Producing compositions comprising two or more antibodies

Abstract

This invention relates to means and methods for generating at least two types of antibodies. The method may include: providing cells carrying nucleic acids encoding the antibodies; culturing the cells; collecting the antibodies from the culture; and separating the generated antibodies and half-antibodies by ion exchange chromatography (IEX). In some specific examples, under the IEX conditions used, the antibodies exhibit an IEX retention time that deviates from the average retention time of individual antibodies by 10% or less. This invention also relates to compositions of antibodies generated thereby. In some aspects, this invention relates to compositions comprising 2-10 recombinant antibodies, characterized in that, under the IEX conditions, the IEX retention time of at least two of the antibodies deviates from the average retention time of individual antibodies by 10% or less. This invention also relates to compositions comprising 2-10 recombinant antibodies, characterized in that the pI value of at least two of the antibodies differs from the average pI value of the at least two antibodies by 0.4 units or less.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to the field of therapeutic antibodies. Such antibodies can be used in human therapy. More particularly, the present invention relates to the production and / or purification of multiple antibodies. A single host cell can produce such multiple antibodies. Such antibodies can also be produced by a mixture of host cells, each of which produces one of the antibodies. The present invention also relates to methods for producing compositions containing such antibodies and methods for purifying such antibodies. Prior Art

[0002] Polyclonal antibodies are typically collected from the blood of an individual. One advantage of polyclonal antibodies is that the pathogen is attacked via multiple targets and epitopes. One advantage of monoclonal or recombinant antibodies is the well-characterized specificity and function allowing such antibodies to be used as precision medicines with a well-defined spectrum of action and toxicity.

[0003] The specificity of a single antibody can also be a disadvantage, especially when multiple targets need to be addressed. It is possible to reduce this disadvantage by adding more antibodies to the drug, but given that even a single therapeutic antibody can be expensive, it is expected that the costs of such a polyclonal cocktail could quickly become prohibitive.

[0004] The development of bi-, tri- and other multispecific antibodies has successfully introduced some aspects of polyclonal antibodies into antibody therapeutics. In addition to the increase in the number of targets, it has also successfully introduced other functionalities that were previously unavailable using traditional monospecific monoclonal or polyclonal antibodies. The use of multiple bi-, tri- and other multispecific antibodies in the same therapy can provide further benefits.

[0005] The present invention provides an advance in the art by describing a robust and economical method for purifying multiple antibody therapeutics produced from a single host cell or, alternatively, a mixture of host cells. The present invention is particularly useful for the economical production of collections of two or more antibodies, preferably multispecific antibodies. Summary of the invention

[0006] SUMMARY OF THE INVENTION The present invention provides a method for generating at least two multispecific antibodies, comprising: – providing cells carrying nucleic acids encoding the multispecific antibodies; – culturing those cells; – collecting the multispecific antibodies from the culture; and – separation of the generated multispecific antibodies from half antibodies and selectively monospecific antibodies and / or other undesirable antibody product-related impurities by ion exchange chromatography (IEX); The method is characterized in that, under the IEX conditions used, the multispecific antibodies exhibit an IEX retention time that deviates by 10% or less from the mean of the retention times of the individual multispecific antibodies. The retention times of the individual half antibodies and optionally the monospecific antibodies and / or other undesired antibody product-related impurities preferably fall outside the range spanned by the retention times of the multispecific antibodies.

[0007] The present invention also provides a method for generating at least two multispecific antibodies, comprising: – providing a cell carrying nucleic acid encoding the multispecific antibodies; – culturing the cells; – collecting the multispecific antibodies from the culture; and – separation of the generated multispecific antibodies from half antibodies and selectively monospecific antibodies and / or other undesirable antibody product-related impurities by ion exchange chromatography (IEX); The method is characterized in that: under the IEX conditions used, the multispecific antibodies exhibit substantially the same IEX retention time. The retention times of the respective half antibodies and optionally the monospecific antibodies and / or other undesired antibody product-related impurities preferably fall outside the range spanned by the retention times of the antibodies.

[0008] The present invention further provides a method for generating at least two antibodies, wherein the antibodies comprise a monospecific and / or a multispecific antibody, the method comprising: – providing cells carrying nucleic acids encoding such antibodies; – culturing those cells; – collecting the antibodies from culture; and – Separation of produced antibodies from half-antibodies by ion exchange chromatography (IEX); The method is characterized in that: under the IEX conditions used, the antibodies exhibit substantially the same IEX retention time. The retention times of the respective half antibodies and optionally the monospecific antibodies preferably fall outside the range spanned by the retention times of the antibodies.

[0009] The present invention also provides a composition comprising 2-10 recombinant antibodies obtainable by a method as described herein.

[0010] Also provided is a composition comprising 2-10 recombinant antibodies (such as multispecific antibodies), characterized in that: under the IEX conditions used, the IEX retention times of at least two of the antibodies deviate by 10% or less from the average of the retention times of the individual antibodies.

[0011] Further provided is a composition comprising 2-10 recombinant antibodies, characterized in that the IEX retention times of at least two of the antibodies are substantially the same.

[0012] Also provided is a composition comprising 2-10 recombinant antibodies, characterized in that: the isoelectric point (PI) of at least two of the antibodies preferably differs from the average PI value of the at least two antibodies by 0.4 units, 0.3, 0.2 and preferably 0.1 units or less. The PI value of each of the at least two antibodies preferably differs from the other by 0.25 units or less. [Detailed description of the invention]

[0013] The term "antibody" as used herein refers to a proteinaceous molecule belonging to the immunoglobulin class of proteins, which contains one or more domains that bind to an epitope on an antigen, wherein the domains are either derived from or share sequence homology with the variable regions of an antibody. Antibodies are typically composed of basic structural units - each having two heavy chains and two light chains. Antibodies for therapeutic use are preferably natural antibodies that are as close as possible to the individual to be treated (e.g., human antibodies for human individuals). Antibodies with extended heavy and / or light chain variable regions are also included herein. An antibody according to the present invention is not limited to any particular type or method used to produce it.

[0014] Half antibodies are heavy and light chain combinations that are not associated with another heavy and light chain combination and that do not form an interface with another variable region or variable region-like polypeptide. Other undesirable antibody product-associated impurities may be free light chains that are not associated with a heavy chain, free heavy chains that are not associated with a light chain or another heavy chain, or incompletely assembled antibodies that lack a light chain.

[0015] Suitable cells for antibody production are known in the art and include a hybridoma cell, a Chinese hamster ovary (CHO) cell, a NSO cell or a PER-C6 cell, or a variety of other cell lines known to those having ordinary skill in the art. Various institutions and companies have developed cell lines for large-scale production of antibodies, such as for clinical use. Non-limiting examples of such cell lines are, among others, CHO cells, NSO cells or PER.C6 cells or HEK293 cells. In a particularly preferred embodiment, the cell is a human cell. Preferably, a cell transformed with an adenovirus E1 region or a functional equivalent thereof. In a preferred embodiment, the cell is a CHO cell or a variant thereof. Preferably, a variant that uses a glutamine synthetase (GS) vector system for the expression of an antibody. In a preferred embodiment, the cell is a CHO cell.

[0016] The cells can be provided with nucleic acids encoding the antibodies. The cells will express, assemble and secrete the formed antibodies into the supernatant of the cell culture. The introduction of a single heavy and light chain (or more precisely, the nucleic acids encoding them) results in the generation of a monoclonal antibody having two heavy chains each associated with one light chain.

[0017] A method of the invention can be performed using a cell mixture comprising two or more cells that each produce a different antibody. An advantage of using such a mixture is that downstream processing of the collected antibodies can be streamlined more efficiently. A further advantage of a method is that the antibody product is collected and purified as a whole. Also, a mixture containing two or more antibodies produced by a method of the invention can reduce the number of tests required to obtain regulatory certification when compared to the number of tests required for each of the antibodies separately.

[0018] In a further embodiment, the cell lines are a homogeneous collection of cells consisting essentially of copies of a single cell provided with nucleic acids encoding the respective antibodies. Co-expression of several heavy chains in one cell allows for a variety of heavy chain combinations. Combinations with additional light chains increase the number of combinations. Various methods have been developed to favor the formation of specific combinations over others. Heavy chain variants have been generated that specifically promote the formation of heavy chain heterodimers over homodimers, or vice versa. Heavy chains with specific homo- or heterodimerization domains reduce the number of antibodies being produced by such cells and / or increase the level of a preferred antibody over an alternative combination (e.g., higher production of a heterodimer over a homodimer).

[0019] A method of the present invention is particularly suitable for the production of two or more multispecific antibodies, including bispecific antibodies. Various methods for the production of bispecific antibodies exist in the art. One method uses a common light chain that can form a functional variable region with different heavy chains. A preferred method for producing bispecific antibodies comprises the use of a genetically modified animal that harbors a common chain in its genome, so that such an animal, upon immunization with an antigen, produces an antibody library specific for the antigen based on non-common chains, wherein the antibody library is composed of different antibodies comprising the common chain and a rearranged homologous chain. An animal can be immunized with different antigens, or different animals can be immunized separately with different antigens. Nucleic acids encoding the non-common chains or their variable regions can be obtained from the animal(s), such as B cells, spleen or lymphoid tissue. These can be used to produce nucleic acids expressing the different non-common chains, which can then be introduced into the production cell. The common chain can be introduced at the same or different times. The nucleic acid can be incorporated into the nucleus, and preferably into the genome of the host cell, so that the host cell produces multispecific antibodies or polymers targeting multiple antigens (the transgenic animal(s) have been immunized against these antigens) (for the purpose of generating variable regions that can be combined with a common chain to generate functional variable regions specific for different targets and / or different epitopes, see, for example, WO 2009 / 157771, which is hereby incorporated by reference).

[0020] A cell producing a common light chain and two different heavy chains, each of which can form a functional variable domain with the common light chain, produces, among other things, a bispecific antibody with two different heavy and light chain combinations. Likewise, a cell producing a common light chain and three or more different heavy chains can form a multispecific antibody capable of targeting three or more antigens, or a combination of two or more multispecific antibodies capable of targeting three or more antigens. It is now possible to establish a standard format for antibodies (i.e. a constant part and two variable domains) and to add further binding domains. In this way, multispecific antibodies having one or more single-chain Fvs with additional binding specificities attached to one or more of the constant part or variable domains of an antibody can be produced. It is also possible to produce heavy chains with two or more variable regions. The additional heavy chain regions can advantageously be combined with different or common light chain variable regions. See US 62 / 650467, which is hereby incorporated by reference.

[0021] When the cell produces two or more multispecific antibodies, it may also produce a number of half antibodies and antibodies or homodimers with the same heavy chain in some cases. The number of the latter can be reduced by including modifications that promote heterodimer formation within the heavy chain. As mentioned above, various methods exist for inducing heterodimerization of heavy chains. The individual domains with such modifications are collectively referred to as heterodimerization domains. Heavy chains with heterodimerization domains that are conducive to interaction are said to have compatible heterodimerization domains. Such compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. Various methods are described in the art in which such CH3 heterodimerization of heavy chains can be achieved.

[0022] A preferred method for generating bispecific antibodies is disclosed in US 9,248,181 and US 9,358,286. In particular, the preferred changes used to generate essentially only bispecific full-length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain (the "KK-variant" heavy chain) and the amino acid substitutions L351D and L368E in the second domain (the "DE-variant" heavy chain), or vice versa. As mentioned above, the DE-variant and KK-variant preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE-variant heavy chains (DEDE homodimer) or homodimerization of KK-variant heavy chains (KKKK homodimer) hardly occurs due to the strong repulsion between the charged residues located in the CH3-CH3 interface between the same heavy chains.

[0023] In the present invention, it is preferred that the cells are provided with nucleic acid encoding a common light chain. There are various methods available to those skilled in the art to generate antibodies with different heavy chain variable regions but the same light chain variable regions. WO 2004 / 106375 describes a phage library using a common light chain variable region. Phage selection yields variable regions with the same light chain variable region but different heavy chain variable regions. Furthermore, non-human animals with a common chain and non-identical homologous chains are described in WO 2009 / 157771. Antibody selection in such animals yields variable regions with the same or similar common chain variable regions but non-identical homologous chain variable regions. WO 2004 / 106375 and WO 2009 / 157771 are hereby incorporated by reference. Reference is made to these publications, in particular, with respect to the production of antibodies having identical or similar common chain variable regions and non-identical cognate chain variable regions, preferably a common light chain variable region and non-identical heavy chain variable regions, and nucleic acids encoding such antibodies.

[0024] In a preferred embodiment, the cells produce two or more heavy chains and a common light chain. The individual heavy and light chains may have one or more variable regions associated with the individual chains. In a preferred embodiment, the cells produce three or more heavy chains. One of the three heavy chains preferably contains a portion of a compatible heterodimerization domain. The other two or more heavy chains preferably include another portion of the compatible heterodimerization domain. If the first heavy chain is symbolically represented by the letter "A" and the other two by the letters "B" and "C", a specific combination of heterodimerization domains leads to the predominant formation of combinations AB and AC. The combinations AA, BB, CC and BC are not favored by the inclusion of heterodimerization domains. Such a cell is effective in producing only two bispecific antibodies AB and AC (see Figure 1). In the present invention, it is preferred that the cell produces the two antibodies by producing at least 3 heavy chains. In a preferred embodiment, one of the heavy chains contains a portion of a compatible heterodimerization domain, and the other two or more heavy chains preferably contain another portion of the compatible heterodimerization domain. A heavy chain shared by two or more bi- or multi-specific antibodies or multimers in a composition preferably has the CH3 DE portion of the heterodimerization domain. The other heavy chains in the bi- or multi-specific antibodies preferably have the CH3 KK portion.

[0025] The at least two antibodies are preferably multispecific antibodies, preferably bispecific antibodies. In a preferred embodiment, at least two of the antibodies share the same heavy chain. The cell can generate several series of bispecific antibodies by including different heterodimerization domains in the heavy chains. Such implementation can lead to the dominant generation of antibodies with heavy chain combinations AB and CD. The combination AB can be favored by a DE / KK heterodimerization domain as mentioned above, and the CD is by the incorporation of a "knob in hole" heterodimerization domain, or other heterodimerization features known in the art, such as through charge engineering. A shared heavy chain between different bi- or multispecific antibodies or an antibody combination with the shared heavy chain can be manufactured by providing a shared heavy chain with a portion of a heterodimerization domain and various different combination chains with complementary portions of the heterodimerization domain. For example, a CH3 DE portion in the shared heavy chain and a CH3 KK portion in the combination chains. One shared heavy chain and two combined heavy chains in this case will cause the cell to produce bi- or multispecific multimers with heavy chain combinations AB and AC (or AxBC and AxDE for multispecific multimers). Other possible combinations are AB, AE, CD and CF; or AB, AE, AG and CD, and so on.

[0026] Antibodies generally have a unique isoelectric point (typically in the pH 6-10 range) compared to other host cell proteins. The antibodies, such as multispecific antibodies, and also multispecific multimers, can be purified with a relatively high purity via the methods described herein. The methods can include a number of steps, such as culturing the host cells, undergoing harvest clarification, followed by protein capture. IEX chromatography, such as anion exchange chromatography, can be used to remove host cell DNA, and cation exchange chromatography (CIEX) can be used, for example, to remove host cell proteins, leached protein A, and potential aggregates. Additional steps, such as virus filtration or hydrophobic interaction chromatography, can be included.

[0027] Antibodies are typically produced by producing cells. Harvesting of such antibodies typically involves collection of the cell supernatant, followed by several purification steps to remove cell debris or aggregates (the presence of which is undesirable). Clarification of the harvest may involve filtration, centrifugation, or a combination of these of the culture supernatant of the antibody producing cells. Antibody protein capture is typically accomplished by affinity purification. This can be accomplished in several ways. Usually this involves purification using columns with recombinant protein A, protein G, or protein L, which are bacterial proteins with a known high specific binding capacity for the antibody. Currently, various optimized mutant systems are available that bind the antibody more specifically. For example, a recombinant protein A is available that has had its non-essential domains removed, a recombinant protein G is available that has had its albumin binding site deleted, and modified protein L is available. Bound antibodies can be collected by elution against one or more of these columns. Anion and cation exchange chromatography can be used to further purify the preparation, for example, to purify the bispecific or multispecific antibody from half antibodies and / or homodimeric antibodies and / or other undesirable antibody product-related impurities, if any. Hydrophobic interaction chromatography (HIC) is often used as an alternative polishing step in the antibody purification process. HIC provides an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate removal and host cell protein reduction. In the present invention, HIC can be used for analytical purposes after the purification of the two or more bi- or multispecific antibodies or multimers is completed, so as to quantify the two or more species having similar (preferably substantially the same) retention times and / or similar (preferably substantially the same) pI values ​​on IEX. Thus, HIC is used to quantify the relative amounts of the purified molecules.

[0028] A hydrophobic interaction resin is selected as the stationary phase, and the pH and / or conductivity of the mobile phase is modulated to achieve the desired selectivity. Antibodies typically attract positive charges at lower pH values, which affects their polarity and overall surface hydrophobicity. pH conditions can be selected that allow separation of the two or more antibodies in the preparation.

[0029] In certain embodiments, the collected antibodies are first separated from other proteins by affinity purification, preferably by protein A extraction. The affinity purified antibodies are then run on an anion exchange column under conditions that collect the antibodies in the flow-through fraction. The antibodies can then be run on one or more CIEX columns.

[0030] A preferred method for producing at least two antibodies is accomplished by the cell producing them as a single composition comprising the two or more antibodies.

[0031] A method for producing at least two antibodies preferably comprises culturing host cells that produce the two or more antibodies, collecting supernatants from the cells, and treating the supernatant harvest in a harvest clarification process, the harvest clarification process preferably comprising filtration using a pore size threshold that captures aggregates such as cells or cell debris and allows passage of the antibody product. The antibodies are collected from the cell culture fluid by affinity chromatography using protein A. The antibodies bound to the protein resin are eluted from the chromatography column after exposure to low pH, and the eluate is then neutralized using a suitable buffer.

[0032] As used herein, the term "isoelectric point (pI)" refers to the pH value at which the average net charge on the surface of a protein (i.e., the potential of the protein's electrical double layer) is 0. In other words, the term refers to the point at which one group of the protein is dissociated so that the number of cations and anions is equal and the net charge of the protein is 0.

[0033] As used herein, "pi" is calculated from primary amino acids using the system default parameters as of the earliest filing date (priority date) of the present application according to the ExPASy ProtParam tool. ProtParam is a tool that allows the calculation of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or a user-entered protein sequence. The calculated parameters include theoretical pI values. Gasteiger E., Hoogland C., Gattiker A.,​​ Duvaud S., Wilkins MR, Appel RD, Bairoch A.; Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005) pp. 571-607.

[0034] The net surface charge of a protein varies with pH in a manner that depends on the protein's pI. At a pH equal to the pI of a protein, the protein will carry no net charge. At a pH below the pI, the protein will carry a net positive charge. If the buffer pH is raised above the pI of a protein, it will carry a net negative charge.

[0035] The pI of a protein can be determined by its primary amino acid sequence and thus calculated, and a buffer that ensures a known net charge for a protein of interest can then be selected. When the protein of interest carries a net positive charge at the operating pH, a negatively charged cation exchange resin can thus be selected.

[0036] Proteins with different pI values ​​will have different degrees of charge at a given pH and therefore different affinities for positively charged surface groups on the particles of the anion exchange medium; thus, different proteins will bind to the resin with different strengths, facilitating their separation. Thus, by generating heterodimeric polypeptides with unique pI values ​​relative to the homodimers and half antibodies and / or other antibody product-related impurities, the heterodimeric polypeptides can be readily purified using standard elution techniques, for example, by applying a pH gradient, or by applying a salt or conductivity gradient at a fixed pH, or a combination of a pH and a conductivity gradient. In embodiments of the invention, the constant parts and light chains located in the antibodies have essentially the same sequence in different antibodies. Such antibodies typically differ essentially only in the amino acid sequence of the heavy chain variable region. For such antibodies, it is generally sufficient to calculate the pI value of the heavy chain variable region as shown herein. The calculation and standard are then set by using the pI value of the heavy chain variable region instead of the pI value of the (half) antibody. The average pI value of the heavy chain variable region of the antibody represents the retention time of the antibody in a CIEX-column. The antibodies, if any, may have the same or different heterodimerization domains. They preferably have the same heterodimerization domain. The antibodies may further differ in the correct amino acid sequence of the constant parts.

[0037] Regarding the (iso)ion exchange chromatography (IEX) step, this is a process that separates ions and polar molecules based on their affinity for ion exchangers. It works on a variety of charged molecules - including large proteins, small nucleotides and amino acids. IEX is commonly used in protein purification. Water-soluble and charged proteins form ionic bonds with an insoluble stationary phase. The bound molecules can then be eluted and collected using an eluent with a higher concentration of ions and / or a different pH. The salt concentration or pH can be varied in a stepwise manner; by gradually changing the mobile phase of the chromatography run, or a combination of these. Two types of ion chromatography are anion exchange and cation exchange. Cation exchange chromatography (CIEX) is preferred in the present invention. Antibody CIEX is preferably performed at a physiological pH. The pH is typically in the range of 5-9, preferably 6-8.

[0038] CIEX is typically used when the molecule of interest is positively charged at the pH used for chromatography. The molecule is positively charged because the pH used for chromatography is below the molecule's pI. In this format, the stationary phase is negatively charged and positively charged molecules are loaded so as to be attracted to the stationary phase. Anion exchange chromatography is when the stationary phase is positively charged and negatively charged molecules (meaning the pH used for chromatography is above the pI) are loaded so as to be attracted to the stationary phase.

[0039] An antibody (such as a multispecific antibody) is typically bound to the IEX column in a binding phase under conditions that promote binding of the antibody (such as a multispecific antibody) to a matrix. The IEX column is typically then washed to remove unbound material. Elution from the column is accomplished in an elution phase. The retention time of an antibody (such as a multispecific antibody) is typically calculated from the beginning of the elution phase. It is the amount of time the antibody spends on the column when the elution phase is initiated. If a sample contains several compounds, each compound in the sample will typically spend a different amount of time on the column depending on its chemical composition, i.e., each will have a different retention time. Retention times are usually quoted in seconds or minutes.

[0040] In a method of the invention, the retention times of the antibodies (such as multispecific antibodies) are preferably substantially the same. Different antibodies may have different retention times in the same column and conditions. In the present invention, it has been discovered that antibodies, such as multispecific antibodies, can be selected or designed to have IEX retention times that are close enough to allow co-purification of two or more antibodies (such as two or more multispecific antibodies) in a single IEX chromatography run. Retention times that are 10% or less from the mean of the retention times of the individual antibodies are typically close enough to allow co-purification of two or more antibodies (such as two or more multispecific antibodies) in a single IEX chromatography run.

[0041] A suitable CIEX HPLC method for antibody purification and / or analysis according to the present invention uses the TSKgel SP-STAT (7 µm particle size, 4.6 mM ID x 10 cm L, Tosoh 21964) series of ion exchange columns. These columns are packed with non-porous resin particles for speed and high resolution analysis and separation of biomolecules. The particles in the TSKgel STAT columns contain an open access network structure composed of multiple layers of ion exchange groups for loading capacity, and the particle size makes these columns suitable for use with HPLC and FPLC systems.

[0042] One suitable method involves equilibration of TSKgel SP-STAT (7 µm particle size, 4.6 mM ID x 10 cm L, Tosoh 21964) with buffer A (sodium phosphate buffer, 25 mM, pH 6.0), after which the antibody is expelled from the column by increasing salt concentration and running a gradient of buffer B (25 mM sodium phosphate, 1 mM NaCl, pH 6.0). The flow rate is set at 0.5 mL / min. The injected sample mass for test samples and controls is 10 µg, and the injection volume is 10-100 µL. The chromatograms are analyzed for observed peak patterns, retention times, and peak areas of the major peaks at 220 nm. For larger amounts of antibody, the method can be scaled.

[0043] A typical diagram of a CIEX chromatography run of an antibody preparation is depicted in FIG2 . The antibodies used for this run were collected from transfected cells as shown in the examples, and purified from many other proteins in the culture medium using a protein A column. As shown in the examples, the antibodies were eluted by acid elution, followed by neutralization and buffer exchange to PBS pH 7.4. A sample of the antibody preparation was then loaded onto the CIEX column. After washing, the associated proteins were eluted by applying a salt gradient. The CIEX conditions were the same for the samples in FIG2A and FIG2B . The retention time was calculated for the top of the peak of the bispecific antibody. The retention time of two or more antibodies (such as a multispecific antibody) preferably deviates by 10% or less from the average of the retention times of the two or more antibodies. A deviation of more than 10% typically results in an inefficient separation of the antibodies from half antibodies and, selectively in the case of multispecific antibodies, from homodimers and / or other antibody product-related impurities. In a preferred embodiment, the retention times of two or more antibodies deviate by 9% or less from the average of the retention times of the two antibodies. Preferably, they deviate by 8%, 7%, 6% or 5% or less from the average of the retention times of the two or more antibodies. In a preferred embodiment, the retention times of two or more antibodies deviate by 4% or less from the average of the retention times of the two or more antibodies. Preferably, they are 3% or less, preferably 2% or less. Increasingly similar retention times typically allow increasingly efficient separation of the multispecific antibodies from half antibodies and selectively homodimers and / or other antibody product-related impurities and, therefore, allow cleaner collection of the two antibodies in the fractions of the IEX column.

[0044] In the means and methods of the present invention, the average retention time of the two or more antibodies (such as the two or more multispecific antibodies) is calculated for the antibodies to be co-purified or collected. Thus, in the embodiment in which the antibodies to be purified are multispecific antibodies, the average retention time of the two or more antibodies is calculated based on the multispecific antibodies. The retention time of antibodies not to be collected (such as homodimeric antibodies) is not used in the calculation of the average.

[0045] Antibodies such as multispecific antibodies can be selected for co-purification in a method of the invention by selecting antibodies such as multispecific antibodies that have substantially the same IEX retention time under the conditions used in IEX. Such antibodies such as multispecific antibodies can also be tailored through appropriate modification of one or more variable regions to have substantially the same IEX retention time under the same or similar conditions used in IEX.

[0046] In one embodiment, the antibodies (such as bi- and / or multispecific antibodies) generated to be co-purified have similar pI values. The isoelectric point (pI) of at least two of the antibodies preferably differs from the average pI value of the at least two antibodies by 0.4 units, 0.3, 0.2 and preferably 0.1 units or less. The pI value of each of the at least two antibodies preferably differs from the other by 0.25 units or less.

[0047] A small to no difference among the pI values ​​of the antibodies typically allows for a good co-purification. Advantageously, the pI values ​​of the individual half antibodies within an antibody differ more from the average. This difference promotes good separation of the half antibodies from one of the "co"-migrating intact antibodies in the CIEX chromatography step.

[0048] In embodiments in which the antibodies sought to co-purify are bi- or multispecific antibodies, it is preferred that the pI value of the variable domain in each of the antibodies sought to co-purify differs from the average pI value of the variable domain of the other antibody(ies) to be co-purified by more than 0.2 (preferably 0.3, preferably 0.4, 0.5, 0.6, 0.7, 1.0, 1.2, 1.4, preferably more than 1.8 or 2.0) units. In this embodiment, the difference in the pi values ​​of the variable domains located in one antibody is preferably at least 0.2 units greater than the difference between "x" and "y", preferably it is at least 0.3, 0.4, 0.5 (preferably at least 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 2.0 or 2.5) greater than the difference between "x" and "y", wherein "x" is the average of the pi values ​​of the two variable domains of a first one of the antibodies and "y" is the average of the pi values ​​of the two variable domains of a second one of the antibodies. A difference as mentioned in the pi values ​​of the variable domains located in one antibody typically indicates a good separation of the antibody product-related impurities from one of the antibodies sought to be co-purified and / or from one of the monospecific antibodies.

[0049] Certain compositions comprising two or more bi- or multispecific antibodies have constant regions and light chains that are similar in amino acid sequence or have a substantially identical amino acid sequence. Such two or more bi- and / or multispecific antibodies typically differ from each other substantially only in the amino acid sequence of the variable regions or substantially only in the amino acid sequence of the heavy chain variable regions. In such cases, it is usually necessary to determine the pI value of the whole antibody. Instead, the pI values ​​of the variable regions and / or the pI values ​​of the heavy chain variable regions can be determined. This provides a means to assess whether the antibodies can migrate close together in a CIEX chromatography step (in other words, whether the antibodies have retention times that are sufficiently similar to allow co-purification).

[0050] In one embodiment of a method or composition as disclosed herein, two or more bi- or multi-specific antibodies have constant regions and light chains with the same amino acid sequence or with substantially the same amino acid sequence. The two or more bi- or multi-specific antibodies can be co-purified in a CIEX chromatography step when the average pI value of the variable domains in each antibody differs by 0.7 units or less from the average pI value of the variable domains of the respective antibodies sought to be co-purified. In a preferred embodiment, the average pI value "x" of the two variable domains of a first one of the antibodies and the average pI value "y" of the two variable domains of a second one of the antibodies differ by 0.6 units or less, preferably by 0.5 units or less from the average values ​​of "x" and "y" of the first and second antibodies sought to be co-purified. "x" and "y" preferably differ from the mean value of "x" and "y" of the first and second antibodies sought to be co-purified by 0.4 (preferably 0.3, preferably 0.2 and preferably 0.1) units or less. Such bi- and / or multispecific antibodies typically have substantially identical retention times. In this embodiment, the constant regions of the antibodies are substantially identical. The pI values ​​of such antibodies and in particular the mean values ​​"x" and "y" as a whole represent the pI values ​​of the respective antibodies. In this embodiment, it is preferred that the pI value of the variable region in each of the antibodies sought to be co-purified differs from the mean value of the pI values ​​of the variable regions in the antibodies by more than 0.2 (preferably 0.3, preferably 0.4, 0.5, 0.6, 0.7, 1.0, 1.2, 1.4, preferably more than 1.8 or 2.0) units. In this embodiment, the difference in the pi values ​​of the variable domains located in one antibody is preferably at least 0.2 units greater than the difference between "x" and "y", preferably it is at least 0.3, 0.4, 0.5 (preferably at least 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 2.0 or 2.5) greater than the difference between "x" and "y". A difference as mentioned in the pi values ​​of the variable domains located in one antibody typically indicates a good separation of the half antibody from one of the antibodies sought to be co-purified and / or a good separation from one of the monospecific antibodies.

[0051] In one embodiment of a method or composition as disclosed herein, two or more bi- or multi-specific antibodies have constant regions and light chains with the same amino acid sequence or with substantially the same amino acid sequence. The two or more bi- or multi-specific antibodies can be co-purified in a CIEX chromatography step when the average pI value of the heavy chain variable region in each antibody differs by 0.7 units or less from the average pI value of the heavy chain variable region of the respective antibody sought to be co-purified. In a preferred embodiment, the average pI value "m" of the two heavy chain variable regions of the first one of the antibodies and the average pI value "n" of the two heavy chain variable regions of the second one of the antibodies differ by 0.6 units or less, preferably by 0.5 units or less from the average values ​​of "m" and "n" of the first and second antibodies sought to be co-purified. "m" and "n" preferably differ from the average value of "m" and "n" of the first and second antibodies for which co-purification is sought by 0.4 (preferably 0.3, preferably 0.2 and preferably 0.1) units or less. Such bi- and / or multispecific antibodies typically have substantially identical retention times. In this embodiment, the constant regions of the antibodies are substantially identical. The pI values ​​of such antibodies and in particular the average values ​​"m" and "n" as a whole represent the pI values ​​of the respective antibodies. In this embodiment, it is preferred that the pI value of the heavy chain variable region in each of the antibodies for which co-purification is sought differs from the average value of the pI values ​​of the heavy chain variable regions in the antibodies by more than 0.2 (preferably 0.3, preferably 0.4, 0.5, 0.6, 0.7, 1.0, 1.2, 1.4, preferably more than 1.8 or 2.0) units. In this embodiment, the difference in the pI values ​​of the heavy chain variable regions located in one antibody is preferably at least 0.2 units greater than the difference between "m" and "n", preferably it is at least 0.3, 0.4, 0.5 (preferably at least 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 2.0 or 2.5) greater than the difference between "m" and "n". A difference as mentioned in the pI values ​​of the heavy chain variable regions located in one antibody typically means that the half antibody is well separated from one of the antibodies sought to be co-purified and / or well separated from one of the monospecific antibodies.

[0052] The antibodies (such as multispecific antibodies) may have or be selected to have heavy and light chain combinations (half antibodies) or homodimers (e.g., monospecific antibodies) or other antibody product-related impurities that have retention times under the IEX conditions used that are significantly different from the retention times of the intact antibodies or desired antibodies (such as multispecific antibodies). In an embodiment in which the cells express a common light chain, the selection is typically on the heavy chain. The heavy chain may be modified so that the half antibodies or homodimers have significantly different retention times. In a preferred embodiment, the retention times of the half antibodies and / or homodimers differ by more than 10% from the mean of the retention times of the individual antibodies or multispecific antibodies. In a preferred embodiment, the mean of the pI values ​​of the individual heavy and light chain combinations of an antibody that is sought not to be co-purified differs by more than 0.5 units from the mean of the pI values ​​of the heavy and light chains of the at least two antibodies to be co-purified.

[0053] The present invention further provides a composition comprising 2-10 recombinant antibodies obtainable by a method as described herein. Also provided is a composition comprising 2-10 recombinant antibodies, characterized in that the IEX retention times of at least two of the antibodies are substantially the same.

[0054] The present invention further provides a composition comprising 2-10 recombinant antibodies, characterized in that: the pI value of at least two of the antibodies differs from the average pI value of the at least two antibodies by 0.4 units, 0.3, 0.2 and preferably 0.1 units or less. The pI value of each of the at least two antibodies preferably differs from the other by 0.25 units or less.

[0055] The present invention further provides a composition comprising 2-10 recombinant antibodies, characterized in that the average value "x" of the pI values ​​of the two variable domains of the first antibody and the average value "y" of the pI values ​​of the two variable domains of the second antibody differ from the average value of "x" and "y" of the first and second antibodies to be co-purified by 0.7, 0.6 and preferably 0.5 units or less. "x" and "y" preferably differ from the average value of "x" and "y" of the first and second antibodies to be co-purified by 0.4 (preferably 0.3, preferably 0.2 and preferably 0.1) units or less. Such antibodies (such as multispecific antibodies) typically have substantially the same retention time. In this embodiment, the constant regions of the antibodies are substantially the same. The pI values ​​of the different variable domains of each of the antibodies comprising a heavy chain variable region and a light chain variable region and in particular the average value of the pI values ​​as a whole represent the pI value of the antibody.

[0056] The present invention further provides a composition comprising 2-10 recombinant antibodies, characterized in that the average value "m" of the pI values ​​of the two heavy chain variable regions of the two variable regions of the first antibody and the average value "n" of the pI values ​​of the two heavy chain variable regions of the two variable regions of the second antibody differ from the average value of "m" and "n" of the first and second antibodies to be co-purified by 0.7, 0.6 and preferably 0.5 units or less. "m" and "n" preferably differ from the average value of "m" and "n" of the first and second antibodies to be co-purified by 0.4 (preferably 0.3, preferably 0.2 and preferably 0.1) units or less. Such antibodies (such as multispecific antibodies) typically have substantially the same retention time. In this embodiment, the constant regions and light chain variable regions of the antibodies are substantially identical. The pI values ​​of the different heavy chain variable regions of the antibody and in particular the average value of the pI values ​​as a whole represent the pI value of the antibody.

[0057] In a preferred embodiment, the IEX retention times and / or the pI values ​​are preferably substantially the same for all the antibodies to be collected in the composition. In a preferred embodiment, at least two of the antibodies are bispecific antibodies. Preferably, at least two of the antibodies share an identical heavy chain.

[0058] In certain embodiments, the common light chain variable region of one or both VH / VL combining regions comprises a germline IgVκ1-39*01 variable region V-segment. In certain embodiments, the light chain variable region of one or both VH / VL combining regions comprises a kappa light chain V-segment IgVκ1-39*01. IgVκ1-39 is an abbreviation for immunoglobulin variable kappa 1-39 gene. The gene is also known as immunoglobulin kappa variable 1-39, IGKV139, IGKV1-39. The external identifier for the gene is: HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. The amino acid sequence of the V-region is provided in Sequence ID Number: 25. The V-region may also be combined with one of the five J-regions. The preferred J-regions are jk1 and jk5, and the linked sequences are denoted IGKV1-39 / jk1 and IGKV1-39 / jk5, alternatively IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (based on the IMGT database global website at imgt.org). In certain embodiments, the light chain variable region of one or both VH / VL combining regions comprises a kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ1*05 (SEQ ID NO: 26 and SEQ ID NO: 27, respectively).

[0059] In some embodiments, the light chain variable region of one or two VH / VL binding regions of a bispecific antibody comprises: a LCDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 22), a LCDR2 comprising the amino acid sequence AAS, and a LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 24) (i.e., the CDRs according to IMGT, IGKV1-39). In some embodiments, the light chain variable region of one or two VH / VL binding regions of a bispecific antibody comprises: a LCDR1 comprising the amino acid sequence QSISSY (SEQ ID NO: 22), a LCDR2 comprising the amino acid sequence AASLQS (SEQ ID NO: 23), and a LCDR3 comprising the amino acid sequence QQSYSTP (SEQ ID NO: 24).

[0060] In certain embodiments, one or two VH / VL binding regions of a bispecific antibody comprise a light chain variable region, and the light chain variable region comprises an amino acid sequence that is at least 90% (preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%) identical to or 100% identical to the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, one or two VH / VL binding regions of a bispecific antibody comprise a light chain variable region, and the light chain variable region comprises an amino acid sequence that is at least 90% (preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%) identical to or 100% identical to the amino acid sequence set forth in SEQ ID NO: 27.

[0061] For example, in some embodiments, the variable light chain of one or two VH / VL binding regions of a bispecific antibody may have from 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions, or a combination thereof relative to sequence identification number: 26 or sequence identification number: 27. In some embodiments, the light chain variable region of one or two VH / VL binding regions of a bispecific antibody comprises from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1, and preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof relative to the specified amino acid sequence.

[0062] In other embodiments, the light chain variable region of one or both VH / VL binding regions of a bispecific antibody comprises an amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27. In certain embodiments, the two VH / VL binding regions of a bispecific antibody comprise the same VL region. In one embodiment, the VL of the two VH / VL binding regions of a bispecific antibody comprises an amino acid sequence as described in SEQ ID NO: 26. In one embodiment, the VL of the two VH / VL binding regions of a bispecific antibody comprises an amino acid sequence as described in SEQ ID NO: 27.

[0063] Bispecific antibodies (such as those disclosed in the methods herein) can be provided in many formats. Many different formats of bispecific antibodies are known in the art. For example, bispecific antibody formats that are not typical antibodies with two VH / VL combinations have at least one variable domain that includes a heavy chain variable region and a light chain variable region. This variable domain can be linked to a single chain Fv-fragment, monomer, a VH and a Fab-fragment that provides a second binding activity.

[0064] Bispecific antibodies (such as those disclosed in the methods provided herein) are generally of the human IgG subtype (e.g., for example, IgG1, IgG2, IgG3, IgG4). In some embodiments, the antibodies are of the human IgG1 subtype. Full-length IgG antibodies are preferred because of their favorable half-life and for the sake of low immunogenicity. Thus, in some embodiments, the bispecific antibodies are full-length IgG molecules. In one embodiment, the bispecific antibodies are full-length IgG1 molecules.

[0065] In some embodiments, the antibody comprises a crystallizable fragment (Fc). The Fc region of the bispecific antibody is preferably composed of a human constant region. A constant region or Fc of the bispecific antibody may contain one or more (preferably no more than 10, preferably no more than 5) amino acid differences with the constant region of a naturally occurring human antibody. For example, in some embodiments, each Fab-arm of the bispecific antibodies may further comprise an Fc-region comprising modifications that promote the formation of the bispecific antibody, modifications that affect Fc-mediated effector functions and / or other features described herein.

[0066] In one aspect, provided is a pharmaceutical composition comprising two or more antibodies as defined herein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a governmental regulatory agency or listed in the U.S. Pharmacopeia or another generally recognized pharmacopoeia for use in animals (particularly humans), and includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The term "carrier" means a diluent, adjuvant, excipient or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water or aqueous salt solutions and aqueous dextrose and glycerol solutions can be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intraarticularly, etc.), the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0067] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example, in a liquid carrier, or suitable for reconstitution into a liquid solution or suspension for intravenous administration. The composition may be formulated in dosage unit form for ease of administration and uniformity of dosage.

[0068] Also included are solid preparations which are intended to be converted, shortly before use, to liquid preparations for oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

[0069] A "bispecific antibody" is an antibody as described herein, wherein one domain of the antibody binds to a first antigen and a second domain of the antibody binds to a second antigen, wherein the first and second antigens are not the same. The term "bispecific antibody" also encompasses antibodies wherein one heavy chain variable region / light chain variable region (VH / VL) combination binds to a first epitope located on an antigen, and a second VH / VL combination binds to a second epitope. The term further encompasses antibodies wherein one VH is capable of specifically recognizing a first antigen and the VL paired with the VH in an immunoglobulin variable region is capable of specifically recognizing a second antigen. The resulting VH / VL pair will bind either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008 / 027236, WO 2010 / 108127 and Schaefer et al. (Cancer Cell 20, 472-486, October 2011). A bispecific antibody according to the invention is not limited to any particular format or method used to generate it.

[0070] When referring to nucleic acid or amino acid sequences herein, "percent (%) identity" is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after aligning the sequences for optimal comparison purposes. Percent sequence identity of compared nucleic acid sequences is determined using the AlignX application of Vector NTI Program Advance 10.5.2 software using default values ​​that use a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ (1994) Nuc. Acid Res. 22: 4673-4680), a swgapdnarnt score matrix, a gap opening penalty of 15, and a gap extension penalty of 6.66. Amino acid sequences were aligned using the AlignX application of Vector NTI Program Advance 11.5.2 software using default values ​​that use a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, 1994), the blosum62mt2 score matrix, a gap opening penalty of 10, and a gap extension penalty of 0.1.

[0071] As used herein, the term "common light chain" means the two light chains (or their VL parts) located in a bispecific antibody. The two light chains (or their VL parts) can be identical or have certain amino acid sequence differences without affecting the binding specificity of the full-length antibody. The terms "common light chain", "common VL", "single light chain", "single VL", with or without the addition of the term "rearranged", are used interchangeably herein. "Common" also means that the amino acid sequence of the light chain is not the same functional equivalent. Many variants of such light chains exist, in which mutations (deletions, substitutions, insertions and / or additions) that do not affect the formation of a functional binding region are present. The light chain of the present invention can also be a light chain as specified above herein, having from 0 to 10 (preferably from 0 to 5) amino acid insertions, deletions, substitutions, additions or a combination thereof. For example, making or discovering light chains that are not identical but are still functionally equivalent is within the scope of the definition of a common light chain as used herein, for example, by introducing and testing conserved amino acid changes, amino acid changes located in regions that do not or only partially contribute to binding specificity when paired with the heavy chain, and the like. The term "full-length IgG" or "full-length antibody" according to the present invention is defined as comprising a substantially complete IgG, but it does not necessarily have all the functions of a complete IgG. For the avoidance of doubt, a full-length IgG contains two heavy chains and two light chains. Each chain contains constant regions (C) and variable regions (V), which can be broken down into regions designated as CH1, CH2, CH3, VH and CL, VL. An IgG antibody binds to the antigen via the variable region regions contained in the Fab portion, and after binding can interact with molecules and cells of the immune system through the constant regions (mostly through the Fc portion). Full-length antibodies according to the present invention encompass IgG molecules in which mutations that provide the desired characteristics may be present. A full-length IgG should not have a substantial portion of any of these regions deleted. However, IgG molecules in which one or several amino acid residues are deleted without substantially altering the binding characteristics of the resulting IgG molecule are encompassed by the term "full-length IgG". For example, such an IgG molecule may have a deletion of between 1 and 10 amino acid residues, preferably in the non-CDR regions, wherein the deleted amino acids are not essential for the antigen or epitope binding specificity of the IgG.

[0072] Since an antibody typically recognizes an epitope of an antigen, and such an epitope may also exist in other compounds, an antibody according to the present invention that "specifically recognizes" an antigen may also recognize other compounds if they contain the same epitope. Therefore, the term "specifically recognizes", with respect to the interaction between an antigen and an antibody, does not exclude the binding of the antibodies to other compounds containing the same epitope.

[0073] The term "epitope" or "antigenic determinant" means a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of a protein (so-called linear or conformational epitopes). Epitopes formed from contiguous linear amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding typically lose conformation upon treatment with denaturing solvents. An epitope typically contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope are known to those of ordinary skill in the art and include techniques in the art, such as X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS) and two-dimensional nuclear magnetic resonance, peptide scanning (pepscan), and alanine scanning based on the nature of the epitope (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0074] For the purpose of clarity and a concise description of the invention, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the invention may include embodiments having a combination of all or some of the described features.

[0075] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. Unless otherwise indicated, all technical and scientific terms used herein have the meanings commonly understood by one having ordinary skill in the art, and conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used.

[0076] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The use of the term "including" along with other forms such as "include" in the infinitive form, "includes" in the third person singular, and "included" in the past tense is not limiting. Simple diagram description

[0077] [picture] [1] is a schematic diagram of an embodiment, wherein the composition comprises two bispecific antibodies sharing a common arm. The figure depicts an antibody with a heavy chain (1) and a light chain (4). The four heavy chains have three different variable regions (5, 6 and 7). The heavy chain with the shared variable region (5) has a portion (3) of a heterodimerization domain. The heavy chains with variable regions (6) and (7) have compatible portions (2) of the heterodimerization domain. Preferred pairing of heterodimerization regions (2) and (3) can lead to the formation of a bispecific antibody.

[0078] [picture] [2] shows Figure A: CIEX-curve of bispecific antibody PB4516 generation number 8 (p08) at 220 nm; Figure B: CIEX-curve of bispecific antibody PB6892 generation number 4 (p04) at 220 nm.

[0079] [picture] [3] shows FIG3a: CIEX-curve at 220 nm for bispecific antibody PB4516, generation number 10 (p10); FIG3b: CIEX-curve at 220 nm for bispecific antibody PB11244, generation number 1 (p01); FIG3c: two bispecific antibodies are identified in each box in the vertical PB (PBXXXX(X)). The heavy chain variable regions for PB11244 and PB4516 are indicated in the verticals Target 1 and Target 2. The sequence of the light chain is identical for all antibodies and has the common light chain amino acid sequence of IgKV1*39 / jk1 with sequence identification number: 26. For each heavy chain variable region, the pI value calculated by the ExPASy ProtParam tool is indicated in the vertical pI. The difference in pi values ​​between the two heavy chain variable regions is shown in the last column, demonstrating that the average VHpi difference between PB11244 and PB4516 is 0.08.

[0080] [picture] [4] shows a CIEX-plot at 220 nm of an antibody preparation of the individual colony cp12 of pool FST2. The CIEX-plot shows a sharp peak for the co-eluted antibodies PB4516 and PB11244. The plot shows that the sample contains a limited amount of product-related impurities. It also shows a good separation between the co-eluted bispecific antibodies and the respective migrating product-related impurities.

[0081] [picture] [5] shows a CIEX-curve at 220 nm of an antibody preparation of colony CP07. This colony was selected from a collection of individual colonies of the single colony FST2cp09. A second subcloning was performed to ensure that the FST2cp09-cp07 cell line was a monogenic cell line. Bispecific antibody-specific ELISA showed the presence of 743 µg / mL of the EGFR / HER2 bispecific antibody PB11244 and 1134 µg / mL of the EGFR / HER3 bispecific antibody PB4516.

[0082] [picture] [6] shows the retention time of an antibody homodimer (PGXXXX) having two identical variable domains. The amino acid sequence of the heavy chain variable region has the sequence shown for MF in Figure 8 and a common light chain IgKV1*39 / jk1 with sequence identification number: 26.

[0083] [picture] [7] shows that each box has two bispecific antibodies identified in column PB (PBXXXX(X)). The heavy chain variable region (MFXXXX) of each of these bispecific antibodies is indicated in columns Target 1 and Target 2. The sequence of the light chain is the same for all antibodies and has a common light chain amino acid sequence of IgKV1*39 / jk1 with sequence identification number: 26. For each heavy chain variable region, the pI value calculated by the ExPASy ProtParam tool is indicated in the column pI. The pI value difference between the two heavy chain variable regions and the measured retention time are shown in the last two columns. The measured retention time and the calculated pI value and the average pI value indicate that a pair of bispecific antibodies can be effectively co-eluted in CIEX chromatography. These antibodies have an IgG1 constant region and a common light chain. The heavy chains with CH3 DE heavy chain or KK heavy chain with a shared heavy chain variable region (same MF) are indicated. Retention times for CIEX chromatography performed on each specific antibody are indicated, and an exemplary condition for CIEX chromatography is described in the Materials and Methods section. Many other CIEX chromatography conditions will result in suitable retention times between the listed bispecific antibody pairs with the provided pI values.

[0084] [ Fig. 8 ] shows the amino acid sequences of the heavy chain variable region (MFXXXX) and CDRs of the light chain variable region of each antibody and the amino acid sequence of the common light chain variable region. Implementation

[0085] [Example] Example 1 Materials and Methods Cell lines

[0086] HEK293 and CHO-K1 were maintained in growth medium. Generation of bispecific antibodies

[0087] The bispecific antibodies were generated using proprietary CH3 technology to ensure efficient heterodimerization and formation of a bispecific antibody. As previously described (PCT / NL2013 / 050294; published as WO 2013 / 157954 A1), the CH3 technology utilizes charge-based point mutations located in the CH3 region to allow efficient pairing of two non-identical heavy chain molecules.

[0088] A VH gene is cloned into one of two IgG1 vectors of different architectures. Depending on the binding partner, the VH is cloned into an IgG1 architecture containing a CH3 variant with heterodimerization variant "DE" or an IgG1 architecture containing a complementary CH3 heterodimerization variant "KK". In the case of bi- or multispecific antibodies in which two or more antibodies share one heavy chain, the shared chain preferably has the CH3 heterodimerization variant "DE" (also referred to as the DE-heavy chain), while the two or more unique heavy chains have the CH3 heterodimerization variant "KK" (also referred to as the KK-heavy chain).

[0089] HEK293 cells were transiently transfected with the DNA-FUGENE mixture and further cultured. 7 days after transfection, the supernatants were harvested and the medium was refreshed. 14 days after transfection, the supernatants were pooled and filtered through 0.22 µM. Sterile supernatants were stored at 4°C. Suspension-adapted 293F cells were cultured in T125 flasks on a shaker plate to a density of 3.0x10 6 cells / mL. Cells were seeded into each well of a 24-deep well plate at a density of 0.3-0.5x10 6 viable cells / mL. The cells were transiently transfected with the respective sterile DNA:PEl-MIX and further cultured. 7 days after transfection, the supernatants were harvested and filtered through 0.22 µM. Sterile supernatants were stored at 4°C. Generation of a stable cell line pool expressing two bispecific antibodies

[0090] CHO cells were transfected with 3 heavy chain constructs and a common light chain construct in a molar ratio of 2.5:2:1:1 of common light chain construct (cLC):EGFR heavy chain:HER2 heavy chain:HER3 heavy chain. Ten pools (AJ) of stably transfected cells were obtained. ELISA analysis of anti-EGFR, anti-HER2 and anti-HER3 antibodies was performed on the supernatants of the 10 pools on day 3 and day 6. All three specificities could be determined in all pools. Generation of stable cell line clones expressing two bispecific antibodies

[0091] The pools were plated in semi-solid medium and allowed to grow for 7-10 days. Single colonies were picked and seeded into 24-well plates. Colonies were reseeded before collecting antibodies from the supernatant of the culture. Determination of antibody titers

[0092] The anti-HER2 antibody titer of samples containing a single bispecific antibody was determined by ELISA against Erbb-2 Fc protein (R&D systems). The anti-HER3 antibody titer of samples containing a single bispecific antibody was determined by ELISA against human Erbb-3-Fc protein (R&D systems). The anti-EGFR antibody titer of samples containing a single bispecific antibody was determined by ELISA against human EGFR ECD-Fc protein (R&D systems). Serial 2-fold dilutions of the antigens were used to coat the wells of an ELISA plate, starting at 5 µg / mL.

[0093] ELISA analysis for quantification of EFGRxHER2 and EGFR x HER3 bispecific antibodies in a composition comprising the two bispecific antibodies was performed by coating ELISA plates with EGFR-Fc (R&D systems). After washing, the plates were incubated with samples. After washing, the presence of bound bispecific antibodies with one EFGR arm and one HER2 arm was detected by incubation with labeled HER2-Fc. The presence of bound bispecific antibodies with one EFGR arm and one HER3 arm was detected by incubation with labeled HER3-Fc. IgG purification

[0094] Purification of IgG was performed using affinity chromatography. Purification was performed using vacuum filtration under sterile conditions. First, the pH of the medium was adjusted to pH 8.0 and the product was then incubated with Protein A Sepharose CL-4B beads (50% v / v) (Pierce) for 2 hours at 25°C on a plate shaker set at 600 rpm. Next, the beads were harvested by vacuum filtration. The beads were washed twice with PBS pH 7.4. IgG was eluted using 0.1 M citrate buffer at pH 3.0 and the IgG fraction was immediately neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using Ultracel (Millipore). The samples were finally in a final buffer of PBS pH 7.4. Cation Exchange Chromatography (CIEX)

[0095] CEX-HPLC chromatography is accomplished using the TSKgel SP-STAT (7 µm particle size, 4.6 mM ID x 10 cm L, Tosoh 21964) series of ion exchange columns. These columns are packed with non-porous resin particles for speed and high resolution analysis and separation of biomolecules. The particles in TSKgel STAT columns contain an open access network of multiple layers of ion exchange groups for loading capacity, and the particle size makes these columns suitable for use with HPLC and FPLC systems.

[0096] TSKgel SP-STAT (7 µm particle size, 4.6 mM ID x 10 cm L, Tosoh 21964) was equilibrated with buffer A (sodium phosphate buffer, 25 mM, pH 6.0), after which the antibody was expelled from the column by increasing the salt concentration and running a gradient of buffer B (25 mM sodium phosphate, 1 mM NaCl, pH 6.0). The flow rate was set at 0.5 mL / min. The injected sample mass for all test samples and controls (in PBS) was 10 µg, and the injection volume was 10-100 µL. Chromatograms were analyzed for observed peak patterns, retention times, and peak areas of the major peaks based on the results at 220 nm. result

[0097] The CIEX curves of the bispecific antibodies PB4516p08 and PB6892p04 were compared (see Figure 2). It was observed that the production of PB6892 contained a significant amount of impurities. Also, the retention time of the bispecific antibody fraction of PB6892 was significantly lower than that of the bispecific antibody fraction of PB4516. For co-production by the same cell and subsequent co-purification using CIEX, the retention times of the two bispecific antibodies are preferably closer. For this reason, the variable region of the HER2 arm of PB6892 was replaced with a different variable region. The heavy chain with the variable region MF2032 was selected and used to generate the EGFR x HER2 bispecific antibody PB11244. The CIEX curves of PB4516p10 and PB11244p01 are shown in Figure 3. The retention times of the bispecific antibody fractions were 16.310 and 16.950, respectively. These retention times are sufficiently similar to allow co-purification using CIEX under the conditions shown. In addition, the figure shows that the retention times of the impurities are sufficiently different to allow efficient separation in analytical and preparative columns. The above bispecific antibody preparations were produced in HEK293 cells.

[0098] For co-production, CHO-K1 cells were used. CHO cells were transfected with three heavy chain constructs carrying the respective variable regions of MF3755 (EGFR), M2032 (HER2) and MF3178 (HER3), together with a construct expressing the light chain variable region with sequence identification number: 26. Vector positive cells were selected and pooled. Ten independent pools of transfected CHO-K1 cells (identified as AJ) were generated.

[0099] Table 1 shows the amount of bispecific antibodies PB4516 (EGFR x HER3) and PB11244 (EGFR x HER2) produced by the respective pools. Also, the ratio of these amounts and the total amount of IgG produced are shown. Pools F and J were selected for secondary cloning.

[0100] Table 2 shows the amount of bispecific antibodies PB4516 (EGFR x HER3) and PB11244 (EGFR x HER2) produced by the respective clones.

[0101] Antibodies produced by clone FST2cp12 were used to analyze the CIEX profile (see Figure 4). It can be clearly seen that the two bispecific antibodies effectively co-eluted in the same CIEX elution fraction.

[0102] The clone FST2cp09 was further subcloned to ensure that the cell line was monogenic and a further CIEX plot confirmed the antibodies produced. Figure 5 shows the CIEX plot. It is clearly visible that the two bispecific antibodies effectively co-eluted in the same CIEX elution fraction. The relative contribution of the two bispecific antibodies in the co-elution was analyzed by ELISA and / or by hydrophobic interaction column. The bispecific antibody specific ELISA indicated the presence of 743 µg / mL of the EGFR / HER2 bispecific antibody PB11244 and 1134 µg / mL of the EGFR / HER3 bispecific antibody PB4516. Demonstration Example 2 Generation of a stable cell line pool expressing two bispecific antibodies

[0103] Cell lines expressing the two by two bispecific antibodies listed in FIG7 were generated as follows. CHO cells were transfected with three heavy chain constructs and one common light chain construct. The three heavy chains are recognized by the heavy chain variable region (MFXXXX) indicated in the box. The light chain comprises the light chain variable region sequence of IgVκ1*39 / jk1 with sequence identification number: 26. The two bispecific antibodies have one common heavy chain and one different heavy chain. For example, the first pair indicated in FIG7 shares a common heavy chain (comprising the same HER3 binding arm comprising a heavy chain variable region (MF3178)) and a different second binding arm. PB4528 has an EGFR binding arm with a heavy chain variable region (MF4003), while PB4188 has a HER2 binding arm with a heavy chain variable region (MF3958). The shared heavy chain has a KK CH3 region with compatible DE / KK heterodimerization domains. The shared heavy chain arm may also have a DE CH3 region. For example, in Figures 3-5, two bispecific antibodies were co-purified, PB11244 and PB4516. As shown in Figure 3c, PB11244 and PB4516 share the same EGFR binding arm with a heavy chain variable region (MF3755), and PB11244 has a HER2 binding arm with a heavy chain variable region (MF2032), while PB4516 has a HER3 binding arm with a heavy chain variable region (MF3178). The shared heavy chain arm in this pair of bispecific antibodies has a DE CH3 region, while the different HER2 and HER3 binding arms have KK CH3 regions.

[0104] Molar ratio of common light chain construct (cLC): shared heavy chain construct: different heavy chain construct 1: different heavy chain construct 2 = 2.5:2:1:1. Pools of stably transfected cells were obtained. ELISA analysis of antigen was performed on supernatants collected from the pools. All 3 antigen binding species were determined in the pools. The CIEX retention time of the bispecific antibodies in each pair of Figure 7 was determined under similar CIEX conditions and is shown in the 8th column. The deviation from the mean retention time was calculated using the formula 100x((AB) / (A+B)), where A is the retention time of the bispecific antibody with the longest retention time. For example, the deviation for the first pair is 100x((16.46-16.24) / (16.46+16.24)) = 0.67 or 0.7%.

[0105] The antibodies in the collected supernatant were first separated from other proteins in the supernatant by protein A extraction, followed by acid elution and rapid neutralization. The buffer of the collected antibodies was then exchanged to PBS. The samples were then loaded onto a CIEX column and washed and eluted by applying an increasing salt gradient. The absorbance of the eluate was measured at 220 nm and the retention time was calculated from the onset of the salt gradient and the observation of the peak of the bispecific antibody. The bispecific antibodies were collected and the individual bispecific antibodies in the collected eluate were verified by ELISA. The retention time of the individual bispecific antibodies is indicated in the last column. It is clear that many of the paired pairs have retention times that effectively co-elute in a CIEX column. It is also clear that the CIEX chromatography provides good separation of the co-eluted bispecific antibodies and the individual homodimers (if any). FIG6 lists the retention times of various antibodies with homodimers of heavy chains (including heavy chain variable regions) present in the co-eluted bispecific antibodies. It is clear that the retention times of the homodimers are sufficiently different from the individual bispecific antibodies. For example, in the first frame of FIG7 , the homodimers PG3178, PG3958 and PG4003 can be present in one product. The retention times of the individual homodimers are about 22, 12 and 13 ( FIG6 , rows 1-3), while the retention times of the bispecific antibodies including the heavy chain variable region are about 19 and 19.5 ( FIG7 , rows 1 and 2). [Table 1] Pool concentration α-EGFR x α-HER2 (μg / mL) concentration α-EGFR x α-HER3 (μg / mL) ratio (α-EGFR x α-HER2: α-EGFR x α-HER3) Total IgG (μg / mL) A 153.6 162.0 1:1.1 315.6 B 120.2 336.3 1:2.8 456.3 C 325.5 741.6 1:2.3 167.1 D 510.6 856.8 1.1.7 1367.4 E 273.0 526.5 1:1.9 799.6 F 1824.7 1024.9 1:0.6 2849.6 G 704.2 849.8 1:1.2 1554.0 H 951.1 467.5 1:0.5 1418.6 I 450.7 691.0 1:1.5 1141.7 J 1288.9 1572.1 1:1.2 2861.0 Table 1: Quantification of EGFRxHER2 and EGFRxHER3 bispecific antibody production in cell pools. Culture supernatants of 10 pools (AJ) were assessed. ELISA analysis was based on EGFR-Fc coating, binding of the produced antibodies and detection using either the indicated HER2-Fc or the indicated HER3-Fc. Pool AJ was analyzed using both ELISA assays. Bispecific antibodies PB4516 and PB11244 are IgG1 heavy chain antibodies with compatible DE / KK heterodimerization domains. The heavy chains were combined with the common light chain. The bispecific antibodies shared the MF3755 heavy chain variable region on one heavy chain and each had a different heavy chain variable region on the other IgG1 heavy chain (MF3178 for PB4516 and MF2032 for PB11244). [Table 2] # HER2 (μg / mL) HER3 (μg / mL) HER2 / HER3 FST1cp02 48.7 1449.2 0.03 FST1cp03 103.4 488.9 0.21 FST1cp04 131.4 1675.0 0.08 FST1cp14 259.1 214.6 1.21 FST1cp24 372.3 817.0 0.46 FST1cp26 92.2 706.7 0.13 FST2cp09 1026.2 1509.0 0.68 FST2cp12 725.7 1334.2 0.54 FST2cp13 737.4 1173.0 0.63 FST2cp20 617.6 1759.3 0.35 FST2cp21 993.0 1852.3 0.54 FST2cp23 937.4 1095.5 0.86 JST1cp01 121.3 490.6 0.25 JST1cp04 239.8 387.7 0.62 JST1cp05 187.2 759.0 0.25 JST1cp09 828.6 718.5 1.15 JST1cp13 103.5 175.8 0.59 JST1cp24 481.5 423.7 1.14 Table 2: Selected pools were used for single cell cloning. 18 colonies were selected from 3 pools. Two independent F pools (FST1 and FST2) and one J pool (JST1) were used for single cell cloning. The designation "cp" followed by a number identifies the individual colonies of a pool. The selected colonies were grown and used for antibody collection. Individual colonies from the same pool produced different amounts and different ratios of the respective bispecific antibodies.

[0106] From the above discussion, it will be understood that the present invention can be embodied in many aspects, including but not limited to the following: Aspect 1: A method for generating at least two antibodies, comprising: Providing cells with nucleic acids encoding the antibodies; culturing the cells; collecting the antibodies from the culture; and Separating the generated antibody from the half-antibody by ion exchange chromatography (IEX); The method is characterized in that, under the IEX conditions used, the antibodies exhibit an IEX retention time that deviates from the mean value of the retention times of the individual antibodies by 10% or less. Aspect 2: The method of claim 1, wherein collecting the antibodies from the culture comprises purifying the antibodies from other proteins by antibody affinity purification, preferably by protein A extraction. Aspect 3: The method of claim 2, further comprising subjecting the affinity purified antibody to size-exclusion chromatography (gel filtration chromatography and / or anion exchange chromatography). Aspect 4: The method of claim 1-3, wherein after the IEX, the collected antibodies are quantitatively analyzed for relative expression levels by hydrophobic interaction chromatography (HIC). Aspect 5: The method of claim 4, wherein the specificity of the collected antibodies is verified by ELISA. Aspect 6: The method of claim 1-5, wherein the retention time of each half antibody falls outside the range spanned by the retention times of the antibodies. Aspect 7: The method of claim 6, wherein the cells produce three types of heavy chains. Aspect 8: The method of claim 7, wherein the heavy chains comprise a region for efficient heterodimerization of the heavy chains. Aspect 9: The method of claim 1-8, wherein at least two of the antibodies are bispecific antibodies. Aspect 10: The method of claim 1-9, wherein at least two of said antibodies share an identical heavy chain. Aspect 11: The method of claim 1-10, wherein the antibodies have an isoelectric point (pI) that differs from the average pI value of the at least two antibodies by 0.4 units or less. Aspect 12: The method of claim 1-11, wherein the antibodies are selected to have a heavy chain and light chain combination that has a retention time under the IEX conditions used that is significantly different from the retention time of the intact antibody. Aspect 13: The method of claim 12, wherein the pi value of the heavy chain and light chain combination differs from the average pi value of the at least two antibodies by 0.4 units or less. Aspect 14: The method of claim 1-13, wherein the heavy chains comprise a CH3 domain that facilitates heterodimerization of the heavy chains. Aspect 15: The method of claim 1-14, wherein the heavy chains of said antibodies are IgG heavy chains. Aspect 16: The method of claim 7-15, wherein one heavy chain comprises amino acid substitutions L351K and T366K (EU numbering) in the CH3 region, and the other heavy chain comprises amino acid substitutions L351D and L368E in the CH3 region. Aspect 17: A method for producing at least one antibody, comprising: Providing cells with nucleic acids encoding the antibodies; culturing the cells; collecting the antibodies from the culture; and Separating the generated antibody from the half-antibody by ion exchange chromatography (IEX); The method is characterized in that: under the IEX conditions used, the antibodies exhibit an IEX retention time that deviates from the mean value of the retention time of the individual antibodies by 10% or less, and wherein after the IEX, the collected antibodies are quantitatively analyzed for relative expression levels by hydrophobic interaction chromatography (HIC), and the specificity of the collected antibodies is verified by ELISA. Aspect 18: A composition comprising 2-10 recombinant antibodies obtainable by a method according to any one of claims 1-17. Aspect 19: A composition comprising 2-10 recombinant antibodies, characterized in that: under the IEX conditions, the IEX retention time of at least two of the antibodies deviates from the average value of the retention time of the individual antibodies by 10% or less. Aspect 20: A composition comprising 2-10 recombinant antibodies, characterized in that the pI values ​​of at least two of the antibodies differ by 0.4 units or less from the average pI values ​​of the at least two antibodies. Aspect 21: A composition as claimed in claims 18-20, characterized in that: for all of the antibodies, the IEX retention times and / or the pI values ​​are substantially the same. Aspect 22: The composition of claim 18-21, wherein at least two of said antibodies are bispecific antibodies. Aspect 23: The composition of claim 22, wherein at least two of said antibodies share an identical heavy chain.

[0107] 1: Heavy chain 2: Compatible parts of heterodimerization domain 3: a part of the heterodimerization domain 4: Light chain, common light chain 5: Heavy chain variable region 6: Heavy chain variable region 7: Heavy chain variable region

[0108] Sequence Listing <![CDATA[ <110> MERUS NV]]> <![CDATA[ <120> Techniques for producing compositions comprising two or more antibodies]]> <![CDATA[ <140> TW 113139107]]> <![CDATA[ <141> 2020-02-14]]> <![CDATA[ <150> EP 19178542.7]]> <![CDATA[ <151> 2019-06-05]]> <![CDATA[ <150> EP 19157286.6]]> <![CDATA[ <151> 2019-02-14]]> <![CDATA[ <160> 31 ]]> <![CDATA[ <170> PatentIn Version 3.5]]> <![CDATA[ <210> 1]]> <![CDATA[ <211> 124]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> HER3-MF3178]]> <![CDATA[ <400> 1]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Gly Ser Arg His Phe Trp Ser Tyr Trp Gly Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 2]]> <![CDATA[<211> 124]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> HER3-MF3163]]> <![CDATA[<400> 2]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ser Tyr Ser Arg His Phe Tyr Ser Trp Trp Ala Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 3]]> <![CDATA[<211> 124]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<22]]>0>]]> <![CDATA[<223> HER3-MF6061]]> <![CDATA[<400> 3]]> <![CDATA[Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 <![CDATA[Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr]]> 20 25 30 <![CDATA[Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met]]> 35 40 45 Gly Trp Ile Asn Pro Gln Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Gly Ser Arg His Phe Trp Ser Tyr Trp Gly Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 4]]> <![CDATA[<211> 120]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> PD1-MF6930]]> <![CDATA[<400> 4]]> Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Met Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30 Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile Ile Pro Val Phe Glu Thr Ala Thr Tyr Glu Lys Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ile Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Val Glu Ala Thr Leu Leu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 5]]> <![CDATA[<211> 120]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> PD1-MF6256]]> <![CDATA[ <400> 5]]> Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Met Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile Ile Pro Val Phe Asp Thr Ser Ser Tyr Glu Lys Lys Phe 50 55 60 Gln Gly Arg Ile Thr Ile Ile Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Val Glu Ala Thr Leu Leu Phe Asp Phe Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 6]]> <![CDATA[<211> 122]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> HER2-MF1849]]> <![CDATA[<400> 6]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Gly Asp Tyr Gly Ser Tyr Ser Ser Tyr Ala Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 7]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> LGR5-MF7423]]> <![CDATA[<400> 7]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Ile Pro Ile Leu Gly Thr Thr Asp His Ala Gln Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Gly Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Ile Ala Ala Arg Leu Asp Tyr Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 8]]> <![CDATA[<211> 120]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> EGFR-MF3755]]> <![CDATA[<400> 8]]> Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp Phe Thr Asn Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Asn Thr Gly Asp Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Glu Arg Phe Leu Glu Trp Leu His Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 9]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> LGR5-MF7428]]> <![CDATA[<400> 9]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Ile Pro Ile Leu Gly Thr Thr Asp His Ala Gln Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Ser Lys Thr Thr Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Leu Ile Ala Ala Arg Leu Asp Tyr Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 10]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> LGR5-MF7533]]> <![CDATA[<400> 10]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Asn Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Ile Pro Ile Leu Gly Thr Thr Asp His Ala Gln Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Gly Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Ile Ala Ala Arg Leu Asp Tyr Phe Asp Ser Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 11]]> <![CDATA[<211> 127]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> LGR5-MF7532]]> <![CDATA[<400> 11]]> Glu Val Gln Leu Val Gln Ser Gly Ser Lys Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Asp Thr Gly Asp Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Phe 65 70 75 80 Leu Gln Ile Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Cys Asp Ser Thr Ser Cys Tyr Arg Tyr Ser Tyr Gly 100 105 110 Tyr Glu Asp Tyr Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <![CDATA[<210> 12]]> <![CDATA[<211> 125]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> EGFR-MF4280]]> <![CDATA[<400> 12]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Tyr Gly Lys Thr Phe Phe Ala Gln Asn Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Ala Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Gly Tyr Tyr Glu Thr Thr Thr Tyr Tyr Tyr Asn Leu Phe 100 105 110 Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <![CDATA[<210> 13]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> HER2-MF3958]]> <![CDATA[ <400> 13]]> Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Ser Gly Tyr Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Pro Pro Val Tyr Tyr Asp Ser Ala Trp Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 14]]> <![CDATA[<211> 119]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> EGFR-MF4003]]> <![CDATA[<400> 14]]> Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Ser Phe 20 25 30 Ala Met Asn Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Thr Thr Asn Thr Gly Asp Pro Thr Tyr Ala Gln Gly Phe 50 55 60 Ser Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Tyr Asn Trp Ile Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 15]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Not applicable]]> <![CDATA[<400> 15]]> Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 <![CDATA[ <210> 16]]> <![CDATA[ <211> 10]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Not applicable]]> <![CDATA[ <400> 16]]> Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 <![CDATA[ <210> 17]]> <![CDATA[ <211> 121]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> HER2-MF3025]]> <![CDATA[<400> 17]]> Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Tyr Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Glu Glu Ser Ser Asn Thr Ala Tyr 65 70 75 80 甲硫氨酸-组氨酸-亮氨酸-丝氨酸-丝氨酸-亮氨酸-苏氨酸-丝氨酸-谷氨酸-天冬氨酸-丝氨酸-丙氨酸-缬氨酸-酪氨酸-苯丙氨酸-半胱氨酸 85 90 95 丙氨酸-精氨酸-脯氨酸-组氨酸-酪氨酸-甘氨酸-酪氨酸-天冬氨酸-天冬氨酸-色氨酸-酪氨酸-苯丙氨酸-丙氨酸-缬氨酸-色氨酸-甘氨酸 100 105 110 苏氨酸-甘氨酸-苏氨酸-苏氨酸-缬氨酸-苏氨酸-缬氨酸-丝氨酸-丝氨酸 115 120 <![CDATA[<210> 18]]> <![CDATA[<211> 120]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> EGFR-MF4010]]> <![CDATA[<400> 18]]> Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Asn 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Ile Thr Gly Asp Pro Ser Tyr Ala Gln Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Glu Phe Leu Glu Trp Leu Phe Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 19]]> <![CDATA[<211> 121]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> TIM3-MF6501]]> <![CDATA[<400> 19]]> Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Asn Ala Trp Asp Ser Met Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 20]]> <![CDATA[<211> 122]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> HER2-MF1898]]> <![CDATA[<400> 20]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Phe Arg Arg Thr Thr Leu Ser Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[<210> 21]]> <![CDATA[<211> 122]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> HER2-MF2032]]> <![CDATA[<400> 21]]> Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Tyr Tyr Arg Arg Thr Ala Arg Ala Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <![CDATA[ <210> 22]]> <![CDATA[ <211> 6]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> VL CDR1]]> <![CDATA[ <400> ]]> 22 Gln Ser Ile Ser Ser Tyr 1 5 <![CDATA[ <210> 23]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> VL CDR2]]> <![CDATA[ <400> 23]]> Ala Ala Ser Ser Leu Gln Ser 1 5 <![CDATA[ <210> 24]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> VL CDR3]]> <![CDATA[ <400> 24]]> Gln Gln Ser Tyr Ser Thr Pro 1 5 <![CDATA[ <210> 25]]> <![CDATA[ <211> 95]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> IgVk1-39*01]]> <![CDATA[ <400> 25]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro 85 90 95 <![CDATA[ <210> 26]]> <![CDATA[ <211> 107]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Common light chain IgKV1*39 / jk1]]> <![CDATA[ <400> 26]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <![CDATA[<210> 27]]> <![CDATA[<211> 108]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial Sequence]]> <![CDATA[ <220> ]]> <![CDATA[ <223> Common light chain IgKV1*39 / jk5]]> <![CDATA[ <400> 27]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <![CDATA[<210> 28]]> <![CDATA[<211> 109]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> IgG1 CH1]]> <![CDATA[<400> 28]]> Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys 100 105 <![CDATA[<210> 29]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> IgG1 CH2]]> <![CDATA[<400> 29]]> Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 1 5 10 15 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 20 25 30 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 35 40 45 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 50 55 60 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 65 70 75 80 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 85 90 95 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 110 <![CDATA[ <210> 30]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> IgG1 CH3 KK]]> <![CDATA[<400> 30]]> Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Lys Pro Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Lys Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <![CDATA[<210> 31]]> <![CDATA[<211> 107]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Synthetic Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> IgG1 CH3 DE]]> <![CDATA[<400> 31]]> Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Asp Pro Pro Ser Arg Glu 1 5 10 15 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Glu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105

Claims

1. A method for generating 2-10 antibodies, wherein the antibodies comprise at least one monospecific and at least one multispecific antibody, comprising: – Provide cells containing nucleic acids encoding the antibodies; – Culture the cells; – Collect the antibodies from the culture; And – the generated antibodies and half antibodies are separated by ion exchange chromatography (IEX); the method is characterized in that, under the IEX conditions used, the antibodies exhibit an IEX retention time that deviates from the average retention time of individual antibodies by 10% or less, wherein the IEX is cation exchange chromatography performed in a pH range of 5 to 9.

2. A method for generating 2-10 antibodies, wherein the antibodies comprise at least one monospecific and at least one multispecific antibody, comprising: – Provide cells containing nucleic acids encoding the antibodies; – Culture the cells; – Collect the antibodies from the culture; And – the generated antibodies and half antibodies are separated by ion exchange chromatography (IEX); the method is characterized in that: under the IEX conditions used, the antibodies exhibit an IEX retention time that deviates from the average retention time of individual antibodies by 10% or less, and the antibodies have an isoelectric point (pI) that differs from the average pI value of the antibodies by 0.4 units or less.

3. The method of claim 1 or 2, further comprising selecting at least two antibodies having substantially the same IEX residence time under the IEX conditions used.

4. The method of claim 1 or 2, wherein collecting the antibodies from the culture includes purifying the antibodies from other proteins by means of antibody affinity purification.

5. The method of claim 4, wherein collecting the antibodies from the culture includes purifying the antibodies from other proteins by extraction of protein A.

6. The method of claim 4, further comprising introducing the affinity-purified antibody to size-exclusion chromatography, gel filtration chromatography, and / or anion exchange chromatography.

7. The method of request item 1 or 2, wherein after the IEX, the collected antibody system is quantitatively analyzed for relative performance levels by hydrophobic interaction chromatography (HIC).

8. The method of claim 7, wherein the specificity of the collected antibodies is verified by ELISA.

9. The method of claim 1 or 2, wherein the residence time of each individual half antibody falls outside the range spanned by the residence time of such antibodies.

10. The method of request item 9, wherein the cells generate three heavy chains.

11. The method of claim 10, wherein the heavy chains include a region for efficient heterodimerization of the heavy chains.

12. The method of claim 1 or 2, wherein at least two of the antibodies are multispecific antibodies.

13. The method of claim 1 or 2, wherein at least two of the antibodies are bispecific antibodies.

14. The method of claim 1 or 2, wherein at least two of the antibodies are monospecific antibodies.

15. The method of claim 1 or 2, wherein at least two of the antibodies share an identical heavy chain.

16. The method of claim 1, wherein the antibodies have an isoelectric point (pI) that differs from the average pI value of the at least two antibodies by 0.4 units or less.

17. The method of claim 1 or 2, wherein the antibodies are selected to have a combination of heavy and light chains, the combination of heavy and light chains having a residence time under the IEX conditions used that is significantly different from the residence time of the intact antibody.

18. The method of claim 17, wherein the pI value of the combination of heavy and light chains differs from the average pI value of the at least two antibodies by 0.4 units or less.

19. The method of claim 1 or 2, wherein the antibody has a heavy chain comprising a CH3 region that facilitates heterodimerization of the heavy chain.

20. The method of claim 1 or 2, wherein the antibodies have an IgG heavy chain.

21. The method of claim 1 or 2, wherein the cells generate two or more heavy chains, one heavy chain comprising amino acid substitutions L351K and T366K (EU number) located in the CH3 region, and the other heavy chain comprising amino acid substitutions L351D and L368E located in the CH3 region.

22. A composition comprising 2-10 recombinant antibodies, wherein the antibodies comprise at least one monospecific and at least one multispecific antibody generated by any one of claims 1-21, and wherein at least two of the antibodies share a common heavy chain.

23. The composition of claim 22 is characterized in that: for all such antibodies, the IEX retention time and / or the pI value are substantially the same.

Images