WO2019153200A1 - 抗pd-1/抗her2天然抗体结构样异源二聚体形式双特异抗体及其制备 - Google Patents

抗pd-1/抗her2天然抗体结构样异源二聚体形式双特异抗体及其制备 Download PDF

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WO2019153200A1
WO2019153200A1 PCT/CN2018/075851 CN2018075851W WO2019153200A1 WO 2019153200 A1 WO2019153200 A1 WO 2019153200A1 CN 2018075851 W CN2018075851 W CN 2018075851W WO 2019153200 A1 WO2019153200 A1 WO 2019153200A1
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chain
antibody
her2
bispecific antibody
amino acid
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PCT/CN2018/075851
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English (en)
French (fr)
Inventor
刘家望
宋楠萌
杨亚平
金孟燮
闫尧
尹晴晴
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北京韩美药品有限公司
信达生物制药(苏州)有限公司
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Priority to PCT/CN2018/075851 priority Critical patent/WO2019153200A1/zh
Priority to TW108103574A priority patent/TWI806961B/zh
Priority to KR1020207024231A priority patent/KR20200120648A/ko
Priority to AU2019219589A priority patent/AU2019219589A1/en
Priority to JP2020565031A priority patent/JP7359784B2/ja
Priority to CA3090507A priority patent/CA3090507A1/en
Priority to EP19751154.6A priority patent/EP3735429A4/en
Priority to PCT/IB2019/051008 priority patent/WO2019155408A1/en
Priority to CN202111073852.XA priority patent/CN113943370A/zh
Priority to ARP190100320A priority patent/AR114102A1/es
Priority to CN201980012471.5A priority patent/CN111699201B/zh
Priority to US16/968,241 priority patent/US11753471B2/en
Publication of WO2019153200A1 publication Critical patent/WO2019153200A1/zh

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Definitions

  • the present invention relates to a bispecific antibody in the form of an anti-PD-1/anti-HER2 natural antibody structural heterodimer and preparation thereof.
  • the present invention provides an anti-PD-1/anti-HER2 bispecific antibody having a highly stable heterodimeric form with native IgG characteristics and no heavy light chain mismatch and a process for its preparation.
  • Monoclonal antibodies are highly specific antibodies that act only on a single antigenic epitope and have been widely used in the treatment of many diseases, such as cancer, inflammation and autoimmune diseases, infectious diseases.
  • diseases such as cancer, inflammation and autoimmune diseases, infectious diseases.
  • therapeutic molecules are used alone, none of them can show sufficient efficacy due to the complexity of the disease, such as cancer or inflammatory diseases usually involving multiple disease-mediated molecular pathways and signaling pathways.
  • the role of the fork In these cases, targeting a single molecule does not provide optimal therapeutic results, while simultaneously blocking multiple targets or molecules at multiple sites of the same target can improve the therapeutic effect.
  • dual-targeted therapy using multispecificity such as bispecific molecules, simplifies the new drug development process because it is a single molecule. It is more convenient for patients and medical workers than with a combination of multiple monospecific molecules.
  • bispecific or bifunctional molecules have been reported in the art.
  • the original bispecific antibody was a chemical method using a bifunctional coupling reagent to join two existing IgG molecules, Fab' or (Fab') 2 fragments.
  • chemically coupled bispecific antibodies have a number of limitations, such as the labor intensity of production, the complexity and low yield of purifying heterologous conjugates, removal of homologous conjugates, and original monoclonal antibodies or fragments.
  • bispecific antibodies Another method for producing bispecific antibodies is to use hybrid-hybridoma (or four-source hybridoma) technology, which is produced by somatic cell fusion of two hybridoma cell lines that secrete different antibodies. Due to the random pairing of immunoglobulin heavy and light chains, only 1/10 of the antibody mixture is the desired functional bispecific antibody, which complicates the purification process and reduces production yield.
  • hybrid-hybridoma or four-source hybridoma
  • WO2013060867 describes a method for the large-scale production of heterodimeric bispecific antibodies, which first reduces the two mixed homodimeric forms of the antibody and then introduces them in the CH3 region of the two homodimeric antibodies. Asymmetric amino acid mutations facilitate the exchange of Fab arms of different antibodies, and finally a stable bispecific antibody is formed by oxidizing the interchain disulfide bonds in the hinge region.
  • WO2009089004 describes a method for preparing a heterodimeric protein by mutating an amino acid at the interface of CH3-CH3 to a charged amino acid, thereby promoting heterodimer formation by electrostatic force, which is not conducive to homologous The polymer is formed.
  • No. 5,731,168 describes a process for the preparation of heterodimeric IgG using the " ⁇ - ⁇ " strategy.
  • the method replaces the small amino acid at the interface of the CH3 region of the first chain with a large amino acid to form a " ⁇ "; and simultaneously mutates the corresponding large amino acid at the CH3 interface of the second chain into a small amino acid, thereby forming a " ⁇ " .
  • the interaction of ruthenium and osmium facilitates the formation of heterodimeric IgG and is not conducive to the formation of homodimers.
  • WO2012058768 describes a stable and highly specific method for the preparation of heterodimeric IgG. This method combines negative and positive design and structural and computer model directed protein engineering techniques to mutate multiple amino acids of the IgGl CH3 domain to form a stable heterodimeric IgG with low homodimer impurity content.
  • PD-1 Programmed death-1
  • PD-1 is a recently-advanced immune checkpoint that is involved in the regulation of T cell activation and regulates the strength and duration of immune responses.
  • PD-1 can mediate and maintain the autoimmune tolerance of the body tissues, prevent the excessive activation of the immune system during the inflammatory reaction and damage its own tissues, and have a positive effect on avoiding the occurrence of autoimmune diseases; It is involved in tumor immunity and the development of various autoimmune diseases (Anticancer Agents Med Chem. 2015; 15(3): 307-13. Hematol Oncol Stem Cell Ther. 2014 Mar; 7(1): 1-17. Trends Mol Med. 2015 Jan; 21(1): 24-33. Immunity. 2013 Jul 25; 39(1): 61-73. J Clin Oncol. 2015 Jun 10; 33(17): 1974-82.).
  • PD-1 belongs to the CD28 family member, but unlike other members of the CD28 family, such as CTLA4, can form a covalent dimer with a disulfide bond, and PD-1 exists as a monomer.
  • the structure of PD-1 mainly includes the extracellular immunoglobulin variable region domain, the hydrophobic transmembrane region and the intracellular region, and the intracellular region contains two independent phosphorylation sites, respectively.
  • ITIM Amino acid inhibition motif
  • ITAM immunoreceptor tyrosine transfer motif
  • PD-1 is mainly induced on the surface of activated T cells and also expressed on B cells, NK cells, monocytes, and DC cells.
  • the ligand of PD-1 includes PD-L1 (programmed death ligand 1) and PD-L2 (programmed death ligand 2), and its ligand belongs to the B7 family, in which PD-L1 is induced to be expressed on various immune cell surfaces including T cells. , B cells, monocytes, macrophages, DC cells, and endothelial cells, epidermal cells, etc., while PD-L2 is only induced in some immune cells, including macrophages, DC cells, B cells (Autoimmun Rev, 2013, 12(11): 1091-1100. Front Immunol, 2013, 4: 481. Nat Rev Cancer, 2012, 12(4): 252-264. Trends Mol Med. 2015 Jan; 21(1): 24-33. ).
  • PD-L1 programmed death ligand 1
  • PD-L2 programmed death ligand 2
  • its ligand belongs to the B7 family, in which PD-L1 is induced to be expressed on various immune cell surfaces including T cells. ,
  • Trastuzumab is a humanized monoclonal antibody against the extracellular domain of HER2 (Carter P, et al, PNAS, 89(10): 4285-4289, 1992). However, its anti-tumor effect in clinical applications is often not as good as in preclinical experiments, so it is usually required to be combined with chemotherapeutic drugs (Slamon DJ, et al, N Engl J Med, 344: 783-792, 2001).
  • Catumomamab is another promising multi-functional antibody, a heterozygous Ig molecule that targets CD3 and EpCAM, and is currently approved for the treatment of ascites (Jager M, et al. Man, Cancer Res, 72: 24-32, 2012).
  • Another multifunctional antibody in clinical phase II is Ertumaxomab, which targets CD3 and HER2.
  • One heavy chain and light chain of this hybrid antibody is derived from rat IgG, targeting CD3; the other heavy and light chain is derived from mouse IgG, targeting HER2.
  • Combination administration requires sequential injection of two or more antibodies, or the same dosage form of the antibody.
  • sequential injection of antibodies reduces the patient's treatment adherence and increases pain.
  • the present invention provides a novel bifunctional antibody capable of simultaneously blocking PD-1 and HER2 and a highly stable heterodimeric form having natural IgG structural features and no heavy light chain mismatch, and a preparation method thereof.
  • the bifunctional antibody tends to selectively bind to tumor cells that simultaneously express PD-1 and HER2, thereby exerting a highly effective and specific killing effect with low toxic side effects.
  • a first aspect of the invention relates to a heterodimeric antibody in the form of a heterodimer comprising a first antigen binding domain capable of specifically binding to PD-1 and a second antigen capable of specifically binding to HER2 Binding to a functional region, wherein the bispecific antibody comprises a first Fc chain and a second Fc chain linked by one or more disulfide linkages, the first Fc chain and the second Fc chain being linked by a covalent bond or a linker Linking to the PD-1 antigen binding domain and the HER2 antigen binding domain, respectively, or the first Fc chain and the second Fc chain are linked to the HER2 antigen binding domain and PD-1 antigen binding by a covalent bond or a linker, respectively.
  • the amino acid sequence of the immunoglobulin light chain variable region in the PD-1 antigen binding functional region is selected from the group consisting of SEQ ID NO. 10, and the immunoglobulin heavy chain variable region amino acid in the PD-1 antigen binding domain
  • the sequence is selected from SEQ ID NO. 12, and the first Fc chain and the second Fc chain comprise a substitution of 5 amino acids at:
  • the first Fc chain and the second Fc chain comprising the above amino acid substitutions are more likely to form heterodimers with each other without tending to form homodimers, respectively.
  • amino acid positions are numbered according to the Kabat EU index numbering system.
  • the first Fc chain and the second Fc chain amino acid are replaced by
  • K409C K409P, K409S, K409F, K409V, K409Q or K409R.
  • amino acid substitutions comprise:
  • the amino acid substitutions of the first Fc chain are T366L and D399R, and the amino acid substitutions of the second Fc chain are L351E, Y407L, and K409V.
  • the Fc chain is derived from IgG.
  • the PD-1 and HER2 antigen binding functional regions are Fab fragments or scFv fragments.
  • both the PD-1 and HER2 antigen binding functional regions are Fab fragments.
  • one of the PD-1 and HER2 antigen binding functional regions is a Fab fragment and the other is a scFv.
  • the Fab fragment comprises a different first heavy chain variable region and a second heavy chain variable region, and a different first light chain variable region and a second light chain variable region.
  • the binding functional region and the second Fc chain and their linked PD-1 antigen binding functional regions have a weight ratio of less than 50% in the formation of homodimers when present alone and simultaneously with a reducing agent.
  • the amino acid sequence of the bispecific antibody is selected from the group consisting of: SEQ ID NO. 2, 4, 6, 8, 10, 12, and 14. In some embodiments, the amino acid sequence of the bispecific antibody is the corresponding combination of SEQ ID NO. 2, 4, 6, 8, 10, 12, and 14.
  • a second aspect of the invention relates to an isolated polynucleotide encoding a bispecific antibody in the form of a heterodimer as described in the first aspect.
  • sequence of the polynucleotide is selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13. In some embodiments, the sequence of the polynucleotide is the corresponding combination of SEQ ID NO. 1, 3, 5, 7, 9, 11 and 13.
  • a third aspect of the invention relates to a recombinant expression vector comprising the isolated polynucleotide of the second aspect.
  • the expression vector is a plasmid vector X0GC engineered based on pC DNA.
  • a fourth aspect of the invention relates to a host cell comprising the isolated polynucleotide of the second aspect, or the recombinant expression vector of the third aspect.
  • the host cell is selected from the group consisting of human embryonic kidney cell HEK293 or HEK293T, HEK293F, HEK293E engineered on HEK293 cell basis; hamster ovary cell CHO or CHO-S, CHO modified based on CHO cells. -dhfr - , CHO/DG44, ExpiCHO.
  • a fifth aspect of the invention relates to a composition
  • a composition comprising the bispecific antibody in the form of a heterodimer according to the first aspect or the isolated polynucleotide of the second aspect or the recombination of the third aspect
  • An expression vector or host cell of the fourth aspect and a pharmaceutically acceptable carrier.
  • a sixth aspect of the invention relates to a method of producing a bispecific antibody in the form of a heterodimer according to the first aspect, comprising the steps of:
  • the host cell is selected from the group consisting of human embryonic kidney cell HEK293 or HEK293T, HEK293F, HEK293F engineered on HEK293 cell basis; hamster ovary cell CHO or CHO-S, CHO modified on CHO cell basis -dhfr - , CHO/DG44, ExpiCHO.
  • the reducing step comprises: 1) performing a reduction reaction selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol, tris(2-carboxyethyl)phosphine or a chemical derivative thereof, Or a combination thereof; 2) removing the reducing agent.
  • the oxidizing step is oxidizing in air, and also includes performing an oxidation reaction in the presence of an oxidizing agent selected from the group consisting of: L-dehydroascorbic acid or other chemical derivative.
  • the method further comprises the step of isolating the purification.
  • a seventh aspect of the invention relates to the bispecific antibody of the heterodimeric form of the first aspect and/or the isolated polynucleotide of the second aspect and/or the recombinant expression vector of the third aspect and The use of the host cell of the fourth aspect and/or the composition of the fifth aspect for the preparation of a medicament for preventing and/or treating a disease in a subject.
  • the eighth aspect of the invention relates to the bispecific antibody of the heterodimeric form of the first aspect and/or the isolated polynucleotide of the second aspect and/or the recombinant expression vector of the third aspect and The host cell of the fourth aspect and/or the composition of the fifth aspect, which is used as a medicament for preventing and/or treating a disease in a subject.
  • a ninth aspect of the invention relates to a method of preventing and/or treating a disease comprising the heterodimeric antibody of the heterologous dimer form of the first aspect and/or the isolated polynucleotide of the second aspect And/or the recombinant expression vector of the third aspect and/or the host cell of the fourth aspect and/or the composition of the fifth aspect are administered to a subject in need thereof.
  • the subject is a mammal, preferably a human subject.
  • the disease is selected from the group consisting of a leukemia, a lymphoma, a myeloma, a brain tumor, a head and neck squamous cell carcinoma, a non-small cell lung cancer, a nasopharyngeal cancer, an esophageal cancer, a gastric cancer, a pancreatic cancer, Gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
  • the present invention devised a novel anti-PD-1/anti-HER2 natural antibody structure-like heterodimeric form bispecific antibody which has the characteristics of natural IgG and has no heavy light chain mismatch and is highly stable heterologous Anti-PD-1/anti-HER2 bispecific antibody in the form of a polymer.
  • the bispecific antibody binds to both target molecules PD-1 and HER2 simultaneously and is more effective in treating complex diseases.
  • Figure 1 shows the elution peak chromatogram of the anti-PD-1 expression product.
  • Figure 2 shows the structure of an anti-PD-1/anti-HER2 heterodimeric antibody molecule.
  • Figure 3 A half-antibody molecular structure diagram showing one heavy chain and one light chain.
  • Figure 4 Results of SEC-HPLC analysis of half-antibody molecules of one heavy chain and one light chain.
  • Panels A and B show the results of anti-PD-1 half antibody molecules and anti-HER2 half antibody molecules, respectively.
  • Figure 5 shows the results of SEC-HPLC analysis of anti-PD-1/anti-HER2 heterodimeric antibody molecules.
  • Figure 6 shows the results of SEC-HPLC purity analysis of purified anti-PD-1/anti-HER2 heterodimeric antibody molecules.
  • Figure 7.A shows the affinity of anti-PD-1/anti-HER2 heterodimer antibody for PD-1
  • B shows the affinity of anti-PD-1/anti-HER2 heterodimer antibody for HER2 .
  • Figure 8 Shows that the combination of PD-1 mAb and HER2 does not bind both PD-1 and HER2, and only the anti-PD-1/anti-HER2 heterodimer antibody has the activity of binding both antigens simultaneously.
  • Figure 9 Shows that anti-PD-1/anti-HER2 heterodimer antibodies are capable of triggering the close proximity of SK-BR-3 and CHO/PD-1 cells.
  • Panels A and B show that anti-PD-1/anti-HER2 heterodimer antibody can block PD-1/PD-L1 binding, PD-1/PD-L2 binding, and better retention.
  • the blocking activity of the bivalent monoclonal antibody is not limited.
  • Figure 11 Shows that anti-PD-1/anti-HER2 heterodimeric antibody shows T cell regulatory activity comparable to PD-1 mAb, significantly promoting cytokine IFN- ⁇ secretion.
  • Figure 12 Shows that anti-PD-1/anti-HER2 heterodimeric antibody shows tumor cell killing inhibitory activity comparable to HER2 mAb.
  • Figure 13 Shows that anti-PD-1/anti-HER2 heterodimer antibody has stronger antitumor efficacy than PD-1 mAb, HER2 mAb, and still shows good tumor after stopping the drug. Control role.
  • Covalent linkage refers to a bispecific antibody in the form of a heterodimer. Between two Fc chains, between any Fc chain and the antigen-binding functional region to which it is linked, is linked by a covalent bond into a molecule.
  • the Fc chain comprises a first antigen binding functional region and a second antigen binding functional region joined by one or more covalent linkages (eg, a disulfide bond chain); the first Fc chain and the second Fc chain are respectively a valency linkage (such as an imine bond or an amide bond) attached to an antigen binding functional region;
  • An antigen-binding domain refers to a region that can specifically interact with a target molecule such as an antigen, and its action is highly selective, and a sequence that recognizes a target molecule generally does not recognize other molecular sequences.
  • Representative antigen binding functional regions include: a variable region of an antibody, a structural allosteric domain of an antibody variable region, a binding domain of a receptor, a ligand binding domain, or an enzyme binding domain.
  • One or more disulfide linkages mean that the first Fc chain and the second Fc chain are joined by one or more disulfide linkages to form a heterodimeric fragment.
  • the formation of one or more disulfide bonds may be the first Fc chain and the second Fc chain or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions are synthesized in the same cell.
  • the first Fc chain and the second Fc chain or the first Fc chain and the second Fc chain and the antigen-binding functional regions linked thereto are separately synthesized in different cells, and then formed by in vitro reductive oxidation.
  • the first Fc chain and the second Fc chain refer to a binding fragment formed by covalent linkage, the covalent linkage comprising a disulfide bond, each strand comprising at least a portion of an immunoglobulin heavy chain constant region; and the first Fc chain and The second Fc chain differs in amino acid sequence and includes at least one amino acid difference.
  • the first Fc chain and the second Fc chain in the invention there is a strong mutual repulsion between the same chains, and there is an attraction between the different chains, so when co-expressed in cells, the first Fc chain and the first The di-Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions, are more prone to form heterodimers.
  • first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions are expressed in two host cells, respectively, the first Fc chain or the first Fc chain and The linked antigen binding domain does not tend to form a homodimer, and the second Fc chain or the second Fc chain and its associated antigen binding domain do not tend to form homodimers.
  • first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions are expressed in two host cells, respectively, and in the presence of a reducing agent
  • the proportion of homodimers is less than 50%, ie the ratio of monomer (one Fc chain or one Fc chain and its associated antigen binding functional region) is greater than 50%.
  • the immunoglobulin is a symmetrical structure with four polypeptide chains. Two of the same heavy chains with larger relative molecular weights contain 450-550 amino acid residues, and the relative molecular mass is between 55,000 and 70,000 Da. The same light chain (L chain) with a short, relatively small molecular weight, containing about 210 amino acid residues, has a relative molecular mass of about 24,000 Da.
  • the different immunoglobulin heavy and light chains vary greatly in the sequence of about 110 amino acids near the N-terminus, called the variable region (V region), while the remaining amino acid sequences near the C-terminus are relatively stable, called constant Zone (constant region, zone C).
  • variable region in the heavy chain accounts for about 1/4 of the length of the heavy chain, and the constant region accounts for about 3/4 of the length of the heavy chain.
  • IgG ⁇
  • IgA ⁇
  • IgD ⁇
  • IgM ⁇
  • IgE ⁇
  • the constant region is both the backbone of the immunoglobulin molecule and one of the sites that activate the immune response.
  • a portion of the constant region in the present invention includes at least a region in which the first Fc chain and the second Fc chain interact, and the region is a part of amino acids in the CH3 region for IgG, including at least GLN347, TYR349, THR350, LEU 351, SER 354, ARG 355, ASP 356, GLU 357, LYS 360, SER 364, THR 366, LEU 368, LYS 370, ASN390, LYS392, THR394, PRO395, VAL 397, ASP399, SER400, PHE405, TYR407, LYS409, LYS439.
  • the attachment of the first Fc chain and the second Fc chain to an antigen-binding functional region by a covalent bond or a linker, respectively, means that the first Fc chain and the second Fc chain are respectively linked to an antibody antigen by a covalent bond or a linker.
  • a covalent bond is an atom.
  • the atoms of the same element or different elements may be bonded by a covalent bond, and the covalent bond between the first Fc chain and the second Fc chain of the present invention includes, but is not limited to, an amino group of one molecule of amino acid and another molecule.
  • An amide bond formed by a dehydration reaction of an amino acid, or an aldehyde group of ethylene glycol or polyethylene glycol or other compound or a multimer thereof forms an amide bond or an imine bond with an amino group of one molecule of an amino acid
  • the linker is An amino acid sequence in which two polypeptide chains are joined by a covalent bond or a compound or a multimer of a compound, wherein an amino acid sequence includes, but is not limited to, a small peptide such as GGGGSGGGGSGGGGS, and the first Fc chain is passed through an amide bond.
  • the first Fc chain and the second Fc chain are more prone to form a heterodimer without tending to form a homodimer, respectively, since the same polypeptide chain is in the first Fc chain and the second Fc chain.
  • first Fc chain and the second Fc chain, or the first Fc chain and the second Fc chain and their associated antigen-binding functional regions are expressed in two host cells, respectively, the first Fc chain or the first Fc chain and The linked antigen binding domain does not tend to form a homodimer, and the second Fc chain or the second Fc chain and its associated antigen binding domain do not tend to form homodimers.
  • the Kabat EU index numbering system means that Kabat uses a method to assign a number to each amino acid of an antibody sequence and such a method of specifying the numbering of each residue has become a standard method in the art.
  • the Kabat protocol can be extended to other antibodies not present in his study, based on conserved amino acids, the target antibody is aligned to one of the consensus sequences identified by Kabat.
  • the Fc domain refers to a fragment crystallizable (Fc), which corresponds to the CH2 and CH3 domains of Ig, and is a site where Ig interacts with an effector molecule or a cell.
  • Fc fragment crystallizable
  • IgG is an abbreviation for Immunoglobulin G (IgG) and is a major antibody component of serum. According to the difference in antigenicity of the r chain in IgG molecules, human IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4.
  • a semi-antibody molecule refers to a structure in which a heavy chain of an antibody and a light chain are formed, wherein a heavy chain and a light chain may be linked by a covalent bond or may not be linked by a covalent bond, and is a monovalent antibody structure that recognizes an antigen. .
  • a Fab fragment is a molecular recognition sequence, which is a fragment of antigen binding (Fab), which corresponds to two arms of an antibody molecule and consists of a complete light and heavy chain VH and CH1 domains.
  • scFv is a molecular recognition sequence which is a structural isomer of an antibody fragment obtained by genetic engineering of a light chain variable region and a heavy chain variable region of an antibody.
  • the extracellular domain of a membrane receptor is a molecular recognition sequence, and the membrane receptor usually includes an extracellular region located outside the cell that recognizes and binds to the corresponding antigen or ligand, and anchors the receptor to the transmembrane region of the cell surface.
  • a ligand for a cell membrane receptor refers to a protein, small peptide or compound that can be recognized and bound by the extracellular region of the membrane receptor.
  • Cytokines are low-molecular-weight soluble proteins produced by various cells induced by immunogens, mitogens or other stimulators. They regulate innate and adaptive immunity, hematopoiesis, cell growth, APSC pluripotent cells, and damaged tissue repair. Features. Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors and the like.
  • the protein expression tag refers to an amino acid sequence added at the N-terminus or C-terminus of the target protein, which may be a small peptide or a long amino acid.
  • the addition of the tag may facilitate the correct folding of the protein, and may facilitate the separation and purification of the protein. It is beneficial to reduce the degradation of proteins in cells.
  • Commonly used labels include, but are not limited to, HA, SUMO, His, GST, GFP, and Flag.
  • the antibody to be used in the heterodimeric form of the bispecific antibody of the present invention is not limited at all.
  • antibodies known in the art to be useful in the treatment and/or prevention of diseases are useful in the present invention.
  • a bispecific antibody in the form of a heterodimer of the invention may have one or more substitutions, deletions, additions and/or insertions.
  • certain amino acids can replace other amino acids in the structure of the protein without significant loss of ability to bind to other polypeptides (eg, antigens) or cells. Since the binding ability and protein properties determine the biological functional activity of the protein, certain amino acid sequence substitutions can be made on the protein sequence without significant loss of their biological utility or activity.
  • a polypeptide variant contains one or more conservative substitutions.
  • Constant substitution refers to the replacement of an amino acid by another amino acid having similar properties such that one skilled in the art of peptide chemistry can expect substantially no change in the secondary structure and hydrophilic nature of the polypeptide.
  • Amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take into account various of the foregoing features are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine ; and valine, leucine and isoleucine.
  • identity has the meaning commonly known in the art, and those skilled in the art are also familiar with the rules and criteria for determining the identity between different sequences, referring to sequence alignment and introduction of gaps (if necessary, After the maximum percent homology is obtained, the residues of the polynucleotide or polypeptide sequence variant are the same percentage of the non-variant sequence. .
  • identity in the case where the definition of identity is satisfied, it is also required that the obtained variant sequence has the biological activity possessed by the parent sequence. Methods and means for screening variant sequences using the above activities are well known to those skilled in the art. Such variant sequences can be readily obtained by those skilled in the art in light of the teachings of the present disclosure.
  • the polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least the polynucleotide or polypeptide described herein. About 98%, or at least about 99%, or at least about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% of the polynucleotide or polypeptide identity. Due to the redundancy of the genetic code, variants of these sequences encoding the same amino acid sequence will be present.
  • polynucleotide compositions are provided which are capable of hybridizing to a polynucleotide sequence provided by the invention, or a fragment thereof, or a complement thereof, under moderate to high stringency conditions.
  • Hybridization techniques are well known in the art of molecular biology.
  • suitable moderately stringent conditions for testing the hybridization of a polynucleotide of the invention to other polynucleotides include pre-washing in a solution of 5 x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); Hybridization was carried out at 50-60 ° C, 5 x SSC, overnight; and washed twice with 2 x, 0.5 x and 0.2 x SSC containing 0.1% SDS for 20 minutes at 65 °C.
  • suitable high stringency hybridization conditions include those described above, except that the hybridization temperature is increased, for example, to 60-65 ° C or 65-70 ° C.
  • the host cell of the invention may be all cells used for expression of a foreign gene, including but not limited to E. coli, yeast, insect cells, plant cells, mammalian cells.
  • Vectors of the invention include vectors that can replicate in any type of cell or organism, including, for example, plasmids, phages, cosmids, and minichromosomes.
  • a vector comprising a polynucleotide of the invention is a vector suitable for propagation or replication of a polynucleotide, or a vector suitable for expression of a polypeptide of the invention.
  • Such vectors are known in the art and are commercially available.
  • Vector includes both a shuttle vector and an expression vector.
  • plasmid constructs also include an origin of replication (such as the origin of the replicated ColE1) and a selectable marker (such as ampicillin or tetracycline resistance) for plasmid replication and selection in bacteria, respectively.
  • Expression vector refers to a vector comprising control sequences or regulatory elements required for expression of an antibody of the invention, including antibody fragments, in a bacterial or eukaryotic cell.
  • the vector of the present invention may be all vectors for expression of a foreign gene, including but not limited to a plasmid vector, wherein the plasmid vector comprises at least an origin of replication, a promoter, a gene of interest, a multiple cloning site, a selection marker gene, preferably
  • the vector of the present invention includes, but is not limited to, a plasmid vector obtained based on pC DNA engineering, such as a X0GC vector.
  • Subjects of the invention include birds, reptiles, mammals, and the like.
  • the mammal comprises a rodent, a primate, preferably, the primate comprises a human.
  • the scope of the diseases involved in the present invention includes, but is not limited to, tumors.
  • the tumors include: leukemia, lymphoma, myeloma, brain tumor, head and neck squamous cell carcinoma, non-small cell lung cancer, nasopharyngeal carcinoma, Esophageal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
  • a pharmaceutically acceptable carrier means a pharmaceutical carrier conventionally used in the pharmaceutical field, such as diluents, excipients and water, fillers such as starch, sucrose, lactose, microcrystalline cellulose, etc.; binders such as cellulose Derivatives, alginates, gelatin and polyvinylpyrrolidone; wetting agents such as glycerin; disintegrating agents such as sodium carboxymethyl starch, hydroxypropyl cellulose, croscarmellose, agar, calcium carbonate and sodium hydrogencarbonate ; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol, sodium lauryl sulfate; adsorption carriers such as aged soil and soap clay; lubricants such as talc, calcium and magnesium stearate, micronized silica gel and Polyethylene glycol and the like. It is also possible to add other adjuvants such as flavoring agents, sweeteners and the like to the composition.
  • the following experimental methods are conventional methods unless otherwise specified, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified.
  • the various antibodies used in the following examples of the invention are all derived from standard antibodies of the commercial route.
  • X0GC expression vectors containing antibody heavy and light chains against human PD-1, respectively, were constructed.
  • the light chain variable region nucleotide sequence is as shown in SEQ ID NO. 9
  • the amino acid sequence is as shown in SEQ ID NO: 10
  • the light chain constant region nucleotide sequence is as shown in SEQ ID NO. 3, and the amino acid sequence is as SEQ ID NO: 4
  • the heavy chain variable region nucleotide sequence is shown in SEQ ID NO. 11, the amino acid sequence is shown in SEQ ID NO: 12
  • the heavy chain constant region nucleotide sequence is as SEQ ID NO.
  • the amino acid sequence is shown as SEQ ID NO: 14.
  • the light chain variable region and the light chain constant region, the heavy chain variable region, and the heavy chain constant region were respectively amplified by a PCR method.
  • NEC Phusion Ultra-Fidelity DNA Polymerase F-530L was used for all PCR reactions in this application.
  • PCR primers are routinely designed according to the principle of base complementation and the need for restriction sites.
  • the reaction system was: H 2 O 8.9 ⁇ l, 5 ⁇ Phusion ultra-fidelity DNA polymerase buffer 4 ⁇ l, 1 mM dNTP 4 ⁇ l, upstream primer 1 ⁇ l, downstream primer 1 ⁇ l, Phusion ultra-fidelity DNA polymerase 0.1 ⁇ l, template 1 ⁇ l.
  • variable region and constant region PCR products were electrophoresed on a 1.5% agarose gel and the corresponding fragments were recovered using a DNA recovery kit (Promega, A9282, the same below).
  • a DNA recovery kit Promega, A9282, the same below.
  • a variable region upstream primer and a constant region downstream primer are used, and then a round of PCR reaction is carried out, and then the corresponding fragment is recovered to obtain a full-length fragment of the heavy chain and the light chain.
  • the X0GC vector and the full-length fragment were digested with EcoRI (NEB, Cat. No. R3101L) and HindIII (NEB, Cat. No. R3104L).
  • the digestion reaction system was: 10 ⁇ buffer 3 2 ⁇ l, EcoRI and Hind III each 0.5 ⁇ l, and the gel was recovered. The full length fragment was 3 ⁇ l, H 2 O 14.5 ⁇ l. The digestion system was reacted at 37 ° C for 3 hours.
  • the digested product was ligated with T4 DNA ligase (NEB, Cat. No. M0202V).
  • the reaction system was: 10 ⁇ ligase buffer 2 ⁇ l, ligase 0.5 ⁇ l, 3 ⁇ l of the full-length fragment obtained by gel recovery, and 3 ⁇ l of X0GC vector obtained by gel recovery. H 2 O 11.5 ⁇ l. The reaction was allowed to react at room temperature for 12 hours.
  • the ligation product was transformed into E. coli competent cell DH5 ⁇ (Tiangen, Cat. No. CB104) to obtain an X0GC expression vector for the heavy and light chain of the antibody, respectively, for expressing the heavy and light chains of the antibody in euk
  • the X0GC expression vector containing the heavy and light chain of the antibody against human HER2 was separately constructed, wherein the antibody variable region sequence was derived from http://www.drugbank.ca/drugs/DB00072.
  • the light chain variable region nucleotide sequence is shown in SEQ ID NO. 1, the amino acid sequence is shown in SEQ ID NO: 2; the light chain constant region nucleotide sequence is shown in SEQ ID NO. 3, and the amino acid sequence is as SEQ. ID NO: 4; the heavy chain variable region nucleotide sequence is shown in SEQ ID NO. 5, the amino acid sequence is shown in SEQ ID NO: 6; the heavy chain constant region nucleotide sequence is as SEQ ID NO. As shown, the amino acid sequence is shown as SEQ ID NO: 8.
  • the X0GC expression vector of the antibody heavy and light chains was separately obtained by the same method as described above for expressing the heavy and light chains of the antibody in eukaryotic cells.
  • the expression vector containing the respective anti-human PD-1 heavy and light chains of an antibody transfected 293F cells (FreeStyle TM 293F Cells, NO R79007, invitrogen), respectively, further comprising an anti-human HER2 antibody heavy and light chain
  • the expression vector for the strand was also transfected into 293F cells. Cells are seeded one day before transfection, the day of transfection the cells were collected by centrifugation, the cells were resuspended in fresh FreeStyle TM 293 expression medium (FreeStyle TM 293Expression Medium, NO 12338001, Gibco) in a cell density of 200 * 10 5 cells /mL.
  • the plasmid was added to the transfection volume at a final concentration of 36.67 ug/mL and gently mixed; then linear PEI (polyethyleneimine, linear, MW 25000, Cat. No. 43896, Alfa Aesar) was added at a final concentration of 55 ug/mL. Mix well. Thereafter, the cells were placed in a cell culture incubator and incubated at 37 ° C for 1 hour at 120 rpm. A 19-fold transfection volume of fresh medium was then added. Continue to incubate at 37 ° C with a 120 rpm shaker. The cell culture supernatant transfected for 5-6 days was collected by centrifugation.
  • linear PEI polyethyleneimine, linear, MW 25000, Cat. No. 43896, Alfa Aesar
  • the amount of expression was determined by ELISA.
  • the precipitate was removed by filtration through a 0.2 ⁇ m filter before applying column chromatography. This step was carried out at 4 °C.
  • Purification was carried out at 4 °C using an AKTA explorer 100 protein purification system (GE Healthcare) and an affinity chromatography column rProtein A Sepharose Fast Flow (16 mm I.D., 22 ml, GE Healthcare).
  • the column was first equilibrated with mobile phase A (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.4), and the supernatant of the above treated cells was loaded after the baseline was stabilized at a flow rate of 5 ml/min.
  • the sample was equilibrated with mobile phase A.
  • the samples were anti-PD-1 expression products and anti-HER2 expression products, respectively.
  • the anti-PD-1 and anti-Her2 expression products obtained by the above-described rProtein A Sepharose Fast Flow (16 mm I.D., 22 ml, GE Healthcare) method were subjected to in vitro recombination to obtain a heterodimer.
  • PBS phosphate buffer saline
  • the obtained anti-PD-1 and anti-HER2 purified expression product solutions were separately adjusted to 1 mg/ml with the PBS, and 1/200 times the final volume of 1 M DTT was added, and the final concentration of DTT was 5 mM, respectively, and the reduction was carried out at 4 ° C. (3-8 hours), through the process of reduction, the disulfide bond is opened, and the anti-PD-1 and anti-HER2 expression products contain a small amount of antibody dimer molecule hinge region disulfide bond also opens, forming a A half-antibody molecule of a heavy chain and a light chain, the structure is shown in Figure 3.
  • the reduced sample was analyzed by SEC-HPLC (TOSOH, TSKgel superSW3000) containing 1 mM DTT reducing agent in the mobile phase buffer, and the results showed that the ratio of the anti-PD-1 half-antibody molecule was 100% as shown in A and B of FIG.
  • the ratio of the anti-HER2 half-antibody molecule was 89.3%, and the remaining 10.7% was an aggregate, but there was no homodimer in which the disulfide bond could not be opened.
  • the reduced anti-PD-1 and anti-HER2 half-antibody molecules are then mixed in an equimolar ratio and subjected to a recombination reaction at 4 ° C for 24 hours. During the recombination, the anti-PD-1 and anti-HER2 half-antibody molecules pass through CH2 and CH3.
  • the non-covalent interaction forms a bispecific antibody containing both a heterodimer against PD-1 and an anti-HER2 half-antibody molecule, followed by ultrafiltration through a ultrafiltration concentrator (nominal molecular weight cutoff 10KDa)
  • the oxidation reaction was carried out under the conditions of adding an oxidizing agent of 100 mM L-dehydroascorbic acid, a final protein concentration of 1 mg/ml, and a final concentration of oxidizing agent of 1 mM, and oxidizing at 4 ° C for 24 hours.
  • the sample obtained by the above oxidation reaction was subjected to SEC-HPLC analysis, and the results are shown in Fig. 5.
  • the heterodimeric antibody molecule obtained by the reductive oxidation of the anti-PD-1 and anti-HER2 half-antibody molecules is concentrated by ultrafiltration in a ultrafiltration concentrating tube (nominal molecular weight cutoff 10 KDa), and the solution is replaced with 10 mM sodium phosphate buffer, pH 5 .8.
  • Purification was carried out at 4 °C using an AKTA explorer 100 protein purification system (GE Healthcare) and an ion chromatography column Source 15S (16 mm I.D., 17 ml, GE Healthcare). The column was first equilibrated with mobile phase A (10 mM sodium phosphate, pH 7.0).
  • the protein solution treated as described above was loaded at a flow rate of 3 ml/min and equilibrated with mobile phase A after loading. Afterwards, 20 column volumes (0% B-100% B, 170 min, flow rate 2 ml/min) were washed with a gradient of A (10 mM sodium phosphate, pH 5.8) to B (10 mM sodium phosphate, pH 5.8), and the main peaks were collected and collected.
  • the purified product was subjected to purity analysis by SEC-HPLC, and as shown in Fig. 6, the purity was 99.96%.
  • a fully sealed 1 mg/mL anti-PD-1/anti-HER2 heterodimer sample was placed in a 40 °C incubator (BINDER KBF240) at the corresponding time point (baseline (day 0), week 2, 4 weeks)
  • a 20 ⁇ g sample was taken for high performance exclusion liquid chromatography (SEC-HPLC) separation.
  • SEC-HPLC conditions were as follows: (1) Exclusion chromatography column: TSKgel G3000SWxl (Tosoh Bioscience), 5 ⁇ m, 7.8 mm ⁇ 30 cm; (2) Mobile phase: 5 mM PBS, 150 mM NaCl, pH 6.7; (3) Flow rate: 0.6 mL/min; (4) UV detection wavelength: 280 nm; (5) Acquisition time: 30 min.
  • the instrument used was an Agilent 1200 Infinity chromatograph, which was recorded using an Agilent ChemStation and the ratio of remaining monomers was calculated. As shown in Table 1, under the experimental conditions of 40 ° C, the dimer did not undergo significant aggregation; therefore, the anti-PD-1/anti-HER2 heterodimer was considered to have good thermal stability.
  • the ability of the anti-PD-1/anti-HER2 heterodimer antibody to bind to a single antigen was determined by enzyme-linked immunosorbent assay (ELISA).
  • the anti-PD-1/anti-HER2 heterodimer antibody has high affinity for PD-1 and HER2, and maintains the antigen affinity activity of the bivalent monoclonal antibody.
  • the simultaneous binding ability of the anti-PD-1/anti-HER2 heterodimer antibody to two different antigens was determined by enzyme-linked immunosorbent assay (ELISA).
  • a heterodimeric antibody sample diluted in a PBST containing 1% BSA and a control were added, and 100 ⁇ L was added to each well, and incubated at 25 ° C for 1 hour. Wash PBST 5 times. Then, biotin-labeled PD-1-Fc (Beijing Hanmei Pharmaceutical Co., Ltd.) diluted in 1% BSA in PBST was added, 0.5 ⁇ g/mL, 100 ⁇ L per well, and incubated at 25 ° C for 1 hour. Streptavidin-horseradish peroxidase conjugate (BD Pharmingen, Cat. No.
  • PD-1 mAb its heavy chain variable region sequence and light chain variable region sequence and PD-1 binding functional region in anti-PD-1/anti-HER2 heterodimer antibody
  • HER2 mAb trastuzumab
  • HER2 mAb does not bind PD-1, HER2 at the same time, only anti-PD-1/anti-HER2 heterodimer antibody binds both antigens simultaneously Activity.
  • Anti-PD was determined by flow cytometry (FCM, FACS Calibur, purchased from BD Biosciences) on CHO/PD-1 (GenScript, Cat. No. M00529) cells with high expression of PD-1 and SK-BR-3 cells with high expression of HER2. -1 The ability of the anti-HER2 heterodimeric antibody to bind to the dual target antigen simultaneously.
  • CHO/PD-1 cells were stained according to the PKH26 kit (Sigma, Cat. No. SLBH4568V) instructions. Briefly, CHO/PD-1 cells were collected, washed once in serum-free medium, and CHO/PD-1 was separately prepared into 2 ⁇ 10 7 /mL cell suspension using Diluent C in the PKH26 kit. PKH26 dye was prepared. Dilute to 4 ⁇ M, then mix 1:1 together. The mixed suspension has a cell density of 1 ⁇ 10 7 /mL, the concentration of PKH26 is 2 ⁇ M, incubate for 1 minute at room temperature, and the staining is terminated by incubating with the same volume of FBS for 1 minute.
  • PKH26 kit Sigma, Cat. No. SLBH4568V
  • SK-BR-3 cells were stained according to the CFSE kit (Life technology, Cat. No. C34554). Briefly, CFSE was diluted with PBS to a working concentration of 0.5 ⁇ M and pre-warmed at 37 ° C, centrifuged at 1000 rpm for 5 minutes to collect SK-BR-3 cells, and resuspended SK-BR-3 with pre-warmed CFSE working solution at 37 ° C.
  • the heterologous dimer antibody binds both CHO/PD-1 cells with high expression of PD-1 and SK-BR-3 cells with high expression of HER2, and anti-PD-1/anti-HER2 heterologous two.
  • the polymer antibody is capable of initiating the close proximity of SK-BR-3 and CHO/PD-1 cells.
  • Example 8 Blocking activity of anti-PD-1/anti-HER2 heterodimeric antibody molecule on PD-1 binding to ligand PD-L1, PD-L2
  • the blocking activity of anti-PD-1/anti-HER2 heterodimer antibody on PD-1/PD-L1 binding and PD-1/PD-L2 binding was determined by enzyme-linked immunosorbent assay (ELISA).
  • the anti-PD-1/anti-HER2 heterodimer antibody was able to block PD-1/PD-L1 binding and PD-1/PD-L2 binding, and was well maintained. Blocking activity of bivalent monoclonal antibody.
  • the regulatory activity of anti-PD-1/anti-HER2 heterodimer antibody on T cell immune response was determined by mixed lymphocyte reaction (MLR).
  • DC human dendritic cells
  • Human PBMC cells were resuspended in serum-free RPMI 1640 medium at a cell density of 5 ⁇ 10 6 /mL and seeded in cell culture flasks and incubated in a 37 ° C carbon dioxide incubator for 90 minutes. The culture supernatant and suspension cells were discarded, and the adherent cells were cultured in complete medium (RPMI 1640 containing 10% FBS), and 100 ng/mL GM-CSF (Beijing Yishen Shenzhou, Cat. No. 10015-HNAH) and 100 ng/mL IL were added.
  • complete medium RPMI 1640 containing 10% FBS
  • 100 ng/mL GM-CSF Beijing Yishen Shenzhou, Cat. No. 10015-HNAH
  • the collected human DC cells and human T cells were resuspended in complete medium (RPMI 1640 containing 10% FBS), seeded in 96-well plates, and the inoculated DC cells and T cells were 1 ⁇ 10 4 /well, respectively. ⁇ 10 5 /well, mixed culture.
  • a heterodimeric antibody sample diluted with the complete medium sequence and a control were added.
  • the plate was incubated for 5 days in a 37 ° C carbon dioxide incubator. After the incubation was completed, the supernatant in the well was taken out and the cytokine IFN- ⁇ (RayBiotech, article number ELH-IFNg) was detected according to the kit manual.
  • human T cells activate IFN- ⁇ secretion under the stimulation of allogeneic DC cells.
  • the addition of PD-1 mAb enhances T cell activation and promotes cytokine secretion, whereas HER2 mAb does not.
  • the anti-PD-1/anti-HER2 heterodimeric antibody also showed T cell-regulating activity comparable to that of PD-1 mAb, and significantly promoted the secretion of the cytokine IFN- ⁇ .
  • the anti-PD-1/anti-HER2 heterodimer antibody was tested for its killing activity against human breast cancer cell SK-BR-3 in the presence of human PBMC.
  • SK-BR-3 cells were cultured in RPMI 1640 medium (ie, complete medium) containing 10% FBS. SK-BR-3 cells were harvested, resuspended in complete medium at a cell density of 5 x 10 4 /mL, and 100 ⁇ L/well was seeded in 96-well cell culture plates, ie 5000 cells per well. Human PBMC cells (Lonza, Cat. No.
  • CC-2702 were collected by resuscitation, and human PBMC cells were resuspended in RPMI 1640 complete medium at a cell density of 5 ⁇ 10 5 /mL, and 50 ⁇ L/well was seeded into 96-well cell culture plates, ie each The pores were 25,000 cells with a target cell ratio of 5:1. 50 ⁇ L/well of the heterodimeric antibody sample diluted with the complete medium sequence and the control were added. The plates were incubated for 3 days at 37 ° C in a 5% CO 2 incubator.
  • the PBMCs in the cell culture plates were washed with the medium, and then 100 ⁇ L of the complete medium and 20 ⁇ L of MTS (CellTiter 96 Aqueous One Solution, Promega, Cat. No. G358B) were added to detect SK-BR-3 cells.
  • MTS CellTiter 96 Aqueous One Solution, Promega, Cat. No. G358B
  • the cell culture plates were incubated for an additional 3-4 hours in an incubator and then the absorbance at 490 nm was read on a microplate reader.
  • HER2 mAb would kill SK-BR-3, whereas PD-1 mAb did not have this activity in vitro.
  • the anti-PD-1/anti-HER2 heterodimer antibody also showed tumor cell killing inhibitory activity comparable to HER2 mAb.
  • mice The experimental materials were selected from immunodeficient NCG mice (Nanjing Institute of Biomedical Research), female, 6-8 weeks old. After one week acclimatization the mice, each mouse was inoculated subcutaneously in the right side of 5x 10 6 th HCC1954 human breast cancer cells. When the tumor volume was as long as about 100 mm 3 , grouped according to tumor volume, 8 tumor-bearing mice per group.
  • the mouse immune system was partially humanized by human PBMC, and each mouse was inoculated with 5 x 10 6 cells intravenously.
  • the anti-PD-1/anti-HER2 heterodimer antibody had stronger antitumor efficacy than PD-1 mAb and HER2 mAb, and showed good after stopping the drug. Tumor control.

Abstract

提供了一种抗PD-1/抗HER2天然抗体结构样异源二聚体形式的双特异抗体及其制备方法，所述抗体能同时结合两种靶分子，可用于治疗复杂疾病。

Description

抗PD-1/抗HER2天然抗体结构样异源二聚体形式双特异抗体及其制备 技术领域
本发明涉及抗PD-1/抗HER2天然抗体结构样异源二聚体形式双特异抗体及其制备。具体而言，本发明提供了一种具有天然IgG特点、并且没有重轻链错配的高度稳定的异源二聚体形式的抗PD-1/抗HER2双特异抗体及其制备方法。
背景技术
单克隆抗体是只作用于单一抗原表位的高度特异性抗体，已被广泛用于许多疾病的治疗，例如，癌症、炎症和自身免疫性疾病、感染性疾病。但是，此类治疗分子如果单独使用，没有一种能够显示出足够的药效，这是由于疾病的复杂性，如癌症或炎症性疾病通常涉及多种疾病介导的分子通路以及信号通路之间的相叉作用。在这些情况下，靶向单一的分子不能提供最佳的治疗效果，而同时阻断多个靶点或者同一靶点的多个位点的分子能够改善治疗效果。同时，使用多特异性如双特异性分子的双靶向治疗可以简化新药开发过程，因为它是单一分子。和使用多个单特异分子的联合用药相比，对于患者和医疗工作者都是更方便的。
许多不同形式的双特异抗体或双功能分子在该领域已被报道。最初的双特异抗体是应用化学方法使用双功能偶联试剂将两个现有的IgG分子、Fab’或(Fab’)2片段连接起来。但是，这种化学偶联的双特异抗体存在很多的局限性，例如生产的工作强度，纯化异源偶联物、去除同源偶联物及原单特异抗体或片段的复杂性和低收率。
用于产生双特异性抗体的另一种方法是使用杂种-杂交瘤(或四源杂交瘤)技术，其是通过体细胞融合两种分泌不同抗体的杂交瘤细胞系而生产的。由于免疫球蛋白重链和轻链的随机配对，仅有抗体混合物的1/10是所需要的功能性双特异性抗体，这使纯化过程复杂并使生产收率减少。
WO2013060867描述了一种大规模生产异二聚体双特异抗体的方法，该方法先还原两种混合的同源二聚体形式抗体，然后通过在这两种同源二聚体抗体的CH3区引入不对称氨基酸突变从而促使不同抗体的Fab臂发生交换，最后通过氧化铰链区的链间二硫键形成稳定的双特异抗体。
WO2009089004描述了一种制备异源二聚体蛋白的方法，该方法通过突变CH3-CH3 界面处的氨基酸为带电荷氨基酸，从而通过静电作用力促进异源二聚体形成，而不利于同源二聚体形成。
US5731168描述了一种利用“杵－臼”策略来制备异源二聚体IgG的方法。该方法将第一条链CH3区界面处的小氨基酸替换成大氨基酸，从而形成“杵”；同时将第二条链上CH3界面处相应的大氨基酸突变成小氨基酸，从而形成“臼”。杵和臼的相互作用有利于异源二聚体IgG的形成，而不利于同源二聚体的形成。
WO2012058768描述了一种稳定的和高特异的异源二聚体IgG制备方法。该方法组合采用阴性和阳性设计以及结构和计算机模型指导的蛋白工程技术来突变IgG1CH3结构域的多个氨基酸，从而形成稳定的并且同源二聚体杂质含量低的异源二聚体IgG。
程序性死亡受体-1(programmed death-1，PD-1)是近期炙手可热的免疫检查点(immune checkpoint)，主要参与T细胞的活化调控，可以调节免疫应答的强弱程度和持续时间。在正常情况下，PD-1可以介导和维持机体组织的自身免疫耐受，防止在炎症反应过程中免疫***过度活化伤害自身组织，对避免自身免疫性疾病的发生具有积极作用；在病理情况下，其参与肿瘤免疫以及多种自身免疫病的发生发展过程(Anticancer Agents Med Chem.2015；15(3):307-13.Hematol Oncol Stem Cell Ther.2014Mar；7(1):1-17.Trends Mol Med.2015Jan；21(1):24-33.Immunity.2013Jul 25；39(1):61-73.J Clin Oncol.2015Jun10；33(17):1974-82.)。
PD-1属于CD28家族成员，但不同于CD28家族其它成员如CTLA4能以二硫键形成共价二聚体，PD-1以单体形式存在。PD-1的结构主要包括胞外免疫球蛋白可变区样结构域、疏水的跨膜区和胞内区，其胞内区含有两个独立的磷酸化作用位点，分别为免疫受体酪氨酸抑制基序(ITIM)和免疫受体酪氨酸转移基序(ITSM)。PD-1主要诱导性表达于活化的T细胞表面，也表达于B细胞、NK细胞、单核细胞、DC细胞。PD-1的配体包括PD-L1(programmed death ligand 1)、PD-L2(programmed death ligand 2)，其配体属于B7家族，其中PD-L1诱导性表达于多种免疫细胞表面包括T细胞、B细胞、单核细胞、巨噬细胞、DC细胞、以及内皮细胞、表皮细胞等，而PD-L2仅诱导性表达于一些免疫细胞，包括巨噬细胞、DC细胞、B细胞(Autoimmun Rev,2013,12(11):1091-1100.Front Immunol,2013,4:481.Nat Rev Cancer,2012,12(4):252-264.Trends Mol Med.2015Jan；21(1):24-33.)。
在二十世纪80年代，Denis Slamon首次在189个原发性乳腺癌病例中发现HER2(人表皮生长因子受体2)基因在30％的病例中都过度扩增，并表明HER2与总存活率 及复发时间密切相关(Salman DJ,等人，Science,235:177-182,1985)。目前的研究表明，大约25～30％的乳腺癌病人中的HER2都是过表达的(Revillion F,等人，Eur J Cancer,34:791-808,1998)，并且其与肿瘤的恶性生长程度相关(Wright C,等人，Cancer Res,49:2087-2090,1989)。
曲妥珠单抗(Trastuzumab)是一个抗HER2胞外区的人源化单克隆抗体(Carter P,等人，PNAS,89(10):4285-4289,1992)。然而其在临床应用中的抗肿瘤效果往往没有临床前实验那么好，所以通常需要与化疗药等联合用药(Slamon DJ,等人，N Engl J Med,344:783-792,2001)。
设计一种可以募集效应细胞的双功能抗体是提高抗体效能的有效手段。目前为止，研究最多的是利用CD3分子的功能。通过CD3分子将杀伤性T细胞激活，可以有效地清除目的肿瘤(Haas C,等人，Immunobiology,214:441-453,2009)。其中Micormet公司开发的一种重组双功能T细胞激动抗体为BiTE，具有很好的前景，但是其最大的问题在于血浆半衰期非常短，人体半衰期仅为1小时(Loffler A,等人，Blood,95:2098-2103)。这是由BiTE本身的结构所导致的，其由两个单链抗体片段组成，分子量仅60kDa，并且缺失了抗体分子中对延长半衰期有重要作用的Fc片段。
卡妥索单抗(Catumaxomab)是另一种具有前景的多功能抗体，是一种靶向CD3及EpCAM的杂合Ig分子，目前该产品已经被批准用于腹水癌的治疗(Jager M,等人，Cancer Res,72:24-32,2012)。另一种处于临床二期的多功能抗体为厄妥索单抗(Ertumaxomab)，其靶向CD3及HER2。这种杂合抗体的一条重链及轻链来源于大鼠的IgG，靶向CD3；另一条重链及轻链来源于小鼠的IgG，靶向HER2。由此而带来的问题就是这种产品的生产非常困难，因为，为了获得表达双功能厄妥索抗体的细胞系，首先要获得一株表达CD3抗体的二倍体杂交瘤，以及一株表达HER2抗体的二倍体杂交瘤，然后将两株杂交瘤再次杂交，获得四倍体杂交瘤，其可以表达抗CD3以及HER2的双功能抗体。而生产普通的单个靶点的抗体，只需要获得一株二倍体杂交瘤，与此相比较，双功能抗体的生产过程更复杂，而且四倍体杂交瘤的获得更加困难，并且由于其鼠源的来源，导致其免疫原性很高。
另外，由于抗CD3的抗体最明显的副反应是导致体内的细胞因子在短时间内增高，也称为细胞因子风暴。因此需要开发一种新的将免疫细胞招募至肿瘤细胞的双功能抗体。
联合给药需要依次注射两种或多种抗体，或将抗体做成同一种剂型。然而，一方面，依次注射抗体会降低患者的治疗依从性，并增加疼痛。另一方面，由于不同抗体的物化 性质存在差异，很难或几乎不太可能将不同抗体做成同一种剂型。
鉴于此，仍然有必要研究一种同时阻断PD-1和HER2信号通路的新型治疗药物。
发明内容
本发明提供了一种新的具有天然IgG结构特点、并且没有重轻链错配的高度稳定的异源二聚体形式的能同时阻断PD-1和HER2的双功能抗体及其制备方法，该双功能抗体倾向于选择性结合同时高表达PD-1和HER2的肿瘤细胞，从而发挥高效、特异的杀伤效果，同时具有较低的毒副作用。
本发明的第一方面涉及一种异源二聚体形式的双特异抗体，其包含能与PD-1特异性结合的第一个抗原结合功能区和能与HER2特异性结合的第二个抗原结合功能区，其中所述双特异抗体包含通过一个或多个二硫键链间连接的第一Fc链及第二Fc链，该第一Fc链和第二Fc链通过共价键或连接体分别连接到PD-1抗原结合功能区和HER2抗原结合功能区上，或者该第一Fc链和第二Fc链通过共价键或连接体分别连接到HER2抗原结合功能区和PD-1抗原结合功能区上；其中PD-1抗原结合功能区中的免疫球蛋白轻链可变区氨基酸序列选自SEQ ID NO.10，PD-1抗原结合功能区中的免疫球蛋白重链可变区氨基酸序列选自SEQ ID NO.12，并且第一Fc链和第二Fc链在如下位置包含5个氨基酸的替换：
第一Fc链上366位及399位氨基酸替换，第二Fc链上351位、407位及409位氨基酸的替换，
包含上述氨基酸替换的第一Fc链与第二Fc链更倾向于互相形成异源二聚体而不倾向于各自形成同源二聚体，
其中氨基酸位置根据Kabat EU指数编号***编号。
在一些实施方案中，第一Fc链及第二Fc链氨基酸替换如下，
a)L351G、L351Y、L351V、L351P、L351D、L351E、L351K或L351W；
b)T366L、T366P、T366W或T366V；
c)D399C、D399N、D399I、D399G、D399R、D399T或D399A；
d)Y407L、Y407A、Y407P、Y407F、Y407T或Y407H；和
e)K409C、K409P、K409S、K409F、K409V、K409Q或K409R。
在一些实施方案中，氨基酸替换包括：
a)第一Fc链T366L及D399R替换，第二Fc链L351E、Y407L及K409V替换；
b)第一Fc链T366L及D399C替换，第二Fc链L351G、Y407L及K409C替换；
c)第一Fc链T366L及D399C替换，第二Fc链L351Y、Y407A及K409P替换；
d)第一Fc链T366P及D399N替换，第二Fc链L351V、Y407P及K409S替换；
e)第一Fc链T366W及D399G替换，第二Fc链L351D、Y407P及K409S替换；
f)第一Fc链T366P及D399I替换，第二Fc链L351P、Y407F及K409F替换；
g)第一Fc链T366V及D399T替换，第二Fc链L351K、Y407T及K409Q替换；
h)第一Fc链T366L及D399A替换，第二Fc链L351W、Y407H及K409R替换。
在一些实施方案中，第一Fc链的氨基酸替换为T366L和D399R，第二Fc链的氨基酸替换为L351E、Y407L和K409V。
在一些实施方案中，Fc链来源于IgG。
在一些实施方案中，PD-1和HER2抗原结合功能区是Fab片段或scFv片段。
在一些实施方案中，PD-1和HER2抗原结合功能区都是Fab片段。
在一些实施方案中，PD-1和HER2抗原结合功能区一个是Fab片段，另一个是scFv。
在一些实施方案中，Fab片段包含不同的第一重链可变区及第二重链可变区，以及不同的第一轻链可变区及第二轻链可变区。
在一些实施方案中，第一Fc链及其相连接的PD-1抗原结合功能区和第二Fc链及其相连接的HER2抗原结合功能区，或者第一Fc链及其相连接的HER2抗原结合功能区和第二Fc链及其相连接的PD-1抗原结合功能区，在单独存在并且同时存在还原剂时其形成同源二聚体的重量比例均低于50％。
在一些实施方案中，双特异抗体的氨基酸序列选自：SEQ ID NO.2、4、6、8、10、12和14。在一些实施方案中，双特异抗体的氨基酸序列是SEQ ID NO.2、4、6、8、10、12和14的相应组合。
本发明的第二方面涉及一种分离的多核苷酸，其编码如第一方面所述的异源二聚体形式的双特异抗体。
在一些实施方案中，多核苷酸的序列选自：SEQ ID NO：1、3、5、7、9、11和13。在一些实施方案中，多核苷酸的序列是SEQ ID NO.1、3、5、7、9、11和13的相应组合。
本发明的第三方面涉及一种重组表达载体，其包含第二方面所述的分离的多核苷酸。
在一些实施方案中，表达载体为基于pCDNA改造得到的质粒载体X0GC。
本发明的第四方面涉及一种宿主细胞，其包含第二方面所述的分离的多核苷酸，或 第三方面所述的重组表达载体。
在一些实施方案中，宿主细胞选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO。
本发明的第五方面涉及一种组合物，其包含第一方面所述的异源二聚体形式的双特异抗体或第二方面所述的分离的多核苷酸或第三方面所述的重组表达载体或第四方面所述的宿主细胞，及药学上可接受的载体。
本发明的第六方面涉及一种生产如第一方面所述的异源二聚体形式的双特异抗体的方法，其包括步骤：
1)将第二方面所述的分离的多核苷酸或第三方面所述的重组表达载体分别在宿主细胞中进行表达；
2)将在宿主细胞中分别表达的蛋白进行还原；以及
3)将还原的蛋白混合，然后将混合物进行氧化。
在一些实施方案中，宿主细胞选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293F；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO。
在一些实施方案中，还原步骤包括：1)进行还原反应，所述还原剂选自：2-巯基乙胺、二硫苏糖醇、三(2-羧乙基)膦或其化学衍生物，或者其组合；2)去除还原剂。
在一些实施方案中，氧化步骤为在空气中氧化，也包括在氧化剂存在下进行氧化反应，所述氧化剂选自：L-脱氢抗坏血酸或其他化学衍生物。
在一些实施方案中，所述的方法还包括分离纯化的步骤。
本发明的第七方面涉及第一方面所述的异源二聚体形式的双特异抗体和/或第二方面所述的分离的多核苷酸和/或第三方面所述的重组表达载体和/或第四方面所述的宿主细胞和/或第五方面所述的组合物在制备用于预防和/或治疗受试者疾病的药物中的用途。
本发明的第八方面涉及第一方面所述的异源二聚体形式的双特异抗体和/或第二方面所述的分离的多核苷酸和/或第三方面所述的重组表达载体和/或第四方面所述的宿主细胞和/或第五方面所述的组合物，其用做用于预防和/或治疗受试者疾病的药物。
本发明的第九方面涉及一种预防和/或治疗疾病的方法，包括将第一方面所述的异源二聚体形式的双特异抗体和/或第二方面所述的分离的多核苷酸和/或第三方面所述的重组表达载体和/或第四方面所述的宿主细胞和/或第五方面所述的组合物施予有需求的受 试者。
在一些实施方案中，受试者是哺乳动物，优选地，人类受试者。
在一些实施方案中，所述疾病选自如下肿瘤：白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、肝癌、结直肠癌、乳腺癌、卵巢癌、***、子宫内膜癌、子宫肉瘤、***癌、膀胱癌、肾细胞癌、黑色素瘤。
本发明设计了一种全新的抗PD-1/抗HER2天然抗体结构样异源二聚体形式双特异抗体，其具有天然IgG特点，并且没有重轻链错配，是高度稳定的异源二聚体形式的抗PD-1/抗HER2双特异抗体。该双特异抗体能同时结合两种靶分子PD-1和HER2并且在治疗复杂疾病方面会更有效。
附图说明
图1：示出了抗PD-1表达产物的洗脱峰色谱图。
图2：示出了抗PD-1/抗HER2异源二聚体抗体分子的结构。
图3.示出了一条重链和一条轻链的半抗体分子结构图。
图4.示出了一条重链和一条轻链的半抗体分子的SEC-HPLC分析结果。其中A图和B图分别表示抗PD-1半抗体分子和抗HER2半抗体分子的结果。
图5.示出了抗PD-1/抗HER2异源二聚体抗体分子的SEC-HPLC分析结果。
图6.示出了纯化的抗PD-1/抗HER2异源二聚体抗体分子的SEC-HPLC纯度分析结果。
图7.A图示出了抗PD-1/抗HER2异源二聚体抗体对PD-1的亲和力，B图示出了抗PD-1/抗HER2异源二聚体抗体对HER2的亲和力。
图8.示出了PD-1单抗和HER2的组合不能同时结合PD-1和HER2，只有抗PD-1/抗HER2异源二聚体抗体具有同时结合两种抗原的活性。
图9.示出了抗PD-1/抗HER2异源二聚体抗体能够引发SK-BR-3和CHO/PD-1细胞的相互靠近。
图10.A图和B图分别示出了抗PD-1/抗HER2异源二聚体抗体能够阻断PD-1/PD-L1结合、PD-1/PD-L2结合，较好的保持了双价单抗的阻断活性。
图11.示出了抗PD-1/抗HER2异源二聚体抗体显示了与PD-1单抗相当的T细胞调控活性，显著的促进细胞因子IFN-γ的分泌。
图12.示出了抗PD-1/抗HER2异源二聚体抗体显示了与HER2单抗相当的肿瘤细胞杀伤抑制活性。
图13.示出了抗PD-1/抗HER2异源二聚体抗体具有比PD-1单抗、HER2单抗更强的抗肿瘤药效，且在停药后依然显示出了良好的肿瘤控制作用。
具体实施方式
定义：
共价连接是指异源二聚体形式的双特异抗体中，两个Fc链之间，任一个Fc链及与其相连接的抗原结合功能区之间，是通过共价键而连接成为一个分子。其中Fc链包含通过一个或多个通过共价连接(如二硫键链)而连接的第一抗原结合功能区及第二抗原结合功能区；该第一Fc链及第二Fc链分别通过共价连接(如亚胺键或酰胺键)而连接到一个抗原结合功能区上；
抗原结合功能区是指可以与目标分子如抗原发生特异性相互作用的区域，其作用具有高度选择性，识别一种目标分子的序列通常不能识别其他分子序列。代表性的抗原结合功能区包括：抗体的可变区、抗体可变区的结构变构体、受体的结合域、配体结合域或酶结合域。
一个或多个二硫键链间连接是指第一Fc链及第二Fc链通过一个或多个二硫键链间连接，形成异源二聚体片段。在本发明中，一个或多个二硫键的形成可以是第一Fc链及第二Fc链或者第一Fc链及第二Fc链及其相连接的抗原结合功能区在同一个细胞内合成时形成，也可以是第一Fc链及第二Fc链或者第一Fc链及第二Fc链及其相连接的抗原结合功能区在不同细胞内分别合成，之后通过体外还原氧化的方法形成。
第一Fc链及第二Fc链是指通过共价连接而组成结合片段，共价连接包括二硫键，每条链至少包含免疫球蛋白重链恒定区的一部分；并且该第一Fc链及第二Fc链在氨基酸序列上是不同的，至少包括了一位氨基酸的不同。在此发明中的第一Fc链及第二Fc链，相同链之间存在强烈的相互排斥作用，而不同链之间存在吸引作用，因此当在细胞内共同表达时，第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区，更倾向于形成异源二聚体。将第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区分别在两个宿主细胞内表达时，第一Fc链或者第一Fc链及其相连接的抗原结合功能区不倾向于形成同源二聚体，第二Fc链或者第二Fc链及其相连接的抗原结合功能区不倾向于形成同源二聚体。在本发明中，当第一Fc 链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区分别在两个宿主细胞内表达时，并且在存在还原剂时，同源二聚体的比例低于50％，即单体(一条Fc链或者一条Fc链及其相连接的抗原结合功能区)比例大于50％。
免疫球蛋白是具有四条多肽链的对称结构，其中两条较长、相对分子量较大的相同的重链，含450～550个氨基酸残基，相对分子质量在55000～70000Da之间；两条较短、相对分子量较小的相同的轻链(L链)，含约210个氨基酸残基，相对分子质量约24000Da。不同的免疫球蛋白重链和轻链在靠近N端的约110个氨基酸的序列变化很大，称为可变区(variable region，V区)，而靠近C端的其余氨基酸序列相对稳定，称为恒定区(constant region，C区)。重链中可变区约占重链长度的1/4，恒定区约占重链长度的3/4。对于已知五种Ig来说，IgG(γ)、IgA(α)、IgD(δ)、IgM(μ)和IgE(ε)，其中前三类Ig的H链内有三个恒定区，即CH1、CH2和CH3组成。后两类(IgM和IgE)的H链中有一个VH区和四个恒定区，即CH1至CH4。恒定区既是免疫球蛋白分子的骨架，又是激活免疫反应的部位之一。
本发明中的恒定区一部分至少包括了第一Fc链和第二Fc链相互作用的区域，该区域对于IgG来说，是位于CH3区域的一部分氨基酸，至少包括GLN347、TYR349、THR350、LEU 351、SER 354、ARG 355、ASP 356、GLU 357、LYS 360、SER 364、THR 366、LEU 368、LYS 370、ASN390、LYS392、THR394、PRO395、VAL 397、ASP399、SER400、PHE405、TYR407、LYS409、LYS439。
第一Fc链及第二Fc链分别通过共价键或连接体连接到一个抗原结合功能区上是指第一Fc链及第二Fc链分别通过共价键或连接体连接到一个抗体的抗原结合片段，或可以识别抗原的单链抗体，或可以识别抗原的其他抗体片段变构体，或可以识别配体的受体，或可以识别受体的配体，其中所述共价键是指是化学键的一种，两个或多个原子共同使用它们的外层电子，在理想情况下达到电子饱和的状态，由此组成比较稳定的化学结构叫做共价键，或者说共价键是原子间通过共用电子对所形成的相互作用。同一种的元素的原子或不同元素的都可以通过共价键结合，对于本发明的第一Fc链及第二Fc链间的共价键，包括但不限于一分子氨基酸的氨基与另一分子氨基酸的羧基脱水反应形成的酰胺键，或者乙二醇或聚乙二醇或其他化合物或其多聚物的醛基与一分子氨基酸的氨基形成酰胺键或亚胺键，其中连接体是可以将两条多肽链通过共价键连接起来的一段氨基酸序列或者一种化合物或者一种化合物的多聚体，其中一段氨基酸序列包括但不限于一段小肽，如GGGGSGGGGSGGGGS，通过酰胺键将第一Fc链或第二Fc链，以及可 以识别抗原的单链抗体，或可以识别抗原的其他抗体片段结构变构体连接起来
第一Fc链与第二Fc链更倾向于形成异源二聚体而不倾向于各自形成同源二聚体是指，由于在第一Fc链与第二Fc链中，相同的多肽链间存在互相排斥的作用，而不同的多肽链间存在吸引作用，因此当在细胞内共同表达时，第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区，更倾向于形成异源二聚体。将第一Fc链及第二Fc链，或者第一Fc链及第二Fc链及其相连接的抗原结合功能区分别在两个宿主细胞内表达时，第一Fc链或者第一Fc链及其相连接的抗原结合功能区不倾向于形成同源二聚体，第二Fc链或者第二Fc链及其相连接的抗原结合功能区不倾向于形成同源二聚体。
Kabat EU指数编号***是指，Kabat利用一种方法将一个编号指定给抗体序列的每个氨基酸并且这种指定每个残基的编号的方法已经成为本领域的标准方法。Kabat方案可以延伸到不存在于他的研究中的其它抗体，基于保守的氨基酸，将目标抗体与Kabat鉴定的共有序列之一进行比对。
Fc结构域是指可结晶段(fragment crystallizable,Fc)，相当于Ig的CH2和CH3结构域，是Ig与效应分子或者细胞相互作用的部位。
IgG是免疫球蛋白G(Immunoglobulin G，IgG)的缩写，是血清主要的抗体成分，根据IgG分子中的r链抗原性差异，人IgG有四个亚型：IgG1、IgG2、IgG3、IgG4。
半抗体分子是指抗体的一条重链与一条轻链形成的结构，其中重链与轻链间可以通过共价键连接，也可以不通过共价键连接，是一种识别抗原的单价抗体结构。
Fab片段是一种分子识别序列，是抗原结合片段(fragment of antigen binding,Fab)，相当于抗体分子的两个臂，由一个完整的轻链和重链的VH和CH1结构域组成。scFv是一种分子识别序列，是一种由抗体的轻链可变区与重链可变区通过基因工程改造而得到的抗体片段的结构异构体。膜受体的细胞外区是一种分子识别序列，膜受体通常包括位于细胞外部的可以识别并结合相应抗原或者配体的细胞外区域，将受体锚定在细胞表面的跨膜区，以及在胞内的具有激酶活性或者可以传递信号通路的胞内区。细胞膜受体的配体是指能被膜受体胞外区识别并结合的蛋白质，小肽或化合物。细胞因子是免疫原、丝裂原或其他刺激剂诱导多种细胞产生的低分子量可溶性蛋白质，具有调节固有免疫和适应性免疫、血细胞生成、细胞生长、APSC多能细胞以及损伤组织修复等多种功能。细胞因子可被分为白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子等。蛋白表达标签指在目标蛋白的N端或C端加入的一段氨基酸序列，可以是小肽也可以是 长的氨基酸，标签的加入可以有利于蛋白质的正确折叠，可以有利于蛋白质的分离纯化，可以有利于降低蛋白质在胞内的降解，常用的标签包括但不限于HA、SUMO、His、GST、GFP、Flag。
可应用于本发明的异源二聚体形式的双特性抗体中的抗体并无任何限制。优选地，现有技术中已知可以用于治疗和/或预防疾病的抗体均可以用于本发明。
本发明的异源二聚体形式的双特性抗体可具有一个或多个替换、缺失、添加和/或***。例如，某些氨基酸可以替换在蛋白质结构中的其它氨基酸而没有明显损失与其它多肽(如抗原)或细胞结合的能力。由于结合能力和蛋白性质决定了蛋白的生物功能活性，可以在蛋白序列上进行某些氨基酸序列的替换而不会明显损失它们的生物效用或活性。
在许多情况中，多肽变体含有一个或多个保守替换。“保守替换”是指其中氨基酸被其它具有类似性质的氨基酸所替换，使得肽化学领域中技术人员可预期多肽的二级结构和亲水性质基本上不发生变化。
氨基酸替换通常是基于氨基酸侧链取代基的相对相似性，如它们的疏水性、亲水性、电荷、大小等。考虑了各种前述特征的示例性替换是本领域技术人员公知的并包括：精氨酸和赖氨酸；谷氨酸和天冬氨酸；丝氨酸和苏氨酸；谷氨酰胺和天冬酰胺；以及缬氨酸、亮氨酸和异亮氨酸。
本发明使用的术语“同一性”具有本领域通常已知的含义，本领域技术人员也熟知测定不同序列间同一性的规则、标准，是指在序列比对和引入缺口(如果必要的话，以获得最大百分比同源性)后，多核苷酸或多肽序列变体的残基与非变体序列的相同的百分比。。在本发明中，在满足同一性限定的情况下，还需要所获得的变体序列具有母体序列所具有的生物活性。本领域技术人员公知如何利用上述活性筛选变体序列的方法和手段。本领域技术人员可以在本申请公开内容的教导下容易地获得这样的变体序列。在具体实施方式中，多核苷酸和多肽变体与本文所述的多核苷酸或多肽具有至少约70％、至少约75％、至少约80％、至少约90％、至少约95％、至少约98％、或至少约99％，或至少约99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的多核苷酸或多肽同一性。由于遗传密码的冗余性，将存在编码相同氨基酸序列的这些序列的变体。
本发明的另一个实施方式中，提供了能够在中度至高度严格条件下与本发明提供的多核苷酸序列或其片段或其互补序列相杂交的多核苷酸组合物。杂交技术是分子生物学领域公知的。为了说明的目的，用于测试本发明的多核苷酸与其他多核苷酸杂交的合适中等严格条件包括在5×SSC、0.5％SDS、1.0mM EDTA(pH8.0)的溶液中预洗；在50-60℃、5×SSC、 过夜的条件下杂交；再于65℃下20分钟用含0.1％SDS的2×、0.5×和0.2×的SSC各洗涤两次。本领域技术人员理解，可容易地操纵杂交的严格性，例如通过改变杂交溶液的盐含量和/或进行杂交的温度。例如，在另一个实施方式中，合适的高严格杂交条件包括上述的条件，所不同的是杂交温度升高了，例如达到60-65℃或65-70℃。
本发明的宿主细胞可以是用于外源基因表达的所有细胞，包括但不限于大肠杆菌，酵母，昆虫细胞，植物细胞，哺乳动物细胞。
本发明的载体包括可以在任何类型的细胞或生物体中进行复制的载体，包括如质粒、噬菌体、粘粒和迷你染色体。在一些实施方式中，包括本发明多核苷酸的载体是适合于多核苷酸繁殖或复制的载体，或者是适合于表达本发明多肽的载体。这样的载体是本领域已知并可以购买的。
“载体”包括穿梭载体和表达载体。通常，质粒构建体也包括分别用于细菌中质粒复制和选择的复制起点(如复制的ColE1起点)和选择标记(如氨苄青霉素或四环素抗性)。“表达载体”是指包含用于在细菌或真核细胞中表达本发明的抗体包括抗体片段所需要的控制序列或调控元件的载体。
本发明的载体可以是用于外源基因表达的所有载体，包括但不限于质粒载体，其中质粒载体至少包含复制起始位点、启动子、目的基因、多克隆位点、筛选标记基因，优选地，本发明所述载体包括但不限于基于pCDNA改造得到的质粒载体，比如X0GC载体。
本发明的受试者包括禽类、爬行动物、哺乳动物等。优选地，哺乳动物包括啮齿类动物、灵长类动物，优选地，灵长类动物包括人。
本发明所涉及的疾病的范围包括但不限于肿瘤，优选的，所述肿瘤包括：白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、肝癌、结直肠癌、乳腺癌、卵巢癌、***、子宫内膜癌、子宫肉瘤、***癌、膀胱癌、肾细胞癌、黑色素瘤。
药学上可接受的载体是指是指药学领域常规的药物载体，例如：稀释剂、赋形剂和水等，填充剂如淀粉、蔗糖，乳糖、微晶纤维素等；粘合剂如纤维素衍生物、藻酸盐、明胶和聚乙烯吡咯烷酮；润湿剂如甘油；崩解剂如羧甲基淀粉钠，羟丙纤维素，交联羧甲基纤维素，琼脂、碳酸钙和碳酸氢钠；吸收促进剂如季铵化合物；表面活性剂如十六烷醇，十二烷基硫酸钠；吸附载体如高龄土和皂粘土；润滑剂如滑石粉、硬脂酸钙和镁、微粉硅胶和聚乙二醇等。另外还可以在组合物中加入其它辅剂如香味剂、甜味剂等。
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。本发明下述实施例中使用的各种抗体均来源于商业途径的标准抗体。
实施例1抗PD-1/抗HER2异源二聚体抗体分子的载体构建
构建分别含抗人PD-1的抗体重链和轻链的X0GC表达载体。其中轻链可变区核苷酸序列如SEQ ID NO.9所示，氨基酸序列如SEQ ID NO:10所示；轻链恒定区核苷酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO:4所示；重链可变区核苷酸序列如SEQ ID NO.11所示，氨基酸序列如SEQ ID NO:12所示；重链恒定区核苷酸序列如SEQ ID NO.13所示，氨基酸序列如SEQ ID NO:14所示。通过PCR的方法分别扩增轻链可变区以及轻链恒定区，重链可变区以及重链恒定区。本申请中所有PCR反应均使用NEB公司的Phusion超保真DNA聚合酶(F-530L)。PCR引物根据碱基互补原则以及酶切位点的需要进行常规设计。反应体系均为：H 2O 8.9μl，5×Phusion超保真DNA聚合酶缓冲液4μl，1mM dNTP 4μl，上游引物1μl，下游引物1μl，Phusion超保真DNA聚合酶0.1μl，模板1μl。将可变区及恒定区PCR产物，经1.5％琼脂糖凝胶电泳后用DNA回收试剂盒(Promega，A9282，下同)回收相应片段。以回收的可变区片段与恒定区片段作为模板，使用可变区上游引物及恒定区下游引物，再进行一轮PCR反应，然后再回收相应片段，得到重链和轻链的全长片段。将X0GC载体及全长片段，用EcoRI(NEB，货号R3101L)及HindIII(NEB，货号R3104L)酶切，酶切反应体系为：10×缓冲液3 2μl，EcoRI及HindIII各0.5μl，胶回收获得的全长片段3μl，H 2O 14.5μl。酶切体系于37℃条件下反应3小时。将酶切产物用T4DNA连接酶(NEB，货号M0202V)连接，反应体系为：10×连接酶缓冲液2μl，连接酶0.5μl，胶回收获得的全长片段3μl，胶回收获得的X0GC载体3μl，H 2O 11.5μl。连接于室温反应12小时。将连接产物转化大肠杆菌感受态细胞DH5α(天根，货号CB104)，分别获得抗体重链和轻链的X0GC表达载体，用于在真核细胞中表达抗体的重链和轻链。
分别构建含抗人HER2的抗体重链和轻链的X0GC表达载体，其中抗体可变区序列来源于http://www.drugbank.ca/drugs/DB00072。轻链可变区核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO:2所示；轻链恒定区核苷酸序列如SEQ ID NO.3所示，氨基酸序列如SEQ ID NO:4所示；重链可变区核苷酸序列如SEQ ID NO.5所示，氨基酸序列如SEQ ID NO:6所示；重链恒定区核苷酸序列如SEQ ID NO.7所示，氨基酸序列如SEQ  ID NO:8所示。通过如上所述同样的方法分别获得抗体重链和轻链的X0GC表达载体，用于在真核细胞中表达抗体的重链和轻链。
实施例2抗PD-1/抗HER2异源二聚体抗体分子的表达
分别将含抗人PD-1抗体的重链和轻链的表达载体转染293F细胞(FreeStyle TM293-F Cells,货号R79007,invitrogen)，另外分别将含抗人HER2的抗体的重链和轻链的表达载体也转染293F细胞。转染前一天接种细胞，转染当天将细胞离心收集细胞，将细胞重悬于新鲜的FreeStyle TM293表达培养基(FreeStyle TM293Expression Medium，货号12338001，Gibco)中，细胞密度为200*10 5细胞/mL。按照转染体积加入质粒，终浓度为36.67ug/mL，轻轻混匀；然后加入线性PEI(聚乙烯亚胺，线形，M.W.25000，货号43896，Alfa Aesar)，终浓度为55ug/mL，轻轻混匀。之后放入细胞培养箱，120rpm摇床37℃培养1小时。之后加入19倍转染体积的新鲜培养基。继续120rpm摇床37℃培养。离心收集转染5～6天的细胞培养上清。
通过ELISA的方法测定表达量。在应用层析柱纯化之前，以0.2μm滤膜过滤以去除沉淀物。此步骤在4℃下进行。
实施例3抗PD-1/抗HER2异源二聚体抗体分子表达产物的纯化
采用AKTA explorer 100型蛋白纯化***(GE Healthcare)以及亲和色谱柱rProtein A Sepharose Fast Flow(16mm I.D.，22ml，GE Healthcare)于4℃下进行纯化。首先以流动相A(20mM磷酸钠缓冲液，150mM氯化钠，pH 7.4)平衡色谱柱，在基线稳定后将经过上述处理的细胞上清液进行上样，流速为5ml/min，并在上样后以流动相A进行平衡。样品分别为抗PD-1表达产物和抗HER2表达产物。之后，首先以流动相B1(含有0.5M精氨酸的流动相A)冲洗5个柱体积；然后以流动相B2(100mM柠檬酸，pH 3.0)洗脱5个柱体积，收集洗脱峰即为目的蛋白峰；以上洗脱步骤流速都为5ml/min。抗PD-1表达产物的洗脱峰色谱图如图1所示，抗HER2表达产物的洗脱峰与之类似(结果未包括)。收集标示的洗脱峰(图示灰色区域)，并通过滴加1M醋酸钠溶液将pH调至5.0。
实施例4抗PD-1/抗HER2异源二聚体抗体分子的制备和纯化
抗PD-1/抗HER2异源二聚体抗体分子的结构如图2所示。
将上述rProtein A Sepharose Fast Flow(16mm I.D.，22ml，GE Healthcare)方法纯 化获得的抗PD-1和抗Her2表达产物进行体外重组以获得异源二聚体。首先将上述纯化收集的蛋白溶液通过超滤浓缩管超滤浓缩(标称截留分子量10KDa)，将溶液置换为磷酸盐缓冲液(phosphate buffer saline，PBS)PBS(pH＝7.4)。将获得的抗PD-1和抗HER2纯化表达产物溶液分别加所述PBS调整到1mg/ml,加入1/200倍终体积的1M DTT，DTT终浓度分别为5mM，在4℃条件下进行还原(3-8小时)，通过还原的过程，二硫键被打开，抗PD-1以及抗HER2表达产物中含有的少量的抗体同源二聚体分子铰链区二硫键也打开，形成了含有一条重链和一条轻链的半抗体分子，结构如图3所示。还原的样品经流动相缓冲液中包含1mM DTT还原剂的SEC-HPLC(TOSOH，TSKgel superSW3000)分析，结果如图4的A和B所示，抗PD-1半抗体分子的比例为100％，抗HER2半抗体分子的比例为89.3％，其余10.7％为聚集体，但是并不存在二硫键不能打开的同源二聚体。
然后将还原的抗PD-1以及抗HER2半抗体分子等摩尔比例混合，在4℃条件下进行重组反应24小时，在重组的过程中，抗PD-1及抗HER2半抗体分子通过CH2以及CH3间的非共价作用力形成了同时含有抗PD-1以及抗HER2半抗体分子的异源二聚体的双特异抗体，之后将蛋白溶液通过超滤浓缩管超滤浓缩(标称截留分子量10KDa)，将溶液置换为磷酸盐溶液(PBS，pH＝7.4)终止还原，通过空气或者氧化剂进行氧化反应，使异源二聚体的双特异抗体的二硫键重新形成。氧化反应的条件为加入氧化剂100mM L-脱氢抗坏血酸，蛋白终浓度1mg/ml，氧化剂终浓度1mM，在4℃条件下进行氧化，反应进行24小时。将上述氧化反应获得的样品进行SEC-HPLC分析，结果如图5所示。
上述抗PD-1以及抗HER2半抗体分子经还原氧化得到的异源二聚体抗体分子通过超滤浓缩管超滤浓缩(标称截留分子量10KDa)，将溶液置换为10mM磷酸钠缓冲液，pH5.8。采用AKTA explorer 100型蛋白纯化***(GE Healthcare)以及离子色谱柱Source 15S(16mm I.D.，17ml，GE Healthcare)于4℃下进行纯化。首先以流动相A(10mM磷酸钠，pH 7.0)平衡色谱柱，在基线稳定后将经过上述处理的蛋白溶液进行上样，流速为3ml/min，并在上样后以流动相A进行平衡，之后以A(10mM磷酸钠，pH 5.8)到B(10mM磷酸钠，pH 5.8)梯度冲洗20个柱体积(0％B-100％B，170min，流速2ml/min)，收集洗脱主峰，收集的蛋白溶液通过超滤浓缩管超滤浓缩(标称截留分子量10KDa)，将溶液置换为磷酸盐溶液(PBS，pH＝7.4)，过滤除菌4℃保存。将纯化产物通过SEC-HPLC进行纯度分析，结果如图6所示，纯度为99.96％。
实施例5.抗PD-1/抗HER2异源二聚体抗体分子的稳定性
分别将充分密封的1mg/mL的抗PD-1/抗HER2异源二聚体样品放置于40℃恒温箱(BINDER KBF240)，在相应时间点(基线(第0天)、第2周、第4周)取20μg样品进行高效排阻液相色谱(SEC-HPLC)分离。上述SEC-HPLC条件如下：(1)排阻色谱柱：TSKgel G3000SWxl(Tosoh Bioscience)，5μm，7.8mm×30cm；(2)流动相：5mM PBS，150mM NaCl，pH 6.7；(3)流速：0.6mL/min；(4)紫外检测波长：280nm；(5)采集时间：30min。所用仪器是Agilent 1200Infinity色谱仪，利用Agilent ChemStation记录图谱并计算剩余单体的比例。如表1所示，在40℃实验条件下，所述二聚体不会发生明显的聚集；故认为所述抗PD-1/抗HER2异源二聚体具备较好的热稳定性。
表1 抗PD-1/抗HER2异源二聚体抗体分子的稳定性
Figure PCTCN2018075851-appb-000001
实施例6.抗PD-1/抗HER2异源二聚体抗体的体外靶点结合活性
用酶联免疫吸附试验(ELISA)测定抗PD-1/抗HER2异源二聚体抗体与单个抗原的结合能力。
ELISA具体实施过程如下：用pH＝9.6的碳酸盐缓冲溶液(0.05M)在96孔高吸附酶标板(Costar，货号42592)上包被重组人PD-1(北京义翘神州,货号10377-H08H)或者人HER2(北京义翘神州,货号10004-H08H)，包被浓度为1μg/mL，包被量为每孔100μL，包被在4℃过夜进行。PBST洗涤5次。用含1％BSA的PBST按300μL/孔封闭，25℃孵育1小时。PBST洗涤5次。加入序列稀释在含1％BSA的PBST里的异源二聚体抗体样品以及对照，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。然后加入1:2000稀释在含1％BSA的PBST里的辣根过氧化物酶标记的抗人IgG抗体(Chemicon，货号AP309P)，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。加入比色底物TMB，100μL/孔，室温显色10分钟。加入1M H 2SO 4，100μL/孔，终止显色。在酶标仪上读取450nm处的吸光度。
结果如图7的A和B所示，抗PD-1/抗HER2异源二聚体抗体具有对PD-1和HER2的高亲和力，保持了双价单抗的抗原亲和活性。
实施例7.抗PD-1/抗HER2异源二聚体抗体的双靶点同时结合活性
用酶联免疫吸附试验(ELISA)测定抗PD-1/抗HER2异源二聚体抗体与两个不同抗原的同时结合能力。
ELISA具体实施过程如下：用pH＝9.6的碳酸盐缓冲溶液在96孔高吸附酶标板上包被重组人HER2(北京义翘神州,货号10004-H08H)，包被浓度为1μg/mL，每孔100μL，包被在4℃过夜进行。PBST洗涤5次。用含1％BSA的PBST按300μL/孔封闭，25℃孵育1小时。PBST洗涤5次。加入序列稀释在含1％BSA的PBST里的异源二聚体抗体样品以及对照，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。然后加入稀释在含1％BSA的PBST里的生物素标记的PD-1-Fc(北京韩美药品)，0.5μg/mL，每孔100μL，25℃孵育1小时。加入1:1000稀释在含1％BSA的PBST里的链霉亲和素-辣根过氧化物酶偶联物(BD Pharmingen，货号554066)，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。加入比色底物TMB，100μL/孔，室温显色10分钟。加入1M H 2SO 4，100μL/孔，终止显色。在酶标仪上读取450nm处的吸光度。
结果如图8所示，PD-1单抗(其重链可变区序列和轻链可变区序列与抗PD-1/抗HER2异源二聚体抗体中的PD-1结合功能区中的相应序列相同)和HER2单抗(曲妥珠单抗(Trastuzumab))的组合不能同时结合PD-1，HER2，只有抗PD-1/抗HER2异源二聚体抗体具有同时结合两种抗原的活性。
用流式细胞术(FCM，FACS Calibur,购自BD Biosciences)在高表达PD-1的CHO/PD-1(GenScript,货号M00529)细胞和高表达HER2的SK-BR-3细胞上测定抗PD-1/抗HER2异源二聚体抗体与双靶点抗原的同时结合能力。
参照PKH26试剂盒(Sigma，货号SLBH4568V)使用说明书染色CHO/PD-1细胞。简要的说，收集CHO/PD-1细胞，无血清培养基洗涤一次后，用PKH26试剂盒里的Diluent C分别将CHO/PD-1制备成2×10 7/mL细胞悬液，将PKH26染料稀释到4μM,然后1:1混合在一起，此混合悬液的细胞密度为1×10 7/mL，PKH26的浓度为2μM，室温孵育1分钟，再用同等体积的FBS孵育1分钟终止染色。400g离心10分钟，用完全培养基洗涤两次，再重悬于完全培养基备用。参照CFSE试剂盒(Life technology，货号C34554)使用说明书染色SK-BR-3细胞。简要的说，用PBS稀释CFSE到工作浓度0.5μM并在37℃预热, 1000rpm离心5分钟收集SK-BR-3细胞，用预热的CFSE工作溶液重悬SK-BR-3，在37℃孵育15分钟，1000rpm离心5分钟收集细胞，再重悬于完全培养基中，孵育30分钟，用完全培养基洗涤一次，再重悬于完全培养基备用。离心收集以上已经染色的细胞，并用含2％FBS的冷PBS洗涤一次。将细胞重悬于含2％FBS的冷PBS中，细胞密度为5×10 6/mL。将SK-BR-3和CHO/PD-1按1:1混合，然后每个流式管取100μL(即2.5×10 5SK-BR-3和2.5×10 5CHO/PD-1)，再加入100μL稀释于含2％FBS的冷PBS中的异源二聚体抗体样品，对照以及同型对照(人免疫球蛋白，江西博雅生物制药股份有限公司，国药准字S19993012)，终浓度为5nM。流式管于冰上孵育30分钟。用含2％FBS的PBS洗涤两次。再重悬于500μL冷PBS中，该细胞悬液于流式细胞仪上进行检测分析。
结果如图9所示，通过异源二聚体抗体同时结合高表达PD-1的CHO/PD-1细胞和高表达HER2的SK-BR-3细胞，抗PD-1/抗HER2异源二聚体抗体能够引发SK-BR-3和CHO/PD-1细胞的相互靠近。
实施例8.抗PD-1/抗HER2异源二聚体抗体分子对PD-1与配体PD-L1，PD-L2结合的阻断活性
用酶联免疫吸附试验(ELISA)测定抗PD-1/抗HER2异源二聚体抗体对PD-1/PD-L1结合、PD-1/PD-L2结合的阻断活性。
用pH＝9.6的碳酸盐缓冲溶液在96孔高吸附酶标板上包被重组人PD-1-Fc，包被浓度为1μg/mL，包被量为100μL每孔，包被在4℃过夜进行。PBST洗涤5次。用含1％BSA的PBST按300μL/孔封闭，25℃孵育1小时。PBST洗涤5次。加入序列稀释在含1％BSA的PBST里的异源二聚体抗体样品以及对照，并同时加入终浓度为1μg/mL的生物素标记的PD-L1-Fc或者终浓度为4μg/mL的生物素标记的PD-L2，100μL/孔，25℃孵育1小时。PBST洗涤5次。然后加入1:1000稀释在含1％BSA的PBST里的辣根过氧化物酶标记的链霉亲和素(BD，货号554066)，每孔加入100μL，25℃孵育1小时。PBST洗涤5次。加入比色底物TMB，100μL/孔，室温显色10分钟。加入1M H 2SO 4，100μL/孔，终止显色。在酶标仪上读取450nm处的吸光度。
结果如图10的A和B所示，抗PD-1/抗HER2异源二聚体抗体能够阻断PD-1/PD-L1结合、PD-1/PD-L2结合，较好的保持了双价单抗的阻断活性。
实施例9.抗PD-1/抗HER2异源二聚体抗体分子的T细胞调控活性
用混合淋巴细胞反应(MLR)测定抗PD-1/抗HER2异源二聚体抗体对T细胞免疫反应的调控活性。
人树突状细胞(DC)的获取：复苏收集人PBMC细胞(Lonza，货号CC-2702)。将人PBMC细胞按细胞密度为5×10 6/mL重悬于无血清的RPMI 1640培养基并接种在细胞培养瓶中，在37℃二氧化碳培养箱中孵育90分钟。弃除培养上清及悬浮细胞，贴壁细胞在完全培养基(RPMI 1640含10％FBS)中培养，并加入100ng/mLGM-CSF(北京义翘神州,货号10015-HNAH)和100ng/mL IL-4(北京义翘神州,货号11846-HNAE)。孵育3天，换液，再孵育3天。然后将培养基更换为完全培养基(RPMI 1640含10％FBS)中含100ng/mLGM-CSF，100ng/mL IL-4以及20ng/mL TNF-α，孵育1天。即得DC细胞。
人T细胞的获取：复苏收集人PBMC细胞(Lonza，货号CC-2702)，保证此PBMC与诱导DC细胞的PBMC来自不同个体。参照Pan T细胞分离试剂盒(Miltenyi Biotech，货号5150414820)使用说明书分离人T细胞。简要的说，先用PBS洗涤PBMC一次，再将PBMC按10 7细胞每40μL分离缓冲液(PBS含2mM EDTA,0.5％BSA,pH＝7.2)重悬(以下使用量均按10 7细胞计)，并加入10μL Pan T cell Biotin Antibody Cocktail，在4℃孵育5分钟。再加入30μL分离缓冲液和20μL Pan T cell MicroBead Cocktail，在4℃孵育10分钟。过MACS分离柱，即得T细胞。
将收集到的人DC细胞和人T细胞重悬于完全培养基(RPMI 1640含10％FBS)中，接种于96孔板，接种的DC细胞和T细胞分别为1×10 4/孔，1×10 5/孔，混合培养。并加入用完全培养基序列稀释的异源二聚体抗体样品以及对照。将培养板置于37℃二氧化碳培养箱中孵育5天。孵育结束之后，取出孔内上清，按照试剂盒使用手册检测细胞因子IFN-γ(RayBiotech,货号ELH-IFNg)。
如图11所示，人T细胞在同种异体DC细胞的刺激下，会活化分泌IFN-γ。加入PD-1单抗会增强T细胞的活化，促进细胞因子的分泌，而HER2单抗没有此活性。抗PD-1/抗HER2异源二聚体抗体也显示了与PD-1单抗相当的T细胞调控活性，显著的促进细胞因子IFN-γ的分泌。
实施例10.抗PD-1/抗HER2异源二聚体抗体分子的肿瘤细胞抑制活性
在人PBMC的存在下，检测抗PD-1/抗HER2异源二聚体抗体对人乳腺癌细胞SK-BR-3的杀伤抑制活性。
SK-BR-3细胞在含10％FBS的RPMI 1640培养基(即完全培养基)中培养。收集 SK-BR-3细胞，重悬于完全培养基中，细胞密度为5×10 4/mL，100μL/孔接种于96孔细胞培养板，即每孔5000个细胞。复苏收集人PBMC细胞(Lonza，货号CC-2702)，将人PBMC细胞按细胞密度为5×10 5/mL重悬于RPMI 1640完全培养基中，50μL/孔接种96孔细胞培养板，即每孔25000个细胞，效靶细胞比率为5:1。加入50μL/孔用完全培养基序列稀释的异源二聚体抗体样品以及对照。将培养板置于37℃，5％CO2培养箱中孵育3天。在孵育终点，用培养基洗去细胞培养板中的PBMC，再加入完全培养基100μL和MTS(CellTiter96Aqueous One Solution，Promega,货号:G358B)20μL来检测SK-BR-3细胞。细胞培养板在培养箱中继续孵育3-4小时，然后在酶标仪上读取490nm处的吸光度。
如图12所示，加入HER2单抗会杀伤抑制SK-BR-3，而PD-1单抗在体外没有此活性。抗PD-1/抗HER2异源二聚体抗体也显示了与HER2单抗相当的肿瘤细胞杀伤抑制活性。
实施例11.抗PD-1/抗HER2异源二聚体抗体分子在动物体内的抗肿瘤药效研究
实验材料选用免疫缺陷的NCG小鼠(南京生物医药研究院)，雌性，6-8周龄。小鼠适应环境一周后，每只小鼠于右侧背部皮下接种5x 10 6个HCC1954人乳腺癌细胞。待肿瘤体积长至约100mm 3时，根据肿瘤体积进行分组，每组8只荷瘤小鼠。先给予人PBMC将小鼠免疫***部分人源化，每只小鼠接种5x 10 6个细胞，静脉注射。再分别给予溶媒(PBS)，PD-1单抗35nmol/kg(5mg/kg)，HER2单抗35nmol/kg(5mg/kg)，PD-1单抗35nmol/kg加HER2单抗35nmol/kg的组合，以及抗PD-1/抗HER2异源二聚体抗体35nmol/kg(5mg/kg)，每周给药2次，连续给药2周，给药方式为腹腔注射。自给药之日起，每周测量3次肿瘤体积，测量其长径a，短径b，肿瘤体积计算公式为：肿瘤体积(mm3)＝(a x b 2)/2。肿瘤体积测量持续时间为3周，即停药后，再观察一周。
结果如图13所示，抗PD-1/抗HER2异源二聚体抗体具有比PD-1单抗、HER2单抗更强的抗肿瘤药效，且在停药后依然显示出了良好的肿瘤控制作用。

Claims (28)

  1. 一种异源二聚体形式的双特异抗体，其包含能与PD-1特异性结合的第一个抗原结合功能区和能与HER2特异性结合的第二个抗原结合功能区，其中所述双特异抗体包含通过一个或多个二硫键链间连接的第一Fc链及第二Fc链，该第一Fc链和第二Fc链通过共价键或连接体分别连接到PD-1抗原结合功能区和HER2抗原结合功能区上，或者该第一Fc链和第二Fc链通过共价键或连接体分别连接到HER2抗原结合功能区和PD-1抗原结合功能区上；其中PD-1抗原结合功能区中的免疫球蛋白轻链可变区氨基酸序列选自SEQ ID NO.10，PD-1抗原结合功能区中的免疫球蛋白重链可变区氨基酸序列选自SEQ ID NO.12；并且第一Fc链和第二Fc链在如下位置包含5个氨基酸的替换：
    第一Fc链上366位及399位氨基酸替换，第二Fc链上351位、407位及409位氨基酸的替换，
    包含上述氨基酸替换的第一Fc链与第二Fc链更倾向于互相形成异源二聚体而不倾向于各自形成同源二聚体，
    其中氨基酸位置根据Kabat EU指数编号***编号。
  2. 如权利要求1所述的异源二聚体形式的双特异抗体，其中第一Fc链及第二Fc链氨基酸替换如下，
    a)L351G、L351Y、L351V、L351P、L351D、L351E、L351K或L351W；
    b)T366L、T366P、T366W或T366V；
    c)D399C、D399N、D399I、D399G、D399R、D399T或D399A；
    d)Y407L、Y407A、Y407P、Y407F、Y407T或Y407H；和
    e)K409C、K409P、K409S、K409F、K409V、K409Q或K409R。
  3. 如权利要求1或2所述的异源二聚体形式的双特异抗体，其中氨基酸替换包括：
    a)第一Fc链T366L及D399R替换，第二Fc链L351E、Y407L及K409V替换；
    b)第一Fc链T366L及D399C替换，第二Fc链L351G、Y407L及K409C替换；
    c)第一Fc链T366L及D399C替换，第二Fc链L351Y、Y407A及K409P替换；
    d)第一Fc链T366P及D399N替换，第二Fc链L351V、Y407P及K409S替换；
    e)第一Fc链T366W及D399G替换，第二Fc链L351D、Y407P及K409S替换；
    f)第一Fc链T366P及D399I替换，第二Fc链L351P、Y407F及K409F替换；
    g)第一Fc链T366V及D399T替换，第二Fc链L351K、Y407T及K409Q替换；
    h)第一Fc链T366L及D399A替换，第二Fc链L351W、Y407H及K409R替换。
  4. 如权利要求1所述的异源二聚体形式的双特异抗体，其中第一Fc链的氨基酸替换为T366L和D399R，第二Fc链的氨基酸替换为L351E、Y407L和K409V。
  5. 如权利要求1-4任一项所述的异源二聚体形式的双特异抗体，其中Fc链来源于IgG。
  6. 如权利要求1-5任一项所述的异源二聚体形式的双特异抗体，其中PD-1和HER2抗原结合功能区是Fab片段或scFv片段。
  7. 如权利要求1-6任一项所述的异源二聚体形式的双特异抗体，其中PD-1和HER2抗原结合功能区都是Fab片段。
  8. 如权利要求1-6任一项所述的异源二聚体形式的双特异抗体，其中PD-1和HER2抗原结合功能区一个是Fab片段，另一个是scFv。
  9. 如权利要求6-8任一项所述的异源二聚体形式的双特异抗体，其Fab片段包含不同的第一重链可变区及第二重链可变区，以及不同的第一轻链可变区及第二轻链可变区。
  10. 如权利要求1-9任一项所述的异源二聚体形式的双特异抗体，其中第一Fc链及其相连接的PD-1抗原结合功能区和第二Fc链及其相连接的HER2抗原结合功能区，或者第一Fc链及其相连接的HER2抗原结合功能区和第二Fc链及其相连接的PD-1抗原结合功能区，在单独存在并且同时存在还原剂时其形成同源二聚体的重量比例均低于50％。
  11. 如权利要求1-10任一项所述的异源二聚体形式的双特异抗体，其中双特异抗体的氨基酸序列选自：SEQ ID NO.2、4、6、8、10、12和14。
  12. 一种分离的多核苷酸，其编码如权利要求1-11任一项所述的异源二聚体形式的双特异抗体。
  13. 如权利要求12所述的分离的多核苷酸，其序列选自：SEQ ID NO.1、3、5、7、9、11和13。
  14. 一种重组表达载体，其包含权利要求12或13所述的分离的多核苷酸。
  15. 如权利要求14所述的重组表达载体，其中表达载体为基于pCDNA改造得到的质粒载体X0GC。
  16. 一种宿主细胞，其包含权利要求12或13所述的分离的多核苷酸，或权利要求14或15所述的重组表达载体。
  17. 如权利要求16所述的宿主细胞，其选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293E、HEK293F；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr-、CHO/DG44、ExpiCHO。
  18. 一种组合物，其包含权利要求1-11任一项所述的异源二聚体形式的双特异抗体或权利要求12或13所述的分离的多核苷酸或权利要求14或15所述的重组表达载体或权利要求16或17所述的宿主细胞，及药学上可接受的载体。
  19. 一种生产如权利要求1-11任一项所述的异源二聚体形式的双特异抗体的方法，其包括步骤：
    1)将权利要求12或13所述的分离的多核苷酸或权利要求14或15所述的重组表达载体分别在宿主细胞中进行表达；
    2)将在宿主细胞中分别表达的蛋白进行还原；以及
    3)将还原的蛋白混合，然后将混合物进行氧化。
  20. 如权利要求19所述的方法，其中宿主细胞选自人胚肾细胞HEK293或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293F；仓鼠卵巢细胞CHO或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr-、CHO/DG44、ExpiCHO。
  21. 如权利要求19或20所述的方法，其中还原步骤包括：1)进行还原反应，所述还原剂选自：2-巯基乙胺、二硫苏糖醇、三(2-羧乙基)膦或其化学衍生物，或者其组合；2)去除还原剂。
  22. 如权利要求19-21任一项所述的方法，其中氧化步骤为在空气中氧化，也包括在氧化剂存在下进行氧化反应，所述氧化剂选自：L-脱氢抗坏血酸或其他化学衍生物。
  23. 如权利要求19-22任一项所述的方法，其还包括分离纯化的步骤。
  24. 权利要求1-11任一项所述的异源二聚体形式的双特异抗体和/或权利要求12或13所述的分离的多核苷酸和/或权利要求14或15所述的重组表达载体和/或权利要求16或17所述的宿主细胞和/或权利要求18所述的组合物在制备用于预防和/或治疗受试者疾病的药物中的用途。
  25. 权利要求1-11任一项所述的异源二聚体形式的双特异抗体和/或权利要求12或13所述的分离的多核苷酸和/或权利要求14或15所述的重组表达载体和/或权利要求16或17所述的宿主细胞和/或权利要求18所述的组合物，其用做用于预防和/或治疗受试者疾病的药物。
  26. 一种预防和/或治疗疾病的方法，包括将权利要求1-11任一项所述的异源二聚体形式的双特异抗体和/或权利要求12或13所述的分离的多核苷酸和/或权利要求14或15所述的重组表达载体和/或权利要求16或17所述的宿主细胞和/或权利要求18所述的组合物施予有需求的受试者。
  27. 如权利要求24所述的用途，权利要求25所述的异源二聚体形式的双特异抗体、分离的多核苷酸、重组表达载体、宿主细胞或组合物，或权利要求26所述的方法，其中受试者是哺乳动物，优选地，人类受试者。
  28. 如权利要求24所述的用途，权利要求25所述的异源二聚体形式的双特异抗体、分离的多核苷酸、重组表达载体、宿主细胞或组合物，或权利要求26所述的方法，其中所述疾病选自如下肿瘤：白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈部鳞状细胞癌、非小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、肝癌、结直肠癌、乳腺癌、卵巢癌、***、子宫内膜癌、子宫肉瘤、***癌、膀胱癌、肾细胞癌、黑色素瘤。
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WO2021136227A1 (zh) * 2019-12-31 2021-07-08 周易 Ch3 结构域改造诱导形成的异源二聚体及其制备方法和应用
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