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WO2018038469A1 - Procédé de confirmation de production sélective d'anticorps bispécifique souhaité - Google Patents

Procédé de confirmation de production sélective d'anticorps bispécifique souhaité Download PDF

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Publication number
WO2018038469A1
WO2018038469A1 PCT/KR2017/009043 KR2017009043W WO2018038469A1 WO 2018038469 A1 WO2018038469 A1 WO 2018038469A1 KR 2017009043 W KR2017009043 W KR 2017009043W WO 2018038469 A1 WO2018038469 A1 WO 2018038469A1
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WIPO (PCT)
Prior art keywords
antibody
light chain
variable
crab
heavy chain
Prior art date
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PCT/KR2017/009043
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English (en)
Korean (ko)
Inventor
김호언
배소현
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Ibentrus Inc
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Ibentrus Inc
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Publication date
Priority claimed from KR1020170104379A external-priority patent/KR101933656B1/ko
Application filed by Ibentrus Inc filed Critical Ibentrus Inc
Publication of WO2018038469A1 publication Critical patent/WO2018038469A1/fr
Anticipated expiration legal-status Critical
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present invention relates to a method for identifying bispecific antibody production capable of confirming whether a desired bispecific antibody is produced or whether a desired bispecific antibody is selectively made.
  • Bispecific antibodies are antibodies with two antigen binding sites, each of which recognizes a different target antigen, which collectively refers to an antibody or antigen binding fragment thereof that can simultaneously bind to the different target antigen.
  • the dual targeting ability of such bispecific antibodies may provide new fields of application that are not applicable with monospecific antibodies (Monospeci ant ibodies). From a therapeutic point of view, (1) reliably introducing immune cells into the vicinity of the target cell, (2) synergistic effects by simultaneously inhibiting or activating two distant signal transduction pathways of the target cell, (3) Specific and controllable delivery of therapeutic, radioactive materials, medicines, toxins, etc. to target cells has emerged as an important concern.
  • bispecific antibody preparation technology For the development of efficient bispecific antibody production techniques, the evaluation of the production of bispecific antibodies of the desired combination and / or the ratio of production of the bispecific antibodies of the desired combination, among 10 possible combinations as shown in FIG. Thus, the development of a means by which bispecific antibody preparation technology can evaluate the efficiency or selective preparation rate of a desired bispecific antibody is Required.
  • the inventors of the present invention prepared a antibody having one heavy chain and one light chain having no variable region in a wild-type antibody having two heavy chains and two light chains, and then confirming antigen binding, thereby determining the desired heavy chain-light chain and heavy chain-.
  • the present invention has been completed with the discovery that it is possible to easily and accurately identify and / or evaluate whether bispecific enclosures with heavy chain combinations are selectively produced and / or selective production levels (volume or ratio of the desired bispecific antibody).
  • One example provides a method for identifying and / or evaluating the selective production of a desired bispecific antibody.
  • Another example provides a method of identifying and / or evaluating the selective production of a desired bispecific antibody in an antibody preparation method (bispecific antibody preparation method).
  • FIG. 1 When a bispecific antibody is prepared using the heavy chain A and the light chain a that bind one antigen, and the heavy chain B and the light chain b that binds the other antigen, an antibody including two heavy chains and two light chains is as shown in FIG. 1. 10 combinations can be made (in FIG. 1, the antibodies in which heavy chain A and light chain a and heavy chain B and light chain b are correctly paired are indicated by dotted circles). At this time, one heavy and light chains (for example, heavy chain B and light chain b) were prepared in such a manner that the variable portion was removed, thereby producing a combination of antibodies in the case of producing a bispecific antibody is shown in FIG. In FIG.
  • FIG. 3 schematically shows an antibody (indicated by a dotted circle in FIG. 2) in which two heavy and two light chains are correctly paired using a set of heavy and light chains lacking a variable portion. At light weight the antibody combinations illustrated in FIG.
  • the heavy weights are grouped into groups 1 through 5, with the heaviest gains for a combination of two relatively heavy (long chain) heavy chains and two relatively heavy (long chain) light chains.
  • the lightest weight is represented as one group (see FIG. 4).
  • the bispecific antibodies of interest belong to group 3 because the combination of the relatively heavy and heavy light chains and the combination of the relatively light and heavy light chains form a dimer and have a medium weight (indicated by the dashed circle). ). Since the antibodies belonging to the three groups have almost the same molecular weight, they can be distinguished from other groups by conventional methods such as electrophoresis. However, even if it was confirmed that the antibodies belonging to the three groups were produced, since it contains mismatched dimers in addition to the desired bispecific antibody, there is a limit in accurately determining whether the desired bispecific antibody has been formed.
  • the heavy chain (without the variable part) including the variable part in the antibody combination (Fig. 3) corresponding to the desired bispecific antibody (heavy chain of A chain in Fig. 3) and Affinity chromatography was performed using an antigen to which the light chain (light chain of A chain in FIG. 3) binds, and includes a combination of a heavy chain including a variable portion and a light chain including a variable portion among the antibodies belonging to the three groups shown in FIG. 4.
  • step (1) analyzing the antibody contained in the result obtained in step (1).
  • the preparation of the antibody in step (1) can be carried out by conventional antibody preparation methods, such as all methods commonly used for the preparation of bispecific antibodies (eg, recombinant methods, chemical methods, etc.).
  • the preparation of the antibody of step (1) may include a gene encoding the heavy chain, the crab-2 light chain, and the heavy chain which does not include some or all of the variable parts, and the light chains that do not include some or all of the variable parts.
  • the recombinant vector obtained by cloning the vector can be carried out by a recombinant method comprising the step of expressing in a suitable host cell.
  • the second heavy chain, which does not include some or all of the variable parts, and the second light chain, which does not include some or all of the variable parts, include a full-length heavy chain including the variable part by lacking the variable part of the second heavy chain and the crab light chain capable of binding to a second antigen, and It means having a molecular weight difference of a degree distinguishable from the full length light chain.
  • the first and crab antigens may be different proteins or different parts of the same protein that are not overlapping (separated so as not to interfere with each other when combined with different antibodies), preferably different proteins. have.
  • steps (3) and (4) are performed after the chromatography of step (2), and step (5) is performed after the antibody preparation step of step (1) and the chromatography step of step (2) It can be done before, after, or simultaneously.
  • steps (3) to (5) they may be performed simultaneously or sequentially.
  • the method is
  • the eluate of step (3) is obtained by eluting the adsorbent after performing the chromatography of step (2) with a commonly used elution buffer, and includes an antibody adsorbed on the adsorbent.
  • Analyzing the antibody contained in the eluate of step (3) may be performed by detecting an antibody including two heavy chains and two light chains, regardless of whether the eluate contains intact variable parts.
  • the eluate contains an antibody capable of binding a first antigen, i.e., an intact C 1 antibody (half antibody; hal f-ant ibody) comprising a full l-length U heavy chain and a full length first light chain. Only included. At this time, only the antibody having a molecular weight corresponding to the desired bispecific antibody (that is, the antibody of the full length first heavy chain-full length crab 1 light chain and the variable part lacking second heavy chain-lacking variable crab 2 light chain combination) is detected.
  • the higher the level of the antibody having the molecular weight detected ie, the higher the concentration of the antibody having the molecular weight contained in the eluate; for example, as compared to the level (concentration) of the antibody having a molecular weight different from the molecular weight.
  • the selective production rate of the desired bispecific antibody comprising not only intact Crab 1 antibodies but also well-matched 12 antibodies (including half variable chains lacking the variable region and two light chains lacking the variable region) This can be identified as high and / or evaluated.
  • the selective production rate of the paired target bispecific antibody is confirmed to be high and / or
  • an antibody level having a molecular weight corresponding to a desired bispecific antibody in the eluate ie, an antibody concentration having a molecular weight corresponding to a desired bispecific antibody included in the eluate
  • Antibody levels with a molecular weight different from the molecular weight The eluate is higher than the objective antibody concentration having a molecular weight different from the molecular weight of a bispecific antibody comprising a) may be a "premises.
  • the molecular weight of the desired bispecific antibody includes a full length first heavy chain, a full length first light chain, a double heavy chain not including some or all of the variable parts, and some or all of the variable parts.
  • step (3) only the antibody having a molecular weight corresponding to the desired bispecific antibody in the eluate is detected, or the molecular weight at which the level of the antibody having a molecular weight corresponding to the desired bispecific antibody is different.
  • the identification and / or evaluation that the desired bispecific antibody is selectively produced or that the method for producing the antibody used in step (1) selectively produces the desired bispecific antibody may be further included.
  • Analyzing the antibody contained in the flow-through of step (4) is carried out in the flow-through, whether or not including the intact variable region, the antibody comprising two heavy chains and two light chains ('intact ant ibody
  • all antibodies not specifically referred to as 'half antibody' can be performed by detecting an antibody including two heavy chains and two light chains regardless of whether they contain variable regions.
  • the impurities Of the desired bispecific antibody comprising a secret type first antibody (half antibody; including one heavy chain and one light chain) capable of binding to a first antigen with low production rate and a first heavy and crab light pair paired correctly High selective production rates can be identified and / or evaluated.
  • the method after step (4), if the antibody is not detected in the flow-through solution or the level is low, the desired bispecific antibody is correctly paired with the first heavy chain and the first light chain constituting the Crab antibody Is optionally generated or the method for producing the antibody used in step (1) further comprises the step of confirming and / or evaluating the production of the desired bispecific antibody (step (4-1)). can do.
  • step (2) performing chromatography on the antibody prepared in step (1) using an adsorbent to which the first antigen is bound;
  • step (3) may be performed by detecting the antibody in the eluate
  • step (4) may be performed by detecting the antibody in the flow through.
  • the desired duplex comprising not only the first paired antibody (half antibody) correctly paired, but also the second antibody (half antibody) paired correctly with the second heavy chain lacking the variable region and the second light chain lacking the variable region Selective generation and / or selective production rate of the specific antibody can be confirmed and / or evaluated
  • Selective production and / or selective production rate of the desired bispecific antibody comprising can be identified and / or evaluated more accurately (or supplementally).
  • a second antibody (half antibody) paired correctly with a first paired antibody (half antibody) It may be more advantageous to confirm and / or evaluate the selective production and / or evaluation of the desired bispecific antibody, and / or to identify and / or evaluate the selective production or selective production efficiency of the desired bispecific antibody in a method of preparing the bispecific antibody.
  • the test subject in order to confirm and / or evaluate the selective production of the desired bispecific antibody of the test method, the test subject The steps (1), (2), and (3) are performed for the production method and the comparison target production method, respectively, and the results obtained in step (3) are compared with each other.
  • an antibody level having a molecular weight different from that corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be tested ie, the molecular weight corresponding to the desired bispecific antibody included in the eluate.
  • the test subject manufacturing method can identify and / or evaluate that the selective production efficiency of the desired bispecific antibody or the production rate of the desired bispecific antibody is high as compared with the comparative subject manufacturing method.
  • an antibody level ie, having a molecular weight corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be tested
  • Antibody concentration having a molecular weight corresponding to the desired bispecific antibody contained in the eluate ie, the level of the antibody having a molecular weight corresponding to the desired bispecific antibody in the eluate obtained in step (3) using the preparation method of comparison
  • the concentration of the antibody having a molecular weight corresponding to the desired bispecific antibody contained in the eluate ie, having a molecular weight corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be tested
  • an antibody level having a molecular weight different from that corresponding to the desired bispecific antibody in the eluate detected in step (3) of the preparation method to be tested i.e., the molecular weight corresponding to the desired bispecific antibody in the eluate
  • Antibody levels having different molecular weights i.e., the desired duplex contained in the eluate
  • the test subject manufacturing method may further comprise the step (3-2) of confirming and / or evaluating that the selective production efficiency of the desired bispecific antibody or the rate of production of the desired bispecific antibody is high as compared with the comparative subject manufacturing method. Can be.
  • the method of confirming and / or evaluating the selective production of the desired bispecific antibody of the method of producing the bispecific antibody is tested after the step (3) and before the identification and / or evaluation (3-2). Comparing the antibody level having a predetermined molecular weight in the eluate obtained in step (3) using the subject preparation method with the antibody level having a predetermined molecular weight in the eluate obtained in step (3) using the subject preparation method It may further include.
  • the method may include one or more of steps (3-1) and (3-2) in any order.
  • the test target production method may It can be confirmed and / or evaluated that the selective production efficiency of the bispecific antibody or the production rate of the desired bispecific antibody is high.
  • the method of confirming and / or evaluating the selective production of the desired bispecific antibody of the bispecific antibody production method after step (2), the analysis result in step (4) of the test target production method, the pass-through solution
  • no antibody ie, no antibody is present in the flow through
  • the antibody level in the flow through is lower than the antibody level in the flow through obtained in step (4) using the preparation method to be compared.
  • the production method to be tested may further include the step (4-2) of identifying and / or evaluating that the selective production efficiency of the desired bispecific antibody or the production rate of the desired bispecific antibody is high compared to the production method to be compared. have.
  • the method for confirming and / or evaluating the selective production of the desired bispecific antibody of the method for producing a bispecific antibody is tested after the step (4) and before the step of identifying and / or evaluating (4-2).
  • Comparing the antibody levels in the flow-through obtained in step (4) using the subject preparation method may further comprise comparing the antibody levels in the flow-through obtained in step (4) using the subject preparation method.
  • the method may include one or more of steps (4-1) and (4-2) regardless of order.
  • step (1) the method for confirming and / or evaluating the selective production of the desired bispecific antibody of the bispecific antibody production method is described in step (1) to
  • steps (3-1) and (3-2), and one or more of steps (4-1) and (4-2) eg, step (1), ( 2), (3), (3-1), (4) and (4- 1), or include steps (1), (2), (3), (3-2), (4) and ( 4-2)), optionally (when step (3-2) and / or (4-2) are included), and further comparing the results with the results of the corresponding comparison method respectively. It may include. Details are as described above.
  • (5) may comprise the step of analyzing the antibody contained in the result (manufacturing product) obtained in step (1).
  • the product used in step (5) may be a product obtained in step (1), or may be subjected to a conventional antibody purification method (for example, protein A affinity chromatography, etc.) to increase the antibody concentration.
  • a conventional antibody purification method for example, protein A affinity chromatography, etc.
  • the antibody can be carried out by detecting the antibody according to the molecular weight in the result of the step (5).
  • the antibody having a molecular weight corresponding to the desired bispecific antibody as described above, or the higher the detection level of the antibody having the molecular weight (that is, the higher the concentration of the antibody having the molecular weight included in the result) For example, the higher the level (concentration) of an antibody having a molecular weight different from the above molecular weight, the higher the selective production rate of the desired bispecific antibody can be identified and / or evaluated.
  • the method after step (5), if only the antibody having a molecular weight corresponding to the desired bispecific antibody in the result is detected, or if the detection level is higher than the antibody level having a different molecular weight, the desired bispecific Confirming and / or evaluating that the antibody is selectively produced or that the method for producing the antibody used in step (1) selectively produces the desired bispecific antibody (step (5-1)) It can be included as.
  • step (5) is performed on the result before the chromatography using the C antigen 1 of step (2), the antibody having a molecular weight corresponding to the desired bispecific antibody may be combined with other combinations in addition to the desired bispecific antibody. May be included (see group 3 in FIG. 4). However, an antibody having a molecular weight corresponding to the desired bispecific antibody is detected in step (5), and an antibody having a molecular weight corresponding to the desired bispecific antibody in the eluate is detected in step (3), and / or If no antibody is detected in the flow-through in step (4), the probability of selectively producing the desired bispecific antibody is increased. Therefore, step (5) may be further included in addition to steps (1) (2), and (3) and / or (4).
  • Another example is the coding gene of the crab 1 heavy chain, the coding gene of the first light chain, A recombinant vector comprising the coding gene of the second heavy chain lacking part or all of the variable portion, and the coding gene of the second light chain lacking part or all of the variable portion, and the coding gene of the first heavy chain, encoding of the first light chain
  • a recombinant vector comprising the coding gene of the second heavy chain lacking part or all of the variable portion, and the coding gene of the second light chain lacking part or all of the variable portion, and the coding gene of the first heavy chain, encoding of the first light chain
  • a recombinant cell comprising a gene encoding a double heavy chain gene lacking part or all of a variable portion, and a coding gene of a double light chain lacking a portion or all of a variable portion.
  • the recombinant vector may be a transformed host cell with the recombinant vector.
  • compositions for identifying and / or evaluating production include the selective generation and / or evaluation of a bispecific antibody comprising at least one selected from the group consisting of the recombinant vector and the recombinant cell, and / or the selection of the desired bispecific antibody in a method of preparing the bispecific antibody.
  • 'antibody' may be one or more selected from all kinds of immunoglobulins (immunoglobulin) derived from all kinds of mammals or birds.
  • immunoglobulins immunoglobulin derived from all kinds of mammals or birds.
  • antibodies used herein include IgG (eg, IgG type 1 (IgGl), IgG type 2 (IgG2), IgG type 3 (IgG3), and IgG type 4 (IgG4)), IgA (eg, IgA type 1 (IgAl) and I g A type 2 (IgA2)), IgD, IgE, and IgM.
  • the antibody may be an immunoglobulin derived from a mammal, such as a human, a primate including a mouse, a rodent including a mouse, a rat, and the like, for example, an immunoglobulin derived from a human.
  • the antibody comprises human IgGl (constant region; protein: GenBank Accession No. AAC82527.1, gene: GenBank Accession No. J00228.1), human IgG2 (constant region; protein: GenBank Accession No. AAB59393.1, Gene: GenBank Accession No. J00230.1), human IgG3 (constant region; protein: GenBank Accession No. P01860, gene: GenBank Accession No.
  • human IgG4 (constant region; protein: GenBank Accession No. AAB59394. 1, gene: GenBank Accession No. K01316.1), human IgAl (constant region; protein: GenBank Accession No. AAT74070.1, gene: GenBank Accession No. AY647978.1), human IgA2 (constant region; protein: GenBank Accession No. AAB59396.1, Gene: GenBank Accession No. J00221.1), human IgD (constant region; protein: GenBank Accession No. AAA52771.1, AAA52770.1), human IgE (constant region; protein: GenBank Accession No. AAB59395.1, genes: GenBank Accession No. J00222.
  • the antibody may be, for example, one or more selected from the group consisting of human, IgGl, IgG2, IgG3, and IgG4, but is not limited thereto.
  • the amino acid sequences of the heavy and light chain constant regions of the antibody have a very high degree of subtype preservation.
  • 'an intact antibody' includes two heavy chains and two light chains, and includes one light chain and one heavy chain (Hal f-ant), regardless of whether they contain intact variable regions.
  • body two means a disulfide-linked form of antibody in a heavy chain (eg, hinge region), and the term 'antibody' described herein without reference to two heavy chains and two light chains, regardless of whether they contain intact variable regions.
  • the half antibody means a structure in which one heavy chain and one light chain are disulfide-linked regardless of whether the intact variable region is included, and the first antibody or the second antibody is included in the half antibody.
  • 'first antibody' and 'second antibody' are monovalent antibodies each comprising one heavy chain and one light chain, and recognize different antigens or different epitopes. Because of the nature of the methods provided herein, one of the 'first antibody' and 'second antibody' (assuming a second antibody herein) includes heavy and light chains that lack all or part of the variable portion. And substantially lost binding ability to the antigen (assumed herein as the second antigen). As described above, the first antibody or the second antibody is included in the half antibody, regardless of whether the secret variable portion is included.
  • 'bispecific antibody' refers to an antibody that recognizes and / or binds two different antigens or two distinct (non-duplicate) epitopes of one antigen.
  • a 'bispecific antibody of interest' refers to a first antibody (half antibody) that recognizes a desired first antigen or first epitope and a second that recognizes a desired second antigen or second epitope.
  • the antibody refers to an antibody to which an antibody (half antibody) is bound.
  • the first heavy chain and the first light chain, which constitute the first antibody, and the second light It means an antibody in which the second heavy chain and the second light chain, and the first heavy chain and the second heavy chain constituting the antibody are exactly matched.
  • the first desired bispecific antibody in order to confirm the production of the desired bispecific antibody, since the second heavy chain and the second light chain are each used in a state in which all or part of the variable part is missing, the first desired bispecific antibody 'is the full-length first heavy chain. And full length first light chain pairing, between a second heavy chain lacking a variable portion and a second light chain lacking a variable portion, and between a full length first heavy chain and a second heavy chain lacking a variable portion (between Fc domains) Means the antibody formed.
  • the preparation of the bispecific antibody in step (1) may be by conventional recombinant methods.
  • the preparation of the bispecific antibody comprises a recombinant vector (expression vector) comprising a gene encoding the first heavy chain, the first light chain, the second heavy chain, and the second light chain, respectively, or two or more thereof, in a host cell. Expression (eg, co-expression in one cell).
  • the genes may be expressed by culturing a recombinant vector including the recombinant vector.
  • the "host cell 1" refers to a cell into which the recombinant vector is introduced, and the host cell into which the recombinant vector is introduced is called a recombinant cell.
  • the host cell may be a viral cell, a bacterial cell, a eukaryotic cell, a nasal cell, or a plant. Cells, or animal cells, for example, E.
  • mice cells eg, COP, L, C127, S P 2/0, NS-0, NS-1, At 20, NIH3T3, etc.
  • rat cells PC12, PC12h, GH3, MtT, etc.
  • hamster cells e.g., BHK, CHO, GS gene defect CHO, DHFR gene defect CH0, etc.
  • monkey cells e.g., C0S1, C0S3, C0S7, CV1, etc.
  • Vero et al. Human cells
  • Human cells eg, Hela, HEK-293, HEK-293-producing cells, retinal-derived PER-C6, diploid fibroblasts, myeloma cells, HepG2, NS / 0 cells, and lymph Constituent cells, etc.
  • insect cells eg Sf9 cells, Sf21 cells
  • Tn-368 cells such as BTI-TN-5B1-4 cells
  • Such vectors include viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors.
  • the recombinant vector comprises a first heavy chain, a first light chain, a second heavy chain lacking part or all of the variable portion, and
  • plasmids used in the art eg, pcDNA series, pSC10, P GV1106, pACYC177, ColEl, pKT230, P ME290, PBR322, P UC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX series, ET series, pUC19, etc.
  • phage e.g., ⁇ ⁇ 4 ⁇ ⁇ , ⁇ -Charon, ⁇ ⁇ ⁇ , M
  • 'heavy chain (or lacking a variable portion) heavy chain that does not include some or all of the variable portion' and 'light chain (or lacking a variable portion) that does not include some or all of the variable portion' refers to the full-length heavy chain and It means a state in which all or part of the variable part is removed from the light chain of the full length.
  • the reason for the lack or elimination of the variable portion in the heavy and light chains is due to the difference in molecular weight, the full length first heavy chain and the full length first light chain, the second heavy chain lacking the variable portion and the second light chain lacking the variable portion, and
  • the bispecific antibody in which the full length first heavy chain and the second heavy chain lacking the variable portion are correctly paired is intended to be distinguishable from other antibodies.
  • the lack of 'variable portion' may be a variable portion fragment of a size that can cause a molecular weight difference that can be distinguished from full-length antibodies, including full-length heavy chain and full-length light chain, by conventional protein analysis techniques.
  • the 'variable part' lacking in the first heavy chain or the first light chain may be fragments having the same or different sizes, but since the deletion of the variable part must occur in both the first heavy chain and the first light chain, All 11 light chains should be free of variable region fragments comprising at least one amino acid or variable region fragments having a molecular weight greater than 100 Da.
  • the portion of the variable region lacking in the first heavy chain and the first light chain means a contiguous or non-contiguous amino acid fragment in the first heavy chain variable region and / or the first light chain variable region, and includes a total size (molecular weight; The sum of the molecular weight of the heavy chain variable part and the molecular weight of the light chain variable part that is lacking) is at least IkDa, at least 2kDa, at least 4kDa, at least 6kDa, at least 8kDa, at least 10kDa, at least 12kDa, at least 14kDa, at least 16kDa, at least 18kDa, or at least 20kDa.
  • the upper limit may be the total molecular weight of the heavy chain variable part and the light chain variable part), and the molecular weight of the part of the heavy chain variable part or the part of the light chain variable part lacking in the first heavy chain and the first light chain is 0.5 kDa or more, IkDa or more, and 2 kDa, respectively.
  • the upper limit is the molecular weight of the heavy chain variable part (for some heavy chain variable parts) or the molecular weight of the light chain variable part (for some light chain variable parts), and the sum of the molecular weights of each of the variable parts The total molecular weight range of some of the above-described variable parts).
  • variable parts lacking in the first heavy chain and the first light chain have a total number of amino acids of at least about 10, at least about 20, at least about 40, at least about 60, About 80 or more, or about 100 or more (the upper limit is the total number of amino acids in the heavy and light chain variable regions), and each of the variable regions lacking in the first heavy and first light chains has an amino acid number of about 5 At least about 10, at least about 20, at least about 30, at least about 40, or at least about 50 (the upper limit is the total number of amino acids in the heavy chain variable portion (for some of the heavy chain variable portions) or the total of the light chain variable portions
  • the sum of the number of amino acids (for a part of the light chain variable part) and the number of amino acids of each of the part of the variable part may range from the total number of amino acids of the part of the variable part described above).
  • the adsorbent used in step 2 may be selected from all the adsorbents used in the chromatography, for example, may be at least one selected from the group consisting of resin (eg, agarose resin, etc.), porous particles, and the like.
  • the antigen may be a commonly used marker material (protein or peptide of 5 to 50 aa, 5 to 30 aa, 5 to 20 aa, 5 to 15 aa, or 5 to 10 aa).
  • a flag tag, etc. may be tagged
  • the adsorbent may be a conjugated substance (eg, a label-binding antibody) capable of binding to a label tagged with the antigen.
  • the chromatography may be selected from all kinds of chromatography used to separate and / or purify the protein of interest, and may be, for example, affinity chromatography or the like, but is not limited thereto.
  • the 'preparation method to be tested' can be selected from all bispecific antibody preparation methods to confirm the selective production of the desired bispecific antibody.
  • 'Comparative subject manufacturing method' is not the same as the 'test subject manufacturing method', and may be selected from known methods for producing bispecific antibodies, for example, heavy chain constant region and / or light chain constant region of an antibody. Does not introduce mutations into (ie wild-type heavy chain Bispecific antibody production method using the constant region and / or light chain constant region, the existing dual antibody production method to compare the degree or degree of improvement of the production efficiency of the desired bispecific antibody in the development of the bispecific antibody production method, etc. It may be selected.
  • Selective production (or production) of bispecific antibodies refers to the production (or production) or presence of an antibody comprising the exact combination of interest among the various combinations as shown in FIG. 1 in the preparation of the bispecific antibody, or the level (concentration) of the antibody. Or ratio).
  • the methods used for antibody analysis in the above steps (3) to (5) may be independently selected from all protein detection methods and means, for example, may be selected from among methods and means for detecting proteins according to molecular weight differences.
  • the antibody analysis may be performed by a method selected from the group consisting of gel electrophoresis, si ze exclusion chromatography (SEC HPLC) and the like.
  • SEC HPLC si ze exclusion chromatography
  • the level of the antibody can be confirmed and / or evaluated and / or determined and / or determined by the concentration of the band (The thicker the band, the higher the level of the antibody can be identified and / or evaluated and / or determined and / or determined).
  • the second step chromatography and the third to fifth antibody analysis performed in the preparation method to be tested and the second step chromatography and the third to five steps performed for the preparation method to be compared The antibody analysis of the steps can be performed under the same conditions (eg, the total amount of the antibody prepared in step (1), the chromatography conditions, the volumes of the eluate and the pass-through, the antibody analysis (eg, electrophoresis) conditions, etc.).
  • FIG. 2 schematically shows a mutation for removing all of the variable portions of heavy chain B and a corresponding mutation for removing all of the variable portions of light chain b, and shows a total of 10 combinations that may appear according to their combination. It was.
  • the exact paired antibodies are shown as dotted circles.
  • the exactly matched antibody indicated by the dotted circle is shown schematically in FIG. 3. These are grouped by weight (molecular weight) and are shown in FIG. Indicated.
  • the dotted circles in FIG. 4 are the antibodies with the correct pairing. Antibodies with correct pairing are included in group 3.
  • Group 3 contains three different combinations of antibodies other than those with exact pairing, but the remaining antibodies other than those with exact pairing do not have antigen-binding sites made from the combination of heavy chain A and light chain a. It cannot bind to its Daeung antigen. This can be used to determine how precisely the method for producing a bispecific antibody selectively produces the desired bispecific antibody. If the heavy and light chains are randomly bound when the bispecific antibody is prepared, a total of 10 antibodies may be generated as described above, and a total of 4 antibodies may be formed in Group 3 as well.
  • the formation of the bispecific antibody is specific binding of each heavy chain and the light chain only (that is, the binding is formed only between different heavy chains, so that only the bispecific antibody is formed solely, the bispecific antibody shown as circle in Fig. 3 or 4). Only the antibody having a weight corresponding to group 3 is produced, and only one species is included in the antibody having a weight of group 3. If so, the single antibody is not detected in the pass-through when the adsorption chromatography is filled with the resin to which the Daewoong antigen is bound, and only one band is formed in the eluate.
  • bispecific antibodies were prepared using heavy and light chains in which one antigen-binding portion (variable portion) was removed according to the present invention
  • the adsorption chromatography was first performed when the bispecific antibodies were paired correctly.
  • Antibodies are not detected in the pass-through solution of (the size of the detected antibody can be investigated by electrophoresis to predict what combinations are made between the two heavy chains and the two light chains. If you look at what combinations are present a lot), even if the antibody is eluted through the elution buffer to check the size of the eluted antibody can be confirmed whether the bispecific antibody is correctly paired.
  • the present invention it is possible to confirm the efficiency of the method of producing a bispecific antibody.
  • exactly paired bispecific antibodies are generated . Whether it can be easily confirmed. This can be used to determine how good any method of producing a bispecific antibody is.
  • Figure 1 shows the type of combination between the heavy and light chains and heavy and heavy chains that may appear when producing a bispecific antibody
  • a and B represents a heavy chain
  • a is a light chain that binds to heavy chain A to create an antigen binding site
  • b is Each of the light chains that bind to heavy chain B to form another antigen-binding site 3 ⁇ 4 (eg, A: giant U antibody heavy chain; B: second antibody heavy chain; a: first antibody light chain; b: crab 2 antibody light chain).
  • Bispecific antibodies paired exactly in the desired combination are indicated by dotted lines. It is indicated by a circle.
  • FIG. 2 is a diagram showing the case where the heavy and light chains of one half of Aa antibody (half antibody) and Bb antibody (half antibody) are not included in the variable region to determine whether bispecific antibodies are formed.
  • the types of combinations between the heavy and light chains and between the heavy and heavy chains are shown, and the antibodies paired exactly in the desired combination are indicated by dotted circles.
  • FIG. 3 woolly shows an antibody in which a half antibody comprising a full-length heavy chain and a full-length light chain (indicated by A chain) and a half antibody (indicated by a B chain) comprising a heavy and light chain having the variable portion removed.
  • Figure 4 shows the molecular weight of the antibody that can be produced when the antibody is prepared by using the full-length heavy chain and full-length light chain, and heavy and light chains that do not include a variable portion in one embodiment, a total of 5 depending on the molecular weight It can be divided into groups.
  • Figure 5 shows the results when the antibody was prepared using the heavy and light chains, and full-length heavy and light chains that do not include the variable portion of the anti-TNF a antibody according to Example 1,
  • L is condition media (condi t ion medium) was purified by Protein A, followed by electrophoresis, and FT was performed by electrophoresis of TNF a layered affinity chromatography and flow through.
  • E is the result of electrophoresis of the eluate according to the affinity for TNF_alpha bound to ant i-Flag resin.
  • FIG. 6 shows the results obtained when the antibody was prepared using the heavy and light chains from which the variable portion of the anti-TNF ⁇ antibody was removed and the full length heavy and light chains using the Chimps method illustrated in Example 2.
  • L Is the electrophoresis of condition media
  • FT is the result of electrophoresis of flow through after affinity chromatography filled with TNF a
  • E is the result of electrophoresis of eluate eluted with flag. .
  • Full-length heavy chain and full-length light chain of anti-TNF a antibody (adal imumab), heavy chain not including variable region (variable region lacking heavy chain) and variable region Antibodies were prepared by conventional methods (without introducing mutations into the constant regions) using a non-contained light chain (lacking variable region).
  • various kinds of antibodies see schematic diagram of FIG. 5
  • the preparation of bispecific antibodies can be modeled.
  • the antibody corresponding to the desired bispecific antibody is an antibody including a combination of a full length heavy chain and a full length light chain, and a combination of a variable region lacking heavy chain and a variable region lacking light chain.
  • a gene encoding the full-length heavy chain, full-length light chain, heavy chain not including the variable part (variable part lacking heavy chain) and light chain not including the variable part (variable part lacking light chain) of the anti-TNF a antibody respectively After cloning into a pcDNA3 vector (Invi trogen; see FIG.
  • the condition medium of L lane was found to include all five groups of antibodies.
  • the result is full length heavy chain, full length light chain, variable region.
  • the lacking heavy and variable region lacking light chains show random binding.
  • the antibody (lOOkDa (group 5)) containing a combination of a variable region lacking heavy chain and a variable region lacking light chain does not include a combination of a full length heavy chain and a full length light chain, and does not include a full length heavy chain and a variable region missing light chain.
  • Example 2 Test of Antibody Production Method Including Mutation of Constant Region
  • SEQ ID NO: 1 full-length heavy chain
  • SEQ ID NO: 2 full-length light chain
  • An antibody is prepared using a heavy chain (variable lacking heavy chain; SEQ ID NO: 3) and a light chain not including the variable region (variable lack light chain; SEQ ID NO: 4), and Chimps (Correl ated and Antibodies were prepared by introducing mutations into the constant region using a Harmonious Interfaci al Mutat ion between Protein Subuni ts) method (see Korean Patent Application No. 10-2017-0091758, which is incorporated herein by reference).
  • Table 2 Table 2
  • the antibody having a full-length heavy chain-full length light chain and a variable region lacking heavy chain-variable region lacking light chain combination corresponds to the intended bispecific antibody.
  • Example 1 the results of performing SDS-PAGE on the prepared antibody are shown in FIG. 6.
  • L is the result of electrophoresis of the condition medium
  • FT is the result of electrophoresis of the flow (flow through) after performing affinity chromatography filled with TNF a
  • E is a flag tag on condi t ioned medi a TNF a was added and the ant ibody bound to TNF a was precipitated using ant i -Flag resin, followed by electrophoresis of the eluate eluted with flag.
  • the antibody is prepared by the Chimps method of introducing the mutations of Table 1, only bands corresponding to the three groups belonging to the antibody of the desired combination matched exactly, and appear in other groups No band appeared.
  • Example 2 the bispecific antibody production method according to the Chimps method of Example 2 compared with the conventional production method of the bispecific antibody (using the wild-type constant region of the antibody) of Example 1 It can be confirmed or evaluated that the selective production efficiency of the desired bispecific antibody is excellent.

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Abstract

La présente invention concerne un procédé permettant de confirmer la production d'un anticorps bispécifique, qui peut confirmer si un anticorps bispécifique souhaité est produit sélectivement. Grâce à ce procédé, l'efficacité de production de l'anticorps bispécifique souhaité, à partir du procédé de production d'anticorps bispécifique, peut être confirmée.
PCT/KR2017/009043 2016-08-20 2017-08-18 Procédé de confirmation de production sélective d'anticorps bispécifique souhaité Ceased WO2018038469A1 (fr)

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KR20160105837 2016-08-20
KR1020170104379A KR101933656B1 (ko) 2016-08-20 2017-08-17 목적하는 이중특이성 항체의 선택적 생산 확인 방법
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
GB2576914A (en) * 2018-09-06 2020-03-11 Kymab Ltd Antigen-binding molecules comprising unpaired variable domains produced in mammals

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US20090263392A1 (en) * 2006-03-31 2009-10-22 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
WO2013088259A2 (fr) * 2011-10-19 2013-06-20 Novimmune S.A. Procédés de purification d'anticorps
US9018137B2 (en) * 2011-03-17 2015-04-28 Chromacon Ag Method for identification and purification of multi-specific polypeptides
US20160024147A1 (en) * 2014-07-26 2016-01-28 Regeneron Pharmaceuticals, Inc. Purification Platform for Bispecific Antibodies
KR20160044023A (ko) * 2013-08-19 2016-04-22 에프. 호프만-라 로슈 아게 히드록시아파타이트 크로마토그래피를 사용하는 이중특이적 항체 및 이중특이적 항체 생산 부산물의 분리

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Publication number Priority date Publication date Assignee Title
US20090263392A1 (en) * 2006-03-31 2009-10-22 Chugai Seiyaku Kabushiki Kaisha Methods of modifying antibodies for purification of bispecific antibodies
US9018137B2 (en) * 2011-03-17 2015-04-28 Chromacon Ag Method for identification and purification of multi-specific polypeptides
WO2013088259A2 (fr) * 2011-10-19 2013-06-20 Novimmune S.A. Procédés de purification d'anticorps
KR20160044023A (ko) * 2013-08-19 2016-04-22 에프. 호프만-라 로슈 아게 히드록시아파타이트 크로마토그래피를 사용하는 이중특이적 항체 및 이중특이적 항체 생산 부산물의 분리
US20160024147A1 (en) * 2014-07-26 2016-01-28 Regeneron Pharmaceuticals, Inc. Purification Platform for Bispecific Antibodies

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2576914A (en) * 2018-09-06 2020-03-11 Kymab Ltd Antigen-binding molecules comprising unpaired variable domains produced in mammals
WO2020049128A1 (fr) * 2018-09-06 2020-03-12 Kymab Limited Molécules de fixation d'antigènes comportant des domaines variables non appariés
JP2021536478A (ja) * 2018-09-06 2021-12-27 カイマブ・リミテッド 対合していない可変ドメインを含む抗原結合分子
US12473378B2 (en) 2018-09-06 2025-11-18 Kymab Limited Antigen-binding molecules comprising unpaired variable domains

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