Method for selecting cell clones expressing heterologous polypeptides - Patents.com

Abstract

Herein, we report a novel, high-throughput, and suitable method for the characterization and identification and selection of recombinant cell clones expressing bispecific antibodies with high titers and low levels of antibody-related by-products. The method according to the invention uses the binding signals of immunoassay-based screening of recombinant cell clone culture supernatants for individual and simultaneous binding of the antibodies to their respective targets. This data allows the selection of high-producing cell clones with the desired product profile.

Description

[Technical field]

[0001] The present invention relates to the field of cell line engineering. More precisely, a method is reported herein for using ELISA-based data to select cell clones expressing large amounts of correctly assembled and functional heterologous polypeptides. [Background technology]

[0002] 2. Background of the Invention In recent years, recombinant polypeptides, such as antibodies and antibody-based molecules, have found wide application in the field of disease treatment.

[0003] Specifically for antibodies, after the first wave of antibody-based drugs, which includes naturally occurring IgG format, next-generation formats now increasingly see molecules with substantial modifications and derivatives from classical IgG antibody format.When it is desired to target two or more antigens sequentially or simultaneously, this leads to increasingly complex antibody formats.As the complexity of the format increases, the number of possible antibody-related by-products, such as chain mismatching, increases.

[0004] One step of therapeutic antibodies on the way to market is the development of stable production cell lines characterized by high expression combined with good recombinant protein quality, i.e. low content of antibody-related by-products. In general, the search for novel drug molecules in the complex format antibody space is bottlenecked by the lack of high-throughput assays to evaluate product quality and function at low volumes during cell line development, especially at the single clone level.

[0005] Therefore, a high-throughput method requiring a small sample volume is needed to identify rare cell clones capable of producing correctly assembled complex antibodies among the large number of cell clones obtained after transfection. Such a method would provide the ability to move screening to an earlier stage, whereby a larger number of monoclonal cell clones can be characterized and thereby evaluated. This would increase the probability of identifying exceptional cell clones capable of producing high-quality grade complex format antibodies, thereby increasing the probability of technical success and at the same time minimizing the cost of goods.

[0006] Sawyer, WS et al. (Proc. Natl. Acad. Sci. USA 117 (2020) 9851-9856) (Non-Patent Document 1) reported high-throughput antibody screening from complex matrices using intact protein electrospray mass spectrometry.

[0007] WO 2016 / 172485 (Patent Document 1) reported a multispecific antigen-binding protein. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2016 / 172485 [Non-patent literature]

[0009] [Non-Patent Document 1] Sawyer, W.S. et al. (Proc. Natl. Acad. Sci. USA 117(2020)9851-9856) Summary of the Invention

[0010] Herein, we report a novel, high-throughput, and suitable method for the characterization and identification and selection of recombinant cell clones expressing complex antibody formats with high titers and low levels of antibody-related by-products. The method according to the invention uses binding signals of immunoassay-based screening of recombinant cell clone culture supernatants for individual and simultaneous binding of the antibodies to their respective targets. This data allows, for the first time, the selection of high-producing cell clones with the desired product profile at the stage of 500 or more clones.

[0011] One aspect of the present invention is a method for identifying and / or selecting one or more recombinant cell clones from a large number (in one embodiment at least 500) of recombinant cell clones all expressing the same (recombinant) (therapeutic) antibody that is at least bispecific and (specifically) binds to a first antigen and to a second antigen, comprising the following steps: - culturing each single deposited recombinant cell clone (for at least 5 days) to produce a culture supernatant; - determining: i) total protein (and in certain embodiments, antibody) concentration in the supernatant (using an immunoassay); ii) binding signals for each supernatant for binding of the (therapeutic) antibody to the first antigen in an immunoassay for at least two, and in one preferred embodiment (at least) four different concentrations of supernatant; iii) the binding signal for each supernatant for binding of the (therapeutic) antibody to the second antigen in an immunoassay for at least two, and in a preferred embodiment (at least) four, concentrations of supernatants as in ii); iv) the binding signal for each supernatant for simultaneous binding of the (therapeutic) antibody to the first and second antigen in a (bridging) immunoassay for at least two, and in one preferred embodiment (at least) four, concentrations of supernatants as in ii) and iii); v) binding signals for the (isolated) (therapeutic) antibody (having a purity of at least 98% as determined by mass spectrometry) for individual binding to the first antigen and the second antigen and for simultaneous binding to both antigens in the same immunoassay as in ii), iii) and iv), respectively, for at least 4, and in a preferred embodiment at least 14, different concentrations of the (isolated) (therapeutic) antibody; vi) binding signals for a first (major / major) (isolated) (therapeutic) antibody-related by-product (having a purity of at least 70% as determined by mass spectrometry) that may also be expressed / is expressed by said cell clone for the individual binding to the first antigen and the second antigen and for the simultaneous binding to both antigens in the same immunoassays as in ii), iii) and iv), respectively, of at least 4, and in one preferred embodiment at least 14, concentrations of the first (major / major) (therapeutic) antibody-related by-product as in v); vii) binding signals for second (second primary) (isolated) (therapeutic) antibody related by-products (having a purity of at least 70% as determined by mass spectrometry) that may / are also expressed by said cell clones for individual binding to the first antigen and the second antigen and for simultaneous binding to both antigens in the same immunoassays as in ii), iii) and iv), respectively, of at least 4, and in one preferred embodiment at least 14, concentrations of the second (second primary) (therapeutic) antibody related by-products as in v) and vi); - below: - i), ii), iii), iv), and the median of all the ratios between the binding signals determined in iii) and in ii) for the same concentrations (this median is further divided (normalized) by the median of all the ratios between the binding signals determined in v) using the immunoassay in iii) and in v) using the immunoassay in ii) for the same concentrations), and the median of all the ratios between the binding signals determined in iv) and those determined in ii) for the same concentrations (this median is further divided (normalized) by the median of all the ratios between the binding signals determined in v) using the immunoassay in iv) and those determined in v) using the immunoassay in ii) for the same concentrations, and the median of all the ratios between the binding signals determined in iii) and in ii) for the same concentrations (this median is further divided (normalized) by the median of all the ratios between the binding signals determined in vi) and in vi) for the same concentrations using the immunoassay in iii) and the binding signals determined in vi) using the immunoassay in ii), and - the median of all ratios between the binding signals determined in iv) and those determined in ii) for the same concentrations (this median is further divided (normalized) by the median of all ratios between the binding signals determined in vii) using the immunoassay in iii) and those determined in vii) using the immunoassay in ii) for the same concentrations) (single individual dimension) ranking recombinant cell clones of the plurality of recombinant cell clones based on ranking the recombinant cell clones of the multitude of recombinant cell clones, the ranking being due to the respective difference of the above values ​​for each individual cell clone relative to the value obtained for the isolated (therapeutic) antibody; Including, The recombinant cell clone with the smallest overall difference is selected.

[0012] In a particular embodiment, the ranking is by the (absolute) difference (distance) of these values ​​for each individual cell clone relative to the respective values ​​obtained for the isolated (therapeutic) antibody used in v) in the assays of ii), iii) and iv) and the ratio derived therefrom.

[0013] In certain embodiments, the ranking takes into account the individual (absolute) distances for each individual value (dimension) (multidimensional ranking), i.e. is based on a combination of the similarity (smallest (absolute) difference) to the values ​​for the (isolated) (therapeutic) antibody combined with the dissimilarity (largest (absolute) difference) for the first (primary) isolated (therapeutic) antibody related by-product and the second (second primary) isolated (therapeutic) antibody related by-product.

[0014] In certain embodiments, the selection is based on a combination of the minimum difference in the concentration, binding signal and divided ratio of the (therapeutic) antibody and the maximum difference in the divided ratio of the by-products.

[0015] In a particular embodiment, the recombinant cell clone with the smallest (absolute) difference (distance) overall, i.e. in all individual dimensions, is identified / selected, i.e. the clone with the smallest sum of all eight differences is identified / selected.

[0016] In certain embodiments, one or more of the identified / selected cell clones express said recombinant (therapeutic) antibody with a lower fraction or proportion of antibody-related by-products on average across multiple recombinant cell clones.

[0017] In certain embodiments, the difference (distance) is determined using in silico methods.

[0018] In certain embodiments, the difference (distance) is determined using machine learning.

[0019] In certain embodiments, the difference (distance) is determined using an extreme gradient boosting model.

[0020] In certain embodiments, the binding signals are blank-corrected binding signals (raw binding signals subtracted by the respective blank signals).

[0021] In certain embodiments, at least 4 concentrations are used in ii), iii) and iv), in certain embodiments, 4 or 5 or 6 or 7 concentrations are used in ii), iii) and iv).

[0022] In certain embodiments, in v), vi) and vii), a concentration of at least 14 is used. In certain embodiments, in v), vi) and vii), a concentration of 14 or 15 or 16 or 17 or 18 is used.

[0023] In certain embodiments, the bispecific (therapeutic) antibody comprises a heterodimeric Fc region, where one Fc region polypeptide comprises a knob mutation and each other Fc region polypeptide comprises a hole mutation; a first (primary) bispecific (therapeutic) antibody related by-product is a hole-hole homodimer of said bispecific (therapeutic) antibody; and a second (second primary) bispecific (therapeutic) antibody related by-product is a knob-knob homodimer of said bispecific (therapeutic) antibody.

[0024] In certain embodiments, the immunoassay is an enzyme-linked immunosorbent assay.

[0025] In a particular embodiment, the cell is a CHO cell.

[0026] In a preferred embodiment, the (therapeutic) antibody is a bispecific (therapeutic) antibody. In a particular embodiment, the bispecific (therapeutic) antibody is a heterodimer with respect to its heavy chains, one of which comprises a hole mutation and each of the other heavy chains comprises a knob mutation. In a particular embodiment, the first (main / principal) (isolated) (therapeutic) antibody-related by-product comprises two heavy chains, each of which comprises a hole mutation, and the second (second main) (isolated) (therapeutic) antibody-related by-product comprises two heavy chains, each of which comprises a knob mutation.

[0027] A further aspect of the present invention relates to a method for identifying recombinant cell clones expressing said bispecific (therapeutic) antibodies with reduced bispecific antibody-related by-products, comprising: i) total antibody concentration determined in the culture supernatant of a single recombinant cell clone, where the recombinant cell clone was transfected with a nucleic acid encoding a bispecific (therapeutic) antibody; ii) binding signals for the individual binding of the supernatant to the first and second antigens of the bispecific (therapeutic) antibody; iii) Binding signals for simultaneous binding of the supernatant to the first and second antigens of a bispecific (therapeutic) antibody The use of.

[0028] In certain embodiments, iv) binding signals for the separate and simultaneous binding of a bispecific (therapeutic) antibody to its first and second antigens; v) binding signals for the separate and simultaneous binding of the first (primary) bispecific antibody-associated by-products to the first and second antigens of the bispecific (therapeutic) antibody; vi) Binding signals for the separate and simultaneous binding of a second (second primary) bispecific antibody-related by-product to the first antigen and to the second antigen of the bispecific (therapeutic) antibody. is further used to identify recombinant cell clones expressing said bispecific (therapeutic) antibodies with reduced bispecific antibody-related by-products.

[0029] In certain embodiments, - the median of the individual ratios between the binding signal for each concentration obtained in iii) and the binding signal for each of the same concentrations obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in v) using the immunoassay in iii) and the binding signal for each of the same concentrations obtained in v) using the immunoassay in ii), - the median of the individual ratios between the binding signal for each concentration obtained in iv) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in v) using the immunoassay in iv) and the binding signal for each same concentration obtained in v) using the immunoassay in ii), - the median of the individual ratios between the binding signal for each concentration obtained in iii) and the binding signal for each of the same concentrations obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in vi) using the immunoassay in iii) and the binding signal for each of the same concentrations obtained in vi) using the immunoassay in ii), - the median of the individual ratios between the binding signal for each concentration obtained in iv) and the binding signal for each of the same concentrations obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in vii) using the immunoassay in iv) and the binding signal for each of the same concentrations obtained in vii) using the immunoassay in ii) is further used to identify recombinant cell clones expressing said bispecific (therapeutic) antibody with reduced bispecific antibody-related by-products, iv) is the binding signal for the separate and simultaneous binding of the bispecific (therapeutic) antibody to its first and second antigens, v) is a binding signal for the separate and simultaneous binding of the first (primary) bispecific antibody-associated by-product to the first antigen and the second antigen of the bispecific (therapeutic) antibody, vi) Binding signals for the separate and simultaneous binding of the second (second primary) bispecific antibody-associated by-product to the first antigen and the second antigen of the bispecific (therapeutic) antibody.

[0030] A further aspect of the invention is a computer program comprising computer executable instructions for performing the method according to the invention when the program is run on a computer or a computer network.

[0031] A further aspect of the invention is a computer program product comprising program code means for carrying out the method according to the invention when the program is run on a computer or a computer network.

[0032] A further aspect of the invention is a data carrier storing a data structure which is capable of carrying out the method according to the invention after being loaded into a computer or computer network, such as into a working or main memory of the computer or computer network.

[0033] In addition to the various embodiments depicted and claimed, the subject matter of the present disclosure is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, the specific features presented herein, particularly as aspects or embodiments, can be combined with each other in other ways within the scope of the subject matter of the present disclosure, such that the subject matter of the present disclosure includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the subject matter of the present disclosure is presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject matter of the present disclosure to the disclosed embodiments.

[0034] An aspect as used herein relates to an independent subject of the present invention, and an embodiment as used herein provides a more detailed realization of one or more or all of the independent aspects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description of the Invention The present invention is based, at least in part, on the discovery that ELISA-based potency and binding characterization can be used to predict important quality attributes of individual clones at least at the 500 clone stage during the cell line development stage of drug development.

[0036] The present invention is further based, at least in part, on the discovery that a combination of ELISA-based potency and binding characterization and in silico data analysis can be used to predict key quality attributes of individual clones at at least the 500 clone stage during the cell line development stage of drug development.

[0037] The method according to the invention therefore increases the number of clones that can be analysed, reduces the analysis time, while at the same time increasing reproducibility and ease of application, making it possible for the first time to perform product quality screening early during cell line development, especially for complex antibody formats.

[0038] General definition Methods and techniques useful for carrying out the invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Vols. I-III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Vols. I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture-a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0039] The use of recombinant DNA techniques allows the production of derivatives of nucleic acids. Such derivatives can be modified, for example, by substitution, alteration, replacement, deletion or insertion, at individual or several nucleotide positions. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications can be easily carried out by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD, and Higgins, SG, Nucleic acid hybridization-a practical approach (1985) IRL Press, Oxford, England).

[0040] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those of skill in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" may be used interchangeably.

[0041] The term "about" refers to a range of ±20% of the numerical value that follows. In certain embodiments, the term "about" refers to a range of ±10% of the numerical value that follows. In certain embodiments, the term "about" refers to a range of ±5% of the numerical value that follows.

[0042] The term "comprising" also includes the term "consisting of."

[0043] Cell-specific definitions The term "cell clone" as used herein refers to a mammalian cell comprising an exogenous nucleotide sequence capable of expressing a polypeptide, i.e., a recombinant mammalian cell. Such a recombinant mammalian cell is a cell into which one or more exogenous nucleic acid(s) have been introduced, including the progeny of such a cell. In certain embodiments, a cell clone is a mammalian cell comprising a nucleic acid encoding a heterologous polypeptide. Thus, the term "cell clone comprising a nucleic acid encoding a heterologous polypeptide" refers to a recombinant mammalian cell comprising an exogenous nucleotide sequence integrated into the genome of the mammalian cell and capable of expressing a heterologous polypeptide. In certain embodiments, a cell clone is a mammalian cell comprising an exogenous nucleotide sequence integrated into a single site within a locus of the cell's genome. In a preferred embodiment, a cell clone is a mammalian cell comprising an exogenous nucleotide sequence integrated into a single site within a locus of the cell's genome, the exogenous nucleotide sequence comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selection marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and the recombination recognition sequences are all different.

[0044] The term "recombinant cell" as used herein refers to a cell after genetic modification, such as a cell that expresses a heterologous polypeptide of interest and can be used for the production of said polypeptide on any scale. For example, a "cell clone" refers to a cell in which a coding sequence for a heterologous polypeptide of interest has been introduced into the genome. For example, a "recombinant mammalian cell comprising an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE), thereby introducing a coding sequence for a polypeptide of interest into the genome of the host cell, is a particular "cell clone".

[0045] As used herein, a "cell clone" refers to a "transformed cell." It includes both the primary transformed cell and progeny derived therefrom, regardless of the number of transfers. The progeny may not be completely identical to the parent cell, for example in nucleic acid content, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0046] An "isolated cell clone" refers to a cell clone that has been separated from a component of its natural environment.

[0047] An "isolated nucleic acid" refers to a nucleic acid molecule that is separated from a component of its natural environment.

[0048] An "isolated polypeptide" or an "isolated antibody" or an "isolated by-product" refers to a polypeptide molecule or an antibody molecule, respectively, that has been separated from a component of its natural environment. In certain embodiments, the isolated polypeptide or isolated antibody or isolated by-product is purified to greater than 70% purity, e.g., as determined by mass spectrometry. In certain embodiments, the isolated polypeptide or isolated antibody or isolated by-product is purified to greater than 95% or 98% purity, e.g., as determined by mass spectrometry. For a review of methods for assessing antibody purity, see, e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0049] Assay The principles of different immunoassays are described, for example, by Hage, DS (Anal. Chem. 71 (1999) 294R-304R). Lu, B. et al. (Analyst 121 (1996) 29R-32R) report the oriented immobilization of antibodies for use in immunoassays. Avidin-biotin mediated immunoassays are reported, for example, by Wilchek, M., and Bayer, EA, in Methods Enzymol. 184 (1990) 467-469.

[0050] The term "immunoassay" refers to any technique that utilizes a specific binding molecule, such as an antibody, to capture and / or detect a specific target for qualitative or quantitative analysis. In general, an immunoassay is characterized by the following steps: 1) immobilization or capture of the analyte, and 2) detection and measurement of the analyte. The analyte can be captured, i.e., bound, to any solid surface, such as a membrane, a plastic plate, or any other solid surface. A specific form of immunoassay is the ELISA (enzyme-linked immunosorbent assay).

[0051] In general, immunoassays can be performed in three different formats: one with direct detection, one with indirect detection, or one with a sandwich assay.

[0052] Direct detection immunoassays use a detector (or tracer) antibody that can be measured directly. An enzyme or other molecule allows the production of a signal that produces color, fluorescence or luminescence that allows the signal to be visualized or measured (radioisotopes can also be used, although not as commonly used today).

[0053] In an indirect assay, a primary antibody that binds to the analyte is used to provide a defined target for a secondary antibody (called a detector or tracer antibody) that specifically binds to the target provided by the primary antibody and produces a measurable signal.

[0054] Sandwich assays utilize two antibodies, a capture antibody and a tracer (detector) antibody. The capture antibody is used to bind (immobilize) the analyte from solution or to bind to the analyte in solution. This allows the analyte to be specifically removed from the sample. The tracer (detector) antibody is used in a second step to generate a signal (either directly or indirectly as described above). The sandwich format requires two antibodies, each with a different epitope on the target molecule. Furthermore, since both antibodies must bind to the target simultaneously, they must not interfere with each other.

[0055] Monoclonal antibodies and their constant domains contain several reactive amino acid side chains for binding to members of binding pairs such as polypeptides / proteins, polymers (e.g. PEG, cellulose or polystyrol), or enzymes. Chemically reactive groups of amino acids are, for example, amino groups (lysine, alpha-amino group), thiol groups (cystine, cysteine ​​and methionine), carboxylic acid groups (aspartic acid, glutamic acid), and sugar alcohol groups. Such methods are described, for example, in "Bioconjugation", MacMillan Ref. Ltd., 1999, pages 50-100.

[0056] One of the most common reactive groups on antibodies is the aliphatic ε-amine of the amino acid lysine. In general, almost all antibodies contain abundant lysine. Lysine amines are reasonably good nucleophiles above pH 8.0 (pKa=9.18), so they react easily and cleanly with a variety of reagents to form stable bonds. Amine-reactive reagents react primarily with α-amino groups of lysine and proteins. Reactive esters, especially N-hydroxy-succinimide (NHS) esters, are among the most commonly used reagents for the modification of amine groups. The optimal pH for reactions in an aqueous environment is pH 8.0-9.0. Isothiocyanates are amine-modifying reagents that form thiourea bonds with proteins. They react with protein amines in aqueous solutions (optimally at pH 9.0-9.5). Aldehydes react with aliphatic and aromatic amines, hydrazines, and hydrazides under mild aqueous conditions to form imine intermediates (Schiff bases). Schiff bases can be selectively reduced with mild or strong reducing agents (such as sodium borohydride or sodium cyanoborohydride) to induce stable alkylamine linkages. Other reagents that have been used to modify amines are acid anhydrides. For example, diethylenetriaminepentaacetic anhydride (DTPA) is a bifunctional chelating agent that contains two amine-reactive anhydride groups. It can react with the N-terminus and ε-amine groups of amino acids to form amide bonds. The anhydride ring opens to generate multivalent metal chelating arms that can strongly bind metals in coordination complexes.

[0057] Another common reactive group in antibodies is the thiol residue from the sulfur-containing amino acid cystine and its reduction product cysteine ​​(or half-cystine). Cysteine ​​is more nucleophilic than amines and contains a free thiol group, which is generally the most reactive functional group in proteins. Thiols are generally reactive at neutral pH, so they can selectively bind to other molecules in the presence of amines. Because free sulfhydryl groups are relatively reactive, proteins with these groups often exist in their oxidized form as disulfide groups or disulfide bonds. In such proteins, reduction of the disulfide bonds with reagents such as dithiothreitol (DTT) is necessary to generate reactive free thiols. Thiol-reactive reagents are reagents that bind to thiol groups on polypeptides to form thioether-linked products. These reagents react rapidly at slightly acidic to neutral pH, and therefore can react selectively in the presence of amine groups. The literature reports the use of several thiolated cross-linking reagents, such as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio)acetate (SATA) and sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (Sulfo-LC-SPDP), to provide an efficient method to introduce multiple sulfhydryl groups via reactive amino groups. Haloacetyl derivatives, such as iodoacetamide, form thioether bonds and are also reagents for thiol modification. Further useful reagents are maleimides. The reaction of maleimides with thiol-reactive reagents is essentially the same as with iodoacetamide. Maleimides react rapidly at slightly acidic to neutral pH.

[0058] Another common reactive group in antibodies is carboxylic acid. Antibodies contain carboxylic acid groups at the C-terminal position and in the side chains of aspartic acid and glutamic acid. The relatively low reactivity of carboxylic acids in water usually makes it difficult to use these groups to selectively modify polypeptides and antibodies. In this case, the carboxylic acid group is usually converted to a reactive ester using a water-soluble carbodiimide and reacted with a nucleophile such as an amine, hydrazide, or hydrazine. The amine-containing reagent must be weakly basic to selectively react with the activated carboxylic acid in the presence of the more highly basic ε-amine of lysine to form a stable amide bond. If the pH is increased above 8.0, protein cross-linking can occur.

[0059] Sodium periodate can be used to oxidize the alcohol moiety of the sugar in the carbohydrate moiety attached to the antibody to an aldehyde. Each aldehyde group can be reacted with an amine, hydrazide, or hydrazine as described for carboxylic acids. Since the carbohydrate moieties are found primarily on the crystallizable fragment region (Fc region) of the antibody, conjugation can be achieved by site-specific modification of the carbohydrate away from the antigen binding site. A Schiff base intermediate is formed that can be reduced to an alkylamine by reduction of the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strong) water-soluble reducing agents.

[0060] The conjugation of the tracer and / or capture and / or detection antibody with its conjugation partner can be performed by different methods, such as chemical binding or binding via a binding pair. The term "conjugation partner" as used herein refers, for example, to a solid support, a polypeptide, a detectable label, a member of a specific binding pair. In certain embodiments, the binding of the capture and / or tracer and / or detection antibody with its binding partner is performed by chemical binding via the N-terminus and / or ε-amino group (lysine), the ε-amino group of different lysines, the carboxy-, sulfhydryl-, hydroxyl-, and / or phenolic functional groups of the amino acid backbone of the antibody, and / or the sugar alcohol group of the carbohydrate structure of the antibody. In certain embodiments, the capture antibody is conjugated to its conjugation partner via a binding pair. In one preferred embodiment, the capture antibody is conjugated to biotin and the immobilization to the solid support is performed via a solid support with immobilized avidin or streptavidin. In certain embodiments, the tracer antibody is conjugated to its conjugation partner via a binding pair. In a preferred embodiment, the tracer antibody is covalently conjugated to digoxigenin as a detectable label.

[0061] "Solid phase" refers to non-fluid substances, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass, and ceramics; gel substances such as silica, alumina, and polymer gels; capillaries, which may be made from polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; and cuvettes, tubes, or other spectrometer sample containers. Solid phase components are distinguished from inert solid surfaces in that the "solid phase" includes at least one moiety on its surface that is intended to interact with substances in the sample. The solid phase may be a stationary component, such as a chip, tube, strip, cuvette, or microtiter plate, or a non-stationary component, such as beads and microparticles. A variety of microparticles that allow both non-covalent or covalent attachment of proteins and other substances may be used. Such particles include polymer particles, such as polystyrene and poly(methyl methacrylate); gold particles, such as gold nanoparticles, gold colloids; ceramic particles, such as silica, glass, metal oxide particles, and the like. See, for example, Martin, CR et al., Analytical Chemistry-News & Features, 70 (1998) 322A-327A, or Butler, JE, Methods 22 (2000) 4-23.

[0062] Chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups or metal particles, haptens, such as digoxigenin, are examples of "detectable labels". A detectable label can also be a photoactivatable crosslinking group, such as an azide or azirine group. Metal chelates that can be detected by electrochemiluminescence are also signal-emitting groups, such as ruthenium chelates, such as ruthenium(bispyridyl)3. 2+Chelates are particularly preferred. Suitable ruthenium labeling groups are described, for example, in EP0580979, WO90 / 05301, WO90 / 11511 and WO92 / 14138. For direct detection, the labeling group may be selected from any known detectable marker group, such as dyes, luminescent labeling groups, such as chemiluminescent groups, for example acridinium esters or dioxetanes, or fluorescent dyes, such as fluoresceins, coumarins, rhodamines, oxazines, resorufins, cyanines and their derivatives. Other examples of labeling groups are luminescent metal complexes, such as ruthenium or europium complexes, enzymes, such as those used in ELISA or CEDIA (cloned enzyme donor immunoassays, for example EP-A-0061888), and radioisotopes.

[0063] Indirect detection systems include, for example, that the detection reagent, e.g., the detection antibody, is labeled with a first partner of a binding pair. Examples of suitable binding pairs are antigen / antibody, biotin or biotin analogues, e.g., aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analogue / complementary nucleic acid, and receptor / ligand, e.g., steroid hormone receptor / steroid hormone. In a preferred embodiment, the first binding pair member comprises a hapten, an antigen and a hormone. In a particular embodiment, the hapten is selected from the group consisting of digoxin, digoxigenin and biotin and analogues thereof. The second partner of such a binding pair, e.g., an antibody, streptavidin, etc., is usually labeled to allow direct detection, e.g., by labeling as described above.

[0064] antibody General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0065] As used herein, the amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and is referred to herein as the "Kabat numbering." Specifically, the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3, which is further clarified herein by referring to this case as "Kabat EU index numbering").

[0066] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, full length antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-antibody fragment-fusions and combinations thereof.

[0067] The term "natural antibody" refers to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a heavy chain variable region (VH) followed by three heavy chain constant domains (CH1, CH2, and CH3), which positions a hinge region between the first heavy chain constant domain and the second heavy chain constant domain. Similarly, from the N-terminus to the C-terminus, each light chain has a light chain variable region (VL) followed by a light chain constant domain (CL). The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0068] The term "full-length antibody" refers to an antibody that has a structure substantially similar to that of a natural antibody. A full-length antibody comprises two full-length antibody light chains, each of which comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant domain, and two full-length antibody heavy chains, each of which comprises, from N-terminus to C-terminus, a heavy chain variable region, a first heavy chain constant domain, a hinge region, a second heavy chain constant domain, and a third heavy chain constant domain. In contrast to a natural antibody, a full-length antibody may comprise further immunoglobulin domains, such as one or more additional scFvs, or heavy or light chain Fab fragments, or scFabs that are conjugated to one or more ends of the different chains of the full-length antibody, but only one fragment at each end. These conjugates are also encompassed by the term full-length antibody.

[0069] The term "antibody binding site" refers to a pair of heavy and light chain variable domains. To ensure proper binding to the antigen, these variable domains are cognate variable domains, i.e. belong together. An antibody binding site comprises at least three HVRs (e.g. in the case of a VHH) or three to six HVRs (e.g. in the case of a conventional antibody with a naturally occurring, i.e. VH / VL pair). In general, the amino acid residues of an antibody involved in antigen binding form the binding site. These residues are usually contained in a pair of an antibody heavy chain variable domain and a corresponding antibody light chain variable domain. An antibody antigen binding site comprises amino acid residues from "hypervariable regions" or "HVRs". "Framework" or "FR" regions are the variable domain regions other than the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from the N- to C-terminus, the regions FR1, HVR1, FR2, HVR2, FR3, HVR3, and FR4. In particular, the HVR3 region of the heavy chain variable domain is the region that contributes most to antigen binding and defines the binding specificity of the antibody. A "functional binding site" can bind to its target. The term "bind" refers to the binding of a binding site to its target in an in vitro assay, and in a specific embodiment, in a binding assay. Such a binding assay can be any assay, as long as a binding event can be detected. "Binding" can be determined, for example, using an ELISA assay.

[0070] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that comprise the stretches of amino acid residues that are hypervariable sequences ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Typically, antibodies contain six HVRs, three in the heavy chain variable domain VH (H1, H2, H3) and three in the light chain variable domain VL (L1, L2, L3).

[0071] HVR includes: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0072] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0073] The "class" of an antibody refers to the type of constant domain or region, preferably the Fc region, possessed by the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0074] The term "heavy chain constant region" refers to the region of an immunoglobulin heavy chain that includes the constant domains, i.e., the CH1 domain, the hinge region, the CH2 domain, and the CH3 domain. In one embodiment, the human IgG constant region extends from Ala118 to the carboxyl terminus of the heavy chain (numbering according to Kabat EU index). However, the C-terminal lysine (Lys447) of the constant region may or may not be present (numbering according to Kabat EU index). The term "constant region" refers to a dimer that includes two heavy chain constant regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0075] The term "heavy chain Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the hinge region, the CH2 domain, and the CH3 domain. In one embodiment, the human IgG heavy chain Fc region extends from Asp221 or Cys226 or Pro230 to the carboxyl terminus of the heavy chain (numbering according to Kabat EU index). Thus, the Fc region is smaller than the constant region, but the C-terminal portion is identical to it. However, the C-terminal lysine (Lys447) of the heavy chain Fc region may or may not be present (numbering according to Kabat EU index). The term "Fc region" refers to a dimer comprising two heavy chain Fc regions, which can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0076] The term "valency" as used within this application refers to the presence of a particular number of binding sites within an antibody. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, within an antibody.

[0077] "Monospecific antibody" refers to an antibody that has a single binding specificity, i.e., that specifically binds to one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2) or combinations thereof (e.g., full-length antibodies with additional scFv or Fab fragments). Monospecific antibodies need not be monovalent; i.e., they may contain more than one binding site that specifically binds to one antigen. For example, natural antibodies are monospecific but bivalent.

[0078] "Multispecific antibodies" refer to antibodies that have binding specificities for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., Fab bispecific antibodies) or combinations thereof (antibody-antibody fragment-fusions, e.g., full-length antibodies conjugated to additional scFv or Fab fragments). Multispecific antibodies are at least bivalent, i.e., contain two antigen-binding sites. Furthermore, multispecific antibodies are at least bispecific. Thus, bivalent bispecific antibodies are the simplest form of multispecific antibodies. Engineered antibodies with two, three or more (e.g., four) functional antigen-binding sites have been reported (see, e.g., US 2002 / 0004587).

[0079] In certain embodiments, the cell clone produces a multispecific antibody. In certain embodiments, one of the binding specificities is for a first antigen and the other is for a different second antigen. In certain embodiments, the multispecific antibody binds to two different epitopes of the same antigen. In certain embodiments, the second epitopes on the same antigen are non-overlapping epitopes. In certain embodiments, the antibody is a bispecific antibody. In a preferred embodiment, the bispecific antibody is a trivalent bispecific antibody or a bivalent bispecific antibody.

[0080] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by a number of techniques, including the manipulation of electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83); the production of bispecific antibodies using leucine zippers (see, e.g., Kostelny, SA, et al., J. Immunol. 148 (1992) 1547-1553); the use of general light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431); the use of "diabody" technology to create bispecific antibody fragments (see, e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and by the preparation of trispecific antibodies as described, for example, in Tutt, A., et al., J. Immunol. 147 (1991) 60-69.

[0081] Engineered antibodies having three or more antigen binding sites, including, for example, "Octopus antibodies," or DVD-Igs, are also included herein (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual acting Fabs" or "DAFs" (see, for example, U.S. Patent Application Publication No. 2008 / 0069820 and WO 2015 / 095539).

[0082] Multispecific antibodies can also be provided in an asymmetric manner with domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging VH / VL domains (see, for example, WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see WO 2009 / 080253) or complete Fab arms (see WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al., Proc. Natl. Acad. Sci. USA 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-1020).

[0083] In one preferred embodiment, the multispecific antibody comprises Fab fragments in which either the heavy and light chain variable or constant regions have been exchanged, i.e. in one chain the heavy chain VH variable domain is conjugated directly or via a peptide linker to the light chain CL constant domain and in the respective other chain the light chain VL variable domain is conjugated directly or via a peptide linker to the heavy chain CH1 constant domain.

[0084] Thus, a domain-swapped Fab fragment comprises a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL).

[0085] Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing, see, e.g., WO 2016 / 172485.

[0086] The antibody or fragment may also be a multispecific antibody as described in WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 or WO 2010 / 145793.

[0087] The antibody or fragment thereof may also be a multispecific antibody as disclosed in WO 2012 / 163520.

[0088] A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0089] Bispecific antibodies are generally antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two epitopes on different antigens.

[0090] In certain embodiments, the bispecific antibody comprises: Domain-swapped 1+1 bispecific antibodies (CrossMab) (a) a bispecific full-length IgG antibody comprising a first light chain and a first heavy chain pair comprising a first Fab fragment, and a second light chain and a second heavy chain pair comprising a second Fab fragment, In the first Fab fragment, a) only the CH1 and CL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises the VL and CH1 domains, and the heavy chain of the first Fab fragment comprises the VH and CL domains); b) only the VH and VL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises the VH and CL domains and the heavy chain of the first Fab fragment comprises the VL and CH1 domains); or c) the CH1 and CL domains and the VH and VL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises the VH and CH1 domains and the heavy chain of the first Fab fragment comprises the VL and CL domains); the second Fab fragment comprises a light chain comprising a VL and a CL domain, and a heavy chain comprising a VH and a CH1 domain; a bispecific full-length IgG antibody, in which the first and second heavy chains both comprise a CH3 domain, and both CH3 domains are complementarily engineered by respective amino acid substitutions to support heterodimerization of the first and second heavy chains (in one preferred embodiment, one CH3 domain comprises a knob mutation and the respective other CH3 domain comprises a hole mutation); C-terminal Fab domain fused 2+1 bispecific antibody (BS) (below: a) a full-length antibody comprising two pairs of full-length antibody light chains and full-length antibody heavy chains, wherein a binding site formed by each pair of full-length heavy chains and full-length light chains specifically binds to a first antigen; and b) one additional Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, the binding site of the additional Fab fragment specifically binding to a second antigen; A bispecific full-length IgG antibody comprising: a bispecific full-length IgG antibody, in which the additional Fab fragment specifically binds to a second antigen comprises a domain crossover such that a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are replaced by each other, or b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are replaced by each other; Bispecific one-arm single chain antibody A bispecific one-arm single chain antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein each chain is as follows: - a light chain (comprising a variable light domain and a light chain constant domain); - a light / heavy chain combination, which comprises, from N-terminus to C-terminus, a variable light domain, a light chain constant domain, a peptide linker, a variable heavy domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain with a knob or hole mutation. - a bispecific one-arm single chain antibody, which is a heavy chain (comprising, from N-terminus to C-terminus, a variable heavy domain, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain with a hole or knob mutation); Bispecific two-arm single chain antibody A bispecific two-arm single chain antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: - light chain / heavy chain combination 1 (comprising, from N-terminus to C-terminus, variable light chain domain 1, light chain constant domain, peptide linker, variable heavy chain domain 1, CH1 domain, hinge region, CH2 domain, CH3 domain with a knob or hole mutation); - a bispecific two-arm single chain antibody, which is a light / heavy chain combination 2 (comprising from N-terminus to C-terminus: variable light domain 2, a light chain constant domain, a peptide linker, variable heavy domain 2, a CH1 domain, a hinge region, a CH2 domain, a CH3 domain with a hole or knob mutation); Common light chain bispecific antibodies A common light chain bispecific antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein each chain is as follows: - a light chain (comprising, from N-terminus to C-terminus, a variable light domain and a light chain constant domain); - heavy chain 1 (comprising, from N-terminus to C-terminus, variable heavy domain 1, CH1 domain, hinge region, CH2 domain, CH3 domain with a hole or knob mutation); - a common light chain bispecific antibody, which is a heavy chain 2 (comprising, from N-terminus to C-terminus, the variable heavy domain 2, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain with a knob or hole mutation); 2+1 Bispecific Antibody (TCB) with N-Terminal Fab Domain Insertion (A bispecific full-length antibody having an additional heavy chain N-terminal binding site with domain swapping, - a first Fab fragment and a second Fab fragment, the respective binding sites of the first Fab fragment and the second Fab fragment specifically binding to a first antigen, - a third Fab fragment, the binding site of which specifically binds to a second antigen, the third Fab fragment comprising a domain crossover such that the variable light domain (VL) and the variable heavy domain (VH) are replaced by one another, and - an Fc region comprising a first Fc region polypeptide and a second Fc region polypeptide; Including, the first Fab fragment and the second Fab fragment each comprise a heavy chain fragment and a full-length light chain; the C-terminus of the heavy chain fragment of the first Fab fragment is fused to the N-terminus of the first Fc region polypeptide; a bispecific full length antibody, in which the C-terminus of the heavy chain fragment of the second Fab fragment is fused to the N-terminus of the variable light chain domain of a third Fab fragment and the C-terminus of the CH1 domain of the third Fab fragment is fused to the N-terminus of a second Fc region polypeptide; Antibody-multimer fusion (A fusion polypeptide, (a) an antibody heavy chain and an antibody light chain, (b) a first fusion polypeptide comprising, from N-terminus to C-terminus, a first portion of a non-antibody multimeric polypeptide, an antibody heavy chain CH1 domain or an antibody light chain constant domain, an antibody hinge region, an antibody heavy chain CH2 domain, and an antibody heavy chain CH3 domain; and a second fusion polypeptide comprising, from N-terminus to C-terminus, a second portion of a non-antibody multimeric polypeptide, and an antibody light chain constant domain if the first polypeptide comprises the antibody heavy chain CH1 domain, or an antibody heavy chain CH1 domain if the first polypeptide comprises the antibody light chain constant domain. Including, (i) the antibody heavy chain of (a) and the first fusion polypeptide of (b), (ii) the antibody heavy chain of (a) and the antibody light chain of (a), and (iii) the first fusion polypeptide of (b) and the second fusion polypeptide of (b) are each independently covalently linked to each other by at least one disulfide bond; A fusion polypeptide in which the variable domains of an antibody heavy chain and an antibody light chain form a binding site that specifically binds an antigen. The bispecific antibody is selected from the group consisting of:

[0091] The CH3 domains of the heavy chains of the antibody can be modified by the "knob-into-hole" technique. This is described in detail with some examples in, for example, WO 96 / 027011, Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are modified to increase the heterodimerization of these two CH3 domains, and thereby the heterodimerization of the polypeptides containing them. Each of the two CH3 domains (of the two heavy chains) can be a "knob" and each other is a "hole". The introduction of disulfide bridges further stabilizes the heterodimers (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0092] The mutation T366W in the CH3 domain (of the antibody heavy chain) is designated as a "knob mutation" and the mutations T366S, L368A, Y407V in the CH3 domain (of the antibody heavy chain) are designated as "hole mutations" (numbering according to the EU index of Kabat). Additional interchain disulfide bridges between CH3 domains (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681) can also be used, for example by introducing a S354C mutation in the CH3 domain of the heavy chain carrying a "knob mutation" (designated as a "knob-cys-mutation") and a Y349C mutation in the CH3 domain of the heavy chain carrying a "hole mutation" (designated as a "hole-cys-mutation") (numbering according to the EU index of Kabat).

[0093] The term "domain crossover" as used herein refers to the deviation of the domain sequences from that of the native antibody in that in a pair of an antibody heavy chain VH-CH1 fragment and its corresponding cognate antibody light chain, i.e., in the antibody Fab (fragment-antigen binding), at least one heavy chain domain is replaced by its corresponding light chain domain, and vice versa. There are three general types of domain crossovers: (i) crossovers of CH1 and CL domains, where the domain crossover in the light chain results in a VL-CH1 domain sequence and in the heavy chain fragment results in a VH-CL domain sequence (or a full-length antibody heavy chain with a VH-CL-hinge-CH2-CH3 domain sequence); (ii) domain crossovers of VH and VL domains, where the domain crossover in the light chain results in a VH-CL domain sequence and in the heavy chain fragment results in a VL-CH1 domain sequence; and (iii) domain crossovers of a complete light chain (VL-CL) and a complete VH-CH1 heavy chain fragment ("Fab crossover"), where the domain crossover results in a light chain with a VH-CH1 domain sequence and in a heavy chain fragment with a VL-CL domain sequence (all domain sequences above are shown in the N-terminal to C-terminal direction).

[0094] As used herein, the term "replaced by each other" with respect to corresponding heavy and light chain domains refers to the domain crossover described above. Thus, when CH1 and CL domains are "replaced by each other", this term refers to the domain crossover described under item (i) and the resulting heavy and light chain domain sequences. Thus, when VH and VL are "replaced by each other", this term refers to the domain crossover described under item (ii), and when CH1 and CL domains are "replaced by each other" and VH and VL domains are "replaced by each other", this term refers to the domain crossover described under item (iii). Bispecific antibodies containing domain crossovers have been reported, for example, in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, and Schaefer, W., et al, Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192. Such antibodies are generally referred to as CrossMab.

[0095] The multispecific antibody also comprises, in certain embodiments, at least one Fab fragment comprising either the domain crossover of the CH1 and CL domains described in item (i) above, or the domain crossover of the VH and VL domains described in item (ii) above, or the domain crossover of the VH-CH1 and VL-VL domains described in item (iii) above. In the case of a multispecific antibody with domain crossover, the Fabs that specifically bind to the same antigen are constructed to have the same domain sequence in certain embodiments. Thus, when more than one Fab with domain crossover is included in a multispecific antibody, the Fabs specifically bind to the same antigen.

[0096] The term "recombinant antibody" as used herein refers to all antibodies (chimeric, humanized and human) that are prepared, expressed, produced or isolated by recombinant means, such as recombinant cells. This includes antibodies isolated from recombinant cells, such as NS0, HEK, BHK, amniotic cells, CHO cells, etc.

[0097] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds, i.e., it is a functional fragment. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bispecific Fab, diabody, linear antibody, single chain antibody molecule (e.g., scFv or scFab).

[0098] The term "properly assembled antibody" and its grammatical equivalents refer to an antibody assembled from a cognate pair of heavy and light chains. That is, a properly assembled antibody contains a respective pairing partner for each chain. For example, each heavy chain is paired with its cognate light chain, each heavy chain Fab fragment is paired with its cognate light chain, and / or each scFab fragment is paired with itself and not with a different light chain of the heavy chain Fab fragment.

[0099] The term "antibody-related by-product" refers to molecules obtained during or upon expression of a recombinant antibody that are not correctly assembled antibodies but contain fewer or more antibody chains (polypeptides) than correctly assembled antibodies. Antibody-related by-products include, for example, antibody molecules in which one or more chains, such as a light chain, are missing, or in which one or more heavy chains or heavy chain Fab fragments are paired with a light chain that is not the cognate light chain, or in which the antibody contains an additional light chain. A "cognate pair" of an antibody heavy chain or heavy chain Fab fragment and light chain refers to the chains that are intended / selected / designed to pair to form a binding site with the correct binding characteristics for a target.

[0100] Recombinant methods and compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acid(s) encoding the antibody are provided.

[0101] In one aspect of the invention there is provided a method of producing an antibody, comprising culturing a cell clone comprising nucleic acid(s) encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cells (or host cell culture medium), wherein the cell clone has been selected by a method according to the invention.

[0102] For recombinant production of an antibody, nucleic acid encoding the antibody is generated / designed / synthesized and inserted into one or more vectors for further cloning and / or expression in a cell. Such nucleic acid can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody), or can be produced by recombinant methods, or can be obtained by chemical synthesis.

[0103] Typically, for recombinant mass production of a polypeptide of interest, such as a therapeutic antibody, a cell clone that stably expresses and secretes the polypeptide is required. This cell clone is called a "recombinant cell clone" or "recombinant production cell clone," and the overall process used to generate such cells is called "cell line development." In the first step of the cell line development process, a suitable host cell, such as, for example, in certain embodiments, a CHO cell, is transfected with one or more nucleic acid sequences suitable for expressing the polypeptide of interest. In the second step, cell clones that stably express the polypeptide of interest are selected based on the co-expression of a selectable marker that has been co-transfected with the nucleic acid encoding the polypeptide of interest.

[0104] A nucleic acid that codes for a polypeptide, i.e., a coding sequence, is called a structural gene. Such a structural gene is pure coding information. Therefore, further regulatory elements are required for its expression. Therefore, a structural gene is usually incorporated into a so-called expression cassette. The minimum control elements required for an expression cassette to be functional in a mammalian cell are a promoter that is functional in said mammalian cell, located upstream, i.e., on the 5' side, of the structural gene, and a polyadenylation signal sequence that is functional in said mammalian cell, located downstream, i.e., on the 3' side, of the structural gene. The promoter sequence, the structural gene sequence, and the polyadenylation signal sequence are arranged in an operably linked form.

[0105] If the polypeptide of interest is a heteromultimeric polypeptide composed of different polypeptides, such as an antibody or complex antibody format, not only a single expression cassette is required, but multiple expression cassettes, each containing different structural genes, i.e., at least one expression cassette for each of the different polypeptides (chains) of the heteromultimeric polypeptide (heteromultimeric antibody). For example, a full-length antibody is a heteromultimeric polypeptide that contains two copies of a light chain and two copies of a heavy chain. Thus, a full-length antibody is composed of two different polypeptides. Thus, two expression cassettes are required for the expression of a full-length antibody, one for the light chain and one for the heavy chain. For example, if the full-length antibody is a bispecific antibody, i.e., the antibody contains two different binding sites that specifically bind to two different antigens / epitopes on the same antigen, the two light chains as well as the two heavy chains are also different from each other. Thus, such a bispecific full-length antibody is composed of four different polypeptides, and therefore four expression cassettes are required.

[0106] The expression cassette for the polypeptide of interest is then incorporated into one or more so-called "expression vectors". An "expression vector" is a nucleic acid that provides all the elements required to amplify the vector in bacterial cells and to express the contained structural gene in mammalian cells. Typically, an expression vector contains a prokaryotic plasmid propagation unit, which, for example in the case of E. coli, contains an origin of replication and a prokaryotic selection marker, as well as a eukaryotic selection marker, and an expression cassette required for the expression of the structural gene of interest. An "expression vector" is a transport vehicle for introducing the expression cassette into a mammalian host cell to generate a polypeptide-expressing cell clone.

[0107] As outlined in the previous paragraph, the more complex the polypeptide to be expressed, the greater the number of different expression cassettes required. Essentially, the number of expression cassettes increases the size of the nucleic acid to be integrated into the genome of the host cell. At the same time, the size of the expression vector also increases. However, the practical upper limit of the vector size is in the range of about 15 kbp, above which the efficiency of manipulation and processing decreases significantly. This problem can be addressed by using two or more expression vectors. Thereby, the expression cassette can be divided between different expression vectors, each of which contains only a part of the expression cassette, resulting in a reduction in size.

[0108] Cell line development (CLD) for generating recombinant cells expressing heterologous polypeptides, such as multispecific antibodies, uses either random integration (RI) or targeted integration (TI) of nucleic acids containing the respective expression cassettes required for the expression and production of the heterologous polypeptide of interest.

[0109] Using RI, typically multiple vectors or fragments thereof are integrated into the genome of a cell at the same or different loci.

[0110] Using TI, typically a single copy of a transgene containing different expression cassettes is integrated into a defined "hot spot" in the host cell's genome.

[0111] Suitable host cells for the generation of cell clones for the expression of (glycosylated) antibodies are generally derived from multicellular organisms, such as vertebrates.

[0112] host cell Any mammalian host cell line adapted to grow in suspension can be used to generate recombinant cell clones that can be treated with the methods according to the invention. Moreover, regardless of the integration method, i.e., in the case of RI and TI, any mammalian host cell can be used.

[0113] Examples of useful mammalian host cell lines are human amniotic cells (e.g., CAP-T cells as described in Woelfel, J. et al., BMC Proc. 5 (2011) p. 133); monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., HEK293 cells or HEK293T cells as described in Graham, F. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TRI cells, e.g., as described in Mather, JP et al., Annals NYAcad. Sci. 383 (1982) 44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, e.g., Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, in Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0114] In certain embodiments, the mammalian host cell is, for example, a Chinese Hamster Ovary (CHO) cell (e.g., CHO K1, CHO DG44, etc.), a human embryonic kidney (HEK) cell, a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell), or a human amniotic cell (e.g., CAP-T, etc.). In a preferred embodiment, the mammalian (host) cell is a CHO cell. Thus, similarly, the cell clone is a CHO cell.

[0115] For TI, any known or future mammalian host cell suitable for TI that contains a landing site described herein integrated at a single site within a genomic locus can be used in the present invention. Such cells are referred to as mammalian TI host cells. In certain embodiments, the mammalian TI host cell is a hamster cell, a human cell, a rat cell, or a mouse cell that contains a landing site as described herein. In a preferred embodiment, the mammalian TI host cell is a CHO cell. In certain embodiments, the mammalian TI host cell is a Chinese Hamster Ovary (CHO) cell, a CHO K1 cell, a CHO K1SV cell, a CHO DG44 cell, a CHO DUKXB-11 cell, a CHO K1S cell, or a CHO K1M cell that contains a landing site described herein integrated at a single site within a genomic locus.

[0116] In certain embodiments, the mammalian TI host cell comprises an integrated landing site, the landing site comprising one or more recombination recognition sequences (RRS). The RRS can be recognized by a recombinase, such as Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase. The RRS can be selected, independently of each other, from the group consisting of LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence, and φC31 attB sequence. If multiple RRSs must be present, the selection of each sequence is dependent on the other, insofar as non-identical RRSs are selected.

[0117] The present invention is exemplified below in CHO cells, which are presented only to illustrate the invention and should not be construed as limiting, the true scope of the invention being set forth in the following claims.

[0118] Targeted embedding One method for producing recombinant mammalian cell clones to be treated in the methods according to the invention is recombinant cell clones produced by using targeted integration (TI) for the introduction of coding nucleic acid.

[0119] In targeted integration, site-specific recombination is used to introduce exogenous nucleic acid at a specific locus in the genome of a mammalian TI host cell to generate a recombinant cell clone. It is an enzymatic process in which the sequence of the integration site in the genome is exchanged with the exogenous nucleic acid. One system used to perform such a nucleic acid exchange is the Cre-lox system. The enzyme that catalyzes the exchange is Cre recombinase. The sequence to be exchanged is defined by the location of two lox(P) sites in the genome as well as in the exogenous nucleic acid. These lox(P) sites are recognized by the Cre recombinase. Nothing more is needed, i.e. no ATP etc. The Cre-lox system was originally discovered in the bacteriophage P1.

[0120] The Cre-lox system functions in a variety of cell types, including mammalian, plant, bacterial, and yeast cells.

[0121] In certain embodiments, exogenous nucleic acids encoding heterologous polypeptides are integrated into mammalian TI host cells by single or double recombinase-mediated cassette exchange (RMCE), resulting in recombinant mammalian cell clones, such as recombinant CHO cell clones, in which a defined, specific expression cassette sequence is integrated into the genome at a single locus.

[0122] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre recombinase is a 38 kDa site-specific DNA recombinase that recognizes 34 bp LoxP sequences. Cre recombinase is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intra- and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8 bp non-palindromic core region flanked by two 13 bp inverted repeats. Cre recombinase binds to the 13 bp repeats, thereby mediating recombination within the 8 bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. If two LoxP sequences are arranged in the same orientation on the same nucleotide sequence, Cre recombinase-mediated recombination will excise the DNA sequence located between the two LoxP sequences as a covalently closed circle. If two LoxP sequences are located in opposite orientations on the same nucleotide sequence, Cre recombinase-mediated recombination will invert the orientation of the DNA sequence located between the two sequences. If two LoxP sequences are on two different DNA molecules and one DNA molecule is circular, Cre recombinase-mediated recombination will result in the integration of the circular DNA sequence.

[0123] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. The RRS can be used to define the location in a nucleotide sequence where a recombination event is expected to occur.

[0124] The term "matching RRS" indicates that recombination occurs between two RRSs. In certain embodiments, the two matching RRSs are the same.

[0125] In certain embodiments, the RRS can be recognized by Cre recombinase. In certain embodiments, the RRS can be recognized by FLP recombinase. In certain embodiments, the RRS can be recognized by Bxb1 integrase. In certain embodiments, the RRS can be recognized by φC31 integrase.

[0126] In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matching RRSs are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matching RRS is a Bxb1 attP sequence and the second matching RRS is a Bxb1 attB sequence. In certain embodiments, the first matching RRS is a φC31 attB sequence and the second matching RRS is a φC31 attB sequence.

[0127] A "two-plasmid RMCE" strategy or "double RMCE" is used in the method according to the invention when a combination of two vectors is used. For example, but not limited to, the integrated landing site can include three RRSs, e.g., an arrangement in which a third RRS ("RRS3") is present between a first RRS ("RRS1") and a second RRS ("RRS2"), where the first vector includes two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence, and the second vector includes two RRSs that match the third and second RRSs on the integrated exogenous nucleotide sequence.

[0128] The two-plasmid RMCE strategy involves performing two independent RMCEs simultaneously using three RRS sites. Thus, the landing site of a mammalian TI host cell using the two-plasmid RMCE strategy contains a third RRS site (RRS3) that has no cross activity towards either the first RRS site (RRS1) or the second RRS site (RRS2). The two plasmids to be targeted require the same flanking RRS sites for efficient targeting, with one plasmid (front) flanked by RRS1 and RRS3 and the other (back) flanked by RRS3 and RRS2. In addition, two selectable markers are also required in two-plasmid RMCE. One selectable marker expression cassette was split into two parts. The front plasmid contains a promoter followed by a start codon and an RRS3 sequence. The back plasmid lacks a start codon (ATG) and has an RRS3 sequence fused to the N-terminus of the selectable marker coding region. It may be necessary to insert additional nucleotides between the RRS3 site and the selection marker sequence to ensure in-frame translation of the fusion protein, i.e., operative linkage. Only when both plasmids are correctly inserted will the complete expression cassette of the selection marker be assembled, thus conferring resistance to the respective selection agent to the cells.

[0129] Two-plasmid RMCE involves a recombinase-catalyzed double recombination crossover event between two heterospecific RRSs in a target genomic locus and a donor DNA molecule. Two-plasmid RMCE is designed to introduce copies of the combined DNA sequences from the front and back vectors into a predetermined locus of the mammalian TI host cell genome. RMCE can be performed such that no sequences of the prokaryotic vector are introduced into the mammalian TI host cell genome, thus reducing and / or preventing unwanted triggering of the host's immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

[0130] In certain embodiments, targeted integration is achieved by two rounds of RMCE, where two different DNA sequences are both integrated into a predetermined site in the genome of a corresponding RRS of a mammalian TI host cell, where each DNA sequence comprises at least one expression cassette encoding a portion of a heteromultimeric polypeptide and / or at least one selectable marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, targeted integration is achieved by multiple rounds of RMCE, where DNA sequences from multiple vectors are all integrated into a predetermined site in the genome of a mammalian TI host cell, where each DNA sequence comprises at least one expression cassette encoding a portion of a heteromultimeric polypeptide and / or at least one selectable marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, the selectable marker may be partially encoded in a first vector and partially encoded in a second vector, such that expression of the selectable marker is only possible if both are correctly integrated by double RMCE.

[0131] In certain embodiments, targeted integration by recombinase-mediated recombination integrates various expression cassettes of selection markers and / or multimeric polypeptides into one or more predefined integration sites of the host cell genome without sequences derived from the prokaryotic vector.

[0132] An exemplary mammalian TI host cell suitable for use in the methods according to the invention is a CHO cell with a landing site integrated at a single site within a locus in its genome, the landing site containing three heterospecific loxP sites for DNA recombination mediated by Cre recombinase.

[0133] In this example, the heterospecific loxP sites are L3, LoxFas, and 2L (see, e.g., Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), where L3 and 2L are adjacent to the 5' and 3' ends of the landing site, respectively, and LoxFas is located between the L3 and 2L sites.

[0134] This organization of the landing sites as outlined in the previous paragraph allows the simultaneous integration of two vectors, for example the so-called front vector with L3 and LoxFas sites and the back vector with LoxFas and 2L sites inside. The functional elements of the selection marker gene, different from those present in the landing sites, can be distributed between both vectors: the promoter and the start codon can be located on the front vector, whereas the coding region and the polyA signal are located on the back vector. Only the correct recombinase-mediated integration of the nucleic acid from both vectors induces resistance to the respective selection agents.

[0135] Typically, a mammalian TI host cell is a mammalian cell that comprises a landing site for integration into a locus in the genome of the mammalian cell, the landing site comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least a first selectable marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and wherein the recombination recognition sequences are all different.

[0136] An exogenous nucleotide sequence is a nucleotide sequence that is not native to a particular cell, but can be introduced into said cell by a DNA delivery method, such as transfection, electroporation, or transformation. In certain embodiments, the mammalian TI host cell comprises at least one landing site that is integrated into one or more integration sites in the genome of the mammalian cell. In certain embodiments, the landing site is integrated into one or more integration sites within a specific locus of the genome of the mammalian cell.

[0137] In certain embodiments, the integrated landing site comprises at least one selection marker. In certain embodiments, the integrated landing site comprises a first RRS, a second RRS, and a third RRS, and at least one selection marker. In certain embodiments, the selection marker is located between the first RRS and the second RRS. In certain embodiments, the two RRSs are adjacent to the at least one selection marker. That is, the first RRS is located 5' (upstream) of the selection marker, and the second RRS is located 3' (downstream) of the selection marker. In certain embodiments, the first RRS is adjacent to the 5' end of the selection marker, and the second RRS is adjacent to the 3' end of the selection marker. In certain embodiments, the landing site comprises a first RRS, a second RRS, and a third RRS, and at least one selection marker is located between the first RRS and the third RRS.

[0138] In certain embodiments, the selection marker is located between the first RRS and the second RRS, and the two adjacent RRSs are different from each other. In certain preferred embodiments, the first adjacent RRS is a LoxP L3 sequence, and the second adjacent RRS is a LoxP 2L sequence. In certain embodiments, the LoxP L3 sequence is located 5' of the selection marker, and the LoxP 2L sequence is located 3' of the selection marker. In certain embodiments, the first adjacent RRS is a wild-type FRT sequence, and the second adjacent RRS is a mutant FRT sequence. In certain embodiments, the first adjacent RRS is a Bxb1 attP sequence, and the second adjacent RRS is a Bxb1 attB sequence. In certain embodiments, the first adjacent RRS is a φC31 attP sequence, and the second adjacent RRS is a φC31 attB sequence. In certain embodiments, the two RRSs are positioned in the same orientation. In certain embodiments, the two RRSs are both oriented in the forward or reverse direction. In certain embodiments, the two RRSs are positioned in opposite directions.

[0139] Description of specific embodiments of the method of the present invention The method according to the invention is exemplified below for a bispecific antibody format using a set of four ELISA-based assays (one for determining the antibody titer in the supernatant of the culture of one cell clone expressing said bispecific antibody, two for determining the individual binding of the bispecific antibody to antigen 1 and antigen 2, respectively, and one for determining the simultaneous binding to antigen 1 and antigen 2), the readouts of which are processed according to the method according to the invention. This allows, on the one hand, to distinguish cell clones based on the ratio of antibody main products to antibody by-products, and, on the other hand, to accurately classify cell clones with respect to their product (antibody) related by-product profile. Both provide the basis for an improved clone selection process according to the method of the invention. This example is presented only as an illustration of the method according to the invention and should not be interpreted as a limitation thereof. The true scope of the invention is set out in the appended claims.

[0140] In this example, the evaluation of individual cell clones producing bispecific antibodies is based on the results of four assays that determine and quantify antibody titers in the supernatants, individual binding to antigens 1 and 2, and simultaneous binding to both antigens.

[0141] Thus, for cell clone selection during cell line development (CLD), different cell clones obtained by transfection of host cells with one or more nucleic acids encoding the bispecific antibody are analyzed by three combined ELISAs as outlined above and assessed according to the amount of desired main antibody product versus undesired antibody by-products (=main antibody product quality) taking into account the expression yield. The combined readout of these assays makes it possible to distinguish clones expressing a high proportion of the desired main antibody product (=lead) from clones expressing only a low proportion of the desired main product.

[0142] Many bispecific antibody formats are based on knob-into-hole heterodimerization technology. Such formats may form characteristic product-related by-products due to chain mispairing, e.g. hole chain-hole chain dimers, knob chain-knob chain dimers, or 3 / 4 antibodies lacking one light chain. Different cell clones often express different mixtures of the antibody main product (= lead, i.e. correctly assembled bispecific antibody) and product-related antibody by-products (e.g. hole chain-hole chain dimers (= by-product I) and knob chain-knob chain dimers (= by-product II)). Differences in the expression patterns of these molecules in recombinant cell clones necessitate early and high-throughput amenable screening methods to deselect cell clones with inappropriate product profiles, e.g. those with low antibody main product yields. Some of the antibody formats and their by-products used in this example are shown in Figure 15.

[0143] The assay used in this embodiment of the method according to the invention utilizes a different assay principle. This has the effect that different expression products give different ELISA signals. The purified main product (i.e. the correctly assembled knob-into-hole heterodimer) and the most common side products (i.e. hole-hole and knob-knob homodimers) are used as standards. They give a specific signal in the assay based on a) avidity effects due to the format itself and b) avidity effects due to the detection antibody used.

[0144] To determine antigen binding, the following enzyme-linked immunosorbent assay (ELISA) was used in this example: - Biotinylated antigen 1 or 2, respectively, was immobilized on a streptavidin-coated microtiter plate. After washing the plate, a mixture of culture supernatant and detection antibody (anti-human Fab antibody conjugated to POD (horseradish peroxidase)) was applied; 3,3',5,5'-tetramethylbenzidine (TMB) was used for readout; - Simultaneous binding to antigen 1 and antigen 2 was determined by bridging ELISA; antigen 1 or antigen 2 was directly coated onto a microtiter plate. After washing the plate, a mixture of culture supernatant, each other biotinylated antigen 2 or 1, and streptavidin-POD conjugate was applied; TMB was used for readout.

[0145] These ELISA readouts were combined according to the method of the invention to distinguish between the antibody main product, i.e. the monomeric and correctly assembled bispecific antibody, and the antibody by-product. This achieved the classification of the respective clones with respect to their properties as a basis for clone selection. For example, a clone is classified as "lead" if it is predicted to mainly express the main product based on the application of the method of the invention. Such clones are suitable as clones for expressing / producing bispecific antibodies with a low content of antibody-related by-products. These clones need to be identified.

[0146] As a reference, the categorization of ELISA results for three different bispecific antibody formats - domain-swapped 1+1 bispecific antibody (CrossMab), N-terminal Fab domain-inserted 2+1 bispecific antibody (TCB) and C-terminal Fab domain-fused 2+1 bispecific antibody (BS) - is visualized in Figures 1 to 3. ELISA results are shown for clarity only for two of the three ELISAs and only for one of the four analyzed concentrations. In the figures, binding to antigen 2 (AG2) is plotted against binding to antigen 1 (AG1). The different shadings show the categorization of the quality of the cell clones. This shows that there is variability depending on the antibody format and the target and / or target combination (= project), thus demonstrating the complexity of the analysis.

[0147] Purified antibody main product (designated as "main product standard"; e.g., a correctly assembled knob-into-hole heterodimer heavy chain paired with all correctly associated light chains) and the most common undesired by-products (designated as "by-product I standard" and "by-product II standard"; e.g., hole-hole homodimer and knob-knob homodimer, respectively) were used as standards in all assays in a dilution series (14 different concentrations). Culture supernatants of each single cell clone after 5 days of culture were analyzed at 4 different concentrations. The method according to the invention can be performed independently of the absolute signal differences (as long as these differences are statistically significant). This is independent of whether the signals are within the linear signal range of the assay or not. In a particular embodiment, all standards, culture supernatants, coated antigens, detection antibodies and detection reagents are used at concentrations that result in the maximum signal difference between the signal obtained with the antibody main product standard and the signal obtained with the antibody by-product standard. The skilled person can easily perform such optimization of the signal difference based on his knowledge without undue burden.

[0148] The present invention is based, at least in part, on the discovery that the selection of recombinant cell clones expressing a bispecific antibody can be efficiently performed based on the results of immunoassays determining the concentration of the bispecific antibody in the culture supernatant of a single cell clone and the binding of the bispecific antibody to an isolated antigen as well as the simultaneous binding to both antigens. Furthermore, objective data processing has been found to be advantageous.

[0149] The present invention relates, at least in part, to the selection of recombinant cell clones producing bispecific antibodies with the lowest antibody-related by-product content. - the median blank-corrected data (binding signal) of four measured concentrations of culture supernatant in the binding immunoassay for binding to each antigen separately and for binding to both antigens simultaneously; - Median blank-corrected data for at least five measured concentrations of the isolated bispecific antibody and the two major by-products in each binding immunoassay; Based on the finding that a combination of the concentrations of bispecific antibodies in single cell clone culture supernatants (after at least 5 days of culture) can be used.

[0150] The terms "blank-corrected data" or "blank-corrected binding signal", as used interchangeably herein, refer to the binding signal determined in an immunoassay for a test sample or standard sample, i.e. the raw binding signal determined for the test sample or standard sample, minus the background signal determined in the same immunoassay for a blank sample (i.e. a sample that does not contain standards, culture supernatants, (therapeutic) antibodies and any by-products but is otherwise identical to the test sample).

[0151] It has been found that the method according to the invention can be further improved by using either all nine or four of the nine possible standard normalized binding ratios and, optionally, the ratio of the absorbance corrected ELISA signal.

[0152] The invention is based, at least in part, on the discovery that certain ratios of binding signals are specific and, when used in combination and normalized with ratios calculated for standards (100% purified antibody main product, antibody by-product I and antibody by-product II), allow the differentiation of individual culture supernatants (=samples) and thereby clones with good (producing / expressing mainly the antibody main product) and poor (producing / expressing mainly the antibody by-product) product quality.

[0153] The standard ratio is calculated as the median of the signals obtained for the concentrations (in a preferred embodiment, 4 concentrations of culture supernatant and 14 concentrations of standards) and then divided by each other ("normalization"), giving a total of 9 possible ratios.

[0154] The present invention is based, at least in part, on the discovery that selection of cell clones expressing correctly assembled bispecific antibodies requires only the median of the following four: - the median ratio of the blank-corrected binding signal obtained for the cell clone culture supernatant at the same (four) concentrations for binding to antigen 2 to the blank-corrected binding signal obtained for the cell clone culture supernatant at the same (four) concentrations for binding to antigen 1, normalized (i.e. divided) to the median ratio of the blank-corrected binding signal obtained for at least five concentrations of the antibody main product (main product standard) for binding to antigen 2 to the blank-corrected binding signal obtained for at least five concentrations of the main product standard for binding to antigen 1 determined in the same assay, - the median ratio of the blank-corrected binding signal obtained for the cell clone culture supernatant at the same (four) concentrations for simultaneous binding to antigens 1 and 2 to the blank-corrected binding signal obtained for the cell clone culture supernatant at the same (four) concentrations for binding to antigen 1, normalized to the median ratio of the blank-corrected binding signal obtained for at least five concentrations of the antibody main product (main product standard) for simultaneous binding to antigens 1 and 2 to the blank-corrected binding signal obtained for at least five concentrations of the main product standard for binding to antigen 1 determined in the same assay, - the median ratio of the blank-corrected binding signal obtained for the cell clone culture supernatant at the same (four) concentrations for binding to antigen 2 to the blank-corrected binding signal obtained for the cell clone culture supernatant at the same (four) concentrations for binding to antigen 1, normalized to the median ratio of the blank-corrected binding signal obtained for at least five concentrations of antibody-related by-product I (by-product I standard) for binding to antigen 2 to the blank-corrected binding signal obtained for at least five concentrations of by-product I standard for binding to antigen 1 determined in the same assay; and - the median ratio of the blank-corrected binding signal obtained for the cell clone culture supernatant at the same (4) concentrations for simultaneous binding to antigens 1 and 2 to the blank-corrected binding signal obtained for the cell clone culture supernatant at the same (4) concentrations for binding to antigen 1, normalized to the median ratio of the blank-corrected binding signal obtained for at least 5 concentrations of antibody by-product II (by-product II standard) for simultaneous binding to antigens 1 and 2 to the blank-corrected binding signal obtained for at least 5 concentrations of by-product II standard for binding to antigen 1 determined in the same assay.

[0155] In certain embodiments, at least 10 concentrations of each standard are used. In a preferred embodiment, at least 14 concentrations of each standard are used.

[0156] Using at least these parameters, the complexity of cell clone characterization can be reduced, and the resulting method according to the invention therefore encompasses this improved data processing and evaluation.

[0157] It was found that using the above parameter selection in combination with in silico analysis resulted in an antibody format-independent and target-independent method.

[0158] To demonstrate the advantageous properties of the method according to the invention, eight different exemplary data processing variants were compared, which are summarized in the table below.

[0159] TIFF2024513474000001.tif179150

[0160] The performance of each of these eight different processing variants in in silico processing with the extreme gradient boosting (XGB) model for quality category prediction for five different target combinations in different formats is visualized in Figure 4 (TCB-1, 2+1 head-to-tail, TCB-2, TCB-3 and CrossMab). XGB model prediction quality was evaluated by cross entropy loss over the entire project not utilized for model training. The different processing methods shown in the previous table were used as experimental conditions.

[0161] The false negative and false positive rates of different processing variants are shown in Figure 5. The effect of various combinations of input parameters and manipulated features can be clearly seen, whereby processing variants 6, 7, and 8 perform the best.

[0162] Thus, using the method according to the invention, a target- and format-independent categorization of recombinant cell clones can be achieved.

[0163] The quality of the method according to the invention is shown in Figures 6 to 10. In these figures, a data set evaluated using the only manual method is compared with the same data set evaluated with the method according to the invention. The projects used to demonstrate the improved performance of the method according to the invention compared to the only manual evaluation method were not used to train the algorithm. Thus, bias can be excluded.

[0164] In FIG. 6, binding to antigen 2 (AG2) is plotted against binding to antigen 1 (AG1). A manual-only analysis is shown at the top, while the results obtained with the method according to the invention are shown at the bottom. Figures 7 to 10 show, in the same arrangement, the median values ​​of AG2:AG1 normalized to the main product standard, AG12:AG1 normalized to the main product standard, AG2:AG1 normalized to the minor product I standard, and AG12:AG2 normalized to the minor product II standard, each plotted against the number of clones. Due to the normalization to the standard, clones falling into each categorization should have values ​​close to 1. The improved accuracy of the results obtained with the method according to the invention is impressively shown in these graphs. The shading indicates the different quality categories, and since the size of the bottom data set is proportional to the predicted accuracy, it is very easily and quickly shown that this clone is most likely to produce a superior product quality.

[0165] The method according to the invention can be used for clone quality assessment to select clones with the best properties for upscaling as shown in the following example.

[0166] Figures 11 to 14 show examples of clone selection using the method according to the invention. The clone quality categories determined by the method according to the invention are shown in Figures 11 and 12 by different shading. In Figures 13 and 14 the selection process is shown. 160 of the 610 clones were selected to have the desired product quality, i.e. antibody main product yield, based on the results of the method according to the invention.

[0167] Cross-entropy loss function (log loss, logarithmic loss or logistic loss) is a function for ranking data in a model. Each predicted class probability is compared to the actual class desired output 0 or 1 and a score / loss is calculated that penalizes the probability based on its distance from the actual expected value. The penalty is logarithmic in nature, resulting in a large score for large differences that are closer to 1 and a small score for small differences that tend towards 0. Cross-entropy loss is used when adjusting model weights during training. The objective is to minimize loss, i.e. the smaller the loss, the better the model. A perfect model has a cross-entropy loss of 0. Cross-entropy is defined as: TIFF2024513474000002.tif14128

[0168] For n classes, t i is the truth label, and p i is the probability of the i-th class.

[0169] The median is the term of a series of terms that is in the middle of said series. For a series with N terms, where N is an odd number, the median is TIFF2024513474000003.tif8128 is the term, i.e. the middle term, of the series. For a series with N terms, where N is an even number, the median is TIFF2024513474000004.tif12128, i.e. the average of the middle two terms of the sequence. The sequence contains the individual terms in ascending numerical (value) order.

[0170] By using the method according to the invention, the number of clones producing products with poor product quality selected during the initial steps of clone selection is reduced, i.e. the number of false positives is reduced, while at the same time the loss of clones producing products with good product quality, i.e. deselection, is reduced, i.e. the number of false negatives is reduced.

[0171] When using state-of-the-art screening methods that do not include product quality attributes for clone selection, clones producing products with poor product quality are only recognized late in the screening process, for example, once sufficient product is available so that the quality can be analyzed by analytical chromatography.

[0172] Therefore, by reducing the number of false positive clones, better producing clones, i.e. true positives, are carried further in the selection process, thereby reducing the potential loss of clones producing high quality products.

[0173] This is nicely illustrated in Figures 16-20.

[0174] In Figure 16, each bar represents one clone. Clones are ranked by titer, i.e. product concentration, size of bar). Clones were further characterized by the method according to the invention. Clones that would have been selected by the method according to the invention are shown in dark.

[0175] Using state of the art titer based selection a combination of light and dark bars is selected, whereas using the method according to the invention only clones represented by dark bars are selected.

[0176] Thus, using the current technology, titer-based selected clones that do not produce high-quality product (light bars), i.e., false positives, are included, thereby reducing the number of selected true-positive clones.

[0177] All clones, and thus the products produced by them, were analyzed by size exclusion chromatography (SEC) to determine monomer content, and by capillary electrophoresis (CE-SDS) and hydrophobic interaction chromatography (HIC) to determine the major product peak.

[0178] In Figure 17, the relationship between monomer content determined by SEC (x-axis) and the amount of main product determined by CE-SDS (y-axis) is shown for the clones of Figure 16, which are analyzed in early clone selection. The clones that would have been deselected by the method according to the invention are shown in bold, i.e. the light bars in Figure 16. Approximately half of the bold deselected clones can be seen in the top right quadrant, i.e. having a product quality that is considered good based on CE-SDS and SEC (circles in Figure 17). However, these are false positives according to the method according to the invention.

[0179] This has been confirmed by reanalysing the clones from the circles in Figure 17 using HIC. The results are shown in Figure 18. The thick clone circled in the upper right quadrant of Figure 17 is now seen in the lower right quadrant of Figure 18 (circle), i.e., with a low major product peak and therefore a false positive.

[0180] To further confirm the advantageous properties of the method according to the invention, the same analysis was performed on the clones selected by the method according to the invention, and the corresponding results for SEC combined with CE-SDS are shown in Figure 19 and for SEC combined with HIC in Figure 20.

[0181] It can be seen that the clones selected by the method according to the present invention are located in the upper right quadrant in both figures and are therefore true positive clones.

[0182] This indicates that the method according to the present invention can reduce the number of selected false-positive clones and at the same time increase the number of deselected true-positive clones.

[0183] Description of Specific Computer-Implemented Embodiments of the Invention One aspect of the present invention is a computer program product comprising instructions that, when executed on an immunoassay measurement device, which is a suitable system including a computer, causes the product to perform at least the following steps: i) determining a signal proportional to the antibody concentration in the single cell clone culture supernatant for different concentrations of the single cell clone culture supernatant; ii) determining, for different concentrations of single cell clone culture supernatant, a signal proportional to the individual binding of the antibodies contained in the single cell culture supernatant to their primary target; iii) determining, for different concentrations of single cell clone culture supernatant, a signal proportional to the individual binding of the antibodies contained in the single cell culture supernatant to their secondary targets; iv) determining, for different concentrations of single cell clone culture supernatant, a signal proportional to the simultaneous binding of the antibody contained in the single cell culture supernatant to its first antigen and to the second target; v) determining, for each of the different concentrations of standards, a signal proportional to the individual binding of the antibody reference standard, the antibody-related by-product I standard, and the antibody-related by-product II standard to the first target of the antibody; vi) determining, for each of the different concentrations of standards, a signal proportional to the individual binding of the antibody reference standard, the antibody-related by-product I standard, and the antibody-related by-product II standard to the secondary target of the antibody; vii) determining, for each standard at different concentrations, a signal proportional to the simultaneous binding of the antibody reference standard, the antibody-related by-product I standard and the antibody-related by-product II standard to the primary target and the secondary target of the antibody.

[0184] In certain embodiments, the instructions further comprise the step after step vii) of: a) calculating the median ratio of the signal obtained for the same four concentrations of cell clone culture supernatant for binding to antigen 2 to the signal obtained for the same four concentrations of cell clone culture supernatant for binding to antigen 1, normalized to the median signal of each ratio obtained for at least five concentrations of the antibody main product (main product standard); b) calculating the median ratio of the signal obtained for the same four concentrations of cell clone culture supernatant for simultaneous binding to antigens 1 and 2 and the signal obtained for the same four concentrations of cell clone culture supernatant for binding to antigen 1, normalized to the median signal of the respective ratios obtained for at least five concentrations of the antibody main product (main product standard); c) calculating the median ratio of the signal obtained for the same four concentrations of cell clone culture supernatant for binding to antigen 2 to the signal obtained for the same four concentrations of cell clone culture supernatant for binding to antigen 1, normalized to the median signal of each ratio obtained for at least five concentrations of antibody-related by-product I (by-product I standard); d) calculating the median ratios between the signal obtained for the same four concentrations of cell clone culture supernatant for simultaneous binding to antigens 1 and 2 and the signal obtained for the same four concentrations of cell clone culture supernatant for binding to antigen 1, normalized to the median signal of each ratio obtained for at least five concentrations of antibody-related by-product II (by-product II standard).

[0185] In certain embodiments, the instructions further comprise fitting the calculated median value and the determined titer value of i) to a model for predicting the quality category of recombinant cell clones expressing the bispecific antibody. In a preferred embodiment, the model is the XGB model.

[0186] In certain embodiments, the system further comprises a clone picker and a final step viii) of selecting clones based on a combination of concentration, binding signal and the minimum difference in the divided ratio of the bispecific antibody and the maximum difference in the divided ratio of the by-products.

[0187] A further aspect of the present invention is a computer program comprising computer executable instructions for implementing the method according to the present invention in one or more of the embodiments attached hereto when the program is executed on a computer or a computer network. In particular, the computer program may be stored on a computer readable data medium. In particular, therefore, one, more than one or even all of the above method steps i) to vii), optionally one, more than one or even all of the above method steps a) to d), and optionally step vii), may be implemented by using a computer or a computer network, preferably by using a computer program.

[0188] A further aspect of the present invention is a computer program product having program code means for carrying out the method according to the present invention in one or more of the embodiments attached hereto when said program is executed on a computer or a computer network. In particular, said program code means may be stored on a computer readable data carrier.

[0189] A further aspect of the invention is a data carrier storing a data structure which, after being loaded into a computer or computer network, such as into a working or main memory of the computer or computer network, is capable of carrying out the method according to the invention in one or more of its embodiments.

[0190] A further aspect of the present invention is a computer program product having a program code stored on a machine readable carrier for performing the method according to the present invention in one or more of its embodiments when the program is run on a computer or a computer network.

[0191] As used herein, a "computer program product" refers to a program as a tradeable product. The product can generally be present in any format, such as a paper format or on a computer-readable data carrier. In particular, the computer program product may be distributed over a data network.

[0192] Finally, a further aspect according to the invention is a modulated data signal comprising instructions readable by a computer system or computer network for carrying out the method according to the invention in one or more of its embodiments.

[0193] With respect to computer-implemented aspects of the invention, one or more or all of the method steps of the method according to one or more of the embodiments disclosed herein may be performed by using a computer or a computer network. Thus, in general, any of the method steps involving providing and / or manipulating data may be performed by using a computer or a computer network. In general, these method steps may include any method steps, except for those that typically require manual operations, such as providing a sample and / or certain aspects of performing the actual measurement.

[0194] Specifically, the following is further disclosed herein: a computer or a computer network comprising at least one processor, the processor being adapted to execute the method according to the invention in one of its embodiments, a computer-loadable data structure adapted to carry out the method according to the invention in one of its embodiments while said data structure is executed on a computer, a computer program adapted to carry out the method according to the invention in one of its embodiments while the program is running on a computer, a computer program comprising program means for carrying out the method according to the invention in one of its embodiments while said computer program is being run on a computer or a computer network, - a computer program comprising program means according to any of the embodiments presented herein stored on a computer readable storage medium, a storage medium on which a data structure is stored, the storage medium being adapted to execute the method according to the invention in one of its embodiments after the data structure has been loaded into a main and / or working storage device of a computer or a computer network, and a computer program product comprising program code means storable or stored on a storage medium, which program code means, when executed on a computer or a computer network, performs the method according to the invention in one of its embodiments.

[0195] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims.

[0196] It is understood that modifications can be made in the procedures set forth without departing from the spirit and scope of the invention. [Brief description of the drawings]

[0197] [Figure 1] Exemplary categorization of antigen-binding ELISA readouts (AG2 vs. AG1) for bispecific antibodies in CrossMab format for single concentrations. [Diagram 2] Exemplary categorization of antigen-binding ELISA readouts (AG2 vs. AG1) for bispecific antibodies in TCB format for single concentrations. [Diagram 3] Exemplary categorization of antigen-binding ELISA readouts (AG2 vs. AG1) for bispecific antibodies in BS format for single concentrations. [Figure 4] Model prediction quality assessed by cross entropy loss (y-axis) across projects not used in model training. For each preprocessing variant 1-8, from left to right: TCB-1; 2+1 head-to-tail; TCB-2; TCB-3; CrossMab. [Diagram 5] False positive and false negative predictions [%] for categorizing as "correctly assembled bispecific antibody" across all five analyzed projects. [Figure 6] Comparison of a purely manual analysis (top) with the method according to the invention (bottom). In the top graph, binding to antigen 2 (AG2) is plotted against binding to antigen 1 (AG1). The figure shows data of 610 monoclonal cell lines from a single project that were not used to train the algorithm. Size indicates prediction accuracy. [Figure 7] Comparison of only manual analysis (top) and the method according to the invention (bottom). Median ratio AG2:AG1 binding signal normalized to the median ratio AG2:AG1 binding signal of the main product standard plotted against the number of clones. [Figure 8] Comparison of only manual analysis (top) and the method according to the invention (bottom). Median ratio AG12:AG1 binding signal normalized to the median ratio AG12:AG1 binding signal of the main product standard plotted against the number of clones. [Figure 9] Comparison of purely manual analysis (top) and the method according to the invention (bottom). Median ratio AG2:AG1 binding signal normalized to median by-product I standard ratio AG2:AG1 binding signal plotted against clone number. [Figure 10] Comparison of purely manual analysis (top) and the method according to the invention (bottom). Median ratio AG12:AG1 binding signal normalized to median ratio AG2:AG1 binding signal of by-product II standards plotted against clone number. [Figure 11]Use of the method according to the invention for the selection of clones based on product quality (part 1). Categorization of the two analyzed antibody concentrations (50 ng / mL, 1.95 ng / mL) is shown. A total of 610 clones (combined Figures 11 and 12) producing diverse antibody qualities are displayed. [Figure 12] Use of the method according to the invention for the selection of clones based on product quality (part 2). Categorization of the two analyzed antibody concentrations (9.9 ng / mL, 0.39 ng / mL) is shown. A total of 610 clones (combined Figures 11 and 12) producing diverse antibody qualities are displayed. [Figure 13] Use of the method according to the invention for the selection of clones based on product quality (part 3). Results are shown for two antibody concentrations analyzed (50 ng / mL, 1.95 ng / mL). A total of 160 clones selected with the method according to the invention based on antibody quality are displayed (Figures 13 and 14 taken together). [Figure 14] Use of the method according to the invention for the selection of clones based on product quality (part 4). Results are shown for two antibody concentrations analyzed (9.9 ng / mL, 0.39 ng / mL). A total of 160 clones selected with the method according to the invention based on antibody quality are displayed (combined Figures 13 and 14). [Figure 15] Bispecific antibody formats and their common by-products. [Figure 16] Clonal product titers. [Figure 17] FIG. 16 Relationship between monomer content determined by SEC (x-axis) and amount of main product determined by CE-SDS (y-axis) of clones; clones deselected by the method according to the invention are shown in bold. [Figure 18] FIG. 16. Relationship between monomer content determined by SEC (x-axis) and amount of major product determined by HIC (y-axis) for clones; clones deselected by the method according to the invention are shown in bold. [Figure 19]FIG. 16. Relationship between monomer content determined by SEC (x-axis) and amount of main product determined by CE-SDS (y-axis) of clones; clones selected by the method according to the invention are shown in bold. [Figure 20] FIG. 16. Relationship between monomer content determined by SEC (x-axis) and amount of major product determined by HIC (y-axis) for clones; clones selected by the method according to the invention are shown in bold. EXAMPLES

[0198] Example 1 General Technology 1) Recombinant DNA Technology DNA was manipulated using standard methods as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989). Molecular biological reagents were used according to the manufacturer's instructions.

[0199] 2) DNA sequencing DNA sequencing was performed at SequiServe GmbH (Vaterstetten, Germany).

[0200] 3) DNA and protein sequence analysis and sequence data management The EMBOSS (European Molecular Biology Open Software Suite) software package and Invitrogen's Vector NTI version 11.5 were used for sequence creation, mapping, analysis, annotation, and illustration.

[0201] 4) Gene and oligonucleotide synthesis The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

[0202] 5) Reagents Unless otherwise stated, all commercially available chemicals, antibodies, and kits were used as provided according to the manufacturer's protocols.

[0203] 6) Cultivation of host cell lines CHO host cells were cultured at 37°C in a humidified incubator with 85% humidity and 5% CO2. They were cultured in a proprietary DMEM / F12-based medium containing selection agents. Cells were split every 3 or 4 days. For culture, 125 ml non-baffled Erlenmeyer shake flasks were used. Cells were shaken at 150 rpm with a shaking amplitude of 5 cm. Cell numbers were measured with a Cedex HiRes Cell Counter (Roche). Cells were kept in culture until they reached 60 days of age.

[0204] 7) Cloning Cloning of nucleic acids has been carried out according to procedures known to those skilled in the art or according to manufacturer's protocols.

[0205] Generally, cloning at R sites depends on the DNA sequence next to the gene of interest (GOI), which is an equal sequence to that in the next fragment. Thus, assembly of the fragments is possible by overlapping of equal sequences followed by sealing of the nicks in the assembled DNA by DNA ligase. After successful cloning of these preliminary vectors, the gene of interest flanked by the R sites is excised via restriction digestion with an enzyme that cuts immediately adjacent to the R site. For assembly of all DNA fragments in one step, a 5'-exonuclease removes the 5' end of the overlapping region (R site). Annealing of the R sites can then be performed and a DNA polymerase extends the 3' end to fill the gap in the sequence. Finally, a DNA ligase seals the nicks between the nucleotides. Addition of an assembly master mix containing different enzymes such as exonucleases, DNA polymerases and ligases followed by incubation of the reaction mix at 50°C results in the assembly of the single fragments into one plasmid. Competent E. coli cells are then transformed with the plasmids.

[0206] For some vectors, a restriction enzyme-mediated cloning strategy was used. By choosing the appropriate restriction enzyme, the desired gene of interest can be excised and then inserted into another vector by ligation. Therefore, it is preferable to use an enzyme that cuts at a multiple cloning site (MCS) and is selected so that ligation of the fragments in the correct array can be performed. If the vector and the fragment have been previously cut with the same restriction enzyme, the sticky ends of the fragment and the vector will fit each other and can then be ligated with DNA ligase. After ligation, competent E. coli cells are transformed with the newly created plasmid.

[0207] For transformation, 10 beta-competent E. coli cells were used according to the manufacturer's protocol. Only bacteria that successfully integrated the plasmid and carried the resistance gene to ampicillin could grow on each plate. Single colonies were picked and cultured in LB-Amp medium for subsequent plasmid preparation.

[0208] bacterial culture The E. coli was cultured in LB medium, short for Luria Bertani, and 1 ml / L of 100 mg / ml ampicillin was added to give an ampicillin concentration of 0.1 mg / ml.

[0209] A 15 ml tube (with ventilated lid) was filled with 3.6 ml of LB-Amp medium and inoculated evenly with a bacterial colony. The toothpick was not removed but left in the tube during incubation. The tube was incubated at 37 °C and 200 rpm for 23 h. The 15 ml tube was removed from the incubator and 3.6 ml of bacterial culture was divided into two 2 ml Eppendorf tubes. The tube was centrifuged at 6,800 × g in a tabletop microcentrifuge at room temperature for 3 min. Minipreps were then performed using the Qiagen QIAprep Spin Miniprep Kit according to the manufacturer's instructions. Plasmid DNA concentrations were measured with a Nanodrop.

[0210] Ethanol precipitation The volume of the DNA solution was mixed with 2.5 volumes of ethanol 100%. The mixture was incubated at -20°C for 10 minutes. The DNA was then centrifuged at 14,000 rpm at 4°C for 30 minutes. The supernatant was carefully removed and the pellet was washed with 70% ethanol. Again, the tube was centrifuged at 14,000 rpm at 4°C for 5 minutes. The supernatant was carefully removed by pipetting and the pellet was dried. Once the ethanol had evaporated, an appropriate amount of endotoxin-free water was added. The DNA was given time to redissolve in water overnight at 4°C. A small aliquot was taken and the DNA concentration was measured with a Nanodrop device.

[0211] Example 2 Preparation of plasmids Composition of the expression cassette For expression of the antibody chains, transcription units containing the following functional elements were used: - the immediate early enhancer and promoter from human cytomegalovirus, including intron A; - a human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - nucleic acids encoding each antibody chain, - the bovine growth hormone polyadenylation sequence (BGH pA), and - optionally, the human gastrin terminator (hGT).

[0212] Besides the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid may include: - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and - Contains the beta-lactamase gene that confers ampicillin resistance to E. coli.

[0213] Front and back vector cloning To construct the two-plasmid antibody constructs, the antibody HC and LC were cloned into a forward vector backbone containing the L3 and LoxFAS sequences, and a reverse vector containing the LoxFAS and 2L sequences and a selection marker. The Cre recombinase plasmid pOG231 (Wong, E. T. et al., Nucl. Acids Res. 33 (2005) e147; O'Gorman, S. et al., Proc. Natl. Acad. Sci. USA 94 (1997) 14602-14607) was used for all RMCE processes.

[0214] cDNAs encoding each antibody chain were generated by gene synthesis (Geneart, Life Technologies Inc.). Gene synthesis and backbone vectors were digested with HindIII-HF and EcoRI-HF (NEB) for 1 h at 37°C and separated by agarose gel electrophoresis. Insert and backbone DNA fragments were excised from the agarose gel and extracted using a QIAquick gel extraction kit (Qiagen). The purified insert and backbone fragments were ligated at a 3:1 insert / backbone ratio using a rapid ligation kit (Roche) according to the manufacturer's protocol. The ligation approach was then transformed into competent E. coli DH5α by heat shock at 42°C for 30 s and incubated at 37°C for 1 h before plating on agar plates containing selective ampicillin. Plates were incubated at 37°C overnight.

[0215] The next day, clones were picked and incubated overnight at 37° C. with shaking for preparation. This was performed using an EpMotion® 5075 (Eppendorf) or using a QIAprep Spin Miniprep Kit (Qiagen) / NucleoBond Xtra Maxi EF Kit (Macherey & Nagel), respectively. All constructs were sequenced to ensure the absence of any unwanted mutations (SequiServe GmbH).

[0216] In the second cloning step, the precloned vector was digested with KpnI-HF / SalI-HF and SalI-HF / MfeI-HF using the same conditions as for the first cloning. The backbone vector was digested with KpnI-HF and MfeI-HF. Isolation and extraction were performed as described above. Ligation of the purified insert and backbone with T4 DNA ligase (NEB) according to the manufacturer's protocol in a 1:1:1 insert / insert / backbone ratio was performed overnight at 4° C. and inactivated at 65° C. for 10 min. The following cloning steps were performed as described above.

[0217] Example 3 Culture, transfection, selection and single cell cloning Host cells were grown in a proprietary DMEM / F12-based medium in disposable 125 ml vented shake flasks under standard humidified conditions (95% rH, 37°C, and 5% CO2) at a constant agitation speed of 150 rpm. Every 3–4 days, cells were seeded at a concentration of 3x10E5 cells / ml in synthetic medium containing effective concentrations of selection marker 1 and selection marker 2. Culture density and viability were measured with a Cedex HiRes cell counter (F. Hoffmann-La Roche Ltd, Basel, Switzerland).

[0218] For stable transfection, equimolar amounts of the front and back vectors were mixed. 1 μg of Cre expression plasmid was added to the mixture of 5 μg.

[0219] Two days before transfection, host cells were seeded in fresh medium at a density of 4x10E5 cells / ml. Transfection was performed in a Nucleofector device using the Nucleofector Kit V (Lonza, Switzerland) according to the manufacturer's protocol. 3x10E7 cells were transfected with 30 μg of plasmid. After transfection, cells were seeded in 30 ml of medium without selection agent.

[0220] Five days after seeding, the cells were centrifuged and transferred to 80 mL of synthetic medium containing selection agent 1 and selection agent 2 at an effective concentration of 6x10E5 cells / ml for the selection of recombinant cells. The cells were incubated at 37°C, 150 rpm, 5% CO2, 85% humidity, and not split from this day on. The cell density and viability of the cultures were monitored regularly. When the viability of the cultures began to increase again, the concentrations of selection agents 1 and 2 were reduced to approximately half of the amounts used previously.

[0221] Ten days after the start of selection, the success of Cre-mediated cassette exchange was confirmed by flow cytometry measuring the expression of intracellular markers and cell surface-bound bispecific antibodies. For FACS staining, APC antibodies (allophycocyanin-labeled F(ab')2 fragment goat anti-human IgG) against the light and heavy chains of human antibodies were used. Flow cytometry was performed using a BD FACS Canto II flow cytometer (BD, Heidelberg, Germany). Ten thousand events were measured per sample. Live cells were gated on a plot of forward scatter (FSC) versus side scatter (SSC). A live cell gate was defined on untransfected host cells and applied to all samples using FlowJo 7.6.5 EN software (TreeStar, Olten, Switzerland). Fluorescence of intracellular markers was quantified in the FITC channel (excitation at 488 nm, detection at 530 nm). Bispecific antibodies were measured in the APC channel (excitation at 645 nm, detection at 660 nm). CHO parental cells, i.e. the cells used to generate the host cells, were used as negative controls for intracellular markers and bispecific antibody expression. After 14 days from the start of selection, viability was greater than 90% and selection was considered complete.

[0222] Example 4 FACS screening FACS analysis was performed to check the transfection efficiency. 4x10E5 cells of the transfected approach were centrifuged (1200 rpm, 4 min) and washed twice with 1 mL of PBS. After a washing step with PBS, the precipitate was resuspended in 400 μL of PBS and transferred to a FACS tube (Falcon® round-bottom tube with cell strainer cap, Corning). Measurements were performed using FACS CantoII and data were analyzed using the software FlowJo.

[0223] Example 5 Single cell cloning Resistant pools were subjected to single cell cloning by limiting dilution at 0.6 cells / well in 384-well plates. Prior to seeding, pools were stained with 5-chloromethylfluorescein diacetate (CMFDA), a live cell fluorescent dye. After seeding, plates were centrifuged and images were acquired from each well in both fluorescent and bright field modes. Fluorescent imaging allows for detection of live cells and differentiation against cell-like artifacts, as observed in bright field images. Approximately 2 weeks after seeding, confluence was determined by bright field imaging. Colonies identified as derived from single cells were expanded to 96-well plates and analyzed by ELISA for titer and binding to target antigen.

[0224] Example 6 ELISA-based titer determination A mixture of 0.25 μg / mL biotinylated F(ab')2 goat anti-human IgG (Fcy spec.; Jackson ImmunoResearch, #109-066-098) and detection antibody POD-conjugated F(ab')2 goat anti-human IgG (Fcy spec; Jackson ImmunoResearch, #109-036-098) in dilution buffer (=1xPBS (0.01M KH2PO4, 0.1M Na2HPO4, 1.37M NaCl, 0.027M KCl, pH 7.0), 0.5% BSA, 0.05% Tween 20 (Sigma Aldrich, CAS# 9005-64-5)) was added to a streptavidin-coated microtiter plate (Nunc, 384-well, #840010; 20 μL / well). Control antibody (TCB) was diluted in dilution buffer to a starting concentration of 0.2 μg / mL and 12 dilutions (dilution factor 1:2) were transferred (10 μL / well) to the assay plate. Culture supernatant was added to the assay plate (10 μL / well) and the plate was incubated for 1 h at room temperature without agitation. The wells were washed six times with 90 μL / well of wash buffer (=1xPBS (0.01M KH2PO4, 0.1M Na2HPO4, 1.37M NaCl, 0.027M KCl, pH 7.0), 0.1% Tween 20 (Sigma Aldrich, CAS# 9005-64-5)). Afterwards, 30 μL / well of detection substrate TMB (SeraCare, KPL SureBlue Reserve TMB Microwell Peroxidase Substrate, Cat# 5120-0087) was added and incubated for 5 min at room temperature without agitation. Absorbance measurements were performed at a wavelength of 370 nm using a Powerwave HT reader (BioTeK). All ELISA signals of samples and control antibodies (raw data) were blank corrected by subtraction of the signal measured for wells containing only dilution buffer (blank corrected data). A standard curve (linear regression) was calculated using the blank corrected signals of the 12 concentrations of control antibodies. Antibody titers were calculated using the linear regression equation of the control antibodies.

[0225] Example 7 ELISA-based binding assay A premix of 0.03 μg / mL biotinylated Antigen 1-human Fc region (huFc) conjugate or 0.25 μg / mL biotinylated Antigen 2-human Fc region conjugate in dilution buffer (=1×PBS (0.01M KH2PO4, 0.1M Na2HPO4, 1.37M NaCl, 0.027M KCl, pH 7.0), 0.5% BSA, 0.05% Tween 20 (Sigma Aldrich, CAS# 9005-64-5)) and detection antibody anti-human F(ab)2-HRP (Jackson ImmunoResearch, #109-036-006; 1:5000 iA) was added to a streptavidin-coated microtiter plate (Nunc; 384-well, #840010; 20 μL / well). Supernatants and control antibodies were diluted to a starting concentration of 0.05 μg / mL in dilution buffer, transferred to wells (5 μL / well) and incubated for 1 h at room temperature without agitation. Purified antibody main product (knob-into-hole heterodimer) and two antibody side products (hole-hole and knob-knob, homodimers, respectively) were added in 14 dilutions with a dilution factor of 1:1.5, respectively, and samples were applied in 4 dilutions with a dilution factor of 1:5.0625, respectively. The wells were washed with 90 μL / well of wash buffer (=1xPBS (0.01M KH2PO 4、 The plates were washed six times with 0.1M Na2HPO4, 1.37M NaCl, 0.027M KCl, pH 7.0, 0.1% Tween 20 (Sigma Aldrich, CAS# 9005-64-5). Afterwards, 30 μL / well of detection substrate TMB (SeraCare, KPL SureBlue Reserve TMB Microwell Peroxidase Substrate, Cat# 5120-0087) was added and incubated for 5 min at room temperature without agitation. Absorbance measurements were performed at a wavelength of 370 nm using an EnVision reader (Perkin Elmer).

[0226] Example 8 Bridging ELISA AG12 Non-biotinylated antigen 2-huFc conjugate was directly coated onto microtiter plates (Nunc, MaxiSorp, #464718) at a concentration of 0.5 μg / mL in dilution buffer and incubated overnight at 4°C without agitation. Plates were washed (3×90 μL / well each with wash buffer; 1×PBS (0.01M KH2PO4, 0.1M Na2HPO4, 1.37M NaCl, 0.027M KCl, pH 7.0), 0.1% Tween 20 (Sigma Aldrich, CAS# 9005-64-5)) and blocked with 90 μL blocking buffer (2% BSA in dilution buffer) for 30 min. After washing the plates again (3×90 μL / well each with wash buffer), sample and control antibodies were added and incubated for 1 h at room temperature. The purified main product (knob-into-hole heterodimer) and two main minor products (hole-hole and knob-knob, respectively, homodimers) were added in 14 dilutions with a dilution factor of 1:1.5, respectively, and the samples were applied in 4 dilutions with a dilution factor of 1:5.0625, respectively. After washing the plate (3 × 90 μL / well with wash buffer), 25 μL of a mixture of biotinylated antigen 1-huFc conjugate and streptavidin POD conjugate (Roche Diagnostics GmbH, Mannheim, Germany) was applied. The wells were washed six times with 90 μL / well of wash buffer, respectively. Afterwards, 30 μL / well of detection substrate TMB (SeraCare, KPL SureBlue Reserve TMB Microwell Peroxidase Substrate, Cat# 5120-0087) was added and incubated for 5 min at room temperature without agitation. Absorbance measurements were performed at a wavelength of 370 nm using an EnVision reader (Perkin Elmer).

[0227] Example 9 Machine Learning Input data For each cell clone, blank-corrected readings from ELISA assays for antibody titer, binding to antigen 1, binding to antigen 2, and simultaneous binding to antigens 1 and 2 were used as input data.

[0228] As target variable (called labels in machine learning), we used the classification of cell clone supernatants into five different product categories (lead, byproduct I, byproduct II, unbound and undefined) by human experts from a historical project.

[0229] Further manipulation of the data was performed (feature engineering) and fed into the model in addition to the input data.

[0230] Working with Features Eight different data processing variants have been used to build the predictive models. Therefore, the blank-corrected readings from the ELISA assays were transformed in various ways, as shown in the table below.

[0231] TIFF2024513474000005.tif179150

[0232] In detail, the processing variations were as follows:

[0233] Process variant no. 1: Only supernatant binding and bridging ELISA assay data (absorbance corrected ELISA signals of supernatants of candidate clones) and determined total antibody titers were used; this was the baseline experiment; the data utilized was part of all subsequent experiments.

[0234] Processing variants no. 2–8: Assay readouts of three standards lead (100% purified main product), byproduct I (100% purified byproduct I, hole-hole homodimer) and byproduct II (100% purified byproduct II, knob-knob homodimer) were added to the data set (variant 2) or used in modified form (variants 3–8); each standard was determined at 14 different concentrations (standard curve); for variants 2–5, four of these concentrations within the linear assay range were selected for the calculation.

[0235] For variants 6 to 8, no selection step was required since the median of all 14 concentrations analyzed was used.

[0236] Variants no. 3-8 included data normalized to data from one or all three standards. Variant 3 utilized sample values ​​normalized to the lead standard, while variant 4 worked with values ​​normalized to all three standards.

[0237] Variants no. 5-8 included ratios instead of simple values ​​and utilized them as normalized values ​​relative to all three standards. Of the nine possible normalized ratios, variant 7 included all nine, while variant 8 utilized only the selected four.

[0238] training For training, three projects were pooled and the fourth project was reserved for final model evaluation (unseen test dataset). The training data was then used for model building and hyperparameter optimization. Machine learning models were generated automatically using a form of driverless AI tool from H2O.ai. Each processing variant (see table above) was used to generate a dataset on which training and evaluation were performed.

[0239] test Then, only the final model (with the best hyper-parameter set) was scored by applying a cross-entropy loss function to an unseen test dataset (Project 4).

[0240] References Scikit-learn:Machine Learning in Python,Pedregosa et al.,JMLR 12,pp.2825-2830,2011.

Claims

1. 1. A method for selecting one or more recombinant cell clones from a large number of recombinant cell clones that all express the same recombinant bispecific antibody that binds a first antigen and a second antigen, comprising the steps of: - culturing each single deposited recombinant cell clone of said plurality of recombinant cell clones to produce a culture supernatant; - determining: i) the total antibody concentration in the supernatant; ii) the binding signal of each supernatant for binding of the bispecific antibody to the first antigen in an immunoassay for four different concentrations of said supernatant; iii) the binding signal of each supernatant for binding of the bispecific antibody to the second antigen in an immunoassay for the same four concentrations of the supernatant as in ii); iv) the binding signal of each supernatant for simultaneous binding of the bispecific antibody to the first antigen and the second antigen in an immunoassay for the same four concentrations of the supernatants as in ii); v) binding signals of the isolated bispecific antibodies for binding to the first antigen, the second antigen, and both antigens in the same immunoassays as in ii), iii), and iv), respectively, for 14 different concentrations of the bispecific antibody; vi) binding signals of a first isolated bispecific antibody-related by-product also expressed by said recombinant cell clone, for binding of said first bispecific antibody-related by-product at the same 14 concentrations as in v) to said first antigen, to the second antigen and to both antigens in the same immunoassays as in ii), iii) and iv), respectively; vii) binding signals of a second isolated bispecific antibody-related by-product also expressed by said recombinant cell clone, for binding of said second bispecific antibody-related by-product at the same 14 concentrations as in v) to said first antigen, to the second antigen and to both antigens in the same immunoassays as in ii), iii) and iv), respectively; - the following values: - said concentration of i), - the binding signals of ii), iii) and iv), the median of the individual ratios between the binding signal for each concentration obtained in iii) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in v) using said immunoassay in iii) and the binding signal for each same concentration obtained in v) using said immunoassay in ii), - the median of the individual ratios between the binding signal for each concentration obtained in iv) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in v) using said immunoassay in iv) and the binding signal for each same concentration obtained in v) using said immunoassay in ii), the median of the individual ratios between the binding signal for each concentration obtained in iii) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in vi) using said immunoassay in iii) and the binding signal for each same concentration obtained in vi) using said immunoassay in ii), - the median of the individual ratios between the binding signal for each concentration obtained in iv) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in vii) using said immunoassay in iv) and the binding signal for each same concentration obtained in vii) using said immunoassay in ii). ranking the recombinant cell clones of the plurality of recombinant cell clones based on said ranking being according to the respective difference of said values ​​for each individual cell clone relative to the value obtained for said isolated bispecific antibody. Including, The recombinant cell clone with the smallest overall difference is selected.

2. The method of claim 1 , wherein the difference is determined using an in silico method.

3. The method of claim 1 or 2, wherein the difference is determined using machine learning.

4. The method of claim 1 or 2, wherein the difference is determined using an extreme gradient boosting model.

5. 3. The method of claim 1 or 2, wherein the selection is based on a combination of the minimum difference in the concentration, the binding signal, and the divided ratio of the bispecific antibody and the maximum difference in the divided ratio of the by-product.

6. 3. The method of claim 1 or 2, wherein the one or more selected cell clones express the recombinant bispecific antibody that, on average, has a lower proportion of antibody-related by-products than the multiple recombinant cell clones.

7. 3. The method of claim 1 or 2, wherein the bispecific antibody comprises a heterodimeric Fc region, where one Fc region polypeptide comprises a knob mutation and each other Fc region polypeptide comprises a hole mutation; the first bispecific antibody-related by-product is a hole-hole homodimer of the bispecific antibody; and the second bispecific antibody-related by-product is a knob-knob homodimer of the bispecific antibody.

8. The method of claim 1 or 2, wherein the immunoassay is an enzyme-linked immunosorbent assay.

9. 3. The method of claim 1 or 2, wherein the cell is a CHO cell.

10. To identify recombinant cell clones expressing bispecific antibodies with reduced bispecific antibody-related by-products, i) the total antibody concentration determined in the culture supernatant of a single recombinant cell clone, said recombinant cell clone being transfected with a nucleic acid encoding said bispecific antibody; ii) binding signals for the respective binding of the supernatant to the first antigen and to the second antigen of the bispecific antibody; iii) binding signals for simultaneous binding of the supernatant to the first antigen and the second antigen of the bispecific antibody. Use of.

11. iv) binding signals for the separate and simultaneous binding of the isolated bispecific antibody to its first and second antigens; v) binding signals for the separate and simultaneous binding of a first isolated bispecific antibody-related by-product to the first antigen and to the second antigen of the bispecific antibody; vi) binding signals for the separate and simultaneous binding of a second isolated bispecific antibody-related by-product to the first antigen and the second antigen of the bispecific antibody. is further used to identify recombinant cell clones expressing said bispecific antibody with reduced bispecific antibody-related by-products.

12. the median of the individual ratios between the binding signal for each concentration obtained in iii) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in v) using said immunoassay in iii) and the binding signal for each same concentration obtained in v) using said immunoassay in ii), - the median of the individual ratios between the binding signal for each concentration obtained in iv) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in v) using said immunoassay in iv) and the binding signal for each same concentration obtained in v) using said immunoassay in ii), the median of the individual ratios between the binding signal for each concentration obtained in iii) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in vi) using said immunoassay in iii) and the binding signal for each same concentration obtained in vi) using said immunoassay in ii), - the median of the individual ratios between the binding signal for each concentration obtained in iv) and the binding signal for each same concentration obtained in ii) divided by the median of the individual ratios between the binding signal for each concentration obtained in vii) using said immunoassay in iv) and the binding signal for each same concentration obtained in vii) using said immunoassay in ii). is further used to identify recombinant cell clones expressing said bispecific antibody with reduced bispecific antibody-related by-products; iv) binding signals for the separate and simultaneous binding of the bispecific antibody to its first and second antigens, v) binding signals for the separate and simultaneous binding of a first isolated bispecific antibody-related by-product to the first antigen and the second antigen of the bispecific antibody; vi) is a binding signal for the separate and simultaneous binding of a second isolated bispecific antibody-related by-product to the first antigen and the second antigen of the bispecific antibody.

12. Use according to claim 10 or 11.

13. A computer program comprising computer executable instructions for carrying out the method according to claim 1 or 2 when the program is run on a computer or a computer network.

14. A computer program product comprising program code means for carrying out the method according to claim 1 or 2 when the program is run on a computer or a computer network.

15. A data carrier having a data structure stored thereon, which is capable of carrying out the method according to claim 1 or 2 after being loaded into a computer or computer network, such as a working or main memory of the computer or computer network.

16. The method of claim 5, wherein the bispecific antibody comprises a heterodimeric Fc region, where one Fc region polypeptide comprises a knob mutation and each other Fc region polypeptide comprises a hole mutation; the first bispecific antibody-related by-product is a hole-hole homodimer of the bispecific antibody; and the second bispecific antibody-related by-product is a knob-knob homodimer of the bispecific antibody.

17. The method of claim 5 , wherein the immunoassay is an enzyme-linked immunosorbent assay.

18. 6. The method of claim 5, wherein the cell is a CHO cell.

19. 17. The method of claim 16, wherein the immunoassay is an enzyme-linked immunosorbent assay.

20. 17. The method of claim 16, wherein the cell is a CHO cell.

21. 18. The method of claim 17, wherein the cell is a CHO cell.

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