WO2013097430A1 - 异二聚体蛋白以及基于电荷网络的制备方法 - Google Patents

异二聚体蛋白以及基于电荷网络的制备方法 Download PDF

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WO2013097430A1
WO2013097430A1 PCT/CN2012/077671 CN2012077671W WO2013097430A1 WO 2013097430 A1 WO2013097430 A1 WO 2013097430A1 CN 2012077671 W CN2012077671 W CN 2012077671W WO 2013097430 A1 WO2013097430 A1 WO 2013097430A1
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region
charge
polypeptide
mutation
amino acid
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PCT/CN2012/077671
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French (fr)
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徐霆
须涛
郭康平
汪皛皛
吴杰
愠丽红
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苏州康宁杰瑞生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a method for preparing heterodumeric antibody FC-related proteins and polypeptides, and also to heterodimeric antibody FC proteins and polypeptides themselves, including individual polypeptides constituting heterodimeric antibody FC; A nucleic acid sequence encoding these polypeptides, and a pharmaceutical component comprising one or more heterologous FC proteins or polypeptides.
  • Monoclonal antibody drugs have grown rapidly in the last fifteen years and have become the growth point of 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 antibody in 2010 exceeded $30 billion, and the annual growth rate exceeded 10%. Because of the high target specificity of monoclonal antibodies, only a single target can be inhibited. In many diseases, including tumors and autoimmunity, multiple signaling pathways need to be suppressed 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.
  • bifunctional antibodies and proteins are used to specifically activate the human immune system (Wolf, Hofmeister et al. 2005).
  • the Fc region of the antibody forms a homodimer, while Fc plays a key role in maintaining the in vivo stability of the antibody and Fc fusion protein.
  • the formation of heterodimers by engineering Fc is an effective method for producing multifunctional antibodies, proteins, and maintaining their in vivo stability.
  • Bispecific antibody is an immunoglobulin molecule containing two different ligand binding sites. Bispecific antibodies can be active against at least two different antigens (Carter 2001), which replaces the same sequence of the classical antibody fab, but uses two different fab sequences, so the Y-arms can bind to different antigens. .
  • the use of bispecific antibodies in cancer therapy 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.
  • bispecific antibodies For the application of other bispecific antibodies, see US Pat. Nos 5,731, 168 and 7,183,076.
  • the absence of bispecific antibodies in their natural state can only be prepared by special methods.
  • methods for preparing bispecific antibodies include chemical cross-linking, hybrid F b ' ) 2 molecular methods, and murine hybridoma methods.
  • the heterogeneity of bispecific antibodies produced by chemical cross-linking, the batch-to-batch instability, and the specificity of antibody specificity that are altered by some modifications or improper linkages make the bispecific antibodies produced by this method unsuitable. Used in the body.
  • bispecific hybrid molecule produced by the thiol cross-linked protease digestion fragment F Ub' has a relatively uniform composition, but it is time consuming and laborious, and the yield is low.
  • Bispecific antibody produced by hybridoma method come The source is reliable, but the multiple possible antibody forms produced by random pairing of light and heavy chains make bispecific antibody production and purification very difficult.
  • Carter et al. used the "knob into hole” model to engineer a partial amino acid of the antibody heavy chain, which successfully achieved bispecific antibodies (Ridgway, Presta et al. 1996; Carter 2001).
  • the "handle-hole” model was originally proposed by Crick to solve the problem of amino acid side chain folding between adjacent ⁇ -helices (Crick 1952). Carter et al.
  • US 2010/286374 A discloses a process for the preparation of a FC heterodimeric protein or polypeptide, the heterodimeric protein involved comprising a first polypeptide comprising a CH3 region and a second polypeptide comprising a CH3 region, which are in contact with each other Forming an interface that promotes the formation of a heterodimer, ie, the first polypeptide comprising a CH3 region and the second polypeptide comprising a CH3 region comprise one or more charged amino acids at the interface, electrostatic interaction between charged amino acids It is not conducive to the formation of homodimers but facilitates the formation of heterodimers, in particular by replacing the charged amino acids in the first or second polypeptides in the CH3 region with oppositely charged amino acids, using electrostatic action to promote Formation of heterodimers.
  • the method of the invention has two defects.
  • One is that the technical scheme lacks integrity, and the interaction between the interface amino acids is not limited to a pair of amino acids, and the interface amino acid is replaced by an opposite charge, especially in the case of multiple amino acid mutations.
  • electrostatic interactions do not always inhibit homodimers or facilitate the formation of heterodimers, although some specific alternatives in this patent do facilitate heterodimers.
  • the formation of the strategy, or the lack of a strategy or method for evaluating and screening the technical solution, in the case of complex amino acid interaction or multi-point mutation, in order to obtain a sense mutation, the patent can not be guided by those skilled in the art;
  • Second the technical solution is limited to the substitution of charged amino acids for the opposite electrical.
  • Another object of the present invention is to provide a heterodimeric antibody FC protein and a polypeptide itself prepared by the method, which comprise a separate polypeptide constituting a heterodimeric antibody FC.
  • Another object of the invention is also to provide nucleic acid sequences encoding these polypeptides.
  • a final object of the present invention is also to provide a pharmaceutical composition comprising the above polypeptide.
  • the technical scheme of the method is: the first polypeptide comprising a CH3 region in the FC and/or the second region comprising a CH3 region
  • one or more amino acids are mutated to positively or negatively charged amino acids, which are in contact with each other to form an interface that promotes the formation of a heterodimer
  • the mutation is one of the sense mutations selected by the following method:
  • Mutation effect value Total charge after mutation - Total charge before mutation
  • Total charge sum of positive and negative subnetworks of the mutated amino acid
  • the positive charge interaction is 1
  • the negative and negative charge interaction is 1
  • the positive and negative charge interaction is -1
  • positive / non - charge or negative / non - charge action is 0;
  • one or more uncharged amino acids are mutated to positively or negatively charged amino acids in the first polypeptide of the FC comprising a CH3 region and/or a second polypeptide comprising a CH3 region, which are in contact with each other Forming an interface that promotes the formation of a heterodimer, and mutating to one of the sense mutations selected by the following method:
  • Random Mutant Antibody One or more uncharged amino acids in the CH3 region of FC are positively or negatively charged amino acids;
  • Mutation effect value Total charge after mutation - Total charge before mutation
  • Total charge sum of positive and negative subnetworks of the mutated amino acid
  • the positive charge interaction is 1
  • the negative and negative charge interaction is 1
  • the positive and negative charge interaction is -1
  • positive / non - charge or negative / non - charge action is 0;
  • the method specifically includes the following steps:
  • the heterodimeric FC comprises human immunoglobulin FC
  • the heterodimeric FC comprises human immunoglobulin IgG FC
  • the mutation is a single point mutation or a double point mutation.
  • the mutated positively charged amino acid is lysine or arginine
  • the mutated negatively charged amino acid is aspartic acid or glutamic acid.
  • the heterodimeric protein comprises a first polypeptide comprising a CH3 region and a second inclusion
  • the polypeptide of the CH3 region comprises the following steps: 1) cultivating a host cell, the host cell comprising a nucleic acid encoding a first polypeptide comprising a CH3 region polypeptide and a second nucleic acid comprising a CH3 region polypeptide, wherein the cultured host cell expresses the first and second a polypeptide comprising a CH3 region; 2) extracting a heterodimeric protein from the host cell culture;
  • the first polypeptide comprising a CH3 region and/or the second polypeptide comprising a CH3 region comprises one or more amino acids produced by mutation at the interface, which are in contact with each other to form an interface that promotes formation of a heterodimer
  • the mutation is mutated from an uncharged amino acid to a positively or negatively charged amino acid and conforms to one of the sense mutations selected as follows:
  • Random mutations One or more uncharged amino acids in the CH3 region are amino acids with positive or negative charges;
  • Mutation effect value Total charge after mutation - Total charge before mutation
  • Total charge sum of positive and negative subnetworks of the mutated amino acid
  • the positive charge interaction is 1
  • the negative and negative charge interaction is 1
  • the positive and negative charge interaction is -1
  • positive / non - charge or negative / non - charge action is 0;
  • the heterodimeric protein comprises human immunoglobulin IgA, IgD, IgE, IgG or IgM FC
  • the heterodimeric protein comprises human immunoglobulin IgGl FC
  • the IgG1 FC comprises a first polypeptide comprising a CH3 region or a second polypeptide comprising a CH3 region, the sequence of which is different from the wild type human immunoglobulin sequence, which is a partial amino acid mutation to
  • the selective mutation consisting of the IgG sequence of the positively charged amino acid is the positively charged amino acid PHE405, SER364. TYR407. VAL397. SER400- GLN362. VAL363 - LEU398
  • the amino acid selected to be negatively charged is VAL348 - TYR349- THR350.
  • the IgG1 FC comprises a first polypeptide comprising a CH3 region or a second polypeptide comprising a CH3 region, the sequence of which differs from the wild type human immunoglobulin sequence by the first strand
  • a partial amino acid mutation consists of a negatively charged amino acid and an IgG sequence in which the second chain partial amino acid is mutated to a positive charge.
  • the IgG1 FC comprises a first polypeptide comprising a CH3 region and a second polypeptide comprising a CH3 region.
  • the sequence differs from the wild type human immunoglobulin sequence by the first strand.
  • the heterodimeric protein is an antibody, a bispecific antibody, a single monovalent antibody, a single domain antibody, a polypeptide type antibody or a bispecific polypeptide type antibody.
  • a heterodimeric protein of the invention comprises a first polypeptide comprising a CH3 region and a second polypeptide comprising a CH3 region, characterized in that said first one comprises CH3
  • Total charge sum of positive and negative subnetworks of the mutated amino acid
  • the positive charge interaction is 1
  • the negative and negative charge interaction is 1
  • the positive and negative charge interaction is -1
  • positive / non - charge or negative / non - charge action is 0 .
  • the heterodimeric protein comprises the FC region of human immunoglobulin IgA, IgE, IgD or IgM.
  • the FC region comprises human immunoglobulin IgGl FC.
  • the first polypeptide of the IgG1 FC comprising the CH3 region or the second polypeptide comprising the CH3 region differs from the wild-type human immunoglobulin sequence by the CH3 region.
  • the selected mutation to a positively charged amino acid is PHE405, SER364. TYR407. VAL397. SER400- GLN362. VAL363- LEU398; or The amino acids that were mutated to negatively charged were VAL348, TYR349-THR350.
  • the IgG1 FC comprises a first polypeptide comprising a CH3 region or a second polypeptide comprising a CH3 region, the sequence of which differs from the wild type human immunoglobulin sequence by the first strand
  • a partial amino acid substitution consists of a negatively charged amino acid and a second chain partial amino acid replaced with a positively charged IgG sequence.
  • the IgG1 FC comprises a first polypeptide comprising a CH3 region and a second polypeptide comprising a CH3 region.
  • the sequence differs from the wild type human immunoglobulin sequence by the first strand.
  • An uncharged amino acid in the upper CH3 region is replaced by a positively charged amino acid and an uncharged amino acid in the CH3 region of the second strand is replaced by a negatively charged IgG sequence.
  • the heterodimeric protein is an antibody, a bispecific antibody, a single monovalent antibody, a single domain antibody, a polypeptide type antibody or a bispecific polypeptide type antibody.
  • a polypeptide of the invention comprises a CH3 region of an antibody, characterized in that said CH3 region comprises a polypeptide sequence different from the wild-type CH3 region, from one or more of the wild-type CH3 regions
  • the uncharged amino acid mutation is formed by a positively charged amino acid or a negatively charged amino acid.
  • the influence of the mutation on the homodimer is greater than or equal to 0, or the effect on the heterodimer is less than or equal to 0.
  • the influence value is calculated as follows: :
  • Mutation effect value total charge after mutation - total charge before mutation
  • Total charge sum of positive and negative subnetworks of the mutated amino acid
  • the positive charge interaction is 1
  • the negative and negative charge interaction is 1
  • the positive and negative charge interaction is -1
  • positive / non - charge or negative / non - charge action is 0 .
  • the present invention also provides a nucleotide sequence encoding the polypeptide as described above.
  • composition of the invention comprises a polypeptide as described above.
  • the beneficial effects of the present invention over the prior art are:
  • the present invention provides a method of increasing the heterodimer content while reducing other undesirable products such as homodimers.
  • the obtained heterodimeric protein is mainly used in the medical field.
  • the heterodimeric protein can guide drugs, markers, cytotoxic cells, T cells and the like to tumor cells, thereby playing more effectively. Killing effect, providing new methods and ways in the immunodiagnosis and immunotherapy of tumors.
  • Figure 1 is a schematic representation of the structure and different regions of the IgG1 antibody.
  • Figure 2 shows the phenomenon of different heterodimeric protein combinations involving FC single strands.
  • Figure 3 is a network diagram of the interaction of amino acids at the CH3-CH3 interaction interface of human IgGl FC.
  • Figure 4 shows the results of SDS-PAGE analysis of heterodimers prepared based on charge network modification FC.
  • the object of the present invention is to obtain a heterodimerization based on the charge interaction network of the two-armed amino acid of the FC heavy chain CH3 by modifying the amino acid of the CH3 region by the charge repulsion effect to reduce the self-binding between the CH3 regions (formation of homodimer). body.
  • the modification of the relevant amino acid to a charged amino acid results in a charge repulsion effect.
  • a positively charged amino acid (lysine, arginine) on the mutated contact surface is A negatively charged amino acid (aspartic acid, glutamic acid) or vice versa can form a repulsive effect.
  • the present invention describes a method of modifying the CH3 amino acid of FC to attenuate the region's own interactions (favoring the formation of homodimers) and enhancing the interaction between the regions (favoring the formation of heterodimers).
  • an amino acid interaction network on the surface of CH3-CH3
  • any one or more amino acids are selected to analyze the influence of selected amino acids on homodimers and heterodimers.
  • Mutating the selected amino acid into a charged amino acid examining the effect of the mutation on the homodimer and heterodimer, comparing the mutation to the pre-mutation, if the mutation is appropriate, it will produce enhanced heterodimer and weaken The role of homodimer formation.
  • the number of amino acid combinations and the number of adjacent amino acids are examined. (The larger the number, the greater the change in the original properties.)
  • the rational amino acid mutation is selected to maximize the heterodimer and weaken the homodimer.
  • the present invention provides a method for modifying a CH3 amino acid to modify a FC to attenuate the region's own interaction (homodimer) and enhance the interaction between the regions (heterodimer), and further obtain a heterodimer.
  • the steps of the method are described as follows:
  • FC homodimer structure is derived from 1DN2 (PDB numbering).
  • Two screening strategies can be used 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 chosen to be 4.5 people.
  • Table 1 is a list of amino acids for the CH3 interaction of the first strand of the antibody and the second strand of the antibody. From this result, it can be seen that the interaction between the first strand and the second strand of the antibody CH3 amino acid is not only a one-to-one relationship, but a one-to-many or many-to-many relationship, and this conclusion can also be derived from the amino acid network diagram. (Figure 2) is more intuitive to see. Listed in Table 1 are the 34 interface amino acids screened by the amino acid contact screening rules. Table 1 CH3-CH3 interface amino acid list
  • an amino acid interaction network was constructed by pairing the interactions between amino acids (see Figure 2).
  • the network of amino acid interactions consists of the interaction between all CH3-CH3 interfacial amino acids and any of the interfacial amino acids contained in the interface with other interfacial amino acids.
  • charged amino acids are labeled with charge properties, aspartic acid and glutamic acid are negative, and arginine and lysine are positive.
  • Node degree refers to the number of adjacent nodes (interacting amino acids) in a node (amino acid). Based on the charge interaction network, the number of adjacent amino acids in each node is calculated separately (see Table 2). Any one of the interface amino acids (nodes) with which the interacting amino acids (adjacent nodes) constitute a subnetwork of the interface amino acids. In general, if the amino acid of an amino acid is adjacent, the amino acid is more likely to affect the stability of the protein.
  • one or more amino acids on the first strand and/or the second strand are randomly mutated, including random single point mutations, random double point mutations, and multiple point mutations. Calculate the subnet containing the mutated amino acid The sum of the charge before the mutation, and the sum of the charges after the mutation.
  • positive and positive charge interaction is 1
  • negative and negative charge interaction is 1
  • positive and negative charge interaction is -1
  • positive / non - charge or negative / non - charge effect is 0
  • Mutation effect total number of charges after mutation - total number of charges before mutation.
  • a reasonable amino acid (the smaller the amino acid degree, the better) and favorable mutations (the beneficial effects on the heterodimer and the adverse effects on the homodimer), select a reasonable mutation.
  • the positively charged amino acids after mutation in this patent are lysine and arginine, and the negatively charged amino acids after mutation are aspartic acid and glutamic acid.
  • the mutations at the CH3-CH3 contact interface were selected to be positively charged, and the mutations having a negative effect on the homodimer (impact value > 0) are shown in Table 3.
  • the heterodimer may also have a negative impact, and the influence value is the same as the effect on the homodimer.
  • No. 1-6 is a negatively charged amino acid mutated to a positively charged amino acid, as disclosed in US 2010/286374A.
  • 7-22 is an uncharged amino acid mutated to a positively charged amino acid.
  • the homodimer can be inhibited by a single point mutation of the uncharged amino acid, and any one of the following methods can be selected, that is, in the first strand of the polypeptide comprising the CH3 region, the mutation is selected to be a positively charged amino acid, PHE405, SER364. TYR407. VAL397. SER400- GLN362. VAL363 or LEU398.
  • the mutations at the CH3-CH3 contact interface were selected to be negatively charged, and the mutations having a negative effect on the homodimer (impact value > 0) are shown in Table 4.
  • Serial numbers 1-4 are positively charged amino acid mutations to negatively charged amino acids, as disclosed in US 2010/286374A.
  • 5-10 is an uncharged amino acid mutation to a negatively charged amino acid. It can be seen that according to the method of the present invention, by inhibiting the homodimer by a single point mutation of the uncharged amino acid, one of the following methods can be selected, that is, in the first strand of the polypeptide comprising the CH3 region, the amino acid selected to be negatively negatively charged is VAL348, TYR349 Or THR350.
  • Sequence numbers 1-11 include positively charged amino acid mutations as negatively charged amino acids or as opposite mutations, some of which are known to be mutated in US 2010/286374A (eg 1, 2, 3, 5 and 9). 12-33 mutates two uncharged amino acids into positively or negatively charged amino acids, respectively. It can be seen that according to the method of the present invention, a heterodimeric protein is prepared by double-point mutation of an uncharged amino acid, and there are two options:
  • the sequence of the IgG1 FC comprising the first polypeptide comprising the CH3 region and the second polypeptide comprising the CH3 region is different from the wild-type human immunoglobulin sequence, which is replaced by an uncharged amino acid in the CH3 region of the first strand.
  • a negatively charged amino acid and an IgG sequence in which an uncharged amino acid in the CH3 region of the second strand is replaced with a positive charge includes one of the following methods:
  • the first strand mutation TYR349 of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation SER354 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation SER354 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation SER408 of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation TYR407 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation THR366 of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation TYR407 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation ASN390 of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation SER400 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation THR399 of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation VAL397 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation VAL397 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation THR394 of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation TYR407 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation of the polypeptide comprising the CH3 region, LEU365, is negatively charged, and the second strand mutation TYR407 of the polypeptide comprising the CH3 region is positively charged;
  • the first strand mutation LEU368 of the polypeptide comprising the CH3 region is negatively charged, and the second strand mutation SER364 of the polypeptide comprising the CH3 region is positively charged.
  • the sequence of the IgG1 FC comprising the first polypeptide comprising the CH3 region and the second polypeptide comprising the CH3 region is different from the wild-type human immunoglobulin sequence, which is replaced by an uncharged amino acid in the CH3 region of the first strand. It consists of a positively charged amino acid and an IgG sequence in which the uncharged amino acid in the CH3 region of the second strand is replaced with a negative charge. Specifically, it includes one of the following methods:
  • the first strand mutation SER364 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation LEU368 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation VAL397 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation PR0395 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation VAL397 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation PR0395 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation TRY407 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation SER408 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation TRY407 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation THR366 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation SER400 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation ASN390 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation TYR407 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation LEU365 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation TYR407 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation THR394 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation VAL397 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation THR394 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation VAL397 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation THR393 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation SER354 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation TYR349 of the polypeptide comprising the CH3 region is negatively charged;
  • the first strand mutation SER354 of the polypeptide comprising the CH3 region is positively charged, and the second strand mutation THR350 of the polypeptide comprising the CH3 region is negatively charged.
  • the method of modifying a heterodimeric FC according to the present invention and preparing a heterodimeric antibody is not limited to the single point mutation and double point mutation described above, and those skilled in the art can according to the gist of the present invention. Mutation of more than three amino acids to form a heterodimeric protein.
  • the invention can also improve the "handle-hole” model to suppress the defects of the homodimer by introducing a charge effect, and further transform the heterodimer FC by introducing the charge on the basis of the "hole” chain. Repulsive action, the repulsive effect of the "hole-hole” chain will be strengthened, and finally the formation of "hole-hole” homodimers will be completely suppressed.
  • the heterodimeric molecule can be purified from the host cell using standard experimental means.
  • the protein can be purified with protein A.
  • Purification methods include, but are not limited to, color techniques such as size exclusion, ion exchange, affinity color transfer, and ultrafiltration.
  • the method for separating and purifying the heterodimer of the present invention also includes a suitable combination of the above various methods.
  • the heterodimeric protein comprises FC, preferably human immunoglobulin FC.
  • FC preferably human immunoglobulin FC.
  • the CH3 region polypeptide of the human immunoglobulin FC region is derived from the wild-type human immunoglobulin FC region.
  • Wild-type human immunoglobulin FC refers to the amino acid sequence that occurs in the human population, and of course the FC sequence will have some subtle differences in the individual.
  • the human immunoglobulin FC of the present invention also includes individual amino acid changes to the wild-type human immunoglobulin FC sequence, for example, changes in certain amino acids in the FC region, such as certain amino acids that are mutated at the glycosylation site, Or other nonsense mutations; Includes changes in individual amino acids that are mutated according to the "handle-hole" model.
  • human immunoglobulin FC refers to the constant region of a human immunoglobulin chain, particularly the carboxy terminus of a constant region of an immunoglobulin heavy chain or a portion thereof.
  • an immunoglobulin FC region can include a combination of two or more domains of heavy chains CH1, CH2, CH3, CH4 and an immunoglobulin hinge region.
  • 9 immunoglobulins can be divided into different types, mainly five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes).
  • immunoglobulin FC region from a particular immunoglobulin class and subclass is well within the purview of those skilled in the art.
  • the human immunoglobulin FC used in the present invention comprises at least one immunoglobulin hinge region, a CH2 domain and a CH3 domain, specifically human IgGl FC (Fig. 1).
  • the CH3 of the human immunoglobulin FC region is encoded by the sequence set forth in SEQ ID NO: 1.
  • Mammalian host cells to which the invention relates include, but are not limited to, CHO, 293, myeloma cells.
  • the host cell can also be a yeast or a prokaryotic cell such as E. Coli.
  • the heterodimeric protein of the present invention is not only an antibody, but also a bispecific antibody, a single monovalent antibody, a single domain antibody, a polypeptide type antibody, a bispecific polypeptide type antibody or the like (see Fig. 2).
  • This example will demonstrate that engineering a CH3 domain by charge analysis based on an interaction network can inhibit homodimers while forming heterodimers.
  • the fusion proteins IL-IR-Fc and Fc were constructed as follows:
  • the expression of homodimers and heterodimers was detected by SDS-PAGE (polyacrylamide gel electrophoresis).
  • SDS-PAGE polyacrylamide gel electrophoresis
  • the principle of detection is that the fusion protein IL-IR-Fc has a larger molecular weight than Fc, then in the process of mixing IL-IR-Fc and Fc, homodimer (IL-1R-Fc/IL-1R-Fc, Fc /Fc) and heterodimer ((IL-1R-Fc/Fc) have different band positions on SDS-PAGE.
  • the ratio of homodimer to heterodimer can be detected. The results are shown in Figure 4.
  • the human Fc gene was obtained by artificial synthesis, and the synthesized Fc (SEQ ID ⁇ : 1) was subcloned into mammalian cell expression plant.
  • Volume pcDNA3.1 In the sequencing, the accuracy of constructing the plasmid was verified; using the Tiangen extraction kit, the recombinant plasmid DNA, pcDNA3.1-Fc, was obtained according to the instructions.
  • the human IgG1 Fc sequence is shown below (SEQ ID ⁇ : 1), and the nucleotide sequence encoding the sequence is shown in SEQ ID NO: 2:
  • the human cell interleukin receptor (IL-1R) gene sequence searched in the gene bank the human IL-1R gene was obtained by artificial synthesis, and the synthesized IL-1R gene was subcloned into the recombinant expression vector pcDNA3.1-Fc.
  • the IL-IR-Fc fusion protein SEQ ID NO: 2, underlined for Fc
  • the IL-IR-Fc sequence is shown below (SEQ ID NO: 3, the underlined portion indicates Fc), and the nucleotide sequence encoding the sequence is shown in SEQ ID NO: 4:
  • the recombinant plasmid pcDNA3.1-Fc and pcDNA3.1-IL-lR-Fc were co-transfected into suspension cultured 293H cells, and after 3-4 days of culture, the cell supernatant was collected. The supernatant was immunoprecipitated with proteinA agarose resin, and the homodimer (IL-1R-Fc/IL-1R-Fc, Fc/Fc) and heterodimer were detected by SDS-PAGE electrophoresis under non-reducing conditions. (Formation of (IL-IR-Fc/Fc).
  • IL-IR-FC and FC expression vectors are co-transfected, and finally homodimers (IL-IR-Fc/IL-IR-FC, FC/FC) and heterodimers (IL-IR-Fc) /FC), in the case of wild type is IL-IR-Fc/IL-IR-FC, the ratio of FC/FC and IL-IR-Fc/FC is close to 1: 1 : 1

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Abstract

一种基于电荷网络的异二聚体FC改造方法和异二聚体蛋白的制备方法，以及蛋白和多肽本身。在FC的第一个包含CH3区域的多肽和/或第二个包含CH3区域的多肽上，一个或多个氨基酸突变为正电荷或负电荷氨基酸，它们相互接触形成促使异二聚体形成的界面，突变为按以下方法选择得到的有义突变之一：1）随机突变抗体FC的CH3区域的一个或多个氨基酸为带有正电荷或负电荷的氨基酸；2）基于界面氨基酸之间的电荷网络作用，计算突变对于同二聚体及异二聚体的影响值：突变影响值=突变后电荷总数-突变前电荷总数；电荷总数=所突变的氨基酸的子网络电荷之和；其中，正正电荷相互作用为1，负负电荷相互作用为1，正负电荷相互作用为-1，正/非电荷或者负/非电荷作用为0；3）筛选对于同二聚体影响值大于等于0的突变，或对异二聚体影响值小于等于0的突变。该方法能够增强异二聚体形成，同时减弱同源二聚体的形成。

Description

异二聚体蛋白以及基干电荷网络的制备方法
本申请要求了申请日为 2012年 01月 04日， 申请号为 201110459100.7, 发明名称为"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"的中国专利申请的优先权， 其 全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种制备异二聚体抗体 FC相关的蛋白和多肽的方法， 也涉及异二聚体抗体 FC蛋白及多肽本身， 包括组成异二聚体抗体 FC的单独的多肽； 本发明还涉及编码这些多 肽的核酸序列， 以及包含一个或者多个异质 FC蛋白或者多肽的药物组分。
背景技术
单克隆抗体药物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， 自体免疫， 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。另外， 双功能抗体、 蛋白被用于特异激活人体免疫系统 (Wolf, Hofmeister et al. 2005)。 抗体的 Fc区 形成同二聚体， 同时 Fc对维持抗体和 Fc融合蛋白的体内稳定性起关键作用。通过改造 Fc使之 形成异二聚体是产生多功能抗体、 蛋白， 并且维持其体内稳定性的有效方法。
异二聚体的一个典型应用的例子是双特异抗体，双特异抗体（ Bispecific antibody,BsAbs ) 是含有两个不同配体结合位点的免疫球蛋白分子。双特异抗体至少可以对两个不同抗原具有 活性 (Carter 2001), 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。 双特异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。 其他双特异抗体的应用可以查看美国专利 U.S. Pat. Nos 5,731, 168和 7,183,076 自然状态下不存在双特异性抗体，只能通过特殊方法进行制备。 以往双特异抗体的制备 方法有化学交联法， 杂合 F b' ) 2分子法和鼠杂交瘤法等。 化学交联法生产双特异抗体的 异源性， 批与批之间的不稳定性， 以及抗体特异性易受某些修饰或不当连接而改变的特性， 使得该法生产的双特异抗体不适于体内使用。 以巯基交联蛋白酶消化片断 F Ub' )生产的双 特异杂交分子， 成分虽较均一， 但费时费力， 且产量很低。 杂交瘤法生产的双特异抗体， 来 源可靠， 但由轻链、 重链随机配对产生的多种可能抗体形式， 使得双特异抗体生产、 纯化变 得非常困难。
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抗体 (Ridgway, Presta et al. 1996; Carter 2001) 。 "把手-孔洞" 模型最初是由 Crick提出用来解决相邻的 α -螺旋之间的氨基酸侧链折叠问题的 (Crick 1952)。 Carter等在 FC第一条重链的 CH3区域上通过将一个侧链小的氨基酸突变成了一个侧链大的 氨基酸从而创造出了一个 "把手" （如 T366Y), 并将第二条重链上的 CH3上的某些氨基酸突 变成了侧链小的氨基酸创造出了 "孔洞" （Y407T等）。 "把手-孔洞" 模型的原理是 "把手- 孔洞" 相互作用是支持异二聚体形成的， 而 "把手-把手" 模型及 "孔洞-孔洞" 模型是阻碍 同二聚体的形成的。 他们进一步在 "把手-孔洞" 突变的基础上引入了 CH3区域内二硫键来 进一步巩固异二聚体的结合能力。 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 -噬菌体展示等方法一步提高异二聚体的含量。 但结果也没有解决根本问题。
US 2010/286374A公开了一种 FC异二聚体蛋白或多肽的制备方法， 所涉及的异二聚体蛋 白包括第一个包含 CH3区域的多肽和第二个包含 CH3区域的多肽， 它们相互接触形成促使异 二聚体形成的界面， 即在第一个包含 CH3区域的多肽和第二个包含 CH3区域的多肽在界面上 包含一个或多个带电荷的氨基酸，带电荷的氨基酸间的静电作用不利于同二聚体的形成但有 利于异二聚体的形成，具体是将第一个或第二个包含 CH3区域中的多肽中带电荷的氨基酸替 换为相反电荷的氨基酸， 利用静电作用促进异二聚体的形成。 该发明方法存在两个缺陷， 其 一是该技术方案缺乏完整性，界面氨基酸之间的作用并不局限于一对氨基酸之间，界面氨基 酸替换为相反电荷，特别是多个氨基酸突变的情况下，基于界面氨基酸之间复杂的作用关系， 静电作用并不总是能抑制同二聚体或有利于异二聚体的形成，虽然该专利中包括部分具体的 替换方案的确有利于异二聚体的形成，但其技术方案中缺少对此进行评价和筛选的策略或方 法， 在界面氨基酸作用复杂或多点突变的情形下， 为获得有义突变， 该专利对本领域技术人 员无法予以指导； 其二是该技术方案局限于电荷氨基酸作相反电性的替换， 实际上， 界面氨 基酸之间形成一个电荷作用网络， 任何一个氨基酸（电荷或非电荷氨基酸）电性的变化均会 引起两条多肽链之间静电作用的改变，局限于将电荷氨基酸作相反电性的替换会忽略可能存 在的大量的有义突变。
发明内容
本发明的目的在于提供一种异二聚体抗体 FC的改造方法。 本发明的另一目的在于提供一种基于异二聚体抗体 FC的？丈造进一步制备异二聚体抗体 FC相关的蛋白和多肽的方法。
本发明的另一目的还在于提供所述的方法制备的异二聚体抗体 FC蛋白及多肽本身， 包 括组成异二聚体抗体 FC的单独的多肽。
本发明的另一目的还在于提供编码这些多肽的核酸序列。
本发明的最后目的还在于提供一种包含上述多肽的药物组合物。
为了实现上述目的之一， 本发明的一种异二聚体抗体 FC的改造方法， 该方法的技术方 案是： 在 FC的第一个包含 CH3区域的多肽和 /或第二个包含 CH3区域的多肽上，一个或多 个氨基酸突变为正电荷或负电荷氨基酸， 它们相互接触形成促使异二聚体形成的界面， 突变 为按以下方法选择得到的有义突变之一：
1 )随机突变抗体 FC的 CH3区域的一个或多个氨基酸为带有正电荷或负电荷的氨基酸；
2 )基于界面氨基酸之间的电荷网络作用，计算突变对于同二聚体及异二聚体的影响值： 突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0;
3 ) 筛选对于同二聚体影响值大于等于 0的突变， 或对异二聚体影响值小于等于 0的突 变。
作为本发明的进一步改进， 在 FC的第一个包含 CH3区域的多肽和 /或第二个包含 CH3 区域的多肽上，一个或多个不带电氨基酸突变为正电荷或负电荷氨基酸，它们相互接触形成 促使异二聚体形成的界面， 突变为按以下方法选择得到的有义突变之一：
1 )随机突变抗体 FC的 CH3区域的一个或多个不带电氨基酸为带有正电荷或负电荷的 氨基酸；
2 )基于界面氨基酸之间的电荷网络作用，计算突变对于同二聚体及异二聚体的影响值： 突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0;
3 ) 筛选对于同二聚体影响值大于等于 0的突变， 或对异二聚体影响值小于等于 0的突 变。 作为本发明的进一步改进， 所述的方法具体包括以下步骤：
1 ) FC的 CH3区域氨基酸序列及结构获得；
2 )界面氨基酸获取；
3 )构建电荷相互作用网络；
4 )相互作用网络中节点度的计算；
5 ) 随机突变 CH3区域的一个或多个氨基酸， 筛选有义突变。
作为本发明的进一步改进， 所述的异二聚体 FC包括人免疫球蛋白 FC
作为本发明的进一步改进， 所述的异二聚体 FC包括人免疫球蛋白 IgG FC
作为本发明的进一步改进， 所述的突变为单点突变或双点突变。
作为本发明的进一步改进，所述的突变后正电荷氨基酸为赖氨酸或精氨酸， 突变后负电 氨基酸为天冬氨酸或谷氨酸。
为实现上述另一目的， 本发明的一种异二聚体蛋白的制备方法， 该方法的技术方案是： 所述的异二聚体蛋白包括第一个包含 CH3区域的多肽和第二个包含 CH3区域的多肽， 包括 以下步骤： 1 )培养宿主细胞， 宿主细胞包含编码第一个包含 CH3区域多肽的核酸和第二个 包含 CH3区域多肽的核酸， 其中培养的宿主细胞表达第一和第二个包含 CH3区域的多肽； 2 )从宿主细胞培养物中提取异二聚体蛋白；
所述的第一个包含 CH3 区域的多肽和 /或所述的第二个包含 CH3 区域的多肽在界面上 包含一个或多个突变产生的氨基酸， 它们相互接触形成促使异二聚体形成的界面， 突变由不 带电氨基酸突变为正电荷或负电荷氨基酸， 并符合按以下方法选择得到的有义突变之一：
1 ) 随机突变 CH3区域上的一个或多个不带电氨基酸为带有正电荷或负电荷的氨基酸；
2 )基于界面氨基酸之间的电荷网络作用，计算突变对于同二聚体及异二聚体的影响值： 突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0;
3 ) 筛选对于同二聚体影响值大于等于 0的突变， 或对异二聚体影响值小于等于 0的突 变。
作为本发明的进一步改进， 所述的异二聚体蛋白包括人免疫球蛋白 IgA, IgD, IgE, IgG 或 IgM FC
作为本发明的进一步改进， 所述的异二聚体蛋白包括人免疫球蛋白 IgGl FC 作为本发明的进一步改进， 所述 IgGl FC包括第一个包含 CH3区域的多肽或者第二个 包含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序列， 它是由部分氨基酸突变为 正电荷的氨基酸的 IgG序列所组成的选择突变为带正电的氨基酸为 PHE405、 SER364. TYR407. VAL397. SER400- GLN362. VAL363 - LEU398 选择突变为带负电的氨基酸为 VAL348 - TYR349- THR350。
作为本发明的进一步改进， 所述的 IgGl FC包括第一个包含 CH3区域的多肽或者第二 个包含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序列， 它是由第一链部分氨基 酸突变为负电荷的氨基酸和第二链部分氨基酸突变为正电荷的 IgG序列所组成。
作为本发明的进一步改进， 所述的 IgGl FC包括第一个包含 CH3区域的多肽和第二个 包含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序列， 它是由第一链上 CH3区域 一个不带电氨基酸突变为正电荷的氨基酸和第二链上 CH3区域一个不带电氨基酸突变为负 电荷的 IgG序列所组成。
作为本发明的进一步改进， 所述的异二聚体蛋白为抗体、双特异性抗体、单一的单价型 抗体、 单域抗体、 多肽型抗体或双特异性多肽型抗体。
为实现上述另一发明目的， 本发明的一种异二聚体蛋白， 包括第一个包含 CH3区域的 多肽和第二个包含 CH3 区域的多肽， 其特征在于， 所述的第一个包含 CH3 区域的多肽和 / 或所述的第二个包含 CH3区域的多肽在界面上包含一个或多个突变氨基酸， 它们相互接触 形成促使异二聚体形成的界面， 突变由不带电氨基酸突变为正电荷或负电荷氨基酸，并且对 于同二聚体影响值大于等于 0, 或对异二聚体影响值小于等于 0, 影响值按以下方法计算： 突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0。
作为本发明的进一步改进， 所述的异二聚体蛋白包括人免疫球蛋白 IgA, IgE, IgD或者 IgM的 FC区域。
作为本发明的进一步改进， 所述的 FC区域包括人免疫球蛋白 IgGl FC。
作为本发明的进一步改进， 所述的 IgGl FC第一个包含 CH3区域的多肽或者第二个包 含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序列， 它是由 CH3区域上的一个不 带电氨基酸替换为正电荷或负电荷的氨基酸的 IgG序列所组成的， 选择突变为带正电的氨 基酸为 PHE405、 SER364. TYR407. VAL397. SER400- GLN362. VAL363- LEU398; 或 者选择突变为带负电的氨基酸为 VAL348、 TYR349- THR350。
作为本发明的进一步改进， 所述的 IgGl FC包括第一个包含 CH3区域的多肽或者第二 个包含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序列， 它是由第一链部分氨基 酸替换为负电荷的氨基酸和第二链部分氨基酸替换为正电荷的 IgG序列所组成的。
作为本发明的进一步改进， 所述的 IgGl FC包括第一个包含 CH3区域的多肽和第二个 包含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序列， 它是由第一链上 CH3区域 一个不带电氨基酸替换为正电荷的氨基酸和第二链上 CH3区域一个不带电氨基酸替换为负 电荷的 IgG序列所组成的。
作为本发明的进一步改进， 所述的异二聚体蛋白是抗体、双特异性抗体、单一的单价型 抗体、 单域抗体、 多肽型抗体或双特异性多肽型抗体。
为实现上述又一发明目的， 本发明的一种多肽， 包括抗体的 CH3区域， 其特征在于所 述的 CH3区域包含不同于野生型 CH3区域的多肽序列， 由野生型 CH3区域中的一个或多 个不带电荷的氨基酸突变为带正电荷氨基酸或负电荷的氨基酸所形成，突变对于同二聚体影 响值大于等于 0, 或对异二聚体影响值小于等于 0, 影响值按以下方法计算：
突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0。
为实现上述再一发明目的， 本发明还提供一种编码如上所述多肽的核苷酸序列。
为实现上诉最后一发明目的， 本发明的一种药物组合物包含如上所述的多肽。
与现有技术相比，本发明的有益效果是： 本发明提供了一个增加异二聚体含量的同时降 低其他不需要的产物如同源二聚体的方法。所获得的异二聚体蛋白主要用于医疗领域，在某 些情况下， 异二聚体蛋白可以将药物、 标记物、 细胞毒细胞、 T细胞等靶向导至肿瘤细胞， 从而更有效地发挥杀伤作用， 在肿瘤的免疫诊断及免疫治疗方面提供新的方法和途径。 附图说明
图 1为 IgGl抗体结构及不同区域示意图。
图 2为包含 FC单链的不同异二聚体蛋白组合现象。
图 3为人 IgGl FC的 CH3-CH3作用界面上氨基酸的相互作用网络图。
图 4为基于电荷网络改造 FC制备异二聚体的 SDS-PAGE分析检测结果。 具体实施方式
本发明的目的是基于 FC重链 CH3两臂氨基酸的电荷相互作用网络， 通过电荷排斥效 应修改 CH3区域氨基酸从而减少 CH3区域之间自身结合的能力 （形成同二聚体）， 从而获 得异二聚体。 一般来说， 在 CH3-CH3接触面， 修改相关氨基酸为带电荷氨基酸就会形成电 荷排斥效应， 在某些情况下突变接触面上某个带正电氨基酸（赖氨酸， 精氨酸）为负电氨基 酸（天冬氨酸， 谷氨酸）或相反， 就能形成排斥作用。
本发明描述了一个修改 FC的 CH3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）的方法。 通过建立 CH3-CH3作 用表面的氨基酸相互作用网络，获得网络中电荷氨基酸之间的作用情况，选择任意一个或者 多个氨基酸，分析所选氨基酸对同二聚体及异二聚体的影响情况，将所选氨基酸突变为带电 氨基酸， 考察突变后对于同二聚体及异二聚体的影响情况， 将突变后与突变前进行对比， 如 果突变合适，那么将会产生增强异二聚体及削弱同二聚体形成的作用。最后考察所有的氨基 酸组合及邻近氨基酸的数目 （数目越大， 则突变导致原属性的变化越大）， 选择合理的氨基 酸突变， 最大化的增强异二聚体及削弱同二聚体的作用。
本发明提供了一种修改 CH3氨基酸对 FC进行改造， 来减弱区域自身相互作用 （同二 聚体）并增强区域之间的相互作用 （异二聚体）， 并进一步获得异二聚体的方法， 所述方法 的步骤描述如下：
序列及结构获得
从蛋白质数据库（PDB, www.pdb.org )共获得 48个包含 FC区域的抗体晶体结构， 通 过结构相似性搜索算法 (Ye and Godzik 2004)。 FC同二聚体结构来自 1DN2(PDB编号）。 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。
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 4.5人。 在某些文献中也可以 选择 5.5 A(Bahar and Jernigan 1997)。 表 1为抗体第一链及抗体第二链的 CH3相互作用的氨 基酸列表。 从此结果可以看出， 第一链及抗体第二链 CH3氨基酸的相互作用不仅仅是一对 一的关系， 而是一对多或者是多对多的关系， 此结论也可以从氨基酸作用网络图 （图 2 )更 直观地看出。 表 1所列的为通过氨基酸接触筛选规则所筛选出的 34个界面氨基酸。 表 1 CH3-CH3界面氨基酸列表
链 A 中的接触氨 链 B中的接触 酸
基酸
(1Ι.Νλ47.\ i圍圖圖圍謹画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画議
VAL348A GLU356B
Ι、'Κ;4,λ、 SI R 4B,GI .11 5 B 1I Λ I357BJ .YS3ftOB
THR350A SER354B,GLU356B
1 1 1 1 ,151 L\ 1 E,VR{ m2E,VR{ ) 3B,Si;R 54B,rn IR366B
PR0352A LEU351B,PR0352B
ΙΊ");5;Λ 隱醫圍圖議謹画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画圖
SER354A TYR349B ,THR350B ,LEU351B
(;Ι Ι.ό(、,、 VAI 4 Β;1ΎΚ34 Η,Ί ΙΚ350Β,Ι
GLU357A TYR349B ,LEU368B ,LYS370B
1、、；(、< ).\ (<Ι,Ν347Β,ΊΎΚ34 ,Ι 8370Β
GLN362A LYS370B
\.\Ι .V、； Λ 議國議議議議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議議 1議議議 1議議圖議
SER364A LEU368B ,LYS370B ,TYR407B
I.I I 、5.\ 議 III!驪議画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画議
THR366A LEU351B,LEU368B,TYR407B
1 1.「；(、 iI,H357B,Si':R364B;i'HR36fiB,LYS409B
LYS370A GLU357B ,LYS360B ,GLN362B ,SER364B,LYS409B ,THR411B
.\S.\ .\ SI R400
LYS392A VAL397B ,LEU398B , ASP399B ,SER400B,PHE405B
Ι ΙΙΚ;υ;.\ 議國議謹議國議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議議議 1議議 1議議議 1議議圖
THR394A THR394B , VAL397B 'PHE405B 'TYR407B
ΙΊ");'λ；.、 )3
VAL397A LYS392B ,THR393B ,THR394B ,PR0395B
1.1 1 III圍圍圍画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画画議
ASP399A LYS392B,LYS409B,THR411B SI-.I OOA A'SW難、 LYS¾>2B
PHE405A LYS392B ,THR394B ,TYR407B,LYS409B
Figure imgf000011_0001
TYR407A THR366B,THR394B ,PHE405B ,TYR407B ,LYS409B
SI.K4()S.\ 圍議||¾國圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議圖議 1議議 1議議圖議 1
LYS409A LEU368B ,LYS370B ,ASP399B ,PHE405B ,TYR407B
ΊΙΚ4ΙΙΛ
LYS439A GLU356B
1. 构建氨基酸相互作用网络
在 CH3-CH3界面氨基酸的基 ftH上， 对氨基酸之间的相互作用配对构建氨基酸相互作用 网络（见图 2 )。氨基酸作用网络由所有 CH3-CH3界面氨基酸及任何一个界面氨基酸所包含 的与其它界面氨基酸之间的相互作用所构成。 网络图中，分别对带电氨基酸进行电荷属性标 注， 天冬氨酸、 谷氨酸为负， 精氨酸、 赖氨酸为正。
2. 相互作用网络中节点 （氨基酸）度的计算
节点度是指一个节点（氨基酸）拥有相邻节点（相互作用的氨基酸）的数目， 基于电荷 相互作用网络， 分别计算每个节点临近氨基酸的个数（见表 2)。 任何一个界面氨基酸（节 点）与其相互作用的氨基酸（相邻节点）构成该界面氨基酸的子网络。一般来说如果， 某个 氨基酸临近的氨基酸越多的话， 则突变此氨基酸更容易对蛋白稳定性产生影响。
表 2 CH3-CH3界面氨基酸节点度列表
链 A中的接触氨 节点度 链 Β中的接触氨 节点度
基酸 基酸
(;l N. ^A 圍画画画國画画 1 (;l Ν 47Ι! 圍画画画謹画画画画 III
VAL348A 1 VAL348B 1
Ί VK. ' .\ 圍謹隱隱隱國謹圖謹 1、Ί ;柳 圍謹隱隱隱議圖謹隱隱隱議
THR350A 2 THR350B 2
1 1 :01.、 謹隱隱 1謹圍謹隱隱議 圖画画画 i議画画画画 1
PR0352A 2 PR0353B 1
ΙΊ")ό;.\ 圍画画画謹画画 II SI I<;54B 圍画画画議画画画画 1
SER354A 3 GLU356B 4 (;l l';5i,.、 画画画画 II画画 1 (;ΐ.Γ 7Η 圍画画画議 1画画画画 1
GLU357A 3 LYS360B 3
1 VS.ViUA 議議議議圖薩画画 1 (;I 、WI 圍隱隱 1謹隱圖謹隱隱隱隱 i
GLN362A 1 SER364B 3
. 、； ,\ 謹 1謹隱隱隱圖謹隱隱 11 I 、(、515 圖画画画画薩画画画画 1
SER364A 3 THR366B 3
1 Ι.Γ. όΛ 画画画画議薩画画 1 l-l.l .V、Sli 圍画画画 II画画画画 1
THR366A 3 LYS370B 7
1 l r.v,s.、 隱隱隱 1謹隱國謹隱隱 圖画画画画薩画画画画 1
LYS370A 6 LYS392B 5
ASN JOA 謹 1謹隱 1111謹隱 111 Κ.^Λ^Κ 圍隱隱 1謹隱國謹隱隱 1謹
LYS392A 5 THR394B 5
1 IIKWA 画画画画議議議議 1 圖画画画画議画画画画 1
THR394A 5 VAL397B 4
l'K().^5.\ 議議議議圖議画画 1 1.1:1 Hi 圖画画画画薩画画画画 1
VAL397A 4 ASP399B 3
1 l.i ;s.\ 画画画画議薩画画 1 SI.U4(K)li 圖画画画画議画画画画 1
ASP399A 3 PHE405B 4
SI K 0n.\ 隱隱隱 1謹隱圖謹隱隱 11 I 4( if! H 圍隱隱 1謹隱圖謹隱隱隱隱 1
PHE405A 4 TYR407B 8
I.I 14(> .\ 謹 1謹隱隱隱圖謹隱隱 SI K4(»S 圖画画画画薩画画画画 1
TYR407A 8 LYS409B 5
M.K40S.\ 画画画画議薩画画 1 MII MIi 圖画画画画議画画画画 1
LYS409A 5 LYS439B 1
ΙΊΙΚ4Ι1Λ 議議議議圖議画画 1
LYS439A 1
3. 随机突变及筛选有义突变
在 CH3-CH3界面氨基酸的相互作用网络图中，随机突变第一链和 /或第二链上的一个或 多个氨基酸， 包括随机单点突变、 随机双点突变及多点突变。 计算包含所突变氨基酸的子网 络的突变前电荷总和， 及突变后电荷总和。
电荷总和=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0
计算突变对同二聚体和异二聚体的形成在电荷意义上所带来的影响：
突变影响=突变后电荷总数 -突变前电荷总数。
筛选对于对同二聚体影响大于等于 0的突变（即影响值 >=0 ), 对异二聚体影响小于等 于 0的突变 （影响值<=0 )。
综合氨基酸度（氨基酸度越小越好）和有利突变（对异二聚体所造成有利影响及对同二 聚体所造成的不利影响）， 选择合理突变。
本专利中突变后的正电荷氨基酸为赖氨酸和精氨酸，突变后的负电氨基酸为天冬氨酸和 谷氨酸。
单点突变
在 CH3-CH3接触界面上选择氨基酸突变为正电荷， 对同二聚体有负面影响的突变（影 响值 >0 )见表 3。同时对异二聚体有可能也会造成负面影响的，影响值同对同二聚体的影响。
表 3 单点突变为正电荷氨基酸所造成的影响
序号 氨基酸 正电荷氨基酸影响值
1 AS} '. WI S 4
2 ASP399A 4
3 GLU356B 2
Figure imgf000013_0001
5 GLU356A 2
議議議議議議議 i議圍議圖圖 il画画画画画画画画画画画画画議画画画画画画画画：
7 Ι Ί Ι Ι 405 H 1
8 PHE405A 2
( il N. I
10 VAL397B 1
圖画画画画画画圖謹國圍圍画画画画画画画画画画画画画画謹議議議議議議議議 I
12 SER364B 1
Figure imgf000014_0001
22 LEU398A 1
序号 1-6为负电荷氨基酸突变为正电荷氨基酸， 已为 US 2010/286374A所披露。 7-22 为不带电氨基酸突变为正电荷氨基酸。 可见，根据本发明的方法， 通过不带电氨基酸单点突 变抑制同二聚体， 可以选择以下任一方法， 即在包含 CH3区域的多肽第一链， 选择突变为 带正电的氨基酸为 PHE405、 SER364. TYR407. VAL397. SER400- GLN362. VAL363或 LEU398。
在 CH3-CH3接触界面上选择氨基酸突变为负电荷， 对同二聚体有负面影响的突变（影 响值 >0 )见表 4。
表 4 单点突变为负电荷氨基酸所造成的影响
序号 ^&酸 负电荷氨
基酸影响值
Figure imgf000014_0002
10 VAL348B 1
序号 1-4为正电荷氨基酸突变为负电荷氨基酸， 已为 US 2010/286374A所披露。 5-10 为不带电氨基酸突变为负电荷氨基酸。 可见，根据本发明的方法， 通过不带电氨基酸单点突 变抑制同二聚体， 可以选择以下任一方法， 即在包含 CH3区域的多肽第一链， 选择突变为 带负电的氨基酸为 VAL348、 TYR349或 THR350。
双点突变
在 CH3-CH3接触界面上随机突变两个氨基酸， 选择氨基酸突变为对同二聚体有负面影 响的突变 （影响值>=0)和 /或对异二聚体有正面影响的突变 (影响值<=0), 见表 5。
序号 氨基酸 突变的 氨基酸 突变的 对 A链 对 B链 异二聚 带电氨 带电氨 同二聚 同二聚 体影响 基酸 基酸 体影响 体影响
圍謹隱 1謹 圖画画薩画画 1 1 YS4()"IS 圍議圖議議 1霞圍画画議画画 1議議議隱醫議議 1圍画画謹画|
2 GLU356A 1 LYS439B -1 2 2 0 議画画画 1 1.1 1 . )SI5圍画画画画議 (ϋ.Γ 57.\圍画謹画画 1圖画画議議画画 1画画画議醫議議圖画画 1謹議1
4 PHE405A 1 LYS409B -1 2 0 0 麵画画 II 1 驪画謹謹議 1 (;l ；5(、Β圖画画議画画 1圖画画圖画画 1議議議隱議議議 1圍画画謹画 1 議画画画 1 Ι、Ί<4<17.、圖画画薩画画 1 1 YS4()"IS 圍議圖議議 1霞圍隱隱 1謹謹隱隱議議議隱醫議議 1圍画画 ill
7 ΡΗΕ405Β 1 LYS409A -1 0 2 0
8 LYS409A -1 TYR407B 1 0 1 -1 議画画画 1 1 VS4(W.\ 圍隱隱國隱 II ASI'.WIi 圍画謹画画 1圍隱隱 1薩隱隱隱 1謹隱隱議國謹隱隱圖画画 1謹議1
10 GLN347A -1 GLU357B 1 0 2 0
11 LEU368A -1 GLU357B 1 0 2 0
12 TYR349A -1 SER354B 1 1 0 0
Ι SI.R.v^h 圖画画 1議議 1 mw).\圍画画 1議圖 1圖画画画謹画画 1画画画議議議議圍画画謹議|
14 LEU368B -1 SER364A 1 1 0 0
1 圍謹隱圍謹隱 I \ ； 7.\圍謹隱圖謹隱議圍謹圖謹圖謹隱隱議議議隱議議議 1圍画画謹画|
16 SER408B -1 TYR407A 1 1 0 0
17 1 IIK.WR圖画圍 1画 II 1 VK407A謹隱隱隱圖謹 II圍隱隱 1謹謹隱隱議議議隱醫議議 1圍画画謹画|
18 SER400A 1 ASN390B -1 1 0 0 1 LI . 0 -I ΥΚ4(»λ\ 隱圖画圖圖議圍圖圖隱圖圖圖誦圖圖薩圖圖圍 II
20 TYR407A 1 THR394B -1 1 0 0
21 THR394B -1 VAL397A 1 1 0 0
22 IIIKWIi ■1議 1 \.\Ι 圖画薩画画 II圍画謹画画画 1画画画 1画画 II圖画画議
2; SI.K40SA 1 VK4H7 圖画薩画画 II圍謹隱 II謹隱議謹隱隱隱圖謹隱隱 i圖画画薩
24 THR366A -1 TYR407B 1 0 1 0
Figure imgf000016_0001
26 ASN390A -1 SER400B 1 0 1 0
2? SI-K 54A 驪画画 1 ΊΎΙ ;4υ|{ 謹隱1画画 1圍画 II画画画画画画薩画画 II圖画画議
28 SER354A 1 THR350B -1 0 1 0
2" \.\I .W7| ιιιι<;υ.ν\ ■III議 1圍謹隱國謹隱隱隱謹隱隱隱圖謹隱隱圖画画議
30 VAL397B 1 PR0395A -1 0 1 0
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序号 1-11 包括正电荷氨基酸突变为负电荷氨基酸或作相反突变， 其中部分带电氨基酸 突变已为 US 2010/286374A所披露（如 1、 2、 3、 5和 9 )。 12-33为两个不带电氨基酸分别 突变为正电或负电荷氨基酸。 可见，根据本发明的方法， 通过不带电氨基酸双点突变制备异 二聚体蛋白， 有以下两种选择：
1、 IgGl FC包括第一个包含 CH3区域的多肽和第二个包含 CH3区域的多肽的序列有 别于野生型的人免疫球蛋白序列， 它是由第一链上 CH3区域一个不带电氨基酸替换为负电 荷的氨基酸和第二链上 CH3区域一个不带电氨基酸替换为正电荷的 IgG序列所组成的。 具 体包括以下方式之一：
在包含 CH3区域的多肽第一链突变 TYR349为负电荷，在包含 CH3区域的多肽第二链 突变 SER354为正电荷；
在包含 CH3区域的多肽第一链突变 THR350为负电荷，在包含 CH3区域的多肽第二链 突变 SER354为正电荷；
在包含 CH3区域的多肽第一链突变 SER408为负电荷， 在包含 CH3区域的多肽第二链 突变 TYR407为正电荷； 在包含 CH3区域的多肽第一链突变 THR366为负电荷，在包含 CH3区域的多肽第二链 突变 TYR407为正电荷；
在包含 CH3区域的多肽第一链突变 ASN390为负电荷，在包含 CH3区域的多肽第二链 突变 SER400为正电荷；
在包含 CH3区域的多肽第一链突变 THR399为负电荷，在包含 CH3区域的多肽第二链 突变 VAL397为正电荷；
在包含 CH3区域的多肽第一链突变 PR0395为负电荷， 在包含 CH3区域的多肽第二链 突变 VAL397为正电荷；
在包含 CH3区域的多肽第一链突变 THR394为负电荷，在包含 CH3区域的多肽第二链 突变 TYR407为正电荷；
在包含 CH3区域的多肽第一链突变 LEU365为负电荷， 在包含 CH3区域的多肽第二链 突变 TYR407为正电荷；
在包含 CH3区域的多肽第一链突变 LEU368为负电荷， 在包含 CH3区域的多肽第二链 突变 SER364为正电荷。
2、 IgGl FC包括第一个包含 CH3区域的多肽和第二个包含 CH3区域的多肽的序列有 别于野生型的人免疫球蛋白序列， 它是由第一链上 CH3 区域一个不带电氨基酸替换为正电 荷的氨基酸和第二链上 CH3区域一个不带电氨基酸替换为负电荷的 IgG序列所组成的。 具 体包括以下方式之一：
在包含 CH3区域的多肽第一链突变 SER364为正电荷， 在包含 CH3区域的多肽第二链 突变 LEU368为负电荷；
在包含 CH3区域的多肽第一链突变 VAL397为正电荷， 在包含 CH3区域的多肽第二链 突变 PR0395为负电荷；
在包含 CH3区域的多肽第一链突变 VAL397为正电荷， 在包含 CH3区域的多肽第二链 突变 PR0395为负电荷；
在包含 CH3区域的多肽第一链突变 TRY407为正电荷， 在包含 CH3区域的多肽第二链 突变 SER408为负电荷；
在包含 CH3区域的多肽第一链突变 TRY407为正电荷， 在包含 CH3区域的多肽第二链 突变 THR366为负电荷；
在包含 CH3区域的多肽第一链突变 SER400为正电荷， 在包含 CH3区域的多肽第二链 突变 ASN390为负电荷； 在包含 CH3区域的多肽第一链突变 TYR407为正电荷，在包含 CH3区域的多肽第二链 突变 LEU365为负电荷；
在包含 CH3区域的多肽第一链突变 TYR407为正电荷，在包含 CH3区域的多肽第二链 突变 THR394为负电荷；
在包含 CH3区域的多肽第一链突变 VAL397为正电荷， 在包含 CH3区域的多肽第二链 突变 THR394为负电荷；
在包含 CH3区域的多肽第一链突变 VAL397为正电荷， 在包含 CH3区域的多肽第二链 突变 THR393为负电荷；
在包含 CH3区域的多肽第一链突变 SER354为正电荷， 在包含 CH3区域的多肽第二链 突变 TYR349为负电荷；
在包含 CH3区域的多肽第一链突变 SER354为正电荷， 在包含 CH3区域的多肽第二链 突变 THR350为负电荷。
此外， 在更为复杂的情况下， 可以根据本发明的方法， 对 CH3-CH3接触界面上随机突 变 3个以上、至少包括 1个不带电氨基酸的电荷，选择氨基酸突变后对同二聚体有负面影响 的突变 （影响值>=0 )和 /或对异二聚体有正面影响的突变 (影响值<=0), 以制备异二聚体蛋 白。
根据本发明方案对异二聚体 FC进行改造， 并制备异二聚体抗体的方法， 并不局限于上 文所述的单点突变和双点突变，本领域技术人员可根据本发明的宗旨，对 3个以上氨基酸进 行突变， 以形成异二聚体蛋白。
本发明也可以通过引入电荷效应来改善"把手-孔洞"模型抑制同二聚体的缺陷，在 "孔 洞" 链的基础上， 进一步通过所述的方法对异二聚体 FC进行改造， 引入电荷排斥作用， 则 "孔洞-孔洞" 链的排斥作用将加强， 最终完全的抑制 "孔洞-孔洞" 同二聚体的形成。
异二聚体分子可以用标准的实验手段从宿主细胞中纯化。例如， 当异二聚体蛋白包含了 FC,蛋白则可以用蛋白 A来纯化。纯化方法包括但不限于色 技术如体积排阻、离子交换、 亲和色傳法及超滤法。 本发明的异二聚体的分离纯化方法也包括上述各种方法的适当组合。
所述的异二聚体蛋白包括 FC,优选人免疫球蛋白 FC。在一般情况下，人免疫球蛋白 FC 区域的 CH3区域多肽来源于野生型的人免疫球蛋白 FC区域。野生型的人免疫球蛋白 FC指 发生在人群中的氨基酸序列， 当然 FC序列在个体中会有一些细微的差异。 本发明中人免疫 球蛋白 FC也包括对于野生型人免疫球蛋白 FC序列的氨基酸个别的改变， 比如， FC区域局 部某些氨基酸的改变， 如包括某些在糖基化位点突变的氨基酸， 或者其他无义的突变； 也包 括根据 "把手-孔洞" 模型突变的个别氨基酸的改变。
术语 "人免疫球蛋白 FC" 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 CH1、 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， 9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-1, IgG-2, IgG-3 , IgG-4, IgA-1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。
在具体实施方式中， 本发明所用的人免疫球蛋白 FC 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， 具体为人 IgGl FC (图 1)。 在实施例中， 所述人免疫 球蛋白 FC区域的 CH3则由 SEQ ID NO: 1所示的序列编码。
本发明涉及的哺乳动物宿主细胞包括但不限于 CHO, 293 , 骨髓瘤细胞。 宿主细胞也可 以是酵母或者原核细胞如 E. Coli。
本发明所述的异二聚体蛋白不仅仅是抗体，也可以是双特异性抗体,单一的单价型抗体， 单域抗体， 多肽型抗体， 双特异性多肽型抗体等（见图 2 )。
下面通过具体的实施例对本发明的技术方案进行详细说明。
实施例 1
该实施例将证明通过基于相互作用网络的电荷分析改造 CH3结构域可以在形成异二聚 体的同时抑制同二聚体。
融合蛋白 IL-IR-Fc和 Fc按以下方式构建：
K439D E356K 1 : 2共转
K439D E356K 1 : 4共转
K439D E356K / F405K 1 : 2共转
K439D E356K / F405K 1 : 4共转
同二聚体和异二聚体的表达情况用 SDS-PAGE (聚丙烯酰胺凝胶电泳）进行检测。检测 的原理是融合蛋白 IL-IR-Fc比 Fc具有更大的分子量， 那么在 IL-IR-Fc和 Fc混合过程中， 同二聚体 (IL-1R-Fc/IL-1R-Fc, Fc/Fc)和异二聚体 ((IL-1R-Fc/Fc)在 SDS-PAGE则具有不同的条 带位置， 通过这个原理就可以检测同二聚体与异二聚体的比例情况， 检测结果见图 4。
根据基因库中搜索到的人 IgGl的 Fc片段（ hing-CH2-CH3 )基因序列， 人工合成方法 获得人的 Fc基因， 合成好的 Fc ( SEQ ID ΝΟ: 1 )亚克隆到哺乳动物细胞表达栽体 pcDNA3.1 中， 测序验证构建质粒的准确性； 采用天根的中抽试剂盒， 按照说明书获得重组质粒 DNA 即 pcDNA3.1-Fc
人的 IgGl Fc序列如下所示（ SEQ ID ΝΟ: 1 ),编码该序列的核苷酸序列见 SEQ ID NO:2:
Figure imgf000020_0001
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
根据基因库中搜索到的人细胞白介素受体（IL-1R )基因序列， 人工合成方法获得人的 IL-1R基因， 合成好的 IL-1R基因亚克隆到重组表达栽体 pcDNA3.1-Fc中， 获得 IL-IR-Fc 融合蛋白 （SEQ ID NO:2, 下划线部分表示 Fc ), 测序验证构建质粒的准确性； 采用天才艮的 中抽试剂盒， 按照说明书获得重组质粒 DNA即 pcDNA3.1-IL-lR-Fc
IL-IR-Fc序列如下所示 （SEQ ID NO:3, 下划线部分表示 Fc ), 编码该序列的核苷酸序 列见 SEQ ID NO:4:
Figure imgf000020_0002
重组质粒 pcDNA3.1-Fc和 pcDNA3.1-IL-lR-Fc共同转染至悬浮培养的 293H 细胞， 培 养 3-4天后， 收集细胞上清。 将上清液与 proteinA琼脂糖树脂进行免疫沉淀反应， 通过非还 原条件下 SDS-PAGE电泳检测同二聚体 (IL-1R-Fc/IL-1R-Fc, Fc/Fc)和异二聚体 ((IL-IR-Fc/Fc) 的形成情况。
通过分析电荷相互作用网络修改 IL-IR-Fc或 Fc的 CH3氨基酸来减弱同二聚体的形成， 并增强异二聚体的形成，具体的突变位点详见表 6,突变体是通过重叠 PCR的方法获得的。
表 6. 突变体的具体突变位点 Fc IL-IR-Fc
Fc(WT) IL-lR-Fc(WT )
E356K K439D
E356K I F405K K439D
IL-IR-FC和 FC表达栽体共转染，最终会同时出现同二聚体 (IL-IR -Fc/ IL-IR -FC, FC/FC) 和异二聚体 (IL-IR -Fc/FC), 在野生型的情况下是 IL-IR -Fc/ IL-IR -FC, FC/FC和 IL-IR -Fc/FC的比例接近 1 : 1 : 1
当在 FC上引入 E356K, IL-IR-Fc融合蛋白上引入 K439D突变位点后， IL-1R-FC/FC 异二聚体的比例有大幅度的上升， 而同二聚体 (IL-IR-Fc/ IL-IR-FC, FC/FC)比例则大幅度下 降（见图 4 );当在 FC上引入两个突变位点 E356K/F405K , IL-IR-Fc融合蛋白上引入 K439D , 表达后蛋白基本上以 IL-1R-FC/FC异二聚体的形式存在，进一步证明电荷作用对于异二聚体 的形成至关重要。
序列表
补充完整的序列表
<110> 苏州康宁杰瑞生物科技有限公司
<120>人免疫球蛋白异二聚体 FC
<160>2
<210>1
<211> 227
<212>PTR
<213>人种 ( homo sapiens )
<220>
<221> misc—feature
<222> (58,70)
<223>人免疫球蛋白编码序列
<400>1 p ( ) homo saiens
Figure imgf000022_0001
U SU SO GS 221 LEER LEER PRLY LY
cSSNS GNS S 211 ALA rm HI A HI TYR THRL LYER
0SN V S CS S VS oe 21 AAL PHEERYERAL MET HIr
VS SG GN GN G THRAL A LYER AR TRPLLLY G SU SSU ALYER PHE PHE LE TYRER LY LE
S〇〇 VUs S TYR LY THR THR ¾R ¾RAL LE A¾ER
GU SSN G GN〇 GUSNSN TRPLER ALYL ¾RL A A
G〇 Ss o GULY 1ΉΕ TYR ¾RER A¾ ILE >r>rL
SN GN V SU CSU VS s ALALER LE THRY LEAL LY
U〇O SGs GUUS LE ¾R PRER AR A¾L LE THR LY
G GNOG GUO GN V 121LYL PR ARL PRLAL TYR THR
O GUS SSS 111 PR ILEL LY THR ILEER LY ALA LY
0 CSS V SSNSU〇 11Y LYALER A LY ALA LE ¾R ALA
GNUSN GS GUSL A TRP LE ALY LYL TYR LY
G o o S oU ocS ARrrERr LE ΊΉ»·r rm PH
OG GU GU GNSN S PR ARLLL TYR AER THR TYR G V GU VSSNSSLYALLAL HI A ALA LY THR LY
5〇 GU VSSN Vs 1 ¾RLAL LY 1ΉΕ A TRP TYRAL A¾
CS V V Vs V SS GUsYALALAL A¾ALER HIL A¾
U SG〇 GU V LE MET ILEER AR THR ¾RLAL THR
UOOSOSs 211ΉΕ LE 1ΉΕ PR PR LY PR LY A¾ THR
〇 GUUU G GO S V 11 ALA ¾RL LE LELYLY PRERAL sSS〇SOO〇SO 1 A¾ LY THR HI THRY PR PRY PR <221> 编码序列
<222> (1)..(681)
<223>人免疫球蛋白核酸序列
<400>2
Figure imgf000023_0001
ACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG
<210>3
<211> 545
<212>PTR
<213>人种 ( homo sapiens )
<223>白介素受体 1-FC融合序列 IL-IR-Fc
<400>3
I ASP LYS CYS LYS GLU ARG GLU GLU LYS ILE
II ILE LEU VAL SER SER ALA ASN GLU ILE ASP
21 VAL ARG PRO CYS PRO LEU ASN PRO ASN GLU 31 HIS LYS GLY THR ILE THR TRP TYR LYS ASP GUcU G G〇 S ocL rm LELYLY ¾RERr 1ΉΕ rm
3S CSOO CSOO21 THR HI THRY PR PRY PR ALA PR
VSN G S G G GSAL THR ALYERLYLYLY A LY
30s GNU〇1 A¾ ALA ALA TYR ILEL LE ILE TYR ¾R
9 CSSSNS G 21 THRY ¾HE ALA LY A THR HILY ILE
8 oe SGSS〇 21 ILErER AR 1ΉΕ TYR LY HI ¾R 1ΉΕ
U VUSN S GU 271 THR LE ILE THRAL LE A ILEERL
6 V GUSN〇SNSGG S 21ALL A ¾R ALA A LY AR ARER
5s〇 VU G GUs S 21 A¾ ¾RAL LELYL A¾ TYR TYRER
SSN G S oS oes LY TRP ALYERr FE APr A¾
3 G OSU Ss 21 THRLY LEER A¾ ILE >r> TYR TRP
G S GN GNU〇SSN V 221LYERL ILEL LE ILEY AAL
〇SN GU GU VsU 211 ¾R ALA AL THR METLAL A¾ LE
0SNSOG〇 V V S 21 A LY PR THR AR ¾RAL ILEALER
G V GUU GU GU ARAL ILEL PHE ILE THR LELL U GS GNO THR TYR LELY LYL TYR PR ILE THR G GSN〇SS S ARLY A TYR THRY HI ALAER TYR
U oSN o GUSS LE ILEr MET Ar ALAL LY HI
SNS S G VSG A ILE HI PHEERLYAL LY A AR
Ss〇SSOUUUs TYR LY A¾Y LY PR LE LE LE A¾
GUSNSN GUU〇SU GNL A AL LE ¾R LY LEL TRP
o CSO GUSSN 121rY PR TYR METL ΙΉΕ 1ΉΕ LY A
SU〇 V Gs G GU 111 LY LE ¾RAL ALALY A¾LYLY LE
〇SSN GNS GNY TYR A ALAL ALA ILE ¾HE LYL
S V GUSN GU〇SNU ALA LY PHEALL AL ¾R A LE
S S CSUGS SERER TYRY LE AR ILE LY ILEER S GS CS V VGSNERLY HI TYR TYRYALAL AR A U V〇S V GUs LE TRP 1ΉΕAL ¾R ALA LYALL A¾
SGS GNSS GUS ALAER AR ILE HIL HI LYL LY
s SS〇 V S GU GN41 A¾ER LY THR ¾RALER THRLL -
Figure imgf000025_0001
53SSNS GNS SU S1 HI A HI TYR THRL LYER LEER
5 S〇S S VS GUU21 PHEERYERAL MET HIL ALA LE
5S SG GN GN GSN V11 A LYER AR TRPLLLY AAL
50 SU SSc o1ER ΙΉΕ 1ΉΕ LE TYRER LY rm THRr
§〇〇 VUs Ss G THR THR ¾R ¾RAL LE A¾ER A¾LY
§ SSN G GN〇 GUSNSNSER ALYL ¾RL A A TYR LY
〇 Ss V GU GU 471 TYR ¾RER A¾ ILE ALAALL TRPL
§ V SU CSU VS GALER LE THRY LEAL LYLY ¾HE
〇 SGs GUUSSN GN ¾RER AR A¾L LE THR LY AL
OG GUO GN VUO 441 PR ARL PRLAL TYR THR LE PR
§ GUS SSS G GNL LY THR ILEER LY ALA LYLYL
V SSNSU〇〇 421ALER A LY ALA LE ¾R ALA ¾R ILE
USN GS GUS〇SS s TRP LE ALY LYL TYR LYY LY
§ o S oU oUS o¾srERr LE ΊΉΚ·r LE HIr A¾
39 GU GU GNSN SG V1LLL TYR AER THR TYR ARAL
38 GU VSSNSS〇G1LAL HI A ALA LY THR LY ¾R AR
3 oSSN os G o71r LY 1ΉΕ A TRP TYRr A¾rYr
36 o os o SS GUS〇 oe1rr A¾rER HIL A¾ ¾Rr
35 SG〇 GU o CS o1 ILEER AR THR ¾RLr THRYr
3OOSOSSU41 PHE PR PR LY PR LY AP THR LE MET <223>白介素受体 1-FC融合序列 IL-1R-FC核苷酸序列 <400>4
9VVD99DDDD9V9XDD9V9XDD9V9VV9VDDDVDVXD
Figure imgf000027_0001
U9LLO/ZlOZ l3/13d oci7.60/cioz OAV

Claims

权利 要求 书
1.一种基于电荷网络的异二聚体 FC？丈造方法， 其特征在于， 在 FC的第一个包含 CH3 区域的多肽和 /或第二个包含 CH3区域的多肽上， 一个或多个氨基酸突变为正电荷或负电荷 氨基酸， 它们相互接触形成促使异二聚体形成的界面， 突变为按以下方法选择得到的有义突 变之一：
1 )随机突变抗体 FC的 CH3区域的一个或多个氨基酸为带有正电荷或负电荷的氨基酸；
2 )基于界面氨基酸之间的电荷网络作用，计算突变对于同二聚体及异二聚体的影响值： 突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0;
3 ) 筛选对于同二聚体影响值大于等于 0的突变， 或对异二聚体影响值小于等于 0的突 变。
2.根据权利要求 1所述的异二聚体 FC？丈造方法，其特征在于，在 FC的第一个包含 CH3 区域的多肽和 /或第二个包含 CH3区域的多肽上， 一个或多个不带电氨基酸突变为正电荷或 负电荷氨基酸， 它们相互接触形成促使异二聚体形成的界面， 突变为按以下方法选择得到的 有义突变之一：
1 )随机突变抗体 FC的 CH3区域的一个或多个不带电氨基酸为带有正电荷或负电荷的 氨基酸；
2 )基于界面氨基酸之间的电荷网络作用，计算突变对于同二聚体及异二聚体的影响值： 突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0;
3 ) 筛选对于同二聚体影响值大于等于 0的突变， 或对异二聚体影响值小于等于 0的突 变。
3.根据权利要求 1或 2所述的异二聚体 FC？丈造方法， 其特征在于， 所述的方法具体包 括以下步骤：
1 ) FC的 CH3区域氨基酸序列及结构获得； 2 )界面氨基酸获取；
3 )构建电荷相互作用网络；
4 )相互作用网络中节点度的计算；
5 ) 随机突变 CH3区域的一个或多个氨基酸， 筛选有义突变。
4. 根据权利要求 1或 2所述的异二聚体 FC ?丈造方法， 其特征在于， 所述的异二聚体 FC包括人免疫球蛋白 FC。
5.根据权利要求 4所述的异二聚体 FC？丈造方法， 其特征在于， 所述的异二聚体 FC包 括人免疫球蛋白 IgG FC。
6.根据权利要求 1或 2所述的异二聚体 FC改造方法， 其特征在于， 所述的突变为单点 突变、 双点突变或者多点突变。
7.根据权利要求 1或 2所述的异二聚体 FC？丈造方法， 其特征在于， 所述的突变后正电 荷氨基酸为赖氨酸或精氨酸， 突变后负电氨基酸为天冬氨酸或谷氨酸。
8.—种异二聚体蛋白的制备方法， 所述的异二聚体蛋白包括第一个包含 CH3 区域的多 肽和第二个包含 CH3区域的多肽， 包括以下步骤： 1 )培养宿主细胞， 宿主细胞包含编码第 一个包含 CH3区域多肽的核酸和第二个包含 CH3区域多肽的核酸，其中培养的宿主细胞表 达第一和第二个包含 CH3区域的多肽； 2 )从宿主细胞培养物中提取异二聚体蛋白；
其特征在于：
所述的第一个包含 CH3 区域的多肽和 /或所述的第二个包含 CH3 区域的多肽在界面上 包含一个或多个突变产生的氨基酸， 它们相互接触形成促使异二聚体形成的界面， 突变由不 带电氨基酸突变为正电荷或负电荷氨基酸， 并符合按以下方法选择得到的有义突变之一：
1 ) 随机突变 CH3区域上的一个或多个不带电氨基酸为带有正电荷或负电荷的氨基酸；
2 )基于界面氨基酸之间的电荷网络作用，计算突变对于同二聚体及异二聚体的影响值： 突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0;
3 ) 筛选对于同二聚体影响值大于等于 0的突变， 或对异二聚体影响值小于等于 0的突 变。
9.根据权利要求 8所述的异二聚体蛋白的制备方法， 其特征在于， 所述的异二聚体蛋白 包括人免疫球蛋白 IgA, IgD, IgE, IgG或 IgM FC。
10.根据权利要求 9所述的异二聚体蛋白的制备方法， 其特征在于， 所述的异二聚体蛋 白包括人免疫球蛋白 IgGl FC。
11.根据权利要求 10所述的异二聚体蛋白的制备方法， 其特征在于， 所述 IgGl FC包括 第一个包含 CH3区域的多肽或者第二个包含 CH3区域的多肽的序列有别于野生型的人免疫 球蛋白序列， 它是由 CH3区域上的一个不带电氨基酸突变为正电荷的氨基酸的 IgG序列所 组成的选择突变为带正电的氨基酸为 PHE405、 SER364. TYR407. VAL397. SER400- GLN362. VAL363 - LEU398选择突变为带负电的氨基酸为 VAL348、 TYR349- THR350。
12.根据权利要求 10所述的异二聚体蛋白的制备方法， 其特征在于， 所述的 IgGl FC包 括第一个包含 CH3区域的多肽或者第二个包含 CH3区域的多肽的序列有别于野生型的人免 疫球蛋白序列，它是由第一链部分氨基酸突变为负电荷的氨基酸和第二链部分氨基酸突变为 正电荷的 IgG序列所组成。
13.根据权利要求 10所述的异二聚体蛋白的制备方法， 其特征在于， 所述的 IgGl FC包 括第一个包含 CH3区域的多肽和第二个包含 CH3区域的多肽的序列有别于野生型的人免疫 球蛋白序列， 它是由第一链上 CH3区域一个不带电氨基酸突变为正电荷的氨基酸和第二链 上 CH3区域一个不带电氨基酸突变为负电荷的 IgG序列所组成。
14.根据权利要求 8所述的异二聚体蛋白的制备方法， 其特征在于， 所述的异二聚体蛋 白为抗体、 双特异性抗体、 单一的单价型抗体、 单域抗体、 多肽型抗体或双特异性多肽型抗 体。
15.—种异二聚体蛋白，包括第一个包含 CH3区域的多肽和第二个包含 CH3区域的多肽， 其特征在于， 所述的第一个包含 CH3 区域的多肽和 /或所述的第二个包含 CH3 区域的多肽 在界面上包含一个或多个突变氨基酸， 它们相互接触形成促使异二聚体形成的界面， 突变由 不带电氨基酸突变为正电荷或负电荷氨基酸， 并且对于同二聚体影响值大于等于 0, 或对异 二聚体影响值小于等于 0, 影响值按以下方法计算：
突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0。
16.根据权利要求 15所述的异二聚体蛋白， 其特征在于， 所述的异二聚体蛋白包括人免 疫球蛋白 IgA, IgE, IgD或者 IgM的 FC区域。
17.根据权利要求 16所述的异二聚体蛋白， 其特征在于， 所述的 FC区域包括人免疫球 蛋白 IgGl FC。
18.根据权利要求 17所述的异二聚体蛋白， 其特征在于， 所述的 IgGl FC第一个包含 CH3区域的多肽或者第二个包含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序列， 它是由 CH3区域上的一个不带电氨基酸替换为正电荷或负电荷的氨基酸的 IgG序列所组成 的，选择突变为带正电的氨基酸为 PHE405、 SER364. TYR407. VAL397. SER400- GLN362. VAL363 - LEU398; 或者选择突变为带负电的氨基酸为 VAL348、 TYR349- THR350。
19.根据权利要求 17所述的异二聚体蛋白， 其特征在于， 所述的 IgGl FC包括第一个包 含 CH3区域的多肽或者第二个包含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序 列， 它是由第一链部分氨基酸替换为负电荷的氨基酸和第二链部分氨基酸替换为正电荷的 IgG序列所组成的。
20.根据权利要求 17所述的异二聚体蛋白， 其特征在于， 所述的 IgGl FC包括第一个包 含 CH3区域的多肽和第二个包含 CH3区域的多肽的序列有别于野生型的人免疫球蛋白序列， 它是由第一链上 CH3区域一个不带电氨基酸替换为正电荷的氨基酸和第二链上 CH3区域一 个不带电氨基酸替换为负电荷的 IgG序列所组成的。
21.根据权利要求 15所述的异二聚体蛋白， 其特征在于， 所述的异二聚体蛋白是抗体、 双特异性抗体、 单一的单价型抗体、 单域抗体、 多肽型抗体或双特异性多肽型抗体。
22.—种多肽， 包括抗体的 CH3 区域， 其特征在于所述的 CH3 区域包含不同于野生型 CH3区域的多肽序列， 由野生型 CH3区域中的一个或多个不带电荷的氨基酸突变为带正电 荷氨基酸或负电荷的氨基酸所形成， 突变对于同二聚体影响值大于等于 0, 或对异二聚体影 响值小于等于 0, 影响值按以下方法计算：
突变影响值 =突变后电荷总数-突变前电荷总数
电荷总数=所突变的氨基酸的子网络正负电荷之和
其中， 正正电荷相互作用为 1 , 负负电荷相互作用为 1 , 正负电荷相互作用为 -1 , 正 /非 电荷或者负 /非电荷作用为 0。
23.编码权利要求 22所述多肽的核苷酸序列。
24.—种药物组合物， 其特征在于其中包含权利要求 23所述的多肽。
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