用SDS-PAGE进行初步的检测。可以看到，如图9所示，KN026双特异性抗体蛋白在还原胶上能看到清晰的两条带，分别为位于25kDa与35kDa之间的轻链条带及位于85kDa和50kDa之间的重链条带。同时，非还原条件下，KN026为一条带。蛋白样品的纯度通过SE-HPLC进行检测，结果见图10，纯度约95％。
3.用Bridging ELISA法验证KN026抗体蛋白能同时识别Trastuzumab和Pertuzumab各自的特异性抗原
用Trastuzumab特异性抗原蛋白HER2m1包被酶标板，4℃过夜。之后加入3％BSA溶液，室温封闭2小时。待检样品自5μg/mL起，1:3梯度稀释，直至1.06ng/uL，共八个梯度。酶标板中加入梯度稀释的待检样品，室温反应2小时。之后生物素化的Pertuzumab特异性抗原蛋白HER2m2-Biotin加入酶标板，和待检样品室温反应2小时。随后加入HRP标记的链霉亲和素，与HER2m2-Biotin室温作用1.5小时，最后催化底物显色读数。得到的数据通过四参数法拟合，得到亲和曲线。
如图11所示，只有同时能识别两种抗原的双特异性抗体KN026才能够得到亲和曲线，而对于只能特异性识别一种抗原的Trastuzumab或Pertuzumab，则即使在最高浓度，也不能看到明显的显色读值。
实施例6 Ptmab抗体混合物的制备和鉴定
1.Ptmab抗体混合物的瞬时表达及纯化
对pcDNA4m-Tmab-CLC1中的TmabHC的Fc片段进行点突变，使TmabHC成为TmabHC-mix1(其中重链序列如SEQ ID NO：21所示)，并进一步构建pcDNA4m-Tmabmix1-CLC1，具体突变方案参照专利CN 103388013A实施例1。这两个新构建的质粒将在“电荷排斥”混合物模型上，利用共同轻链模型构建可在单一重组细胞株中表达的Pmab、Tmab抗体混合物。
2.Pmab、Tmab抗体混合物的瞬时表达及纯化
转染前两天，准备600mL经悬浮驯化的HEK293(ATCC，CRL-1573TM)细胞用于瞬时转染，接种密度为0.8×106cells/mL。两天后对待转染细胞悬液进行计数细胞密度为3.5～4×106cells/mL，取细胞悬液1000rpm离心5min，弃上清液。用100mL的新鲜的Freestyle293培养基重新悬浮细胞，再次 1000rpm离心5min，弃上清液。用600mL Freestyle293培养基重新悬浮293细胞。取pcDNA4m-Tmabmix1-CLC1及pcDNA4m-Pmab-CLC1(其中重链序列如SEQ ID NO：22所示)各300μg混匀后用3mL Freestyle293培养基稀释。随后用5mL Freestyle293培养基稀释1.5mL Polyethylenimine，配制转化所需PEI溶液，并在稀释好的3mL混合质粒中加入3mL的PEI溶液并混匀，室温静置5分钟。将质粒/PEI混合物加入600mL的细胞悬液中，放置在37℃，10％CO2，90rpm中培养；同时补加50μg/L IGF-1。四小时后于转化样品中再分别补加600mL EX293培养基，2mM Glutamine和50μg/L IGF-1，135rpm培养。二十四小时后加3.8mM VPA。
培养6～7天后，收集1200mL的Pmab、Tmab抗体混合物细胞瞬时表达培养上清液，通过ProteinA亲和层析法，初步纯化得到Pmab、Tmab抗体混合物蛋白样品，命名为KN010。根据OD280计算得到KN010瞬时表达水平可达到100mg/L。
一步亲和层析纯化得到的蛋白样品利用SDS-PAGE进行初步的检测。可以看到，如图12所示，共同轻链混合单抗蛋白产物在还原胶上能看到清晰的两条带，分别为位于25kDa与35kDa之间的轻链条带及位于85kDa和50kDa之间的重链条带。同时，非还原条件下，KN010为一条带。蛋白样品的纯度通过SE-HPLC进行检测，结果见图13，纯度约95％。
3.用同抗原Bridging ELISA法验证KN010抗体蛋白能分别识别Trastuzumab和Pertuzumab各自的特异性抗原；用异抗原Bridging ELISA法验证KN010不能同时识别这一对抗原。
用Trastuzumab特异性抗原蛋白HER2m1包被酶标板，4℃过夜。之后加入3％BSA溶液，室温封闭2小时。待检样品自2.5μg/mL起，1:4梯度稀释，直至0.61ng/μL，共七个梯度。酶标板中加入梯度稀释的待检样品，室温反应2小时。之后用生物素化的Pertuzumab特异性抗原蛋白HER2m2-Biotin，或者生物素化的HER2m1-Biotin加入酶标板，和待检样品室温反应2小时。随后加入HRP标记的链霉亲和素，与HER2m2-Biotin或者HER2m1-Biotin室温作用1.5小时，最后催化底物显色读数。得到的数据通过四参数法拟合，得到亲和曲线。
用Perstuzumab特异性抗原蛋白HER2m2包被酶标板，4℃过夜。之后加入3％BSA溶液，室温封闭2小时。待检样品自2.5μg/mL起，1:4梯度稀释，直至0.61ng/μL，共七个梯度。酶标板中加入梯度稀释的待检样品，室温反应2小时。之后用生物素化的Pertuzumab特异性抗原蛋白HER2m2-Biotin加入酶标板，和待检样品室温反应2小时。随后加入HRP 标记的链霉亲和素，与HER2m2-Biotin室温作用1.5小时，最后催化底物显色读数。得到的数据通过四参数法拟合，得到亲和曲线。
如图14所示，KN010蛋白的两臂通过同抗原桥式ELISA证明能同时识别Trastuzumab的抗原，或者同时识别Pertuzumab的抗原。这说明KN010蛋白中至少包括了两种能识别不同抗原靶标的抗体。但是异抗原桥式ELISA没有得到显色，证明KN010的双臂不能同时识别两种抗原，即KN010中不含有Ptmab异二聚体的成分。
实施例7 Ptmab双特异性抗体的对细胞表面HER2蛋白的结合作用
1.Ptmab双特异性抗体对人乳腺癌BT474细胞表面HER2蛋白的结合作用
利用流式细胞仪观察HER2高表达乳腺癌细胞BT474与Ptmab双特异性抗体、Pertuzumab、Trastuzumab等HER2抗体的结合情况，并考察其作用的浓度依存性。
取BT474细胞，消化后用5％BSA/PBS重悬，并于每个1.5mL离心管中加入3×105个/管细胞；待测样品自100μg/mL起3倍稀释，至0.001694μg/mL，共11个浓度。将样品与细胞反应，然后加入FITC-兔抗人IgG检测与细胞结合待测抗体，并利用流式细胞仪读取平均荧光值(MFI)。用MFI对抗体浓度的对数值做曲线，并通过四参数法拟合，得到待检抗体与BT474细胞结合的浓度依存性曲线。由图15可知，Ptmab双特异性抗体(KN026)以及Pertuzumab、Trastuzumab都与BT474有明显结合，且此种作用存在浓度依存性。从结合曲线的EC50可以看出，KN026对BT474细胞表面HER2蛋白的亲和力接近Trastuzumab。
2.Ptmab双特异性抗体对人胃癌N-87细胞表面HER2蛋白的结合作用
利用流式细胞仪观察HER2高表达胃癌细胞N-87与Ptmab双特异性抗体KN026、Pertuzumab、Trastuzumab等HER2抗体的结合情况，并考察其作用的浓度依存性。
取N-87细胞，消化后用5％BSA/PBS重悬，并于每个1.5mL离心管中加入3×105个/管细胞；待测样品自40μg/mL起，2倍稀释至0.009766ug/mL，共13个浓度。将样品与细胞反应，然后加入FITC-兔抗人IgG检测与细胞结合待测抗体，并利用流式细胞仪读取平均荧光值(MFI)。用MFI对抗体浓度的对数值做曲线，并通过四参数法拟合，得到待检抗体与N-87细胞结合的浓度依存性曲线。由图16可知，Ptmab双特异性抗体(KN026)以及 Pertuzumab、Trastuzumab都与N-87有明显结合，且此种作用存在浓度依存性。从结合曲线的EC50可以看出，KN026对N-87细胞表面HER2蛋白的亲和力略低于Trastuzumab和Pertuzumab。
实施例8 Pmab、Tmab抗体混合物对细胞表面Her2蛋白的结合作用
Pmab、Tmab抗体混合物对人乳腺癌BT474细胞表面HER2蛋白的结合作用
利用流式细胞仪观察HER2高表达乳腺癌细胞BT474与Pmab、Tmab抗体混合物、Pertuzumab、Trastuzumab等HER2抗体的结合情况，并考察其作用的浓度依存性。
取BT474细胞，消化后用5％BSA/PBS重悬，并于每个1.5mL离心管中加入15×106个/管细胞；待测样品自1000μg/mL起3倍稀释，至0.01694μg/mL，共11个浓度。将样品与细胞反应，然后加入FITC-兔抗人IgG检测与细胞结合待测抗体，并利用流式细胞仪读取平均荧光值(MFI)。用MFI对抗体浓度的对数值做曲线，并通过四参数法拟合，得到待检抗体与BT474细胞结合的浓度依存性曲线。由图17可知，Pmab、Tmab抗体混合物(KN010)以及Pertuzumab、Trastuzumab都与BT474有明显结合，且此种作用存在浓度依存性。从结合曲线的EC50可以看出，KN010对BT474细胞表面HER2蛋白的亲和力介于Trastuzumab和Pertuzumab之间。
实施例9 Ptmab双特异性抗体的癌细胞增殖的抑制作用
1.Ptmab双特异性抗体对人乳腺癌BT474细胞增殖的抑制作用
利用CKK-8法观察HER2高表达乳腺癌细胞BT474在Ptmab双特异性抗体、Pertuzumab、Trastuzumab等HER2抗体存在的情况下其增殖情况的变化，从而比较并评价Ptmab双特异性抗体对BT474癌细胞增殖作用的抑制效果。
BT474细胞用于96孔板，密度为10000cells/well，37℃贴壁培养16h。用assay medium(DMEM培养基，补充1％的胎牛血清)分别配制不同浓度的样品：最高10μg/ml～0.0015μg/ml，3倍稀释，共9个浓度。每个细胞孔中加入150μl样品，72h后CKK-8试剂盒(DOJINDO)测定细胞活力。将得到的细胞活力值对样品浓度对数作图，并通过四参数法拟合，得到待测样品(Ptmab双特异性抗体KN026)以及参比品(Trastuzumab以及 Trastuzumab+Pertuzumab联合给药)的细胞杀伤曲线。
由图18所示，KN026与Trastuzumab及Trastuzumab+Pertuzumab联合用药都对BT474有明显杀伤效果，且此效果具有浓度依存性。而双特异性抗体KN026在高浓度的情况下，其对BT474的抑制效果要明显优于Trastuzumab单独使用或联合Pertuzumab使用。
2.Ptmab双特异性抗体对人胃癌N-87细胞增殖的抑制作用
利用MTT法观察HER2高表达胃癌细胞N-87在Ptmab双特异性抗体、Pertuzumab、Trastuzumab等HER2抗体存在的情况下其增殖情况的变化，从而比较并评价Ptmab双特异性抗体对N-87癌细胞增殖作用的抑制效果。
N-87细胞用于96孔板，密度为10000cells/well，37℃贴壁培养16h。用assay medium(RPMI-1640培养基，补充1％的胎牛血清)分别配制不同浓度的样品：最高10μg/ml～0.0015μg/ml，3倍稀释，共9个浓度。每个细胞孔中加入150μl样品，72h后CKK-8试剂盒(DOJINDO)测定细胞活力。将得到的细胞活力值对样品浓度对数作图，并通过四参数法拟合，得到待测样品(Ptmab双特异性抗体)以及参比品(Trastuzumabd以及Trastuzumab+Pertuzumab联合给药)的细胞杀伤曲线。
由图19所示，KN026与Trastuzumab及Trastuzumab+Pertuzumab联合用药都对有明显杀伤效果，且此效果具有浓度依存性。而双特异性抗体在高浓度的情况下，其对N-87的抑制效果要明显优于Trastuzumab单独使用或联合Pertuzumab使用。
实施例10 Ptmab双特异性抗体的热稳定性的评价
1.Ptmab双特异性抗体的Tm值的测定
采用DSC(差式扫描热量仪)的方法测定Ptmab双特异性抗体KN026以及参考抗体(此处用Trastuzumab作为参比品)的Tm值，并据此初步判断Ptmab双特异性抗体的热稳定性。
样品蛋白于1×PBS缓冲液(pH7.4)中，制备成2mg/mL浓度的溶液。自10℃开始，以60℃/hr的速率对样品或空白缓冲液的比热容(Cp)进行扫描。将样品扫描的结果分别扣除相应缓冲液的结果，利用得到的Cp值对温度作图，其中，Cp值明显升高的峰值所对应的温度即为样品的Tm值。
由图20可知，与传统抗体相似，Ptmab双特异性抗体KN026以及Trastuzumab参比样品均显示两个明显的Tm值，包括60℃左右的CH2溶解温度以及位于80℃左右的CH3的溶解温度。同时可以看出60℃左右的 Tm值，双特异性抗体和Trastuzumab参比品无显著差异；80℃左右的Tm值，双特异性抗体略低，但是仍高于80℃，与参比品相比差别并不明显，并不认为会对抗体的热稳定性造成影响。
实施例11 Ptmab双特异性抗体的小鼠药代实验
1.Ptmab双特异性抗体的在小鼠中的代谢情况考察
选取6～7周ICR小鼠随机分为两组，实验组单次腹腔注射Ptmab双特性抗体KN02610mg/kg，参比组单次腹腔注射Trastuzumab标准品10mg/kg进行实验。各组动物分为三个梯队，每个梯队4只动物按时间点取血。各动物非终点取血(5min–96h)时眼眶静脉丛取血约0.2ml；终点取血时(192h–576h)，异氟烷吸入麻醉后下腔静脉取血安乐死。血样采集后，分离血清，-80℃冰箱暂存。
血清样品利用Tmab以及Ptmab特异性ELISA检测血药浓度，检测出的血清中抗体含量对采血时间做曲线，得到双特异性抗体(KN026)以及参比抗体(赫赛汀)的药代曲线(见图21)，并进一步计算相应药代参数(表7)。可以看到Ptmab双特异性抗体(KN026)在小鼠体内的半衰期比Trastuzumab略低一些，但是仍然大于10天，与大部分单抗药物在小鼠内的半衰期相似，可以认为Ptmab在小鼠体内稳定性与常规单抗药物类似。
表7 KN026和Trastuzumab的药代参数
Figure PCTCN2016070447-appb-000027
实施例12 Ptmab双特异性抗体对人卵巢癌SKOV3裸鼠移植瘤模型的药效作用
Balb/c裸鼠皮下接种人卵巢癌SKOV3细胞，剂量为5×106cells+50％基质胶(matrigel)/只，将成瘤小鼠随机分组，每组6只(雌雄各半)。肿瘤大小长到约直径100-150mm3时，开始注射抑瘤药物进行实验。腹腔给药,每周给药两次；连续给药2周。一周两次测量肿瘤的大小。实验组每次给药Ptmab双特性抗体KN02620mg/kg，参比组每次给药Trastuzumab标准品，或Pertuzumab标准品20mg/kg，空白对照组每次给药相同体积PBS缓冲液。
如图22所示，实验组与参比组相对空白对照组，对SKOV3的裸鼠移 植瘤模型均表现出一定的肿瘤抑制效果。其中PTmab双特异性抗体表现出比母本参比品Trastuzumab标准品，或Pertuzumab标准品单独使用时更强的抑瘤效果。
实施例13 Ptmab双特异性抗体对人胃癌N-87裸鼠移植瘤模型的药效作用
Balb/c裸鼠皮下接种人胃癌N-87细胞，剂量为4×106cells/只，将成瘤小鼠随机分组，每组6只(雌雄各半)。肿瘤大小长到约直径100-130mm3时，开始注射抑瘤药物进行实验。IP给药，每周给药两次；连续给药4-5周。一周两次测量肿瘤的大小。
实验组每次给药Ptmab双特性抗体KN0265mg/kg，参比组每次给药Trastuzumab标准品，或Pertuzumab标准品5mg/kg，空白对照组每次给药相同体积PBS缓冲液。
如图23所示，实验组与参比组相对空白对照组，对N-87的裸鼠移植瘤模型均表现出一定的肿瘤抑制效果。其中Ptmab双特异性抗体表现出明显优于参比品Trastuzumab标准品，或Pertuzumab标准品单独使用时的抑瘤效果。
实施例14 Ptmab双特异性抗体对人胃癌N-87裸鼠移植瘤模型的药效作用
Balb/c裸鼠皮下接种人胃癌N-87细胞，剂量为4×106cells/只，将成瘤小鼠随机分组，每组6只(雌雄各半)。肿瘤大小长到约直径100-120mm3时，开始注射抑瘤药物进行实验。IP给药，每周给药两次，连续给药3周。一周两次测量肿瘤的大小。
实验组给药PTmab双特性抗体KN0262.5mg/kg，参比组每次给药Trastuzumab标准品与Pertuzumab标准品联合用药，2.5mg/kg，空白对照组每次给药相同体积PBS缓冲液。
如图24所示，实验组与参比组相对空白对照组，对N-87的裸鼠移植瘤模型均表现出一定的肿瘤抑制效果。其中PTmab双特异性抗体表现出比同等摩尔数给药参比品Trastuzumab标准品加Pertuzumab标准品联合使用时更强的抑瘤效果。
实施例15 PTmab双特异性抗体对HER2低表达肿瘤株人非小细胞肺癌NCI-H522小鼠移植瘤模型的剂量依赖性
NOD/SCID免疫缺陷型小鼠皮下接种非小细胞肺癌NCI-H522细胞与matrigel混合物(细胞：matrigel比例为1:1)建立模型，每只接种剂量为5×106cells/只，将成瘤小鼠随机分组，每组6只(雌雄各半)。肿瘤大小长到约体积100mm3时，开始注射抑瘤药物进行实验。首次IP(腹腔)给药当天标记为第0天。之后每周IP给药一次，给药浓度为首次剂量减半；连续给药7周。一周两次测量肿瘤的大小。
实验组共分三组，分别按照如下剂量给药PTmab双特性抗体KN026：首次给药30mg/kg，之后每周每次给药15mg/kg；首次给药10mg/kg，之后每周每次给药5mg/kg；首次给药3mg/kg，之后每周每次给药1.5mg/kg。空白对照组每次给药相同体积PBS缓冲液。
如图27所示，三组实验组相对于空白对照组，对NCI-H522小鼠移植瘤模型均表现出显著的肿瘤抑制效果，且该效果呈现剂量依赖性。
实施例16 PTmab双特异性抗体对HER2低表达肿瘤株人非小细胞肺癌NCI-H522小鼠移植瘤模型的药效作用
NOD/SCID免疫缺陷型小鼠皮下接种非小细胞肺癌NCI-H522细胞与matrigel混合物(细胞：matrigel比例为1:1)建立模型，每只接种剂量为5×106cells/只，将成瘤小鼠随机分组，每组6只(雌雄各半)。肿瘤大小长到约体积100mm3时，开始注射抑瘤药物进行实验。首次IP(腹腔)给药当天标记为第0天，给药剂量为5mg/kg。之后每周IP给药一次，给药浓度为首次剂量减半，即2.5mg/kg；连续给药7周。一周两次测量肿瘤的大小。
实验组给药PTmab双特性抗体KN026，参比组每次给药Trastuzumab标准品与Pertuzumab标准品联合用药，每种药都前述的剂量给，即首次给药5mgP+5mgT/kg，之后每周每次给药2.5mgP+2.5mgT/kg，空白对照组每次给药相同体积PBS缓冲液。
如图28所示，实验组与参比组相对空白对照组，对NCI-H522小鼠移植瘤模型均表现出较为显著的肿瘤抑制效果。同时PTmab双特异性抗体单独给药与同等摩尔数给药参比品Trastuzumab标准品加Pertuzumab标准品联合使用时具有相当的抑瘤效果。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (45)

  1. 双特异性抗体或其抗原结合部分，其特征在于所述双特异性抗体或其抗原结合部分具有共同轻链，所述共同轻链是指两条轻链具有相同的序列。
  2. 权利要求1的双特异性抗体或其抗原结合部分，其重链能够分别与所述轻链在生理条件或体外的蛋白表达状态下正确结合。
  3. 权利要求1或2的双特异性抗体或其抗原结合部分，其中所述的共同轻链能够分别与帕妥珠单抗和曲妥株单抗的重链结合。
  4. 权利要求3的双特异性抗体或其抗原结合部分，其中所述的共同轻链选自帕妥珠单抗或曲妥株单抗的轻链或者它们的突变体。
  5. 权利要求4的双特异性抗体或其抗原结合部分，其中所述共同轻链的可变区的序列包含选自如SEQ ID NO：1～SEQ ID NO：6中第1～107位氨基酸所示的序列。
  6. 权利要求3的双特异性抗体或其抗原结合部分，其重链可变区分别为帕妥珠单抗和曲妥株单抗的重链可变区，例如分别包含如SEQ ID NO：23和SEQ ID NO：24所示的序列。
  7. 权利要求3任一项的双特异性抗体或其抗原结合部分，其重链Fc段的序列分别包含如SEQ ID NO：25和SEQ ID NO：26所示的序列。
  8. 权利要求3任一项的双特异性抗体或其抗原结合部分，其两条重链的序列分别包含如SEQ ID NO：19和SEQ ID NO：20所示的序列。
  9. 能够在一个细胞中正确产生的抗体或其抗原结合部分的混合物，所述混合物包括至少两种抗体或其抗原结合部分，所述抗体或其抗原结合部分具有共同轻链，所述共同轻链是指两条轻链具有相同的序列。
  10. 权利要求9的混合物，其中所述抗体或其抗原结合部分的重链能够分别与所述轻链在生理条件或体外的蛋白表达状态下正确结合。
  11. 权利要求9或10的混合物，其中所述的共同轻链能够分别与帕妥珠单抗和曲妥株单抗的重链结合。
  12. 权利要求11的混合物，其中所述的共同轻链选自帕妥珠单抗或曲妥株单抗的轻链或者它们的突变体。
  13. 权利要求11的混合物，其中所述共同轻链的可变区的序列包含选自如SEQ ID NO：1～SEQ ID NO：6中第1～107位氨基酸所示的序列。
  14. 权利要求11的混合物，其中所述抗体或其抗原结合部分的重链可变区分别为帕妥珠单抗和曲妥株单抗的重链可变区，例如分别包含如SEQ ID NO：23和SEQ ID NO：24所示的序列。
  15. 权利要求11的混合物，其中所述抗体或其抗原结合部分的重链Fc段的序列分别包含如SEQ ID NO：27和SEQ ID NO：28所示的序列。
  16. 权利要求11的混合物，其中所述抗体或其抗原结合部分的重链序列分别包含如SEQ ID NO：21和SEQ ID NO：22所示的序列。
  17. HER2蛋白胞外区域的变体蛋白，其与野生型HER2蛋白胞外区域的序列相比，具有选自如下一组的突变：
    1)第558位谷氨酸的突变和第573位苯丙氨酸的突变；
    2)第288位丝氨酸的突变和第296位组氨酸的突变。
  18. 权利要求17的HER2蛋白胞外区域的变体蛋白，其特征在于以下(1)～(4)项中的一项或多项：
    (1)将第558位谷氨酸突变为丙氨酸；
    (2)将第573位苯丙氨酸突变为丙氨酸；
    (3)将第288位丝氨酸突变为丙氨酸；
    (4)将第296位组氨酸突变为丙氨酸。
  19. 权利要求18的HER2蛋白胞外区域的变体蛋白，所述HER2蛋白胞外区域的变体蛋白包含选自如SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的氨基酸序列。
  20. 核酸分子，其编码权利要求1-8中任一项的双特异性抗体或其抗原结合部分或者权利要求9-16中任一项的混合物中所述的抗体或其抗原结合部分、或者所述抗体或其抗原结合部分的部分序列(例如轻链和/或重链)，或者编码权利要求17-19任一项的HER2变体蛋白。
  21. 重组载体，其含有权利要求20的核酸分子。
  22. 重组细胞，其含有权利要求21的重组载体或权利要求20的核酸分子。
  23. 一种根据两株针对不同抗原表位的单克隆抗体或其抗原结合部分制备双特异性抗体或其抗原结合部分的方法，其包括以下步骤：
    根据两株单克隆抗体的轻链序列得到能够分别与两株单抗重链结合的共同轻链序列，所述共同轻链是指两条轻链具有相同的序列，优选地，该共同轻链为其中一株单抗的轻链或者为其中一株单抗轻链的突变体。
  24. 权利要求23的方法，其进一步包括以下步骤：
    分别将两株单抗的重链序列和共同轻链序列构建于表达载体中，得到两个重组表达载体；优选地，对重链序列特别是Fc段进行突变，以更有利于具有不同重链的两株单抗的Fc段的结合；
    将两个重组表达载体转入同一宿主细胞，诱导表达，得到双特异性抗体或其抗原结合部分。
  25. 权利要求23的方法，其中所述共同轻链的可变区的获取方法为，首先确定两株单抗的轻链可变区与各自抗原或抗原表位之间接触的界面氨基酸，然后确定以任意其中一株单抗的轻链可变区为候选共同轻链的可变区时，该共同轻链与该株单抗的抗原或抗原表位之间接触的界面氨基酸与另一株单抗轻链可变区的界面氨基酸相比较时的差异氨基酸，选取差异氨基酸数量较少的轻链可变区为共同轻链的可变区；优选地，对该共同轻链 的可变区进一步突变以获得与抗原或抗原表位亲合力更好的共同轻链的可变区。
  26. 一种制备包括至少两种单克隆抗体或其抗原结合部分的混合物的方法，所述方法包括以下步骤：
    根据两株单克隆抗体的轻链序列得到能够分别与两株单抗重链结合的共同轻链序列，所述共同轻链是指两条轻链具有相同的序列，优选地，该共同轻链为其中一株单抗的轻链或者为其中一株单抗轻链的突变体。
  27. 权利要求26的方法，其进一步包括以下步骤：
    分别将两株单抗的重链序列和共同轻链序列构建于表达载体中，得到两个重组表达载体；优选地，对重链序列特别是Fc段进行突变，以更有利于具有相同重链的单抗的Fc段的结合；
    将两个重组表达载体转入同一宿主细胞，诱导表达，得到抗体或其抗原结合部分的混合物。
  28. 权利要求26的方法，其中所述共同轻链的可变区的获取方法为，首先确定两株单抗的轻链可变区与各自抗原或抗原表位之间接触的界面氨基酸，然后确定以任意其中一株单抗的轻链可变区为候选共同轻链的可变区时，该共同轻链与该株单抗的抗原或抗原表位之间接触的界面氨基酸与另一株单抗轻链可变区的界面氨基酸相比较时的差异氨基酸，选取差异氨基酸数量较少的轻链可变区为共同轻链的可变区；优选地，对该共同轻链的可变区进一步突变以获得与抗原或抗原表位亲合力更好的共同轻链的可变区。
  29. 一种检测抗体或其抗原结合部分是否为双特异性抗体或其抗原结合部分和/或对其定量的方法，所述方法包括以下步骤：
    1)分别制备能够和双特异性抗体或其抗原结合部分中的抗原结合部分1结合而不和抗原结合部分2结合的特异性抗原1，以及能够和抗原结合部分2结合而不和抗原结合部分1结合的特异性抗原2；
    2)取特异性抗原1(或者特异性抗原2)包被酶标板，加入待检抗体，反应一段时间，再加入标记的特异性抗原2(或者特异性抗原1)，反应一段时间，最后加入能够与前述标记分子结合的检测分子，反应一段时间， 所述检测分子带有可检测的标记，根据检测原理读数，判断为反应阳性或阴性；
    3)当反应为阳性，并且该反应具有浓度依赖性时，则判断该抗体或其抗原结合部分为双特异性抗体或其抗原结合部分；任选地，根据所得阳性数值进一步对双特异性抗体或其抗原结合部分进行定量。
  30. 一种检测抗体或其抗原结合部分的混合物中是否为同二聚体蛋白的方法，所述混合物包括两种抗体(抗体1和抗体2)或其抗原结合部分，所述方法包括以下步骤：
    1)分别制备能够和抗体1结合而不和抗体2结合的特异性抗原1，以及能够和抗体2结合而不和抗体1结合的特异性抗原2；
    2)取特异性抗原1(或者特异性抗原2)包被酶标板，加入待检混合物，反应一段时间，再加入标记的特异性抗原1(或者特异性抗原2)，反应一段时间，最后加入能够与前述标记分子结合的检测分子，反应一段时间，所述检测分子带有可检测的标记，根据检测原理读数，判断为反应阳性或阴性；
    3)另取特异性抗原1(或者特异性抗原2)包被酶标板，加入待检混合物，反应一段时间，再加入标记的特异性抗原2(或者特异性抗原1)，反应一段时间，最后加入能够与前述标记分子结合的检测分子，反应一段时间，所述检测分子带有可检测的标记，根据检测原理读数，判断为反应阳性或阴性；
    4)当步骤2)反应阳性，并且该反应具有浓度依赖性，同时步骤3)反应阴性时，则判断混合物中为同二聚体蛋白并且不含异二聚体蛋白；当步骤2)反应阳性同时步骤3)反应阳性时，则判断混合物中既含有同二聚体蛋白也含有异二聚体蛋白。
  31. 组合物(例如药物组合物)，其含有权利要求1-8任一项的双特异性抗体或其抗原结合部分、或者权利要求9-16任一项的混合物，以及任选的药学上可接受的载体或赋形剂。
  32. 权利要求31的组合物，其还含有其它化疗药物和/或其它抗体。
  33. 试剂盒，其含有权利要求1-8任一项的双特异性抗体或其抗原结合 部分、或者权利要求9-16任一项的混合物，以及任选的缓冲液和/或说明书。
  34. 权利要求3-8任一项的双特异性抗体或其抗原结合部分在制备预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)的药物中的用途。
  35. 权利要求11-16任一项的混合物在制备预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)的药物中的用途。
  36. 权利要求3-8任一项的双特异性抗体或其抗原结合部分在制备诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的试剂或试剂盒中的用途。
  37. 权利要求11-16任一项的混合物在制备诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的试剂或试剂盒中的用途。
  38. 权利要求17-19任一项的HER2蛋白胞外区域的变体蛋白用于检测权利要求3-8任一项的双特异性抗体或其抗原结合部分或者用于检测权利要求11-16任一项的混合物的用途。
  39. 预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)的方法，所述方法包括给有需要的受试者预防或治疗有效量的权利要求3-8任一项的双特异性抗体或其抗原结合部分的步骤。
  40. 预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)的方法，所述方法包括给有需要的受试者预防或治疗有效量的权利要求11-16任一项的混合物的步骤。
  41. 诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的方法，所述方法包括使用权利要求3-8任一项的双特异性抗体或其抗原结合部分的步骤。
  42. 诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的方法，所述方法包括使用权利要求11-16任一项的混合物的步骤。
  43. 检测权利要求3-8任一项的双特异性抗体或其抗原结合部分或者检 测权利要求11-16任一项的混合物的的方法，所述方法包括使用权利要求17-19任一项的HER2蛋白胞外区域的变体蛋白的步骤。
  44. 权利要求3-8任一项的双特异性抗体或其抗原结合部分，其用于预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)。
  45. 权利要求11-16任一项的混合物，其用于预防和/或治疗HER2阳性肿瘤(例如乳腺癌、胃癌)。
PCT/CN2016/070447 2015-01-08 2016-01-08 具有共同轻链的双特异性抗体或抗体混合物 WO2016110267A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
ES16734927T ES2811267T3 (es) 2015-01-08 2016-01-08 Anticuerpo biespecífico o mezcla de anticuerpos con cadenas ligeras comunes
DK16734927.3T DK3243840T3 (da) 2015-01-08 2016-01-08 Bispecifikt antistof eller antistofblanding med fælles lette kæder
CN201680005167.4A CN107108747B (zh) 2015-01-08 2016-01-08 具有共同轻链的双特异性抗体或抗体混合物
US15/541,921 US10808043B2 (en) 2015-01-08 2016-01-08 Bispecific antibody or antibody mixture with common light chains
JP2017536858A JP7041516B2 (ja) 2015-01-08 2016-01-08 共通軽鎖を有する二重特異性抗体又は抗体混合物
EP16734927.3A EP3243840B8 (en) 2015-01-08 2016-01-08 Bispecific antibody or antibody mixture with common light chains
CN202111616341.8A CN114276458A (zh) 2015-01-08 2016-01-08 具有共同轻链的双特异性抗体或抗体混合物
US17/022,545 US11396557B2 (en) 2015-01-08 2020-09-16 Bispecific antibody or antibody mixture with common light chains

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510008045.8A CN105820251B (zh) 2015-01-08 2015-01-08 具有共同轻链的双特异性抗体或抗体混合物
CN201510008045.8 2015-01-08

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US15/541,921 A-371-Of-International US10808043B2 (en) 2015-01-08 2016-01-08 Bispecific antibody or antibody mixture with common light chains
US17/022,545 Division US11396557B2 (en) 2015-01-08 2020-09-16 Bispecific antibody or antibody mixture with common light chains

Publications (1)

Publication Number Publication Date
WO2016110267A1 true WO2016110267A1 (zh) 2016-07-14

Family

ID=56355539

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/070447 WO2016110267A1 (zh) 2015-01-08 2016-01-08 具有共同轻链的双特异性抗体或抗体混合物

Country Status (7)

Country Link
US (2) US10808043B2 (zh)
EP (1) EP3243840B8 (zh)
JP (1) JP7041516B2 (zh)
CN (5) CN110655582B (zh)
DK (1) DK3243840T3 (zh)
ES (1) ES2811267T3 (zh)
WO (1) WO2016110267A1 (zh)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018094300A1 (en) * 2016-11-19 2018-05-24 Potenza Therapeutics, Inc. Anti-gitr antigen-binding proteins and methods of use thereof
EP3327038A3 (en) * 2013-12-20 2018-06-13 F. Hoffmann-La Roche AG Bispecific her2 antibodies and methods of use
WO2018191188A1 (en) * 2017-04-09 2018-10-18 Xiao Shouhua Biparatopic and multiparatopic antibodies with common light chain and method of use
WO2019028125A1 (en) * 2017-08-01 2019-02-07 Ab Studio Inc. BISPECIFIC ANTIBODIES AND USES THEREOF
EP3487888A4 (en) * 2016-07-22 2020-04-01 Beijing Mabworks Biotech Co. Ltd. BISPECIFIC ANTI-HER2 ANTIBODIES
WO2021244553A1 (zh) * 2020-06-02 2021-12-09 三生国健药业（上海）股份有限公司 一种抗pd-l1和egfr的四价双特异性抗体

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106248971B (zh) * 2016-08-19 2017-10-03 合肥瀚科迈博生物技术有限公司 用于检测人血清中her2含量的elisa试剂盒、使用方法及用途
CN107064092B (zh) * 2017-05-08 2019-12-13 上海药明生物技术有限公司 一种双特异性抗体生物学活性与滴度检测方法及其应用
WO2019042153A1 (zh) * 2017-09-01 2019-03-07 四川科伦博泰生物医药股份有限公司 重组双特异性抗体
EP3988574A4 (en) 2019-02-03 2022-10-12 Shanghai Bao Pharmaceuticals Co., Ltd. AGAINST HER2 BI-SPECIFIC ANTIBODIES AND THEIR USE
JP2022520632A (ja) * 2019-02-15 2022-03-31 インテグラル・モレキュラー・インコーポレイテッド 共通軽鎖を含む抗体及びその使用
WO2020206033A1 (en) * 2019-04-02 2020-10-08 Kenjockety Biotechnology, Inc. Efflux pump-cancer antigen multi-specific antibodies and compositions, reagents, kits and methods related thereto
WO2021110107A1 (en) * 2019-12-04 2021-06-10 Jiangsu Alphamab Biopharmaceuticals Co., Ltd. COMBINATION OF BISPECIFIC FUSION PROTEIN AND ANTI-Her2 ANTIBODY FOR TUMOR TREATMENT
MX2022009355A (es) * 2020-01-29 2022-09-02 Kenjockety Biotechnology Inc Anticuerpo anti-mdr1 y uso del mismo.
WO2021190636A1 (en) * 2020-03-27 2021-09-30 Jiangsu Alphamab Biopharmaceuticals Co., Ltd. Efficacious dose for her2 bispecific antibody
WO2021190637A1 (en) * 2020-03-27 2021-09-30 Jiangsu Alphamab Biopharmaceuticals Co., Ltd. Combination of anti-her2 antibody and cdk inhibitior for tumor treatment
CN113563473A (zh) * 2020-04-29 2021-10-29 三生国健药业（上海）股份有限公司 四价双特异性抗体、其制备方法和用途
EP4143236A1 (en) * 2020-05-01 2023-03-08 Novartis AG Engineered immunoglobulins
US20240067728A1 (en) * 2020-11-06 2024-02-29 Bio-Thera Solutions, Ltd. Bispecific antibody and use thereof
CN114478770A (zh) * 2020-11-12 2022-05-13 迈威(上海)生物科技股份有限公司 抗体及其制备方法
CN114539413A (zh) * 2020-11-25 2022-05-27 三生国健药业（上海）股份有限公司 结合her2的多价双特异性抗体、其制备方法和用途
WO2022179507A1 (en) * 2021-02-23 2022-09-01 Jiangsu Alphamab Biopharmaceuticals Co., Ltd. Methods of preventing, alleviating or treating tumors
WO2023280092A1 (zh) * 2021-07-05 2023-01-12 江苏康宁杰瑞生物制药有限公司 抗体药物偶联物及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1668636A (zh) * 2002-07-18 2005-09-14 克鲁塞尔荷兰公司 抗体混合物的重组生产
CN103796678A (zh) * 2011-04-20 2014-05-14 根马布股份公司 针对her2的双特异性抗体

Family Cites Families (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6129914A (en) * 1992-03-27 2000-10-10 Protein Design Labs, Inc. Bispecific antibody effective to treat B-cell lymphoma and cell line
US6485943B2 (en) * 1995-01-17 2002-11-26 The University Of Chicago Method for altering antibody light chain interactions
US6410690B1 (en) * 1995-06-07 2002-06-25 Medarex, Inc. Therapeutic compounds comprised of anti-Fc receptor antibodies
US7951917B1 (en) * 1997-05-02 2011-05-31 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
US20020062010A1 (en) * 1997-05-02 2002-05-23 Genentech, Inc. Method for making multispecific antibodies having heteromultimeric and common components
AU760048B2 (en) * 1998-05-06 2003-05-08 Genentech Inc. Protein purification by ion exchange chromatography
JP3803790B2 (ja) * 2003-02-17 2006-08-02 株式会社東北テクノアーチ 新規なダイアボディ型二重特異性抗体
JP2009529331A (ja) * 2006-03-10 2009-08-20 マクロジェニクス，インコーポレーテッド 変異型重鎖を有する抗体の同定および工学的改変ならびにその使用方法
US8652466B2 (en) * 2006-12-08 2014-02-18 Macrogenics, Inc. Methods for the treatment of disease using immunoglobulins having Fc regions with altered affinities for FcγRactivating and FcγRinhibiting
PT2235064E (pt) * 2008-01-07 2016-03-01 Amgen Inc Método de preparação de moléculas heterodiméricas de fc de anticorpos utilizando efeitos de indução eletrostática
JP2012515540A (ja) * 2009-01-26 2012-07-12 ゲンマブ エー／エス 抗体混合物を産生するための方法
PL2606064T3 (pl) * 2010-08-16 2015-07-31 Novimmune Sa Sposoby wytwarzania wieloswoistych i wielowartościowych przeciwciał
CN102558355B (zh) * 2011-12-31 2015-02-25 苏州康宁杰瑞生物科技有限公司 基于电荷网络的异二聚体fc改造方法及异二聚体蛋白的制备方法
PT2838918T (pt) * 2012-04-20 2019-08-23 Merus Nv Métodos e meios para a produção de moléculas heterrodiméricas do tipo ig
CN102851338A (zh) * 2012-07-25 2013-01-02 苏州康宁杰瑞生物科技有限公司 利用电荷排斥作用制备同二聚体蛋白混合物的方法
CN112079929A (zh) * 2012-11-21 2020-12-15 武汉友芝友生物制药有限公司 双特异性抗体
JP6510532B2 (ja) * 2013-12-20 2019-05-08 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト 二重特異性ｈｅｒ２抗体及び使用方法
UY37758A (es) * 2017-06-12 2019-01-31 Novartis Ag Método de fabricación de anticuerpos biespecíficos, anticuerpos biespecíficos y uso terapéutico de dichos anticuerpos
CN107789631B (zh) * 2017-11-03 2021-03-16 合肥瀚科迈博生物技术有限公司 抗人ErbB2双表位抗体-药物偶联物及其应用
CN113121696A (zh) * 2019-12-31 2021-07-16 周易 Fab改造诱导形成的双特异性抗体及其制备方法和用途
CN114478770A (zh) * 2020-11-12 2022-05-13 迈威(上海)生物科技股份有限公司 抗体及其制备方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1668636A (zh) * 2002-07-18 2005-09-14 克鲁塞尔荷兰公司 抗体混合物的重组生产
CN103796678A (zh) * 2011-04-20 2014-05-14 根马布股份公司 针对her2的双特异性抗体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIN, GUIGAO: "Identification of natural bispecific antibody against hepatitis C virus NS3 and NS5 proteins C patients and the study of producing mechanism of the NS3/NS5 bispecific antibody", MEDICINE & PUBLIC HEALTH, CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 15 February 2014 (2014-02-15), pages 21 - 26, XP009504575, ISSN: 1674-022X *
LIN, GUIGAO: "Identification of natural bispecific antibody against hepatitis C virus NS3 and NS5 proteins C patients and the study of producing mechanism of the NS3/NS5 bispecific antibody", MEDICINE & PUBLIC HEALTH, CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 15 February 2014 (2014-02-15), XP009504575, ISSN: 1674-022X *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3327038A3 (en) * 2013-12-20 2018-06-13 F. Hoffmann-La Roche AG Bispecific her2 antibodies and methods of use
EP3487888A4 (en) * 2016-07-22 2020-04-01 Beijing Mabworks Biotech Co. Ltd. BISPECIFIC ANTI-HER2 ANTIBODIES
WO2018094300A1 (en) * 2016-11-19 2018-05-24 Potenza Therapeutics, Inc. Anti-gitr antigen-binding proteins and methods of use thereof
CN110248960A (zh) * 2016-11-19 2019-09-17 波滕扎治疗公司 抗-gitr抗原结合蛋白及其使用方法
US10988545B2 (en) 2016-11-19 2021-04-27 Potenza Therapeutics, Inc. Anti-GITR antigen-binding proteins and methods of use thereof
WO2018191188A1 (en) * 2017-04-09 2018-10-18 Xiao Shouhua Biparatopic and multiparatopic antibodies with common light chain and method of use
EP3609580A4 (en) * 2017-04-09 2021-03-24 Combio Pharmaceutical, Inc. BIPARATOPIC AND MULTIPARATOPIC ANTIBODIES WITH COMMON LIGHT CHAIN AND METHOD OF USE
US11325982B2 (en) 2017-04-09 2022-05-10 Xuanzhu Biopharmaceutical Co., Ltd. Biparatopic and multiparatopic antibodies with common light chain and method of use
WO2019028125A1 (en) * 2017-08-01 2019-02-07 Ab Studio Inc. BISPECIFIC ANTIBODIES AND USES THEREOF
US11440972B2 (en) 2017-08-01 2022-09-13 Ab Studio Inc. Bispecific antibodies and uses thereof
US11566083B2 (en) 2017-08-01 2023-01-31 Ab Studio Inc. Bispecific antibodies and uses thereof
WO2021244553A1 (zh) * 2020-06-02 2021-12-09 三生国健药业（上海）股份有限公司 一种抗pd-l1和egfr的四价双特异性抗体

Also Published As

Publication number Publication date
EP3243840A1 (en) 2017-11-15
US20210070885A1 (en) 2021-03-11
CN110658340B (zh) 2023-10-31
CN105820251A (zh) 2016-08-03
JP2018504113A (ja) 2018-02-15
US10808043B2 (en) 2020-10-20
CN110655582A (zh) 2020-01-07
US11396557B2 (en) 2022-07-26
EP3243840A4 (en) 2018-09-12
CN110658340A (zh) 2020-01-07
EP3243840B1 (en) 2020-07-15
CN114276458A (zh) 2022-04-05
DK3243840T3 (da) 2020-08-24
CN107108747B (zh) 2022-02-08
EP3243840B8 (en) 2020-08-26
CN110655582B (zh) 2022-12-16
ES2811267T3 (es) 2021-03-11
CN107108747A (zh) 2017-08-29
JP7041516B2 (ja) 2022-03-24
US20190031782A1 (en) 2019-01-31
CN105820251B (zh) 2019-10-15

Similar Documents

Publication Publication Date Title
WO2016110267A1 (zh) 具有共同轻链的双特异性抗体或抗体混合物
JP7080213B2 (ja) 新規抗ｐｄ－ｌ１抗体
US11680100B2 (en) B7-H3 antibody, antigen-binding fragment thereof and medical use thereof
CN104271602B (zh) 双特异性抗体
US20210230269A1 (en) Immune-stimulating monoclonal antibodies against human interleukin-2
WO2019042119A1 (zh) 抗人cd47抗体及其用途
CN112135626A (zh) 抗tigit抗体及其用途
CN112243443B (zh) 抗trop-2抗体、其抗原结合片段及其医药用途
CN112166125B (zh) 全人源的抗lag-3抗体及其应用
CN114181310B (zh) 抗tigit抗体、其药物组合物及用途
CN114478769B (zh) 抗tigit抗体、其药物组合物及用途
JP2015526071A (ja) ヒトＧｎＲＨ受容体に対するモノクローナル抗体のヒト化形態
CN113227148B (zh) 抗gpc3抗体、其抗原结合片段及其医药用途
WO2022267936A1 (zh) 特异性结合糖基化ceacam5的抗体
KR102314451B1 (ko) 단백질 키나아제 a의 촉매 서브 유닛 알파에 특이적으로 결합하는 단일클론항체 및 이를 활용한 암 진단 용도
JP7392200B2 (ja) グリコシル化ｃｅａｃａｍ５に特異的に結合した抗体
WO2023001303A1 (zh) 药物组合物及用途
WO2024052922A1 (en) Anti-klk4 antibodies and uses thereof
TW202229343A (zh) Fgfr3抗體及使用方法
CN116813786A (zh) 抗cd73抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16734927

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017536858

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2016734927

Country of ref document: EP