Variant domains for multimerizing proteins and separation thereof

Abstract

To provide means and methods for producing and isolating immunoglobulin proteins comprising a first and a second immunoglobulin polypeptide.SOLUTION: The present invention relates to means and methods for producing and isolating immunoglobulin proteins comprising a first and a second immunoglobulin polypeptide, in particular to means and methods for producing and separating proteins comprising the first and second immunoglobulin polypeptide. By incorporating into amino acids variations and variant separation domains from cells producing the desired immunoglobulin protein, a desired immunoglobulin protein as produced can be separated from mixtures of immunoglobulin proteins.SELECTED DRAWING: None

Description

[Background technology]

[0001] introduction An important class of therapeutic molecules in recent decades has been the monoclonal antibody class. Monoclonal antibodies have demonstrated efficacy in treating a variety of diseases, including cancer. In the last decade, it has been discovered that targeting two or more epitopes, such as two or more epitopes on tumor cells, can also be effective. Patients can be administered a combination of independently developed monoclonal antibodies, or a combination of monoclonal antibodies derived from a single cell. Such cells can produce antibodies with two or more distinct specificities that form part of a mixture of antibodies developed to target two or more targets on one or more cell types. When two antibodies are expressed in a single cell, various combinations of antibodies can be produced, including bispecific and monospecific antibodies.

[0002] Techniques for tailoring the association of various immunoglobulin chains are available. Various dimerization domains have been developed to prioritize specific associations of heavy chains in such producing cells. A common light chain can be used to avoid mispairing of cognate heavy and light chains. In certain applications, bispecific antibodies can replace the use of a combination of two antibodies. Bispecificity can also be used to combine two cells in a subject, such as a tumor cell and an immune cell, such as a T cell. One example is the combinatorial targeting of epitopes present on CD3 and cancer cells. Similarly, multivalent multimeric or multispecific antibodies capable of binding three or more of the same or different antigens or epitopes are emerging. While a combination of two antibodies represents a mixture of two different immunoglobulins that bind to different epitopes on the same or different targets, in bispecific antibodies, this is achieved by a single immunoglobulin. Multispecific multimeric or multispecific antibodies can target three or more different epitopes on the same or different antigens.

[0003] By binding to two epitopes on the same or different targets, bispecific antibodies can have similar or superior efficacy compared to a combination of two antibodies that bind to the same epitope. This also applies to multispecific multimers that can bind to three or more targets. Bispecific or multispecific immunoglobulin proteins can recruit two or more surface proteins on cells or bring immune effector cells closer to abnormal cells, in either case triggering cellular apoptosis. Furthermore, isolated bispecific antibodies that combine two different binding domains in a single molecule also exhibit beneficial effects compared to mixtures of two antibodies targeting two different targets. From a technical and regulatory perspective, the development of a single bispecific antibody or multispecific multimer or antibody can be less complex because a single molecule is involved in manufacturing, preclinical testing, and clinical trials. Therefore, therapies based on bispecific antibodies or multispecific multimers / antibodies can facilitate a less complex and cost-effective drug development process while potentially providing more effective therapies.

[0004] Bispecific antibodies, such as those based on the IgG format, have been produced by a variety of methods. For example, bispecific antibodies can be produced by expressing two antibody components in a single cell using recombinant DNA technology. As previously described herein, in some embodiments, these approaches can generate multiple antibody species, for example, when two different heavy chains and 72 different light chains are produced by a cell. Unless specifically tailored, heavy chains can typically pair with any light chain produced by the cell, typically resulting in a non-functional binding site if they are not the correct cognate pair. In the above example, one such heavy chain pairs with one of the light chains.

[0005] Unless specifically regulated, heavy chains can typically pair with any other heavy chain produced by a cell. In an unregulated setting, up to 10 different immunoglobulin molecules can be produced by a cell. The complexity of antibody mixtures and the existence of non-functional heavy and light chain combinations can be addressed by selecting heavy and light chain combinations that share a common light chain. This also applies to the production of multispecific multimers or antibodies and the use of recombinant DNA technology to incorporate three or more variable domains into a single antibody.

[0006] When a common light chain is used in combination with the expression of two or more heavy chains containing modifications that drive specific heterodimerization of different heavy chains by a single production cell, several homodimers will nevertheless be produced, in which heavy chains with the same binding domains are paired, resulting in a mixture of monospecific and bispecific antibodies. This is also true when a common light chain is used in combination with the expression of two or more heavy chains, one or more of which contain two or more heavy chain variable regions, resulting in a single production cell producing multispecific multimers or antibodies and additional homodimers. Where specific homodimers are desired, several heterodimers may be produced. Thus, in each situation where a mixture of proteins is produced, it may be necessary to isolate the desired dimer from the resulting mixture. Thus, there is a need in the art for improved and / or alternative techniques for producing and separating monospecific or bispecific antibodies, or multivalent antibodies or multimers.

[0007] A variety of separation methods are available that utilize the charge and / or isoelectric point (pI) of the antibody or fragment thereof, or that use isoelectric focusing, or that use peaks unique to the protein species of interest generated by ion exchange chromatography. Disclosed herein are new products that facilitate separation from mixtures, and new methods for separating such products. DETAILED DESCRIPTION OF THE INVENTION

[0008] When charged amino acids are referred to herein, they refer to their charge at physiologically relevant pH, such as under in vivo conditions.

[0009] In one embodiment, the present invention provides an immunoglobulin region, preferably a CH1 region, comprising an amino acid mutation compared to an original immunoglobulin region, preferably an original CH1 region, more preferably a human wild-type CH1 region, wherein the original amino acid is not surface exposed in the original immunoglobulin region, and the mutation is Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and The mutation is selected from a negatively charged amino acid to a positively charged amino acid.

[0010] In one embodiment, the present invention provides an immunoglobulin region, preferably an immunoglobulin CH1, CH2, CH3 region, which comprises a mutation of an amino acid that is not surface exposed in the immunoglobulin or combination of said regions compared to the original said immunoglobulin region, preferably the original CH1, CH2 or CH3 region, more preferably a human wild-type CH1, CH2 or CH3 region, wherein the mutation is Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and The mutation is selected from a negatively charged amino acid to a positively charged amino acid.

[0011] The CH1 region of the immunoglobulin according to the invention preferably comprises one or more non-surface exposed or preferably buried amino acid mutations compared to the human wild-type CH1 region, the mutations being Mutation of a neutral amino acid to a negatively charged amino acid, Mutation of positively charged amino acids to neutral amino acids, Mutation of neutral amino acids to positively charged amino acids, and Mutation of a negatively charged amino acid to a neutral amino acid.

[0012] The present invention also discloses an immunoglobulin CH1 region comprising an amino acid mutation at a position selected from N159, N201, T120, K147, D148, Y149, V154, A172, Q175, S190, and K213 (EU numbering) compared to a human wild-type CH1 region. The amino acid mutations are preferably at positions D148, Y149, V154, N159, A172, S190, and N201. In a preferred embodiment, the mutation is at an amino acid position selected from N159 and / or N201. The CH1 region may comprise two or more of the amino acid mutations. The two or more mutations are preferably Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, two or more mutations from positively charged amino acids to negatively charged amino acids; or Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, It contains two or more mutations of negatively charged amino acids to positively charged amino acids.

[0013] Preferred combinations of two or more mutations in the CH1 region include amino acid mutations selected from the group A172 / S190 / N201, T197 / K213, D148 / Q175, N159 / Q213, K147 / Q175, Y149 / V154 / A172 / S190, N201 / K213, T120 / N201, N201 / N159, T120 / N159, T120 / N201 / N159, and N201 / K213 / N159.

[0014] The present invention also discloses an immunoglobulin CH2 region comprising an amino acid mutation at position V303 (EU numbering) compared to a human wild-type CH2 region. In one embodiment, the immunoglobulin CH2 region is an Fc silent CH2 region, preferably comprising the amino acid mutations L235G and G236R.

[0015] The present invention also discloses an immunoglobulin CH3 region comprising one or more amino acid mutations at positions K370, E382, and / or E388 (EU numbering) relative to a human wild-type CH3 region. In one embodiment, the immunoglobulin CH3 region comprises mutations of residues to promote heterodimerization at the CH3 / CH3 interface, preferably comprising mutations L351D and L368E, or alternatively comprising mutations T366K and L351K.

[0016] In one embodiment, the CH1, CH2, CH3 region of the immunoglobulin, or a combination thereof, comprises two or more mutations of amino acids, at least one of which is a mutation of an amino acid that is not exposed on the surface of the immunoglobulin. In one embodiment, the CH1, CH2, CH3 region of the immunoglobulin, or a combination thereof, comprises two or more mutations of amino acids that are not exposed on the surface of the immunoglobulin. The mutations are preferably Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and The one or more mutations are preferably selected from mutations of one or more non-surface exposed or preferably buried amino acids. Mutation of a neutral amino acid to a negatively charged amino acid, Mutation of positively charged amino acids to neutral amino acids, Mutation of neutral amino acids to positively charged amino acids, and and mutation of a negatively charged amino acid to a neutral amino acid. The at least one amino acid mutation is preferably to a buried amino acid.

[0017] A CH region containing a mutation from a neutral amino acid to a negatively charged amino acid, a positively charged amino acid to a neutral amino acid, and / or a positively charged amino acid to a negatively charged amino acid is said to have a negative charge difference relative to the original CH region, preferably compared to a human wild-type CH region. The mutation itself is said to provide a negative charge difference to the CH region. A CH region containing a mutation from a neutral amino acid to a positively charged amino acid, a negatively charged amino acid to a neutral amino acid, and / or a negatively charged amino acid to a positively charged amino acid is said to have a positive charge difference relative to the original CH region, preferably compared to a human wild-type CH region. As described herein, when a CH region contains two mutations of amino acid residues, it is preferred that both mutations provide a charge difference in the same direction to the CH region. As described herein, when a CH region contains three or more mutations of amino acid residues, it is preferred that the net result of the mutations provides a charge difference to the CH region. The immunoglobulin region is preferably a human immunoglobulin region. In some embodiments, the immunoglobulin region is an IgG region, preferably an IgG1 region. The immunoglobulin regions disclosed above can be effectively used, for example, as part of an antibody that needs to be separated from an antibody mixture.

[0018] The present invention further discloses antibodies comprising heavy and light chains comprising the CH regions of the immunoglobulins described herein. For example, if such antibodies are produced as part of a mixture, alterations in the charge provided in the CH regions can facilitate separation of the antibodies from the mixture. In preferred embodiments, the antibodies comprise different heavy chains. In preferred embodiments, the antibodies are multispecific antibodies, such as bispecific or trispecific antibodies. In this case, alterations in the charge provided in the CH regions can facilitate separation of the bispecific or trispecific antibodies from the mixture. The different heavy chains preferably comprise interchangeable heterodimerization regions, preferably interchangeable heterodimerization CH3 regions. In one embodiment, one of the heavy chains comprises CH3 mutations L351D and L368E, and the other heavy chain comprises CH3 mutations T366K and L351K. The antibodies of the present invention are preferably IgG antibodies, preferably IgG1 antibodies. In some embodiments, the antibodies comprise first and second heavy chains, each comprising one or more of the CH regions of the immunoglobulins described herein. Preferably, a heavy chain containing CH3 mutations L351D and L368E contains one CH region as described herein, and a heavy chain containing CH3 mutations T366K and L351K contains another CH region as described herein. In such cases, it is preferable that one and the other CH region contain CH regions with different charges. In such cases, the difference in isoelectric points of the resulting antibodies in the mixture is greater, thereby facilitating separation of the antibody from other immunoglobulin molecules or portions thereof in the mixture. In other words, if one CH region has a negative charge difference compared to the original CH region, the other CH region preferably has a positive charge difference compared to the original CH region. Similarly, if one CH region has a positive charge difference compared to the original CH region, the other CH region preferably has a negative charge difference compared to the original CH region.

[0019] Antibodies containing interchangeable heterodimerization regions, such as the interchangeable CH3 heterodimerization regions described herein with CH regions described herein, typically separate better from respective antibodies and / or half-antibodies, if present, that have the same heavy chain, in a separation step that utilizes the charge and / or isoelectric point (pI) of the antibody or fragment thereof. The antibodies preferably contain one or more light chains, which preferably contain the same light chain. The light chain is preferably a common antibody light chain described herein. The common light chain preferably comprises the light chain variable region of Figure 13, e.g., Figure 13B or Figure 13D. In one embodiment, the light chain has a light chain constant region as shown in Figure 13C. In a preferred embodiment, the light chain comprises the amino acid sequence of the light chain shown in Figure 13A or Figure 13E. In a preferred embodiment, the light chain comprises the amino acid sequence of the light chain as shown in Figure 13A. The common light chain is preferably a light chain having CDRs as shown in Figure 13F.

[0020] The antibodies, CH regions or CH domains described herein are preferably human antibodies or human immunoglobulin CH regions or domains, preferably human antibodies, CH domains or CH regions that contain a CH region that has a mutation at an amino acid position that is not surface-exposed or that is preferably buried within a wild-type human CH region.

[0021] Immunoglobulin regions, preferably CH regions or antibodies comprising mutations of non-surface-exposed amino acids as described herein, have mutations selected from amino acids that are not present at the CH1 / CL interface, not present at the CH2 / CH2 interface, and / or not present at the CH3 / CH3 interface. Amino acids at the CH3 / CH3 interface are listed in Figure 22 according to Traxlmayer et al. (2012; J Mol Biol. 26;423(3):397-412. discussion and Figure 3).

[0022] Immunoglobulin regions, preferably CH1, CH2, or CH3 regions or antibodies, comprising the non-surface-exposed amino acid mutations described herein do not substantially adversely affect the stability of the resulting CH1 / CL domain, CH2 domain, or CH3 domain or antibody, including any heavy and light chain interfaces. Immunoglobulin regions, preferably CH1, CH2, or CH3 regions or antibodies comprising the non-surface-exposed amino acid mutations described herein, may also comprise additional mutations that enhance the stability of the charge differential-generating mutations. Immunoglobulin regions, preferably CH1, CH2, or CH3 regions or antibodies comprising the non-surface-exposed amino acid mutations described herein, may also comprise additional mutations that generate a charge differential.

[0023] The present invention also discloses immunoglobulin CH1 / CL domains, CH2 domains, or CH3 domains comprising the immunoglobulin regions described herein. The CH2 domain may further comprise an Fc silent mutation, preferably with CH3 mutations at 235 and / or 236. The CH3 domain may further comprise a CH3 heterodimerization domain, preferably with CH3 mutations L351D and L368E in one CH3 region and CH3 mutations T366K and L351K in the other.

[0024] The present invention further discloses proteins comprising one or more CH1, CH2, CH3 regions, or combinations thereof, as described herein. Also disclosed are proteins comprising one or more CH1 / CL, CH2, CH3 domains, or combinations thereof, as described herein.

[0025] The present invention further discloses multispecific antibodies, such as bispecific antibodies, comprising one or more CH1 / CL, CH2, CH3 domains, or combinations thereof, as described herein.

[0026] Two or more mutations in the CH1, CH2, CH3 regions, or combinations thereof, in one immunoglobulin chain, polypeptide, or protein preferably all include mutations that direct charge in the same direction, i.e., all of the CH regions or combinations thereof toward a more positive charge, or all of the CH regions or combinations thereof toward a more negative charge.

[0027] The present invention further discloses compositions comprising the immunoglobulin regions or antibodies described herein and a pharmaceutical carrier or excipient. Also disclosed are pharmaceutical compositions comprising the immunoglobulin regions or antibodies described herein. The pharmaceutical compositions preferably include a pharmaceutical carrier or excipient.

[0028] Further disclosed are nucleic acids that encode the immunoglobulin regions or antibodies described herein. Further disclosed are combinations of nucleic acids that together encode antibodies or multimeric proteins incorporating the immunoglobulin regions described herein. The nucleic acids may or may not be physically linked.

[0029] Also disclosed are recombinant host cells containing the nucleic acids or combinations of nucleic acids.

[0030] The present invention further provides a method for producing a claimed antibody, the method comprising the steps of: providing a nucleic acid encoding a first heavy chain having a CH1, CH2, CH3 region, or a combination thereof, as described herein; providing a nucleic acid encoding a second heavy chain, wherein the first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and and collecting the antibody from the host cell culture, the method further comprising separating the antibody from other antibodies or antibody fragments based on the charge of the antibody and / or antibody fragment in a separation step. In one embodiment, the first and second heavy chains comprise interchangeable heterodimerization regions, preferably interchangeable CH3 heterodimerization regions.

[0031] The present invention further provides a method for producing a claimed antibody, the method comprising the steps of: providing a nucleic acid encoding a first heavy chain having a CH1, CH2, CH3 region, or a combination thereof, as described herein; providing a nucleic acid encoding a second heavy chain, wherein the first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and harvesting the antibody from the host cell culture, the method comprising: clarifying the harvest; capturing the protein; performing anion exchange chromatography; The method further comprises performing cation exchange chromatography to separate the antibody from other antibodies or antibody fragments. In one embodiment, the first and second heavy chains comprise compatible heterodimerization regions, preferably compatible CH3 heterodimerization regions.

[0032] The present invention further provides a method for producing a claimed antibody, the method comprising the steps of: providing a nucleic acid encoding a first heavy chain having a CH1, CH2, CH3 region, or a combination thereof, as described herein; providing a nucleic acid encoding a second heavy chain, wherein the first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and The method includes the step of harvesting the antibody from the host cell culture, and the method further includes the step of separating the antibody from other antibodies or antibody fragments in a separation step comprising isoelectric electrophoresis on a gel.

[0033] Further provided is a method for producing a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: (a) expressing a nucleic acid encoding a first heavy chain and a nucleic acid encoding a second heavy chain such that the isoelectric point of the encoded first heavy chain and the isoelectric point of the encoded second heavy chain are different, wherein the nucleic acids encode one or more mutations in amino acid positions selected from non-surface exposed positions in the encoded immunoglobulin regions of the first and / or second heavy chains, preferably in the CH1 region, more preferably T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201 and K213, and / or preferably in the CH2 region, preferably V303, and / or preferably in the CH3 region, preferably K370, E382, E388 (EU numbering); (b) culturing the host cells to express the nucleic acid; and (c) harvesting the multispecific antibodies from the host cell culture using the difference in isoelectric points.

[0034] Also provided is a method for separating a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: (a) expressing a nucleic acid encoding amino acid residues of a first heavy chain and / or a nucleic acid encoding amino acid residues of a second heavy chain such that the isoelectric points of the encoded first heavy chain and the encoded second heavy chain are different, wherein the nucleic acids are located at positions that are different from the encoded CH1, CH2, CH3 regions, or combinations thereof, at non-surface exposed residues, preferably T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201 and K213, and / or preferably in the CH2 region, preferably V303, and / or preferably in the CH3 region, preferably K370, E382, E388 (EU numbering); (b) culturing the host cell to express the nucleic acid; and (c) separating the multispecific antibodies from the host cell culture by chromatography. In a preferred embodiment, the nucleic acid encodes the first heavy chain and the second heavy chain such that the retention times of the first heavy chain, the homomultimer of the first heavy chain, the second heavy chain, the homomultimer of the second heavy chain, and the heteromultimer of the first and second heavy chains are different when expressed and separated by an ion exchange chromatography step.

[0035] The variant amino acid at the position encoded by the nucleic acid is preferably selected from amino acids that are not surface-exposed in the human wild-type CH1, CH2, CH3 region, or a combination thereof; and Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral amino acids to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and The amino acids are selected from negatively charged amino acids to positively charged amino acids.

[0036] Also provided is a method for producing a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: providing a nucleic acid encoding a CH1, CH2, CH3 region of a first heavy chain, or a combination thereof, and a nucleic acid encoding a CH1, CH2, CH3 region of a second heavy chain, or a combination thereof, such that the isoelectric points of the first encoded heavy chain and the second encoded heavy chain are different, wherein at least one of the CH regions comprises an amino acid mutation at a position selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, K213, V303, K370, E382, and E388 (EU numbering); and Culturing the host cells to express the nucleic acid; and harvesting the multispecific antibodies from the host cell culture using differences in isoelectric points; harvesting the antibody from the host cell culture; Clarifying the harvest; capturing the protein; performing anion exchange chromatography; and The method further comprises the step of performing cation exchange chromatography to separate the antibody from other antibodies or antibody fragments.

[0037] Further provided is a method for purifying a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: providing nucleic acids encoding a CH1, CH2, CH3 region of a first heavy chain, or a combination thereof, and / or nucleic acids encoding a CH1, CH2, CH3 region of a second heavy chain, or a combination thereof, such that the isoelectric points of the first encoded heavy chain and the second encoded heavy chain are different, wherein at least one of the CH regions comprises an amino acid mutation at a position selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, K213, V303, K370, E382 and E388 (EU numbering); Culturing the host cells to express the nucleic acid; and The method comprises purifying the multispecific antibodies from the host cell culture by isoelectric focusing to separate the multispecific antibodies from other antibodies or antibody fragments.

[0038] The one or more nucleic acids encoding the first heavy chain homomultimer, the second heavy chain homomultimer, and the first and second heavy chain heteromultimer are expressed as proteins with different isoelectric points, resulting in different retention times in ion exchange chromatography.

[0039] The one or more amino acid mutations at positions in the CH region are preferably not surface exposed in the multispecific antibody, and preferably Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and The mutation is selected from a negatively charged amino acid to a positively charged amino acid.

[0040] The amino acid at the mutation position is preferably one or more non-surface exposed or, preferably, buried amino acids. Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, Positively charged to neutral amino acids, and and one or more mutations selected from the group consisting of a negatively charged amino acid to a neutral amino acid mutation. The first and second heavy chains preferably comprise CH3 regions, which preferably comprise interchangeable CH3 heterodimerization regions, one of which preferably comprises L351D and L368E, and the other of which preferably comprises T366K and L351K.

[0041] The variant amino acid at the position encoded by the nucleic acid is preferably selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, K213, V303, K370, E382 and E388.

[0042] Further disclosed are CH1 regions or CH1-containing immunoglobulin polypeptides comprising a first charged amino acid residue at a non-surface-exposed position in human wild-type CH1, preferably at positions 120, 147, 148, 149, 154, 159, 172, 175, 190, 201, or 213. The CH1 region or CH1-containing immunoglobulin polypeptide preferably comprises, in addition to the charged residue, a second charged amino acid residue at a non-surface-exposed position in human, wild-type CH1, preferably at a different position selected from positions 120, 147, 148, 149, 154, 159, 172, 175, 190, 201, or 213, wherein the second charged amino acid has the same charge as the first charged amino acid. The CH1 region or CH1-containing immunoglobulin polypeptide preferably comprises a neutral or negatively charged amino acid residue at positions 147 and / or 213. The CH1 region or CH1-containing immunoglobulin polypeptide preferably comprises a neutral or positively charged amino acid residue in the hinge at positions 148 and / or 216. Further disclosed are CH2 regions or CH2-containing immunoglobulin polypeptides comprising a charged amino acid residue at a non-surface-exposed position in human, wild-type CH2, preferably at position 303. Further disclosed are CH3 regions or CH3-containing immunoglobulin polypeptides comprising a first neutral amino acid residue at a non-surface-exposed position in human, wild-type CH3, preferably at positions 370, 382, or 388. The CH3 region or CH3-containing immunoglobulin polypeptide preferably comprises, in addition to the neutral residue, a second neutral amino acid residue at a non-surface-exposed position in human, wild-type CH3 that is different from the position of the first neutral amino acid, preferably selected from positions 370, 382, or 388. Alternatively, a CH3 region or CH3-containing immunoglobulin polypeptide is disclosed that includes a first negatively charged amino acid residue at a non-surface-exposed position of human, wild-type CH3, preferably at position 370, and a positively charged amino acid at position 382 or 388.

[0043] The mutation at position T120 in the CH1 region is preferably a mutation from a neutral amino acid to a charged amino acid. Examples include T120R, T120K, T120D, and T120E. The mutation preferably includes T120D or T120K.

[0044] The mutation at K147 in the CH1 region is preferably a mutation from a positively charged amino acid to a neutral or negatively charged amino acid. Examples include K147Q, K147T, K147S, K147D, and K147E mutations. The mutation is preferably K147E.

[0045] The mutation at position D148 in the CH1 region is preferably a mutation from a neutral amino acid to a charged amino acid. Examples include D148R, D148K, D148D, and D148E. The mutation preferably includes D148K.

[0046] The mutation at position N159 in the CH1 region is preferably a mutation from a neutral amino acid to a charged amino acid. Examples include N159R, N159K, N159D, and N159E mutations. The mutation preferably includes N159K or N159D mutation.

[0047] The mutation at position Q175 in the CH1 region is preferably a mutation from a neutral amino acid to a charged amino acid. Examples include Q175R, Q175K, Q175D, and Q175E. The mutation preferably includes Q175K or Q175E.

[0048] The mutation at position N201 in the CH1 region is preferably a mutation from a neutral amino acid to a charged amino acid. Examples include N201R, N201K, N201D, and N201E mutations. The mutation preferably includes N201K or N201D mutation.

[0049] The mutation at K213 in the CH1 region is preferably a mutation from a positively charged amino acid to a neutral or negatively charged amino acid. Examples include K213Q, K213T, K213S, K213D, and K213E mutations. The mutation preferably includes K213Q.

[0050] The mutation at V303 in the CH2 region is preferably a mutation from a neutral amino acid to a charged amino acid. Examples include V303K, V303R, V303D, and V303E. The mutation preferably includes V303D or V303E.

[0051] Further disclosed is a CH2-containing immunoglobulin polypeptide comprising a charged amino acid residue at position 303.

[0052] Also disclosed are CH3-containing immunoglobulin polypeptides comprising an uncharged amino acid residue at a position selected from 370, 382, or 388.

[0053] The CH2- and / or CH3-containing immunoglobulin polypeptides described herein may comprise two or more amino acid mutations selected from a charged amino acid residue at position 303, or an uncharged amino acid residue at positions 370, 382, or 388.

[0054] The CH2 region mutations described herein are preferably CH2 mutations at position V303. The mutation is preferably a mutation from a neutral amino acid to a charged amino acid. Examples include V303R, V303K, V303D, or V303E mutations. Preferred mutations are the V303K or V303E mutations described in the examples.

[0055] The mutations in the CH3 region described herein are preferably CH3 mutations at positions K370, E382, E388, or a combination thereof. The mutation at position K370 is preferably a mutation from a charged amino acid to a neutral amino acid. Examples include a K370Q, K370N, K370H, K370S, K370T, or K370Y mutation. A preferred mutation is the K370S or K370T mutation described in the Examples. The mutation at position E382 is preferably a mutation from a charged amino acid to a neutral amino acid. Examples include an E382Q, E382N, E382H, E382S, E382T, or E382Y mutation. A preferred mutation is the E382Q or E382T mutation described in the Examples. The mutation at position E388 is preferably a mutation from a charged amino acid to a neutral amino acid. Examples are the E388Q, E388N, E388L, E388S, E388T or E388M mutations. Preferred mutations are the E388L, E388M or E388T mutations as described in the examples.

[0056] An immunoglobulin polypeptide as described herein is preferably an antibody, preferably a multispecific antibody.

[0057] The antibody may further comprise a positively charged amino acid residue at hinge position 216.

[0058] The antibody may further comprise a mutation at amino acids selected from T197 and E216 in the hinge position.

[0059] Also disclosed are compositions comprising the immunoglobulin domains, immunoglobulin region polypeptides, proteins or antibodies described herein, which further comprise one or more of the following mutations in the CH1 domain: G122P, I199V, N203I, S207T, and V211I.

[0060] The present invention can be used to provide separation between the antibodies or immunoglobulin proteins described herein, between the bispecific antibodies described herein and the monospecific antibodies, and between the multispecific antibodies described herein and other multispecific and monospecific antibodies and half antibodies.

[0061] The present invention can also be used to optimize the simultaneous purification of two or more desired antibodies produced by cells. Two or more bispecific antibodies can be produced by providing three or more heavy chains capable of pairing with a common light chain, where one heavy chain has a member with a compatible heterodimerization domain and the other heavy chain has another member with a compatible heterodimerization domain, e.g., a CH3 DE region on one side and a CH3 KK region on the other side. By adjusting the charge of one or more heavy chains according to the present invention, antibodies containing heterodimeric heavy chains that comigrate in separation methods that utilize charge and / or pI can be provided. Charge can be adjusted so that antibodies containing comigrating heterodimeric heavy chains migrate at a different position than antibodies and / or half-antibodies containing each monomeric heavy chain.

[0062] Further provided is an immunoglobulin protein comprising a first CH1 region or CH1-containing immunoglobulin polypeptide and a second CH1 region or CH1-containing immunoglobulin polypeptide, wherein the first and / or second CH1 region or CH1-containing immunoglobulin polypeptide comprises one or more mutations of one or more amino acids selected from amino acids in the CH1 region that are not surface-exposed, whereby the isoelectric point of the immunoglobulin protein comprising the first CH1 region or CH1-containing immunoglobulin polypeptide and the second CH1 region or CH1-containing immunoglobulin polypeptide is different from the isoelectric point of an immunoglobulin protein containing only the first CH1 region or CH1 immunoglobulin polypeptide or the isoelectric point of an immunoglobulin protein containing only the second CH1 region or CH1 immunoglobulin polypeptide.

[0063] In one embodiment, the present invention relates to proteins comprising at least two different polypeptides, each comprising at least two different heavy chain variable regions and a common light chain, including heavy chain domains, such as bispecific antibodies or multivalent multimers. The present invention further relates to means and methods for producing and isolating such proteins. Proteins comprising two different immunoglobulin variable region polypeptides are generally referred to herein as bispecific proteins, bispecific immunoglobulins, or bispecific antibodies. Based on protein formats comprising two different immunoglobulin variable region polypeptides, multispecific multimers comprising domains specific for three or more targets / epitopes, including trispecific and / or multispecific formats, can also be produced; see, for example, International Application PCT / NL2019 / 050199. Although strategies exist in the art for increasing the yield of desired bispecific or multispecific proteins or antibodies, the generation of undesired species, including monospecific proteins or half antibodies, cannot be easily and completely avoided. Therefore, separating bispecific or multispecific proteins or antibodies from monospecific, half antibodies, or undesired by-product proteins is preferred for isolating the desired bispecific or multispecific proteins or antibodies. Furthermore, such separation of these bispecific or multispecific proteins or antibodies is necessary for the clinical development or commercialization of such proteins.

[0064] The inventors have now surprisingly found that by engineering immunoglobulin regions, preferably CH1, CH2 or CH3, with charged residues at non-surface exposed amino acid positions within the constant region that contain residues that are buried in the immunoglobulin polypeptide, multispecific or bispecific and monospecific proteins, when engineered, can be readily separated and obtained by isoelectric focusing and conventional chromatographic methods, for example non-affinity based chromatography such as ion exchange chromatography.

[0065] Such immunoglobulin regions include the addition, removal, or reversal of charges to one or both of the immunoglobulin polypeptide chains containing the constant regions, preferably CH1, CH2, CH3, or a combination thereof. Prior to the present invention, modifications of non-surface-exposed or buried amino acids of any protein, particularly immunoglobulins, were generally avoided because it was understood that altering the charge of such residues could have potentially deleterious effects on structure and function, including the potential for causing destabilizing effects on the immunoglobulin. Furthermore, such modifications were not expected to alter chromatographic properties because these residues are not readily exposed for interaction with chromatography resins.

[0066] It has been surprisingly discovered that by generating immunoglobulin regions with charged amino acids at non-surface-exposed, buried amino acid positions in the framework or constant regions of immunoglobulin polypeptide chains, preferably in the CH1, CH2, CH3 regions, or a combination thereof, monospecific, bispecific, and multispecific proteins can be generated with distinct charges and different isoelectric points (see, e.g., Figure 1), thereby enabling separation and isolation of the monospecific protein from the bispecific or multispecific protein (or vice versa), or separation of the desired protein from undesired protein by-products. Furthermore, immunoglobulin regions can be generated with charged residues and other mutations at non-surface-exposed or buried positions, which can enhance the stability of such immunoglobulin regions relative to wild-type regions or domains, or wild-type regions or domains with only charge changes.

[0067] It is understood that variant domains, including constant domains, and methods using such domains, are applicable to the production of multimerized proteins and the separation of such proteins. When different protein species are produced in a mixture such that the different species have similar isoelectric points (pIs), making separation difficult, the use of variant domains described herein and the methods described herein can be used to improve the separation of the desired species.

[0068] The present invention discloses methods for selecting mutations that do not adversely affect the structure and function of separating domains and that generate distinguishable isoelectric points between different multimerized protein species incorporating such domains. The invention described herein applies to products containing separating domains applicable to various immunoglobulin regions, e.g., CL, CH1, CH2, and / or CH3 regions, and VH / VL regions (particularly framework regions). The invention is generally applicable to any multimeric protein. For example, the invention is applicable so long as the resulting multimers contain at least two different proteins (e.g., designated A and B) capable of forming different multimerized proteins, whereby the resulting multimeric species can include AA, AB, BA, or BB. In such situations, these multimerizing proteins can employ the mutant domains of the invention in chain A and / or chain B, whereby each multimeric species contains one or more mutant domains bearing a charge at a non-surface-exposed or buried position, generating multimeric species with distinguishable isoelectric points that can be separated by methods known to those skilled in the art, such as by isoelectric focusing and / or based on distinct retention times during ion exchange chromatography.

[0069] The above principles can also be applied to the generation of bispecific antibodies (up to 10 species obtained if two different heavy chains and two different light chains are expressed, or 3 species obtained if two different heavy chains and a common light chain are expressed, or two different light chains and a common heavy chain are expressed). The above principles can also be applied to higher order multimers.

[0070] When the multimer is a bispecific antibody, variant immunoglobulin regions with charge changes at non-surface or buried positions, including adding, subtracting, or reversing charge, can be used. For example, a charged CH1 region can be used (as exemplified in Figures 1A-C), or a charged CH2 region can be used (as exemplified in Figure 1D), or a charged CL region of the light chain can be used (Figure 1E), or a charged CH3 region can be used.

[0071] Such multimers may also be trivalent or tetravalent, e.g., they may comprise three variable domains, e.g., VH and VL, each comprising a mutation in the CH1 or CL region (as illustrated in Figures 2A and 2B).

[0072] Reference herein to a "mutation" of an immunoglobulin region, such as a CH1 region, or any other suitable region or domain, is understood not to imply that the multimeric protein product, e.g., an antibody, is mutated, but rather that the multimeric protein comprises a domain having a separation mutation as described herein that differs from the wild-type domain, e.g., such a domain contains a difference in a non-surface-exposed residue in the wild-type domain, thereby generating a charge difference that can be used to facilitate separation from a mixture of multimerized proteins. Thus, the term mutation refers to the fact that the amino acid sequence of an immunoglobulin polypeptide, such as that contained in a bispecific antibody, has an amino acid sequence that differs from a reference sequence, e.g., a human IgG1 sequence.

[0073] It is understood that an amino acid sequence having a desired residue at a desired position can be selected from a library containing mutations in amino acid sequences, e.g., compared to a reference sequence of the CH1, CH2, CH3 region, or a combination thereof. Thus, the term mutation refers to an amino acid sequence having a desired amino acid residue at a desired position, regardless of the method by which the amino acid sequence is obtained. Instead of referring to amino acid mutations as, e.g., in non-surface amino acids, preferably buried amino acids, as described herein, the amino acid mutations can also be referred to as "separating amino acid residues," since these mutations allow for separation of the desired multimeric species.

[0074] Protein products can be produced from DNA constructs encoding the proteins, and thus mutations in protein products have their origin in the DNA constructs encoding the proteins. Any suitable means of generating such mutations known in the art are encompassed herein, including constructs that can be generated ab initio containing nucleic acids encoding such mutations, e.g., via DNA synthesis, without any necessary mutation, replacement, substitution, insertion, or deletion of the original nucleic acid. Such methods for generating mutant domains are readily available and can be combined, for example, with any suitable nucleic acid encoding any variable region (or, if modified variable regions are used, any combination of CDR sequences contained therein). Constructs encoding selected variable regions can be simply synthesized de novo in combination with encoded constant regions bearing suitable amino acid mutations described herein that provide differentiation in isoelectric point. For example, sequences encoding CH1, CH2, and CH3 can be provided and combined with a selected VH-encoding sequence, and this combination can be performed in silico (and synthesized de novo) and / or in vitro (e.g., using molecular biology techniques such as ligation / cloning) and generated expression cassettes. By providing the sequences of suitable combinations of variable regions (encoded by nucleic acid sequences), these can be readily combined with suitable constant regions according to the invention, e.g., CL or CH1, and CH2 and / or CH3, encoding mutations according to the invention, e.g., non-surface amino acids, preferably buried amino acids, preferably having mutations in the CH1 region.

[0075] For example, cells can be provided that contain suitable expression cassettes encoding components, e.g., polypeptides, comprising a multimeric protein, such as a common light chain and two distinct heavy chains. The cells may initially have stably integrated nucleic acids encoding suitable variant domains for separation. Such cells then need only incorporate nucleic acids encoding selected VL or VH regions, or both (or replace the VL and / or VH regions), and can then produce a mixture of multimeric proteins that can be readily separated based on the variant domains. Thus, one aspect of the invention described herein includes host cells stably integrated into their genomes nucleic acids encoding a common light chain and constant regions comprising one or more domains comprising the separating amino acid residues described herein. Preferably, the invention includes nucleic acids encoding a domain comprising a negatively charged separating amino acid residue for combining with a heavy chain variable region and a domain comprising a positively charged separating amino acid residue for combining with a second heavy chain variable region. Preferably, the two encoded heavy chain variable regions have different pIs, with the more positively charged variable region linked to a domain containing a separating positively charged amino acid residue and the more negatively charged variable region linked to a domain containing a separating negatively charged amino acid residue.

[0076] The present invention also discloses many such mutations or separating amino acid residues. Because these mutations involve non-surface residues of the protein, these mutations may advantageously not result in exposure of potential antigenic motifs at the surface of domains used for separation, such as, for example, the CH1 region in multispecific proteins, particularly multispecific antibodies, thereby reducing undesirable immunological effects. Furthermore, because the mutations are not at the surface of the protein, the selected mutations disclosed herein can generally be beneficially applied to any bispecific or multispecific protein comprising a constant or framework region containing a mutation as disclosed herein and an interchangeable heterodimerization region (e.g., CL, CH2, or CH3 domain), preferably at least two constant region domains, more preferably a CH1 comprising an immunoglobulin polypeptide. Preferably, multispecific proteins according to the present invention having a heavy chain variable domain and a light chain variable domain have one or more selected amino acid changes in the framework or constant region, preferably a non-surface-exposed or buried CH1 region that do not adversely affect the CH / CL interface of the CH1, or a CH2CH3 / CH2CH3 domain in which residues are modified at the Fc interface.

[0077] Alternatively, the multispecific proteins of the invention may comprise a separation domain, such as a CH1 region that does not need to pair with a CL. For example, the CH1 may be the CH1 of a camelid, or may be based on the CH1 of a camelid or other organism lacking light chains, such as a shark, or may be a modified CH1 region that lacks hydrophobic residues and does not pair with light chains, and that contains mutations in non-surface exposed residues to create differences in isoelectric points, facilitating separation of the multispecific protein from other proteins and fragments.

[0078] By including such mutations in the CH1 region, the variants typically do not affect Fc / Fc receptor interactions or multimerization (e.g., hetero- or homo-dimerization) of the peptide at the CH2CH3 / CH2CH3 interface. Preferably, the domains of the invention include one or more selected non-surface-exposed or buried separation residues within the framework or constant region, preferably within the CH1 region, in addition to other mutations that may beneficially improve stability compared to the wild-type domain or compared to a domain containing only one or more separation residues.

[0079] Thus, in one embodiment, there is provided a bispecific protein, particularly an antibody, comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the first and / or second CH1-containing immunoglobulin polypeptide comprises a mutation in one or more non-surface-exposed or buried variant-separating amino acid residues, such that the isoelectric point of the immunoglobulin protein comprising the first CH1-containing immunoglobulin polypeptide and the second CH1-containing immunoglobulin polypeptide is different from the isoelectric point of a protein having only the first CH1-containing immunoglobulin polypeptide and / or the isoelectric point of a protein having only the second CH1-containing immunoglobulin polypeptide (e.g., a parent protein).

[0080] In one embodiment, mutation of a CH1-containing immunoglobulin increases or decreases the retention time of the immunoglobulin on ion exchange chromatography.

[0081] This also applies, for example, to multispecific antibodies comprising immunoglobulin polypeptides comprising a third CH1-containing immunoglobulin polypeptide and immunoglobulin polypeptides having first and second CH1 regions that dimerize, wherein the first and second CH1-containing immunoglobulin polypeptides comprise one or more variant separating residues of one or more amino acids selected from amino acids in the CH1 region that are not surface-exposed or that are buried, such that the isoelectric point of the immunoglobulin protein comprising the first and second CH1-containing immunoglobulin polypeptides and the third CH1-containing immunoglobulin polypeptide is different from the isoelectric point of a protein having only the first CH1- and second CH1-containing immunoglobulin polypeptides and / or from the isoelectric point of a protein having only the third CH1-containing immunoglobulin polypeptide (e.g., the parent protein). See Figure 2B.

[0082] It is understood that bispecific proteins comprising first and second CH1-containing immunoglobulin polypeptides are difficult to separate from the parent protein (e.g., a monospecific, bivalent antibody) using conventional chromatographic methods such as ion exchange due to the similar isoelectric points of each protein. As shown in the Examples section, similarity in isoelectric points is indicated by similar retention times on a selected chromatographic column. Similarity can also be determined using, for example, isoelectric focusing, as shown in the Examples. During the preparation of a mixture of antibodies or proteins containing immunoglobulin domains, retention times may be similar, causing the peaks for each protein to overlap and making separation difficult. It is also understood that the terms "first" and "second" referred to in relation to the first and second CH1-containing immunoglobulin polypeptides do not imply any order or preference, but are used simply to indicate that the chains are different.

[0083] It is understood that mutations in the CH1 region according to the present invention affect the isoelectric point of the bispecific antibody and include the addition, removal, or reversal of a charge. The addition of a charge to a CH1-containing polypeptide or the like may be present in one or each of the CH1 regions of each immunoglobulin polypeptide produced. The addition of a charge can be accomplished by a variety of means. Neutral amino acids can be altered (typically at the encoding DNA level in an expression construct) to either negatively or positively charged amino acids to add a negative or positive charge, respectively. Positively charged amino acids can be changed to neutral or negatively charged amino acids to add a negative charge, and changing an amino acid from a positive charge to a negative charge can result in a relatively large change. Conversely, negatively charged amino acids can be changed to neutral or positively charged amino acids to add a positive charge, and changing an amino acid from a negative charge to a positive charge can result in a relatively large change.

[0084] Thus, in a further embodiment, the immunoglobulin protein according to the invention comprises one or more non-surface exposed or preferably buried amino acids: Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and one or more mutations selected from the group consisting of: a mutation of a negatively charged amino acid to a positively charged amino acid.

[0085] Positively charged amino acids are lysine (Lys, K), arginine (Arg, R) and histidine (His, H). Preferably, when a positively charged amino acid is included in the chain or changed from the parent domain, lysine is selected. Negatively charged amino acids are glutamic acid (Glu, E) and aspartic acid (Asp, D). The remaining amino acids, in terms of isoelectric point, represent neutral amino acids. Preferably, in a further embodiment, the immunoglobulin protein according to the invention comprises one or more non-surface exposed or preferably buried amino acids, Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, Positively charged to neutral amino acids, and and one or more mutations selected from the group consisting of a negatively charged amino acid to a neutral amino acid mutation.

[0086] These mutations may be preferred as conservative design changes.

[0087] As illustrated in Figures 1 and 2, which show schematic representations of monospecific, bispecific, and exemplary multispecific antibodies according to the invention, either or both of the CH1-containing immunoglobulins can be altered. It is understood that the mutations selected for one, etc., of the CH1-containing immunoglobulins are preferably of the same type; i.e., if a positive charge is added to one chain, one or more mutations that add a positive charge (to have an additive effect) will be selected for that chain. It is also understood that if one of the chains has a positive charge and the other chain similarly contains one or more mutations, the mutation or mutations selected for the other chain are preferably selected to include the addition of a negative charge, since otherwise the effect on the isoelectric points of the different paired immunoglobulin proteins containing the first and second CH1-containing immunoglobulins would typically be counteracted or even abolished.

[0088] Thus, in one embodiment, there is provided an immunoglobulin protein comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the first and / or second CH1-containing immunoglobulin polypeptide comprises one or more mutations of one or more amino acids selected from amino acids in the CH1 region that are not surface exposed, and the first CH1-containing immunoglobulin polypeptide comprises: Neutral to negatively charged amino acids, Positively charged to neutral amino acids, and a mutation selected from a mutation of a positively charged amino acid to a negatively charged amino acid; The second CH1-containing immunoglobulin polypeptide is Neutral amino acids to positively charged amino acids, Negatively charged amino acids to neutral amino acids, The present invention includes mutations selected from mutations of negatively charged amino acids to positively charged amino acids.

[0089] In another embodiment, an immunoglobulin protein is provided, comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the first and / or second CH1-containing immunoglobulin polypeptide comprises one or more mutations of one or more amino acids selected from amino acids in the CH1 region that are not surface exposed; The first CH1-containing immunoglobulin polypeptide is Neutral to negatively charged amino acids, and a mutation selected from a positively charged amino acid to a neutral amino acid; The second CH1-containing immunoglobulin polypeptide is Positively charged to neutral amino acids, and The mutations include those selected from negatively charged amino acid to neutral amino acid mutations.

[0090] In one embodiment, the first and second CH1-containing immunoglobulin polypeptides are preferably substantially identical when aligned to the amino acid sequence of their CH1 regions, and preferably only differ with respect to amino acid positions defined herein. Preferably, the amino acid positions that differ between the CH1 regions of the first and second CH1-containing polypeptides differ with respect to amino acid positions that are not surface-exposed. The CH1-containing polypeptides are preferably CH1 regions of human IgG1 immunoglobulins. An example of an amino acid sequence of a CH1 region suitable for generating or comparing separation domains containing variant residues as described herein is shown in Figure 14A.

[0091] In a further embodiment, the immunoglobulin protein according to the invention further comprises a mutation that stabilizes the first and / or second CH1-containing immunoglobulin polypeptide further selected from amino acids within the CH1 region. Further mutations may be introduced to improve the stability of polypeptides and / or bispecific or multispecific proteins comprising domains containing separating residues as described herein.

[0092] Preferably, the separating residues that are not surface exposed or buried in the immunoglobulin polypeptide may provide increased relative stability compared to a reference domain, such as a wild-type domain.

[0093] As used herein, the term "non-surface exposed" refers to a "% Ratio" scoring of 50% or less in the GETAREA 1.0β program using default parameters, where a ratio greater than 50% is scored as "Out" or "Surface Exposed" in this program. As used herein, the term "buried" refers to a "% Ratio" scoring of 20% or less in the GETAREA 1.0β program using default parameters, where this program is scored as "In." Negi et al., "Solvent Accessible Surface Areas, Atomic Solvation Energies, and Their Gradients for Macromolecules," Last modified Wednesday, April 17, 3:00 PM, 2015. The primary amino acid and structural model of the protein domain containing the region are used as input to the GETAREA program to obtain a "% Ratio" in the GETAREA output file as shown in the Examples herein. Herein, the term "buried amino acid" refers to an amino acid or a mutation thereof having a scoring ratio (%) of 20% or less, preferably 15% or less, as shown in Table 1 and Tables 20 to 22. In some embodiments, the term "buried amino acid" refers to an amino acid or a mutation thereof having a scoring ratio (%) of 10% or less, as shown in Table 1 and Tables 20 to 22.

[0094] Structural information for the CH regions can be obtained from the Protein Data Bank, which has several high-resolution structures for each of the CH regions, or by homology modeling (e.g., using a homology modeling tool to model the structure of CH regions including mutations; http: / / swissmodel.expasy.org). Structural information, such as the selected CH1 region provided in pdb format, is imported into the GETAREA program (Protein Data Bank format, which provides a standard representation of macromolecular structural data derived from X-ray diffraction and NMR studies), which records the scoring ratio (%) as input for analysis.

[0095] As used herein, "pi" is calculated based on primary amino acids using ExPASy's ProtParam tool with default parameters. 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. Calculated parameters include the theoretical pI. 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. Full-length polypeptides are used to determine the theoretical pI as shown in the examples herein.

[0096] As shown in the Examples section, for example, Rosetta software (version 3.1<<https: / / www.resettacommons.org / software> By performing an in silico stability analysis using the method described above, further selection of non-surface exposed and buried residues at each amino acid position along the domain of interest can be performed without changing the surface exposed residues. Instead of in silico selection, this can also be performed in vitro. Furthermore, the selection can be initially in silico followed by in vitro confirmation, such as that shown in the Examples section.

[0097] An "antibody" is a proteinaceous molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind to an epitope on an antigen; such domains are derived from or share sequence homology with the variable region of the antibody. Antibody binding has different properties, including specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by a binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. It should be noted that, as used herein, the "specificity" of an antibody refers to its selectivity for a particular antigen, while "affinity" refers to the strength of the interaction between the antigen-binding site of the antibody and the epitope it binds. Therapeutic antibodies are preferably as similar as possible to the natural antibodies of the subject to be treated (e.g., human antibodies in the case of a human subject). Antibodies according to the present invention are not limited to any particular format or method of preparation.

[0098] A "bispecific antibody" is an antibody described herein in which one domain of the antibody binds to a first antigen or epitope and a second domain of the antibody binds to a second antigen or epitope, where the first and second antigens are not identical or the first and second epitopes are not identical. The term "bispecific antibody" also encompasses antibodies in which one heavy chain variable region / light chain variable region (VH / VL) combination binds to a first epitope on an antigen and a second VH / VL combination binds to a second epitope. This term further includes antibodies in which the VH can specifically recognize a first antigen and the VL, paired with a VH in an immunoglobulin variable region, can specifically recognize a second antigen. The resulting VH / VL pair binds to 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 (Cancer Cell 20, 472-486, October 2011). Bispecific antibodies according to the present invention are not limited to any particular bispecific format or method of producing them. Bispecific antibodies are multispecific antibodies. Multispecific multimers or antibodies as referred to herein encompass proteinaceous molecules belonging to the immunoglobulin class of proteins that contain two or more domains that bind to epitopes on antigens, where such domains are derived from or share sequence homology with antibody variable regions, and include proteinaceous molecules that bind to three or more antigens known in the art, such as those described in the previously filed U.S. Patent Application Publication No. 62 / 650,467.

[0099] The domains of the invention described herein comprise a framework or constant domain that differs from the wild-type or reference sequence such that it comprises a negatively charged amino acid, where the corresponding positions in the wild-type or reference sequence are not surface-exposed or buried and contain neutral amino acids. Alternatively, the domains of the invention described herein comprise a framework or constant domain that differs from the wild-type or reference sequence such that it comprises a positively charged amino acid, where the corresponding positions in the wild-type or reference sequence are not surface-exposed or buried and contain neutral amino acids. Alternatively, the domains of the invention described herein comprise a framework or constant domain that differs from the wild-type or reference sequence such that it comprises a neutral amino acid, where the corresponding positions in the wild-type or reference sequence are not surface-exposed or buried and contain positively or negatively charged amino acids. Alternatively, the domains of the invention described herein comprise a combination of the above embodiments, such that the net pI of the domain differs from the wild-type or reference sequence by one or more charges.

[0100] As used herein, the term "charged amino acid residue" or "charged residue" refers to an amino acid residue that has a charged side chain at physiologically relevant pH. These may be either positively charged side chains, such as those present in arginine (Arg, R), histidine (His, H), and lysine (Lys, K), or negatively charged side chains, such as those present in aspartic acid (Asp, D) and glutamic acid (Glu, E). As used herein, the term "neutral amino acid residue" or neutral residue refers to all other amino acids that do not bear a charged side chain at physiologically relevant pH. These neutral residues include serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Glu, Q), cysteine (Cys, C), glycine (Gly, G), proline (Pro, P), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, T).

[0101] Preferred embodiments of the invention described herein include the above-described separation domains and / or proteins comprising such separation domains. The separation domains of the invention described herein can be incorporated into antibodies or proteins having immunoglobulin domains. They can be incorporated into IgG or T cell receptor domains or immunoglobulins of any subclass, monospecific or multispecific.

[0102] A further preferred embodiment of the invention described herein is a protein comprising one or more binding domains, comprising a CH1 separation domain comprising a separation residue of N159K, H or R or N159D or E, more preferably a separation residue of N159K or N159D. A further preferred embodiment of the invention described herein is a protein comprising one or more binding domains, comprising a CH1 separation domain comprising a separation residue of N201K, H or R or N201D or E, more preferably a separation residue of N201K or N201D.

[0103] The monospecific, bispecific or multispecific proteins according to the invention provided incorporating the separation domains of the invention described herein comprise a CH1 region selected to be a CH1 region from human IgG, and in one embodiment, comprise amino acids within the CH1 region selected from the group comprising T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201 and K213. The numbering of these amino acid positions is according to EU numbering.

[0104] The CH1 separation domain of the invention disclosed herein may further comprise a stabilizing mutation corresponding to T197D.

[0105] The CH1 separation domain of the invention disclosed herein may further comprise a stabilizing mutation in the hinge corresponding to E216K.

[0106] The CH1 separation domain of the invention disclosed herein may further comprise stabilizing mutations corresponding to G122P, S157T, I199V, N203I, S207T, and V211I.

[0107] For example, by generating and using separation domains comprising mutations as disclosed herein with mutant amino acid residues selected from the group comprising T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, and K213 in the CH1 region of the human IgG1 immunoglobulin polypeptide chain, it is possible to generate monospecific, bispecific, or multispecific proteins with distinguishable charges, i.e., different isoelectric points (see, e.g., Figure 1), at the pH used during formulation and separation, thereby allowing for the separation and isolation of the monospecific protein from the bispecific or multispecific protein (or bispecific protein from trispecific, etc.).

[0108] In one embodiment, the invention produces a monospecific, bispecific or multispecific protein wherein the CH1 region of an immunoglobulin polypeptide comprises a separating residue in the CH1 region that is a non-surface exposed or buried amino acid selected from the group consisting of D148, Y149, V154, N159, A172, S190 and N20. The protein is preferably a human protein, preferably an IgG protein, preferably an IgG1 protein.

[0109] In one embodiment, in a bispecific protein according to the invention, the CH1 region of the immunoglobulin polypeptide is selected to be a CH1 region from human IgG1, and the amino acids in the CH1 region are selected from the group consisting of T120, K147, D148, N159, Q175, N201, and K213, as these amino acid positions allow for mutations with different charges (changes between neutral, positively charged, and negatively charged amino acids). In a further embodiment, the amino acids in the CH1 region that are not surface-exposed are selected from the group consisting of N159 and N201, which are buried amino acids. More preferably, the immunoglobulin protein is a bispecific antibody or a multispecific protein. Most preferably, the first and second CH1-containing immunoglobulin polypeptides each comprise a heavy chain variable region, and each of the variable regions binds to a different antigen or epitope.

[0110] In another embodiment, the present invention provides a bispecific protein comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the CH1 region is the CH1 region of human IgG1, and wherein the first or second CH1-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from amino acids in the CH1 region, the mutations comprising one or more mutations selected from the group consisting of K147E, N159D, Q175E, N201D, and K213Q, or one or more mutations selected from the group consisting of T120K, D148K, N159K, Q175K, N201K. Most preferably, the bispecific protein is a bispecific antibody.

[0111] In a preferred embodiment, the invention provides an immunoglobulin protein comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the CH1 region is the CH1 region of human IgG1, and wherein one of the first or second CH1-containing immunoglobulin polypeptide comprises the mutations N159K and E216K at the hinge position. In a further embodiment, the other of the first or second CH1-containing immunoglobulin polypeptide comprises no mutations or one or more mutations, e.g., selected from T197D and K213Q. Preferably, the multimerizing protein of the invention is composed of two polypeptides, a first polypeptide comprising a first variable domain that binds to a first antigen or epitope and a second polypeptide comprising a second variable domain that binds to a different antigen or epitope to the first variable domain, the first variable domain being linked via a peptide bond to a separation domain linked to a dimerization domain such as CH3, the dimerization domain forming an interface with the second variable domain and a second dimerization domain such as a second CH3 domain linked via a peptide bond to a second separation domain, preferably having a different charge to the first separation domain, and preferably the protein comprises a bispecific or multispecific protein or an antibody.

[0112] In one embodiment, the invention produces a monospecific, bispecific, or multispecific protein, wherein the CH1, CH2, CH3 regions, or combinations thereof, of an immunoglobulin polypeptide comprise a separating CH region residue that is a non-surface exposed or buried amino acid selected from the group consisting of T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, and K213, V303, K370, EE382, and E388. The protein is preferably a human protein, preferably an IgG protein, preferably an IgG1 protein.

[0113] In one embodiment, in a bispecific protein according to the invention, the CH regions of the immunoglobulin polypeptides are selected to be CH regions from human IgG1, and the amino acids in the CH regions are selected from the group consisting of T120, K147, D148, N159, Q175, N201, K213, V303, K370, E382, and E388, since these amino acid positions allow for mutations with different charges (changes between neutral, positively charged, and negatively charged amino acids). In a further embodiment, the amino acids in the CH regions that are not surface-exposed are selected from the group consisting of N159 and N201 for CH1, V303 for CH2, and E382 and E388 for CH3, which are buried amino acids. More preferably, the immunoglobulin protein is a bispecific antibody or a multispecific protein. Most preferably, the first and second CH-containing immunoglobulin polypeptides each comprise a heavy chain variable region, and each of the variable regions binds to a different antigen or epitope.

[0114] In another embodiment, the present invention provides a bispecific protein comprising a first CH-containing immunoglobulin polypeptide and a second CH-containing immunoglobulin polypeptide, wherein the CH region is a CH region of human IgG1, and the first or second CH-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from those in the CH1 region, the mutations comprising one or more mutations selected from the group consisting of K147E, N159D, Q175E, N201D, K213Q and V303E, or one or more mutations selected from the group consisting of T120K, D148K, N159K, Q175K, N201K, V303K, E382Q, E382T, E388L, E388M, E388T. Most preferably, the bispecific protein is a bispecific antibody.

[0115] It is known in the art that bispecific or multispecific antibodies can be generated preferentially over monospecific antibodies (or undesired protein by-products) by having a bispecific or multispecific antibody comprising a first polypeptide ("DE arm") having a CH3 region comprising the mutations L351D and L368E (EU numbering), and a second polypeptide ("KK arm") having a second CH3 region comprising the mutations T366K and L351K (hereinafter collectively referred to as "DEKK"), whereby two polypeptides that form DEKK preferentially pair over two polypeptides comprising either a DE / DE homodimer or a KK / KK homodimer. Other forms of charge engineering are known in the art to promote heterodimerization.

[0116] In the embodiments described herein, when a negatively charged separation domain such as a CH1 region is used, it is preferentially combined with a DE CH3 domain, and when a positively charged separation domain such as a CH1 region is used, it is preferentially combined with a KK arm or any combination of the foregoing (e.g., a negatively charged separation domain and a DE CH3 domain on one polypeptide and a positively charged separation domain and a KK CH3 domain on the other polypeptide will preferentially form a heterodimer that is more easily separated from either a doubly positively charged separation domain and a KK / KK homodimer or a doubly negatively charged separation domain and a DE / DE homodimer). To the extent other heterodimerization techniques are used, as known to those skilled in the art, the present invention can be applied in the same manner by combining a negatively charged separation domain with a negatively charged heterodimerization domain and / or a positively charged separation domain with a positively charged heterodimerization domain to facilitate heterodimer formation and separation of the heterodimers.

[0117] In a further embodiment, the present invention provides a multimerized protein, preferably a bispecific or multispecific protein, comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the CH1 region is the CH1 region of human IgG1, and wherein the first CH1-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from the amino acids in the CH1 region, the mutations comprising one or more mutations selected from the group consisting of K147E, N159D, Q175E, N201D, and K213Q, and the second CH1-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from the amino acids in the CH1 region, the mutations comprising one or more mutations selected from the group consisting of T120K, D148K, N159K, Q175K, and N201K. Most preferably, the bispecific protein is a bispecific antibody.

[0118] In another embodiment, the present invention provides a multimerized protein, preferably a bispecific or multispecific protein, comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the CH1 region is the CH1 region of human IgG1, and the first or second CH1-containing immunoglobulin polypeptide comprises one or more amino acid mutations selected from amino acids in the CH1 region and an amino acid mutation at hinge residue E216K, wherein the former mutations are selected from the group consisting of K147E and Q175E, N201D and K213Q, T197D and K213Q, N159D and K213Q, and K213Q, or the former mutations are selected from the group consisting of T120K, N201K, D148K and Q175K, and N159K. Most preferably, the protein is a bispecific antibody.

[0119] In an even further embodiment, the present invention provides a multimerized protein, preferably a bispecific or multispecific protein, comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the CH1 region is the CH1 region of human IgG1, the first CH1-containing immunoglobulin polypeptide comprising one or more amino acid mutations selected from the amino acids in the CH1 region, the mutations being selected from the group consisting of K147E and Q175E, N201D and K213Q, T197D and K213Q, N159D and K213Q, and K213Q, and the second CH1-containing immunoglobulin polypeptide comprising one or more amino acid mutations selected from the amino acids in the CH1 region and an amino acid mutation at hinge residue E216K, the former mutation being selected from the group consisting of T120K, N201K, D148K and Q175K, and N159K. Most preferably, the immunoglobulin protein is a bispecific antibody.

[0120] In one embodiment, the present invention provides a multimerizing protein, preferably a bispecific or multispecific protein, comprising a CH region having a CH1, CH2, or CH3 region sequence shown in Table 14, Part B. A multimerizing protein, preferably a bispecific or multispecific protein, may have a CH1, CH2, or CH3 region that is a combination of two or three of the CH region sequences in Table 14, Part B. When it has two CH1, two CH2, or two CH3 sequences as shown in Table 14, Part B, it is preferred that the two have opposite charge differences compared to the wild-type CH region. Thus, if one has a more positive charge compared to the wild-type CH, it is preferred that the other has a more negative charge compared to the wild-type CH. A heavy chain can have two or three of the sequences in Table 14, for example, by having two or three of the CH1, CH2, and CH3 sequences in Table 14, Part B. In such cases, the two or three may have the same charge difference compared to the wild-type CH. Two or all three are more positively charged compared to the wild type, or two or all three are more negatively charged. Again, a multimerizing protein, preferably a bispecific or multispecific protein, may have two of such heavy chains, in which case it is preferred that the two have opposite charge differences compared to the wild type heavy chain.

[0121] The multimerizing protein, preferably a bispecific or multispecific protein, is preferably a multispecific antibody, preferably a bispecific antibody.

[0122] Various approaches have been reported in the art to promote the formation of targeted multispecific proteins, such as bispecific antibodies, thereby reducing the content of monospecific bivalent (parent) proteins. In antibodies, CH3CH3 interactions drive Fc dimerization. Amino acid mutations in the CH3 region at the interface between the two CH3 regions can be introduced to promote bispecific formation and / or inhibit monospecific parent formation (e.g., by introducing compatible / repulsive charges or steric (in)compatibility). Such approaches can be advantageously combined with the CH1 region mutations described herein.

[0123] Thus, in a further embodiment, there is provided an immunoglobulin protein according to the invention, wherein the first and second CH1-containing immunoglobulin polypeptides comprise a CH3 region, and one of the first and second CH1-containing immunoglobulin polypeptides comprises the CH3 mutations L351D and L368E (in EU numbering), and the other comprises the CH3 mutations T366K and L351K (also referred to as "DEKK").

[0124] These so-called DEKK mutations are understood to be consistent with the addition of a charge to the first and / or second CH1-containing immunoglobulin polypeptide. This means that if a CH1-containing immunoglobulin polypeptide contains a mutation that adds a negative charge, that chain preferably has CH3 residues L351D and L368E, and the other chain, which need not (but can) be a variant CH1 region, has CH3 residues T366K and L351K. Conversely, this means that if a CH1-containing immunoglobulin polypeptide contains a mutation that adds a positive charge, that chain preferably has CH3 mutations T366K and L351K, and the other chain, which need not be a variant CH1 region, has CH3 residues L351D and L368E. Preferably, the immunoglobulin protein according to the invention comprises a human immunoglobulin Fc region, and most preferably, the human immunoglobulin Fc region is an IgG1 Fc region. As described above, while other techniques for altering the charge of CH3 may be used to form heterodimers, preferred embodiments of the multimerizing proteins of the present invention include negatively charged separation domain-containing immunoglobulin polypeptides that further comprise a multimerization domain, such as a negatively charged CH3, to facilitate heterodimerization and separation of the multimerizing proteins, and positively charged separation domain-containing immunoglobulin polypeptides that further comprise a multimerization domain, such as a positively charged CH3.

[0125] The terms "CH1 region," "CH2 region," and "CH3 region" are known in the art. The IgG structure has four chains, two light chains and two heavy chains. Each light chain typically has two domains, a variable light chain and a constant light chain (VL and CL), and each heavy chain typically has four domains, a variable heavy chain (VH) and three constant heavy chain domains (CH1, CH2, and CH3). The CH2 and CH3 regions of the heavy chains are referred to as the Fc (fragment crystallizable) portion, Fc fragment, Fc main chain, or simply Fc. An IgG molecule is a heterotetramer with two heavy chains held together by disulfide bonds (-SS-) between the hinge region and CH1 and CL. Heavy chain dimerization involves interactions within the interface of the CH3CH3 domains and through interactions at the hinge region. Examples of suitable amino acid sequences for CH2, CH3, and hinge regions are shown in Figure 14.

[0126] In one embodiment, the immunoglobulin proteins according to the invention described herein comprise a first CH1-containing polypeptide that is an antibody heavy chain. In one embodiment, the immunoglobulin proteins according to the invention described herein comprise a second CH1-containing polypeptide that is an antibody heavy chain. In a further preferred embodiment, both the first and second CH1-containing polypeptides are antibody heavy chains, such as human IgG1 heavy chains. The immunoglobulin proteins according to the invention can further comprise one or more antibody light chains. Most preferably, the antibody light chain is a common light chain.

[0127] Thus, as used herein, the term "common light chain" refers to a light chain that may be identical or may have some amino acid sequence differences while retaining the binding specificity of the resulting antibody after pairing with a heavy chain. For example, by introducing and testing conservative amino acid changes and / or amino acid changes in regions that do not contribute, or only partially contribute, to binding specificity when paired with a heavy chain, light chains that are not identical in amino acid sequence but are still functionally equivalent can be prepared or found. The combination of a specific common light chain and such functionally equivalent variants is encompassed by the term "common light chain." For a detailed description of the use of common light chains, see International Publication Nos. 2004 / 009618 and 2009 / 157771. Preferably, the present invention uses a consensus light chain that is a germline-like light chain, more preferably a germline light chain, preferably a rearranged germline human κ light chain, most preferably a rearranged germline human κ light chain, either IgVκ1-39 / Jκ or IgVκ3-20 / Jκ. Other light chains encompassed by the invention disclosed herein include IgKV3-15 / JK1 and surrogate light chains, which are also known in the art to constitute consensus light chains.

[0128] As an alternative to the use of a common light chain, means for forced pairing of heavy and light chains can be considered to avoid mispairing of mismatched heavy and light chains, such as those described in WO 2009 / 080251, WO 2009 / 080252, and / or WO 2009 / 080253. Examples of common light chain variable regions, amino acid sequences of common light chains, and / or CDR sequences of common light chains are shown in Figure 13. A preferred common light chain has the sequence shown in Figure 13a.

[0129] Because the variant residues in the CH1 region are selected from among amino acids in the CH1 region that are not surface-exposed, or preferably buried, as described above, these mutations enable the bispecific protein to most closely resemble the tertiary structure of a human antibody, making such mutations particularly suitable for human bispecific proteins. It is understood that the term "human" in describing a protein does not imply that the entire amino acid sequence of the first and second CH1-containing polypeptides is of human origin, nor that the amino acid sequences are required to be obtained directly from a human. Reference to a human domain, protein, or antibody is understood to refer to a protein that may include some amino acid sequence modifications, such as CH2 engineering (including Fc silencing), CH3 engineering (including heterodimerization), and / or Fc engineering (including affecting Fc receptor activity), and the inclusion of separating residues. The human domains used to generate bispecific or multispecific proteins may be encoded by nucleic acid sequences obtained from mice bearing features of the human immune system, such as heavy chain, light chain, or hybrid loci encoding human variable region gene segments and / or constant regions, as known in the art. WO 2009 / 157771. Such proteins can also be obtained by identifying nucleic acids encoding human immunoglobulin domains identified from phage display, yeast display, and other techniques known to those skilled in the art.

[0130] The multimerizing proteins according to the present invention may be bispecific or multispecific proteins, preferably antibodies, which may also be non-naturally occurring but human sequences, such as for example the sequences of two heavy chains and two (common) light chains binding to a human bispecific antibody, which may have slight variations in terms of amino acid sequence as described herein, including for example mutations in the CH1 region and / or preferably CH3 engineering.

[0131] In one embodiment, the immunoglobulin protein according to the present invention, further comprising a light chain, preferably comprises one or more mutations of one or more amino acids in the CH1 region that are not surface-exposed and are located away from the CH1 / CL interface. In this way, any potential effects on the function of the antigen-binding domain, including the pairing of the heavy and light chains, can be avoided. Preferably, the bispecific protein according to the present invention is a bispecific antibody. More preferably, the bispecific antibody is a human bispecific antibody. Most preferably, the bispecific antibody of a human bispecific antibody is an IgG1 antibody.

[0132] In one embodiment, a nucleic acid is provided encoding an isolation domain of the invention, such as an immunoglobulin CH1-containing polypeptide comprising one or more mutations selected from amino acids in a CH1 region that are not surface-exposed. Yet another embodiment of the invention described herein is a cell or recombinant host cell comprising a nucleic acid encoding an isolation domain of the invention. Yet another embodiment provides a cell or recombinant host cell comprising one or more nucleic acids encoding first and second CH1-containing immunoglobulin polypeptides according to the invention. Such isolated nucleic acids, cells, and recombinant host cells are particularly suitable for producing immunoglobulin proteins according to the invention disclosed herein, and for methods of isolating such immunoglobulin proteins.

[0133] Also provided is a host animal or transgenic animal according to the invention comprising a nucleic acid encoding a mutant isolation domain disclosed herein. In one embodiment, the host animal or transgenic animal encodes an immunoglobulin region comprising one or more isolation residues corresponding to non-surface-exposed amino acid residues in a wild-type immunoglobulin domain according to the invention. Preferably, such a transgenic animal is a rodent or avian, more preferably a mouse, rat, or chicken, wherein at least a portion of the antibody repertoire of the mouse, rat, or chicken is a human or humanized antibody.

[0134] Thus, in one embodiment, a composition is provided comprising an immunoglobulin protein according to the present invention as described herein. It is understood that such a composition may be an intermediate product, such as, for example, a crude cell lysate and / or a filtered crude lysate or a semi-purified product. Further processing of such a composition may include, for example, a separation step that allows obtaining a bispecific protein with a CH1 region mutation according to the present invention. That is, a pharmaceutical composition comprising a bispecific protein according to the present invention and a pharmaceutically acceptable excipient can be obtained. Such a product may be in the form of a liquid or a lyophilized product. Any pharmaceutically acceptable composition can be used. It is understood that such a pharmaceutically acceptable composition does not necessarily need to be administered directly to a patient; for example, a further preparation step may be performed, in which the pharmaceutical product is dissolved or mixed in an appropriate solution for injection into a patient. The above also applies to compositions comprising trispecific proteins and other multispecific proteins that comprise a separation domain other than the CH1 region, as further described throughout.

[0135] Further embodiments described below relate to methods for producing immunoglobulin proteins according to the invention.

[0136] In one embodiment there is provided a method for producing a mutant bispecific protein according to the invention, the method comprising the steps of: a) providing a nucleic acid encoding a first CH-containing immunoglobulin polypeptide and a nucleic acid encoding a second CH-containing immunoglobulin polypeptide, wherein the first and second CH-containing immunoglobulin polypeptides encode a bispecific protein; b) said nucleic acid encoding the first CH-containing immunoglobulin polypeptide comprises one or more mutations in triplets encoding one or more amino acids in the CH region that are not surface exposed, such that the isoelectric point of a variant bispecific protein comprising the first CH-containing immunoglobulin polypeptide and the second CH-containing immunoglobulin polypeptide is different from the isoelectric point of a monospecific protein comprising only the first CH-containing immunoglobulin polypeptide or the isoelectric point of a monospecific protein comprising only the second CH-containing immunoglobulin polypeptide; c) providing a cell comprising a nucleic acid encoding the first CH-containing immunoglobulin polypeptide and a nucleic acid encoding the second CH-containing immunoglobulin polypeptide, and producing the variant bispecific protein.

[0137] As already mentioned, it is understood that in steps a) and b) described above and below, any mutations can be performed in silico. Thus, the first and second CH-containing immunoglobulin polypeptides can be completely altered in silico, compared to a reference sequence. It is understood that such mutations can also include simply providing the (mutant) nucleic acid sequences and ligating them to, for example, suitable variable domains. Any construction method, including standard molecular techniques, DNA synthesis, and / or in silico design, can be used according to the present invention and can be used in steps a) and d) described above and below. It is also understood that providing nucleic acids to cells can include any suitable method, such as transient and stable transfection. It is also understood that the step of providing nucleic acids to cells in step c) can also include providing only a portion of the nucleic acids, as long as the end result is that a nucleic acid encoding a first CH-containing immunoglobulin polypeptide and a nucleic acid encoding a second CH-containing immunoglobulin polypeptide are provided to the cell and the cell is capable of producing the mutant bispecific protein. In another embodiment, a method for producing a mutant bispecific protein according to the present invention is provided, the method comprising: a) providing a nucleic acid encoding a first CH-containing immunoglobulin polypeptide and a nucleic acid encoding a second CH-containing immunoglobulin polypeptide, wherein the first and second CH-containing immunoglobulin polypeptides encode a bispecific protein; b) the nucleic acid encoding the first CH-containing immunoglobulin polypeptide and the nucleic acid encoding the second CH-containing immunoglobulin polypeptide comprise one or more mutations in triplets encoding one or more amino acids in the CH region that are not surface exposed, such that the isoelectric point of the mutant bispecific protein comprising the first CH-containing immunoglobulin polypeptide and the second CH-containing immunoglobulin polypeptide is different from the isoelectric point of the parent protein containing only the first CH-containing immunoglobulin polypeptide or the isoelectric point of the parent protein containing only the second CH-containing immunoglobulin polypeptide; c) providing a cell harboring nucleic acid encoding the first and second CH-containing immunoglobulin polypeptides and producing the mutant immunoglobulin bispecific protein.

[0138] Preferably, the method according to the invention comprises one or more of said mutations: From neutral amino acids to negatively charged amino acids, Positively charged amino acids to neutral amino acids, Positively charged amino acids to negatively charged amino acids, From neutral amino acids to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and The amino acid sequence is selected from the group consisting of changing a negatively charged amino acid to a positively charged amino acid.

[0139] It will be appreciated that preferably, one of the CH-containing immunoglobulin polypeptides has an added positive charge or an added negative charge, and both CH-containing immunoglobulin polypeptides may have added charges, preferably one CH-containing immunoglobulin polypeptide has an added negative charge and the other CH-containing immunoglobulin polypeptide has an added positive charge.

[0140] Thus, in a further embodiment there is provided a method for producing a bispecific protein according to the invention comprising a first CH-containing immunoglobulin polypeptide and a second CH-containing immunoglobulin polypeptide, the method comprising: a) providing a nucleic acid encoding a first CH-containing immunoglobulin polypeptide and a nucleic acid encoding a second CH-containing immunoglobulin polypeptide, The first and / or second CH-containing immunoglobulin polypeptide comprises one or more mutations of one or more amino acids selected from amino acids in the CH region that are not surface exposed, and the first CH-containing immunoglobulin polypeptide comprises: Neutral to negatively charged amino acids, Positively charged to neutral amino acids, and a mutation selected from a mutation of a positively charged amino acid to a negatively charged amino acid; the second CH-containing immunoglobulin polypeptide is Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and a mutation selected from a negatively charged amino acid to a positively charged amino acid; b) providing a cell harboring nucleic acid encoding the first and second CH-containing immunoglobulin polypeptides and producing the bispecific protein.

[0141] It is understood that in step a), any mutation step may be performed in silico. Thus, the first and second CH-containing immunoglobulin polypeptides can be completely altered in silico, compared to a reference sequence. It is understood that the mutation may also include simply providing the nucleic acid sequences and ligating them with, for example, suitable variable domains. Any construction method, including standard molecular techniques, DNA synthesis, and / or in silico design, can be used according to the present invention and can be used in step a). It is also understood that providing to cells can include any suitable method, such as transient and stable transfection. It is also understood that providing nucleic acids to cells in step b) may also include providing only a portion thereof, as long as the end result is that a nucleic acid encoding a first CH-containing immunoglobulin polypeptide and a nucleic acid encoding a second CH-containing immunoglobulin polypeptide are provided to the cell and the cell is capable of producing the mutant bispecific protein.

[0142] In a further embodiment, the step of altering the amino acid sequence of the CH-containing immunoglobulin polypeptide further comprises introducing stabilizing modifications at additional amino acid positions corresponding to one or more amino acids within the CH region.

[0143] The above also applies to methods for producing trispecific and other multispecific proteins, nucleic acids encoding such proteins, including separation domains other than the CH region with mutations in non-surface-exposed residues.

[0144] The present invention provides a cell comprising a nucleic acid encoding at least a first and a second CH domain comprising a polypeptide chain according to the present invention. The cell according to the present invention may further comprise a nucleic acid encoding a light chain, preferably a common light chain. Any cell can be used to produce the immunoglobulin proteins according to the present invention, including bacteria such as Escherichia coli, Enterobacter, Salmonella, Bacillus, Pseudomonas, and Streptomyces; yeasts such as Saccharomyces cerevisiae, K. lactis, P. pastoris, Candida, and Yarrowia; filamentous fungi such as Neurospora, Aspergillus oryzae, Aspergillus nidulans, and Aspergillus niger; insect cells such as Fallopia oryzae SF-9 or SF-21 cells; mammalian cells, preferably Chinese hamster ovary (CHO) cells; murine cells, including BHK cells, SP2 / 0 cells, and NS-0 myeloma cells; COS and Vero cells; MDCK cells; BRL cells; and the like. Any cell capable of expressing a recombinant DNA molecule, including primate cells such as 3A cells, hybridomas, tumor cells, immortalized primary cells, human cells such as W138, HepG2, HeLa, HEK293, and HT1080, or embryonic retinal cells such as PER.C6, may be used.

[0145] In many cases, the expression system of choice involves a mammalian cell expression vector and host to ensure that the protein is appropriately glycosylated. Human cell lines can be used to obtain bispecific antibodies with a fully human glycosylation pattern. Generally, principles, protocols, and practical techniques for maximizing the productivity of mammalian cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach (M. Butler, ed., IRL Press, 1991). The expression of antibodies in cells and recombinant host cells has been widely described in the art. Thus, nucleic acids encoding proteins of the present invention contain all elements that allow expression of the components of a bispecific protein (e.g., two heavy and light chains), such as promoter sequences, 5' / 3' UTRs, and intron sequences. Nucleic acids encoding proteins of the present invention may exist as extrachromosomal (stably) transfected copies and / or may be stably integrated into the host cell chromosome. The latter is preferred.

[0146] The immunoglobulin polypeptides of the present invention are expressed in host cells and harvested from the cells, or preferably from the cell culture medium, by methods generally known to those skilled in the art. After harvesting, immunoglobulin proteins containing first and second CH-containing immunoglobulin peptides (or the like) can be purified using conventional methods known in the art. Such methods include precipitation, centrifugation, filtration, size-exclusion chromatography, and affinity chromatography. For antibody mixtures containing IgG polypeptides, Protein A or Protein G affinity chromatography can be suitably used (see, e.g., U.S. Pat. Nos. 4,801,687 and 5,151,504). After capture using affinity chromatography, orthogonal polishing steps with appropriate process parameters can be used to remove remaining process-related impurities, such as HCPs and DNA. Generally, to obtain purified bispecific antibodies or multivalent multimers, several steps are performed, including host cell culture, harvest clarification, followed by protein capture to remove host cell DNA, anion exchange chromatography, CIEX to remove host cell proteins (HCPs), leached Protein A, and potential aggregates, followed by further steps such as viral filtration. Those skilled in the art will appreciate that the order of these steps may be modified or individual steps may be substituted. For example, alternative polishing steps include hydrophobic interaction chromatography and mixed-mode chromatography.

[0147] In addition to the above treatments, the method for processing such bispecific proteins may further include a separation step in which the produced bispecific protein is separated from the produced monospecific protein (or the multispecific protein is separated from other produced proteins) based on the difference in isoelectric point between the produced bispecific protein and the produced monospecific protein. Any suitable separation step can be used. A suitable separation step selected may be isoelectric focusing. Alternatively, or in addition, the method including a separation step in which the produced bispecific protein is separated from the produced parent protein includes ion exchange or hydrophobic interaction. As shown in the Examples section, mutation of non-surface-exposed, preferably buried, amino acids in the CH region can enable charge differentiation and provide differentiation in isoelectric point and / or chromatographic properties between the bispecific protein and the parent protein. Such differentiation allows the separation of these proteins using conventional chromatography, including ion exchange and hydrophobic interaction. Preferred methods are industrially applicable separation methods for processing biological pharmaceutical products such as antibodies. Alternative separation methods are included within the scope of the present invention that utilize charge and / or isoelectric point (pI) differences created by the use of separation domains and mutations, including, for example, capillary zone, capillary isotachophoresis, and capillary isoelectric focusing, techniques known to those skilled in the art.

[0148] Again, mutation of a separation domain, such as a CH region, as described herein, can itself enable sufficient separation of the parent protein from the bispecific protein in the methods described herein; for example, mutating the CH3 region contained in a CH-containing immunoglobulin polypeptide can facilitate the formation of the bispecific protein during production in cells. Thus, a further method according to the present invention is disclosed, in which the first CH-containing immunoglobulin polypeptide and the second CH-containing immunoglobulin polypeptide comprise a CH3 region, the CH3 region comprising a CH3 mutation that enhances pairing between the first and second CH-containing immunoglobulin polypeptides. Preferably, one of the first and second CH-containing immunoglobulin polypeptides comprises CH3 mutations L351D and L368E, and the other comprises CH3 mutations T366K and L351K. The DEKK residues are located at the interface between the two interacting domains, promoting heterodimerization of the DE and KK chains, while two KK-modified CH3 domains repel each other. As noted above, it is understood that DEKK mutations are preferably selected to align (i.e., add positive or negative charges to both the CH3 and CH regions within the same polypeptide.) Other forms of heterodimerization techniques are known in the art and can be used with the mutations described herein, for example, using knobs-into-holes technology or electrostatic engineering approaches.

[0149] In the above methods according to the invention for generating a bispecific protein, the mutations in the CH region are preferably those as defined throughout this specification as being suitable for a bispecific protein, and preferably the bispecific protein can be selected to comprise further features as also described throughout this specification.

[0150] Preferably, the protein produced by the method of the present invention is a bispecific antibody, more preferably a human bispecific antibody, and most preferably human IgG1. In the bispecific protein produced by the method of the present invention, the CH region of the immunoglobulin polypeptide is selected to be a CH region derived from human IgG1, and the amino acids within the CH region have a charge difference from the human wild-type CH region at positions selected from the group consisting of T120, K147, D148, N159, Q175, N201, K213, V303, K370, E382, and E388, since these amino acid positions allow alternative residues at these positions with different charges (variations between neutral, positively charged, and negatively charged amino acids), as exemplified in the Examples section. Most preferred are the amino acids N159 and N201, which represent buried amino acids. Most preferred are the amino acids V303, E382, and E388, which represent buried amino acids. Most preferably, the first and second CH-containing immunoglobulin polypeptides provide different antigen-binding domains, ie, represent heavy chains that differ primarily in their heavy chain variable regions.

[0151] In another embodiment, the bispecific or multispecific protein produced by the present invention comprises a first CH-containing immunoglobulin polypeptide and a second CH-containing immunoglobulin polypeptide, wherein the CH region is a CH region of human IgG1, and the first or second CH-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from amino acids within the CH region, the mutations comprising one or more mutations selected from the group consisting of K147E, N159D, Q175E, N201D, K213Q, V303E, K370S, K370T, or one or more mutations selected from the group consisting of T120K, D148K, N159K, Q175K, N201K, V303K, E382Q, E382T, E388L, E388M, E388T. Preferably, the isolated immunoglobulin protein is a bispecific or multispecific antibody.

[0152] In a further embodiment, a bispecific or multispecific protein is produced by a method according to the invention, comprising a first CH-containing immunoglobulin polypeptide and a second CH-containing immunoglobulin polypeptide, wherein the CH region is a CH region of human IgG1, and wherein the first CH-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from the amino acids in the CH region, the mutations comprising one or more mutations selected from the group consisting of K147E, N159D, Q175E, N201D, K213Q, V303E, K370S, K370T, and the second CH-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from the group consisting of T120K, D148K, N159K, Q175K, N201K, V303K, E382Q, E382T, E388L, E388M, E388T. Most preferably, the immunoglobulin protein produced is a bispecific or multispecific antibody.

[0153] In another embodiment, a bispecific or multispecific protein is produced by the method according to the invention, comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the CH1 region is the CH1 region of human IgG1, and the first or second CH1-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from amino acids in the CH1 region and an amino acid mutation at hinge residue E216K, wherein the former mutations are selected from the group consisting of K147E and Q175E, N201D and K213Q, T197D and K213Q, N159D and K213Q, and K213Q, or wherein the former mutations are selected from the group consisting of T120K, N201K, D148K and Q175K, and N159K. Most preferably, the bispecific or multispecific protein produced is a bispecific or multispecific human antibody.

[0154] In yet a further embodiment, a bispecific or multispecific protein is produced by the method according to the invention, comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the CH1 region is the CH1 region of human IgG1, and wherein the first CH1-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from the amino acids in the CH1 region, the mutations being selected from the group consisting of K147E and Q175E, N201D and K213Q, T197D and K213Q, N159D and K213Q, and K213Q, and the second CH1-containing immunoglobulin polypeptide comprises one or more mutations of amino acids selected from the amino acids in the CH1 region and an amino acid mutation at hinge residue E216K, the former mutation being selected from the group consisting of T120K, N201K, D148K and Q175K, and N159K. Most preferably, the produced protein is a bispecific or multispecific human antibody.

[0155] A CH region containing a mutation from a neutral amino acid to a negatively charged amino acid, a positively charged amino acid to a neutral amino acid, and / or a positively charged amino acid to a negatively charged amino acid is said to have a negative charge difference relative to the original CH region, preferably compared to a human wild-type CH region. This mutation provides a negative charge difference to the CH region at the relevant pH. A CH region containing a mutation from a neutral amino acid to a positively charged amino acid, a negatively charged amino acid to a neutral amino acid, and / or a negatively charged amino acid to a positively charged amino acid is said to have a positive charge difference relative to the original CH region, preferably compared to a human wild-type CH region. As described herein, when a CH region contains two mutations of amino acid residues, it is preferred that both mutations provide a charge difference in the same direction to the CH region. As described herein, when a CH region contains three or more mutations of amino acid residues, it is preferred that the net result of the mutations provides a charge difference to the CH region. The immunoglobulin region is preferably a human immunoglobulin region. In some embodiments, the immunoglobulin region is an IgG region, preferably an IgG1 region. The immunoglobulin regions disclosed above can be advantageously used as portions of antibodies that need to be separated from antibody mixtures.

[0156] The present invention further discloses antibodies comprising heavy and light chains comprising the CH regions of the immunoglobulins described herein. For example, if such antibodies are produced as part of a mixture, alterations in the charge provided in the CH regions can facilitate separation of the antibodies from the mixture. In preferred embodiments, the antibodies comprise different heavy chains. In preferred embodiments, the antibodies are multispecific antibodies, such as bispecific or trispecific antibodies. In this case, alterations in the charge provided in the CH regions can facilitate separation of the bispecific or trispecific antibodies from the mixture. The different heavy chains preferably comprise interchangeable heterodimerization regions, preferably interchangeable heterodimerization CH3 regions. In one embodiment, one of the heavy chains comprises CH3 mutations L351D and L368E, and the other heavy chain comprises CH3 mutations T366K and L351K. The antibody is preferably an IgG antibody, preferably an IgG1 antibody. In some embodiments, the antibody comprises CH regions of two or more immunoglobulins described herein. Preferably, a heavy chain containing CH3 mutations L351D and L368E contains one CH region described herein, and a heavy chain containing CH3 mutations T366K and L351K contains another CH region described herein. In such cases, it is preferable that one and the other CH region contain CH regions with different charges. In such cases, the difference in isoelectric points of the antibodies obtained in the mixture is further increased, thereby facilitating separation of the antibodies from the mixture. In other words, if one CH region has a negative charge difference compared to the original CH region, the other CH region preferably has a positive charge difference compared to the original CH region. Similarly, if one CH region has a positive charge difference compared to the original CH region, the other CH region preferably has a negative charge difference compared to the original CH region. The CH3 mutations L351D and L368E and the CH3 mutations T366K and L351K preferably match the charge differences of the CH mutations. The mutations L351D and L368E are preferably present in a heavy chain comprising an original CH region or a CH region that has a negative charge difference relative to a comparison residue in the original or native CH region.The mutations T366K and L351K are preferably present in a heavy chain comprising a CH region that has a positive charge difference relative to the original CH region or a comparable residue in the original or native CH region. For example, a polypeptide comprising CH3 mutations L351D and L368E can be combined with one or more of the following mutations that increase the negative charge of the polypeptides described herein: K147E, N159D, Q175E, N201D, K213Q, V303E, K370S, K370T, or other mutations. Similarly, a polypeptide comprising CH3 mutations T366K and L351K can be combined with one or more of the following mutations that increase the positive charge of the polypeptides described herein: T120K, D148K, N159K, Q175K, N201K, V303K, E382Q, E382T, E388L, E388M, E388T, or other mutations.

[0157] Antibodies containing compatible heterodimerization regions, such as the CH3 heterodimerization region described herein, that are compatible with such CH1 regions typically separate better from respective antibodies and / or half-antibodies, if present, that have identical heavy chains, in a separation process that utilizes the charge and / or isoelectric point (pI) of the antibody or fragment thereof. The antibody preferably contains one or more light chains, which preferably contain the same light chain. The light chain is preferably a common antibody light chain described herein. The common light chain preferably contains a light chain variable region as shown in Figure 13B or Figure 13D. In one embodiment, the light chain has a light chain constant region as shown in Figure 13C. In a preferred embodiment, the light chain comprises the light chain amino acid sequence shown in Figure 13A or 13E. The common light chain is preferably a light chain with CDRs as shown in Figure 13F. The antibody or CH region is preferably a CH region of a human antibody or human immunoglobulin, and the human CH region contains mutations at non-surface-exposed, preferably buried, amino acid positions in the wild-type human CH region.

[0158] Immunoglobulin regions, preferably CH1 regions or antibodies comprising the non-surface exposed, preferably buried amino acid mutations described herein, have mutations selected from amino acids not present at the CH1 / CL interface. Position Q175 is exceptionally effective and stable despite being at the CH1 / CL interface.

[0159] Immunoglobulin regions, preferably CH3 regions or antibodies containing non-surface-exposed, preferably buried amino acid mutations described herein, have mutations selected from amino acids not present at the CH3 interface. Position K370 is an exception; it is present at the CH3 / CH3 interface (see Figure 22). Nevertheless, it is a good location to introduce mutations such as those described herein, even if they do not compensate for mutations in the opposite CH3 chain, such as those present in DEKK.

[0160] Immunoglobulin regions, preferably CH1, CH2, or CH3 regions or antibodies, comprising the non-surface-exposed amino acid mutations described herein preferably do not substantially adversely affect the stability of the resulting CH1 region or antibody, including at any heavy and light chain interfaces. Immunoglobulin regions, preferably CH1, CH2, or CH3 regions or antibodies comprising the non-surface-exposed amino acid mutations described herein may comprise additional mutations that enhance the stability of the charge differential-generating mutations. Immunoglobulin regions, preferably CH1 regions or antibodies comprising the non-surface-exposed amino acid mutations described herein may comprise additional charge differential-generating mutations.

[0161] The invention further provides a method for producing any one of the above antibodies, the method comprising: providing a nucleic acid encoding a first heavy chain comprising a CH region as described herein; providing a nucleic acid encoding a second heavy chain, wherein the first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and and collecting the antibody from the host cell culture, the method further comprising separating the antibody from other antibodies or antibody fragments based on the charge of the antibody and / or antibody fragment in a separation step. In one embodiment, the first and second heavy chains comprise interchangeable heterodimerization regions, preferably interchangeable CH3 heterodimerization regions.

[0162] The invention further provides a method for producing any one of the above antibodies, the method comprising: providing a nucleic acid encoding a first heavy chain comprising a CH region as described herein; providing a nucleic acid encoding a second heavy chain, wherein the first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and The method comprises harvesting the antibody from the host cell culture, clarifying the harvest; capturing the protein; performing anion exchange chromatography; and The method further comprises performing cation exchange chromatography to separate the antibody from other antibodies or antibody fragments. In one embodiment, the first and second heavy chains comprise interchangeable heterodimerization regions, preferably interchangeable CH3 heterodimerization regions.

[0163] The invention further provides a method for producing any one of the above antibodies, the method comprising: providing a nucleic acid encoding a first heavy chain comprising a CH region as described herein; providing a nucleic acid encoding a second heavy chain, wherein the first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and The method includes the step of harvesting the antibody from the host cell culture, and the method further includes the step of separating the antibody from other antibodies or antibody fragments in a separation step comprising isoelectric electrophoresis on a gel.

[0164] Further provided is a method for producing a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: (a) expressing a nucleic acid encoding a first heavy chain and a nucleic acid encoding a second heavy chain such that the isoelectric point of the encoded first heavy chain and the isoelectric point of the encoded second heavy chain are different, wherein the nucleic acids encode one or more mutations at amino acid positions selected from non-surface exposed positions in the first and / or second heavy chain, preferably the CH1 region, CH2, CH3, more preferably T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, K213, V303, K370, E382 and E388 (EU numbering in the CH regions); (b) culturing the host cell to express the nucleic acid; and (c) harvesting the multispecific antibodies from the host cell culture using the difference in isoelectric points.

[0165] Also provided is a method for separating a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: (a) expressing both or either one of a nucleic acid encoding amino acid residues of a first heavy chain and a nucleic acid encoding amino acid residues of a second heavy chain such that the isoelectric points of the encoded first heavy chain and the encoded second heavy chain are different, wherein the positions of the nucleic acids are non-surface-exposed residues at positions different from those in the encoded CH region, and preferably the mutations are at one or more amino acids selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, K213, V303, K370, E382 and E388 (EU numbering within the CH region); (b) culturing the host cell to express the nucleic acid; and (c) separating the multispecific antibodies from the host cell culture by chromatography.

[0166] In a preferred embodiment, the nucleic acid encodes the first heavy chain and the second heavy chain such that the retention times of the first heavy chain, the homomultimer of the first heavy chain, the second heavy chain, the homomultimer of the second heavy chain, and the heteromultimer of the first and second heavy chains are different when expressed and separated by an ion exchange chromatography step.

[0167] The variant amino acid at the position encoded by the nucleic acid is preferably selected from amino acids that are not surface-exposed in the human wild-type CH region, and Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and The amino acids are selected from negatively charged amino acids to positively charged amino acids.

[0168] Also provided is a method for producing a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: providing a nucleic acid encoding a CH region of a first heavy chain and a nucleic acid encoding a CH region of a second heavy chain, such that the isoelectric points of the first encoded heavy chain and the second encoded heavy chain differ, wherein at least one of the CH regions comprises an amino acid mutation in the CH region at a position selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, K213, V303, K370, E382, and E388 (EU numbering); Culturing the host cells to express the nucleic acid; and harvesting the multispecific antibodies from the host cell culture using differences in isoelectric points; harvesting the antibody from the host cell culture; Clarifying the harvest; capturing the protein; performing anion exchange chromatography; and The method further comprises the step of performing cation exchange chromatography to separate the antibody from other antibodies or antibody fragments.

[0169] Further provided is a method for purifying a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: providing a nucleic acid encoding a CH region of a first heavy chain and / or a nucleic acid encoding a CH region of a second heavy chain, such that the isoelectric points of the first encoded heavy chain and the second encoded heavy chain are different, wherein at least one of the CH regions comprises an amino acid mutation at a position selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, K213, V303, K370, E382, and E388 3 (EU numbering of the CH region); Culturing the host cells to express the nucleic acid; and The method comprises purifying the multispecific antibodies from the host cell culture by isoelectric focusing to separate the multispecific antibodies from other antibodies or antibody fragments.

[0170] The one or more nucleic acids encoding the first heavy chain homomultimer, the second heavy chain homomultimer, and the first and second heavy chain heteromultimer are expressed as proteins with different isoelectric points, resulting in different retention times in ion exchange chromatography.

[0171] The present invention further provides a method for producing or purifying an antibody, such as a multispecific antibody, described herein, further comprising determining the difference in charge or pI of the heavy chains relative to one another and selecting the heavy chain with the more negative charge / pI as the first heavy chain and the heavy chain with the more positive charge / pI as the second heavy chain. This embodiment further facilitates the separation of multispecific antibodies from homodimers and half antibodies in charge-based separation methods such as CIEX. As described herein above, the first heavy chain comprises one or more CH1, CH2, or CH3 regions described herein that provide an additional negative charge to the heavy chain. Similarly, as described herein above, the second heavy chain comprises one or more CH1, CH2, or CH3 regions described herein that provide an additional positive charge to the heavy chain. In addition, and as mentioned herein above, the first heavy chain preferably comprises a DE mutation in the CH3 heterodimerization domain, while the second heavy chain preferably comprises a KK mutation in the CH3 heterodimerization domain.

[0172] In these embodiments, the natural charge differences between the heavy chains, the amino acid mutations in the CH1, CH2 and / or CH3 regions described herein, and optionally the charge differences introduced by the DEKK CH3 heterodimerization domain described herein all work together to improve charge separation of antibodies, such as the bispecific and multispecific antibodies described herein.

[0173] The difference in the relative charge between two heavy chains can be attributed to the difference in the amino acid sequence of the variable domain. For example, when the same light chain is used for both heavy chain variable regions, the difference in the amino acid sequence of the heavy chain variable region can be attributed to the difference in the charge or pI of the variable domain or heavy chain variable region. In such cases, it is often sufficient to determine the difference in charge or pI between the variable domain or heavy chain variable region, since they may be relative to each other.

[0174] Charge or pI differences in the variable domains can be used to improve production and / or purification, as described above. In some embodiments, the variable domains are significantly different heavy chains and identical light chains. Examples of light chains that can be used in this manner are described elsewhere herein, and some are listed, for example, in Figure 13. Heavy chain variable regions that can be used in this manner are typically selected to pair well with a selected light chain. A heavy chain variable region selected to pair well with the light chain of Figure 13a is described in the Examples. Other examples of such heavy chain variable regions are described in WO 2015 / 130172, International Application PCT / NL2020 / 050081, WO 2019 / 031965, WO 2019 / 009726, WO 2019 / 009728, and WO 2019 / 009727, which are incorporated herein by reference for this purpose. The heavy chain variable regions described herein and in the above references should be considered as preferred examples of heavy chains, and not as an exclusive list. The present invention can be applied to a wide variety of variable domains and / or heavy and light chain combinations. Some examples of such variable domain and / or heavy chain combinations are shown in Figures 1 and 2 and the accompanying description. Other examples of heavy and light chain combinations are described, for example, in International Publication No. WO2019190327, which is incorporated by reference for that purpose. [Brief explanation of the drawings]

[0175] [Figure 1]Schematic diagrams of bispecific and monospecific antibodies with separating domains of the present invention are shown. It should be noted that other features and aspects of the present invention will become apparent from the detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, features according to embodiments of the present invention. Each figure is illustrative and is not intended to, and does not, limit the scope of the present invention provided, as defined by the full scope of the claims and the detailed disclosure that describes and enables the invention described herein. In Figures 1A-C, the first CH1-containing immunoglobulin is shown in black, representing the first heavy chain; the second CH1-containing immunoglobulin is shown in gray, representing the second heavy chain; and, in a common light chain scenario, the light chain is shown in white. In these figures, the first heavy chain contains a separating CH1 region (Figure 1A), the second heavy chain contains a separating CH1 region (Figure 1B), and both the first and second heavy chains contain separating CH1 regions with distinct charges (Figure 1C). Again, it is understood that the present invention does not require the use of a common light chain, as shown by way of example of an embodiment of the present invention. In Figure 1D, the first CH2-containing immunoglobulin is shown in black to represent the first heavy chain, the second CH2-containing immunoglobulin is shown in gray to represent the second heavy chain, and the light chain is shown in white, with the second heavy chain containing a separating CH2 domain. In Figure 1E, a single heavy chain is used, shown in black, and two different light chains are shown in gray and white. Mutations are indicated with either + or - to indicate the relative change in charge compared to the unmodified or reference domain and to indicate the incorporation of the + and - labels, respectively, for the unmodified or reference antibody. In Figures 1A-C), the CH1 region contains the separating residues of the invention described herein, while in 1D, the CH2 region and in 1E, the CL region of the light chain, are the mutant light chains described according to the invention. A) In this scenario, the first heavy chain is provided with a positive charge (indicated by + in the CH1 region), resulting in two monospecific antibodies with either a ++ or neutral charge, while the bispecific antibody has a + charge. The charges shown represent the change in charge compared to an antibody lacking the separating domain. B) In this scenario, the second heavy chain is provided with two negative charges (indicated by -- in the CH1 region), resulting in two monospecific antibodies with either a -- or neutral charge, and the bispecific antibody with a -- charge.C) In this scenario, the first heavy chain is provided with a positive charge (indicated by a + in the CH1 region) and the second heavy chain is provided with a negative charge (indicated by a - in the CH1 region), resulting in two monospecific antibodies with either a - or ++ charge, and the bispecific antibody has a neutral charge. D) In this scenario, the first heavy chain lacks a separating domain and the second heavy chain contains a negatively charged separating CH2 domain with a -2 charge change. This results in two monospecific antibodies with a neutral or ---- charge, and the bispecific antibody has a - charge. E) In this scenario, two CL domains are used, one containing a positively charged CL separating domain and one without a separating domain that has no mutation. The format shown here utilizes a common heavy chain format. This results in monospecific antibodies with a ++ or neutral charge, and the bispecific antibody has a + charge. [Figure 2] Schematic diagrams of monospecific, trispecific or trivalent, and tetraspecific or tetravalent antibodies according to the present invention are shown. In Figures A and B, the first CH1-containing immunoglobulin is shown in black, representing the first heavy chain, with the second CH1 VH domain (black and striped) via a linker. The second heavy chain is shown in gray. The common light chain is shown in white. Again, it is understood that the present invention does not require the use of the common light chain shown as an example of an embodiment of the invention. Mutations are shown as either + or -, indicating the relative change in charge compared to the unmodified chain or unmodified antibody. In Figure 2A), the CL region of the light chain is the separating domain, while in 2B), the CH1 region of the first heavy chain is the separating domain. In Figure 2A), in this scenario, a tetraspecific or tetravalent antibody with a --- charge and a monospecific antibody with a -- charge are formed, and a trispecific antibody with a --- charge are formed. In Figure 2B), tetraspecific or tetravalent antibodies are formed with a charge, monospecific antibodies have a neutral charge, while trispecific or trivalent antibodies are formed with a charge. [Figure 3] Melting curves of monospecific antibodies are shown, showing two peaks associated with such antibodies having wild-type CH1 and those containing mutated CH1 regions. [Figure 4]Isoelectric focusing of bivalent monospecific antibodies generated with CH1 mutations is shown, demonstrating band separation based on charge. These data show a correlation between separating domains that increase or decrease charge and the corresponding ability to separate bands for antibodies containing these domains during isoelectric focusing. [Figure 5] CIEX chromatography of DE, KK, and DE and K arms. The top graph shows a chromatogram of a monospecific bivalent antibody generated using a DE arm with a wild-type CH1 sequence and heavy chain variable region (MF1516). The bottom graph shows a chromatogram of a monospecific bivalent antibody generated using a KK arm with a wild-type CH1 sequence and a different heavy chain variable region (MF3462). The middle graph shows a chromatogram of a bispecific antibody generated using the above-mentioned KK arm with a wild-type CH1 sequence and the above-mentioned DE arm with a wild-type CH1 sequence. In the top graph, the arrow indicates the generated bivalent monospecific antibody (DE / DE), and in the bottom graph, the arrow indicates the generated monovalent monospecific "half antibody" (KK). The light chains of each antibody are the same. [Figure 6] CIEX chromatography of DE, KK, and DE and KK arms with separate CH1 regions. The top graph shows a chromatogram of a monospecific bivalent antibody generated using a DE arm with a CH1 sequence containing T197D and K213Q mutations and a heavy chain variable region (MF1516). The bottom graph shows a chromatogram of a monospecific bivalent antibody generated using a KK arm with a CH1 sequence containing N159K and a hinge residue E216K mutation and a heavy chain variable region (MF3462). The middle graph shows a chromatogram of a bispecific antibody (MF1516 / MF3462) generated using a combination of KK and DE arms, demonstrating the separation of the bivalent DE, T197D, K213Q / KK, N159K, and E216K peaks from other proteins formed. The light chains of each antibody are the same. [Figure 7]Separation of Bispecific Antibodies. CIEX Retention Time. CIEX chromatography of DE and KK arms with wild-type CH1 containing the CH1 region for separation. The top graph shows a chromatogram of an antibody generated using DE and KK arms with wild-type CH1 sequences. The second graph shows a chromatogram of an antibody generated using a wild-type CH1 region with a KK arm containing T120K and a DE arm. The third graph shows a chromatogram of an antibody generated using a wild-type CH1 region with a KK arm containing N201K and a DE arm. The bottom graph shows a chromatogram of an antibody generated using a wild-type CH1 region with a DE arm and a KK arm containing a CH1 sequence containing N159K and hinge residue E216K. The white arrow indicates the generated bivalent monospecific antibody (DE / DE). The black arrow indicates the generated bivalent bispecific antibody (DE / KK). The gray arrow indicates the generated bivalent monospecific antibody (KK / KK). The light chains of each antibody are the same. [Figure 8] Separation of Bispecific Antibodies. CIEX Retention Time. CIEX chromatography of a DE arm containing a separation CH1 region and a KK arm with a wild-type CH1. The top graph shows a chromatogram of an antibody generated using DE and KK arms with a wild-type CH1 sequence. The middle graph shows a chromatogram of an antibody generated using a DE arm with a CH1 sequence containing T197D and K213Q and a wild-type CH1 region with a KK arm. The bottom graph shows a chromatogram of an antibody generated using a DE arm with a CH1 sequence containing K213Q and a wild-type CH1 region with a KK arm. The white arrow indicates the generated bivalent monospecific antibody (DE / DE). The black arrow indicates the generated bivalent bispecific antibody (DE / KK). The gray arrow indicates the generated bivalent monospecific antibody (KK / KK). The light chains of each antibody are the same. [Figure 9]Separation of Bispecific Antibodies. CIEX Retention Time. CIEX chromatography of DE and KK arms with wild-type or separate CH1 regions. The top graph shows chromatograms of antibodies generated using DE and KK arms with wild-type CH1 sequences. The second graph shows chromatograms of antibodies generated using a KK arm with a CH1 sequence containing T120K and a DE arm with a CH1 sequence containing T197D and K213Q. The third graph shows chromatograms of antibodies generated using a KK arm with a CH1 sequence containing N201K and a DE arm with a CH1 sequence containing T197D and K213Q. The bottom graph shows chromatograms of antibodies generated using a KK arm with a CH1 sequence containing N159K and hinge residue E216K and a DE arm with a CH1 sequence containing T197D and K213Q. The white arrows indicate the bivalent monospecific antibodies (DE / DE) generated. The black arrows indicate the bivalent, bispecific antibody (DE / KK) generated. The gray arrows indicate the bivalent, monospecific antibody (KK / KK) generated. The light chains of each antibody are the same. [Figure 10]Separation of Bispecific Antibodies. CIEX Retention Time. CIEX chromatography of DE and KK arms with wild-type or separate CH1 regions. The top graph shows a chromatogram of an antibody generated using DE and KK arms with wild-type CH1 sequences. The second graph shows a chromatogram of an antibody generated using a KK arm with a CH1 sequence containing T120K and a DE arm with a CH1 sequence containing K213Q. The third graph shows a chromatogram of an antibody generated using a KK arm with a CH1 sequence containing N201K and a DE arm with a CH1 sequence containing K213Q. The bottom graph shows chromatograms of an antibody generated using a KK arm with a CH1 sequence containing N159K and hinge residue E216K, and a DE arm with a CH1 sequence containing K213Q. The white arrow indicates the generated bivalent monospecific antibody (DE / DE). The black arrow indicates the generated bivalent bispecific antibody (DE / KK). The gray arrows indicate the bivalent monospecific antibodies (KK / KK) that were generated, each with the same light chain. [Figure 11] CIEX retention time of monospecific bivalent antibody (MF1122 / MF1122). CIEX chromatography of monospecific antibodies with mutations in the CH1 region. Each variant was tested separately, and the graph shows the CIEX retention time of each variant that exhibits a different retention time compared to a monospecific bivalent antibody containing two human wild-type CH1 regions. [Figure 12] Structure of constructs used for cloning: Constructs used for cloning to prepare constructs for antibody expression with separate CH1 regions. The CH2 and CH3 domains are obtained from the MV1708 construct. This construct contains a unique BspEI site at the N-terminus of CH2. The heavy chain variable domain (VH) was obtained from the MF1122 construct. The CH1 region was cloned into the final construct flanked by BstEII and BstEI restriction sites. [Figure 13]A) Amino acid sequence of the common light chain. B) DNA and amino acid sequence of the common light chain variable domain (IGKV1-39 / jk1). C) DNA and amino acid sequence of the common light chain constant region. D) Amino acid sequence of the IGKV1-39 / jk1 common light chain variable domain. E) Amino acid sequence of the V region of IGKV1-39A. F) CDR1, CDR2, and CDR3 of the common light chain. G) Amino acid sequence of the human common light chain IGKV3-15 / jk1. H) Amino acid sequence of the human common light chain IGKV3-20 / jk1. I) Amino acid sequence of the human common light chain IGKV3-21 / jI1. J) Amino acid sequence of the V region of IgKV3-15. K) Amino acid sequence of the V region of IgKV3-20. L) Amino acid sequence of the human common light chain IGKV1-39 / jk5 and the kappa constant region. M) Amino acid sequence of the human common light chain IGKV3-15 / jk1 and the kappa constant region. N) Amino acid sequence of human common light chain IGKV3-20 / jk1 and κ constant region O) Amino acid sequence of human common light chain IgVλ3-21 / IGJλ3 and λ constant region P) Amino acid sequence of V region of IGLV3-21 [Figure 14] IgG heavy chain for the generation of bispecific molecules. A) CH1 region B) hinge region C) CH2 region D) CH3 domain containing mutations L351K and T366K (KK) E) CH3 domain containing mutations L351D and L368E (DE) [Figure 15] 3D model of the human wild-type CH1 region with the arrow in dark grey and the 201 at position 84 according to EU numbering indicated by a sharp line, indicating its buried position in the core of the protein and lack of solvent accessibility. [Figure 16]ELISA results. Fibrinogen and PD-L1 binding of fibrinogen- or PD-L1-specific IgG1 antibodies containing the indicated CH1 variants. PG1122 is a monospecific, bivalent fibrinogen-binding antibody with two identical heavy and light chains. The two variable domains have a heavy chain variable region containing the amino acid sequence of MF1122 and a light chain as shown in Figure 13a. Numbers such as p113 and p118 indicate which amino acid mutations are present in the CH1 region of the antibody. This information is shown in Table 16. PG PD-L1 is a monospecific, bivalent antibody with two identical PD-L1-binding variable domains. Numbers p06 to p13 indicate which amino acid mutations are present in the CH1 region of the antibody. This information is shown in Table 16. [Figure 17] 1 is an IMGT table containing the EU numbering of each amino acid in the CH1, hinge, CH2, and CH3 regions of IgG1. The numbering of amino acid residue positions is included. [Figure 18] Summary of ELISA results for bispecific antibodies and the indicated monospecific antibodies in Figure 19. All bispecific antibodies tested bind to c-MET and tetanus toxoid in a dose-dependent manner. [Figure 19]Summary of the characteristics of the tested antibodies. Each column lists one antibody. PB indicates an antibody containing two different variable domains, while PG indicates an antibody containing two identical variable domains. The number after PB identifies the combination of the two variable domains, of which the heavy chain variable region is identified by the MG designation followed by a number. MG1516... and MG3462... in the following columns indicate that one variable domain has the VH of MF1516 and the VH of MF3462. The light chain region was the light chain in Figure 13A. NA stands for not applicable. Column MG1 without NA indicates that this antibody has a heavy chain containing a DE CH3 domain. Column MG2 without NA indicates that this antibody has a heavy chain containing a KK CH3 domain. Wild-type IgG1 indicates that these antibodies have all of the wild-type IgG1 constant region, the light chain in Figure 13A, and the heavy chain variable region of MF1516 or MF3462. DEDE indicates that these antibodies have only heavy chains containing a DE CH3 domain. KK indicates that these antibodies have only heavy chains containing a KK CH3 domain. [Figure 20] CIEX profiles of bispecific and monospecific antibodies. The antibody codes are indicated above or below the corresponding panel. The arrow on the left indicates the DEDE homodimer. The arrow on the right indicates the KK half antibody. The antibody codes are decoded in Figure 19 and Table 24. [Figure 21] CIEX profiles of bispecific and monospecific antibodies. The antibody codes are indicated above or below the corresponding panel. The arrow on the left indicates the DEDE homodimer. The arrow on the right indicates the KK half antibody. The antibody codes are decoded in Figure 19 and Table 24. [Figure 22] These are residues in CH3 that were identified as the interface of the CH3 / CH3 homodimer according to Traxlmayer et al. (2012), J Mol Biol. Oct 26;423(3):397-412 (see discussion and Figure 3). [Example]

[0176] Example 1: Identification of non-surface exposed residues for separation design Based on the structural information of the IgG1 CH1 sequence, including the VL domain, the positions of surface and non-surface-exposed amino acid residues buried within the CH1 region were identified using the GETAREA 1.0 program with default parameters (Negi et al., "Solvent Accessible Surface Areas, Atomic Solvation Energies, and Their Gradients for Macromolecules," last modified Wednesday, April 17, 2015, 3:00 PM). A model of the CH1CL domain with the sequence shown in Table 1 and Figure 13C was entered into the SWISS-model website (Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for homology modeling of protein structures. Bioinformatics. 2006 Jan 15;22(2):195-201). A high-quality homology model was obtained by aligning (with >95% identity over the entire length of the CH1 region) with the PDB structure 6C6X.pdb (a 1.99 Å crystal structure of the neutralizing antibody JC57-14 of Middle East Respiratory Syndrome coronavirus isolated from a vaccinated rhesus macaque). Many other CH1 regions in the PDB could provide high-quality starting structures (with >95% sequence identity and high-quality structures with the CH1 region used herein). The pdb file was uploaded, and the structure was processed with GETAREA1.0β to determine the percent of surface area of each residue predicted to be solvent accessible.

[0177] Based on the default parameters of GETAREA1.0β, amino acids with more than 50% surface exposure are referred to as "Out" or surface, while residues that are not OUT or surface exposed are greater than 50% to 20% and less than 20% accessible are referred to as "In" by GETAREA1.0β or buried amino acids herein (see Table 1).

[0178] [Table 1-1]

[0179] [Table 1-2]

[0180] CH1 sequence and modeling information. Positions are indicated by arbitrary numbers. For example, residue number 1 corresponds to EU number 118, and residue number 2 corresponds to EU number 119. The column In / Out indicates whether the amino acid is considered buried (i) or surface-exposed (o). A blank indicates the value of an amino acid that is neither surface-exposed nor buried.

[0181] Below is listed the amino acid sequence of the modeled human CH1 region according to EU numbering, with non-surface exposed amino acid positions underlined and in italics, and buried amino acids in bold.

[0182] [Table 2]

[0183] Using Rosetta software (version 3.1 https: / / www.rosettacommons.org / software) (design mode), mutations of non-surface residues were modeled in conjunction with in silico stability analysis to assess the impact of mutations at these positions and their effect on protein stability. From the Rosetta design run, the following mutations at residues with a SASA of <20% in the starting model were predicted to improve stability: A172P, S190A, Y149A, and V154I. Rosetta also predicted the following mutations to improve stability: G122P, S157T, I199V, N203I, S207T, and V211I. After the first round of designing mutations from identified non-surface residues, two additional Rosetta designs were performed: 1) positions where non-surface residues could only be mutated if the predicted positive charge was increased (either changing the residue to positive or removing D or E), and 2) positions where the residue could only be changed if the predicted negative charge was increased (neutral to D or E, or K and R to uncharged).

[0184] We found that buried residues N159 (N42) and N201 (N84) tolerated mutations of positively (K) and negatively (D) charged residues while maintaining good stability. Other non-surface-exposed residues that could support charge changes without a significant predicted decrease in CH1 stability were identified, including specific mutations predicted by bioinformatic analysis to improve stability (higher negative numerical scores indicate greater stability).

[0185] [Table 3]

[0186] Example 1b: Construct Design Non-surface-exposed and buried positions within CH1 are mutated to alter the charge of the multimerized proteins into which these immunoglobulin regions are incorporated. A total of 13 exemplary mutant CH1 regions are generated and incorporated into monospecific and multispecific antibodies for comparison with monospecific and multispecific antibodies containing wild-type CH1 regions. Constructs expressing these molecules containing these separate CH1 regions are prepared as follows.

[0187] Fragments encoding the CH2 and CH3 domains were obtained from the MV1708 construct. MVI708 was chosen because it contains a unique BspEI site at the N-terminus of CH2. A fragment encoding the variable heavy chain MF1122 with BstEII at its C-terminus was used. MF1122 was chosen because it had no problems with purification, purification, or CIEX and had an average retention time in CIEX of approximately 13.4 minutes at a pI(VH) of 8.64. The constructs used for cloning and the cloning strategy are shown in Figure 12.

[0188] Vector MV1708 (containing a DE mutation in CH3) was modified to contain the wild-type CH3 region. The VH gene from MF1122 was inserted into the vector using Sfil and BstEII-HF restriction enzymes. Correct colonies were selected by colony PCR and sequencing.

[0189] In the construct, the sequence encoding the CH1 region is flanked by the restriction sites BstEII and BspEI, which allow for the exchange of the CH1-encoding sequence. Plasmids containing wild-type or mutant CH1 regions were generated. The sequences specific to each mutant CH1 region are listed below.

[0190] The CHI coding sequence (363 bp) was excised from the plasmid using BstEII and BspEI (2 μg each of the CHI-encoding construct). Simultaneously, the prepared vector was excised from the plasmid using BstEII and BspEI restriction enzymes (20 μg of vector). The plasmid was incubated with BspEI (0.25 μL enzyme / μg DNA) in NEBuffer 3.1 buffer at 37°C for at least 1 hour, then the mixture was heated to 60°C and BstEII was added. The digested DNA was purified by gel electrophoresis and gel extraction. The digestion removed a 748 bp fragment from the backbone (approximately 10 kb) and a 363 bp fragment from the CHI domain and hinge-containing construct.

[0191] After ligation of the vector with the sequence encoding CH1, DH5a cells were transformed and plated on LB agar plates containing ampicillin. Correct constructs were identified by colony PCR and sequencing, which allowed identification of the correct CH1 and the correct CH2CH3. The identity of the final construct was confirmed by sequencing.

[0192] Example 1c: Expression and purification of antibodies containing CH1 variants All buffers used were made using Versylene (endotoxin-free and sterile) water. Endotoxins were removed from glassware, Quixstands, and Akta explorers by incubation with 0.1 M NaOH for at least 16 hours. Hek293 cells were transfected with endotoxin-free plasmid DNA. Six days after transfection, conditioned medium containing recombinant antibodies was harvested by low-speed centrifugation (10 min, 1000 g) followed by high-speed centrifugation (10 min, 4000 g). 100 μL samples were stored at 4°C.

[0193] Purification was performed using MabSelectSureLX (GE Healthcare Life Sciences). Antibodies were bound to 2 mL of MabSelectSureLX per batch for 4 hours. The MabSelectSureLX Sepharose containing the bound antibody was collected by centrifugation and transferred to a gravity-flow column. Nonspecifically bound proteins were removed by washing the column with PBS, which contained 1 M NaCl. Bound antibodies were eluted using 100 nM citrate, pH 3.5, and collected in 5 mL fractions in 12 mL tubes containing 4 mL of 1 M Tris, pH 8.0, neutralized to pH 7. Protein-containing fractions were pooled. The MabSelectSureLX pool was concentrated to 2.0–3.0 mL using a Vivaspin 20 10 KDa spin filter. Aggregates in the concentrated pool were removed by centrifugation. The concentrated sample was stored at 4°C before gel filtration.

[0194] Gel filtration: The recombinant antibody was further purified by gel filtration using a Superdex200 16 / 600 column equilibrated in PBS. Protein-containing fractions were analyzed by LabChip (PerkinElmer), and the correct antibody-containing fractions were pooled. The pool was sterilized by filtration using a 0.22 μm syringe filter. The product was stored at 4°C in aliquots containing 1.8 mL. The product was analyzed by LabChip capillary electrophoresis (PerkinElmer) and LAL assay (endotoxin assay).

[0195] LabChip analysis was performed under reducing and non-reducing conditions. HP-SEC analysis of the samples showed only one major antibody peak, indicating that the samples did not contain aggregates or half-antibodies.

[0196] Example 1d: Generation of constructs producing CH1-engineered bispecific antibodies Exchange of the DE arm of the heavy chain with the KK arm A second vector was constructed that encodes a heavy chain. The heavy chain encoded by this vector contains a KK arm to distinguish it from heavy chains containing a DE arm. The production of two different heavy chains allows for the preferential formation of bispecific antibodies. The vector encoding the KK heavy chain was constructed as follows.

[0197] The fragment encoding the DE arm of the antibody was exchanged for the fragment encoding the KK arm, which was exchanged using the cloning techniques described above, using the flanking restriction sites BspEI and AfIII in the construct.

[0198] The DE heavy chain was then combined with the heavy chain variable domain VH region of MF1516, and the KK heavy chain was combined with the heavy chain variable domain VH region of MF3462. This cloning step was performed using the restriction enzymes SfiI and BstEII and the cloning techniques described above. The identity of the final constructs was confirmed by sequencing.

[0199] This cloning procedure yields a vector encoding two heavy chains with different binding specificities. When the heavy chains are expressed together, they preferentially form bispecific antibodies. By applying the cloning steps described above, CH1 variants can be inserted into each of the two heavy chains.

[0200] Example 2: Demonstration of the ability to separate identical monospecific antibodies based on pI separating residues in the CH1 separating domain. A combination of nucleic acid constructs is used to express the antibody. The constructs encode a common light chain (Figure 13a) and a heavy chain comprising a fibrinogen-targeting heavy chain variable region (MF1122) (described below). The heavy chain further comprises a CH1 separating domain with a negative or positive charge differential compared to wild-type human CH1. Expression of the constructs results in the preferential formation of monospecific IgG1 human antibodies. Rearranged germline human kappa light chain IgVκ1-39 * 01 / IgJκ1 * 01 is used as the common light chain.

[0201] [Table 4]

[0202] The amino acid sequence of the heavy chain variable region (MF1122) capable of binding to fibrinogen used in these experiments is listed below. The CH1, CH2, and CH3 regions are human IgG1 (FIG. 14).

[0203] The target of the heavy chain variable domain is fibronectin, and the isoelectric point of the heavy chain variable domain is 8.64 (pI), while the isoelectric point of the full-length heavy chain is 8.54 (pI).

[0204] [Table 5]

[0205] The following CH1 mutants tested are shown below with the residue mutations identified according to EU numbering:

[0206] [Table 6]

[0207] Sufficient and similar amounts of each antibody were produced with volume yields ranging from 10 to 25 mL with a concentration of approximately 1.7 mg / mL.

[0208] The CIEX retention time was determined for each antibody.

[0209] CIEX-HPLC chromatography was performed using a TSKgel SP-STAT (7 μm particle size, 4.6 mM ID x 10 cm L, Tosoh 21964) series ion exchange column. CIEX assays use a hydrophilic polymer-based column material packed with non-porous resin particles, the surface of which consists of an open-access network of multilayered cation exchange groups (sulfonic acid groups), forming a strong cation exchanger and therefore suitable for separating charge isomers of monoclonal antibodies using a NaCl salt gradient. Positively charged antibodies bind to negatively charged columns.

[0210] A TSKgel SP-STAT column (7 μm particle size, 4.6 mm inner diameter x 10 cm length, Tosoh 21964) was equilibrated for at least 30 minutes at approximately 50 bar pressure using Buffer A (25 mM sodium phosphate buffer, pH 6.0). Following this, control and sample IgG were injected. The injected sample mass for all test samples and controls (in PBS) was 10 μg, and the injection volume ranged from 10 to 100 μl. The antibody was eluted from the column using a gradient of increasing salt concentration, Buffer B (25 mM sodium phosphate, 1 mM NaCl, pH 6.0). The flow rate was set at 0.5 mL / min. The chromatograms were analyzed for the peak pattern, retention time, and peak area of the observed major peaks based on the results at 220 nm.

[0211] In this study, retention time correlated with the total charge difference compared to wild type, ie, the more positive charge added, the longer the retention time, and the more negative charge added, the shorter the retention time.

[0212] [Table 7]

[0213] The CIEX retention times of all CH1 variants are shown in Table 6 and Figure 11. These data demonstrate that antibodies with otherwise the same pI, such as the bivalent monospecific human IgG antibody described above, containing a CH1 separation domain for each heavy chain, wild-type human CH2 and CH3 domains, and a common light chain, can be adequately separated based solely on the use of the separation residues provided above, thereby generating a retention time difference of 0.1 to 7.6 compared to the wild-type CH1 region by incorporating one or more positively or negatively charged residues per CH1 separation domain.

[0214] Example 3: Stability analysis of antibodies incorporating separation moieties that exhibit suitable stability for development The stability of the bivalent monospecific antibodies in PBS was determined by freezing and thawing the antibodies, which showed that all bivalent monospecific antibodies had similar stability to the wild-type monoclonal antibody.

[0215] The composition of the samples was analyzed by HP-SEC after one freeze / thaw cycle. Samples were stored at -80°C overnight and thawed at room temperature the next day. For each antibody, 21 μg dissolved in PBS was analyzed by HP-SEC. All antibodies eluted as one major peak, indicating that the produced antibodies were stable after freeze / thaw cycles. Thus, the samples maintained their composition when stored at -80°C.

[0216] The antibodies incorporating the separation domain were further evaluated by determining the temperature melting curves using differential scanning calorimetry (DSC). To perform DSC, the antibodies were diluted to 0.5 mg / mL in PBS and dialyzed in dialysis buffer. The antibodies were then filtered through a 0.45 μm filter. After dialysis, the samples were diluted to a concentration of 0.25 mg / mL and subjected to DSC analysis to obtain the temperature melting curves for each antibody.

[0217] The temperature melting (TM) curves are shown in Figure 3. TM1 and TM2 determined from the temperature melting curves are shown in Table 7 below (DSC).

[0218] In a second stability assay, TM2 was determined using UNcle (Unchained Labs) as described in Table 8. The results are listed in the table below. The stability of IgGS for TM2 was also ranked and presented. Samples were heated in PBS buffer from 25 to 95°C at 0.5°C / min and tested at pH 7.4 using protein samples ranging from 0.2 to 1 mg / mL. Tm / Tagg temperatures were then calculated from the fluorescence signal and performed in triplicate.

[0219] Thermal stability studies were performed using UNCLE (Unchained Labs) by differential scanning fluorometry (DSF) and static light scattering (SLS). DSF is based on the detection of intrinsic amino acid fluorescence between 250 and 720 nm and is used to infer protein unfolding during denaturation. SLS detects changes in aggregate content by changes in light scattering from a 266 nm laser. Briefly, proteins are analyzed at 50 μg / mL and the temperature is increased from 25 to 95°C (0.3 or 0.5°C / min). Thermal denaturation induces changes in the protein's fluorescence (monitored between 250 and 720 nm) and light scattering (266 nm laser light), which are detected and analyzed. The changes in fluorescence are displayed as barycentric mean (BMC) versus temperature (the detected fluorescence spectrum is divided into two equal regions). UNCLE analysis software is used to calculate the difference in fluorescence change over the temperature graph to identify the presence of a melting point (the temperature at which a change in TM fluorescence occurs) and temperature-induced aggregation (the temperature at which the static light scattering signal at TAGG 266 nm increases by approximately 10% above baseline).

[0220] [Table 8] [Table 9]

[0221] Example 4: Isoelectric focusing Upon production, the IgG was run on an SDS-page gel under reducing and non-reducing conditions. All proteins were of the expected size, and all bands for each mutant were of the same height. The produced IgG was also run on a gel using isoelectric focusing. The results are shown in Figure 4. The relative migration of the bands on the gel correlated with the calculated pIs listed below (Figure 4).

[0222] [Table 10]

[0223] Example 5: Separation of bispecific and monospecific antibodies by use of a CH1 separation domain (containing separation residues) Bispecific antibodies are produced by expressing two different heavy chains together, which are combined with a common light chain as described above to form the antibody.

[0224] The experiments are performed with heavy chains with DE arms and heavy chains with KK arms. The cloning of these constructs is described in Example 1D. The DE or KK modification is located in the CH3 domain of the heavy chain.

[0225] Each heavy chain consists of a CH3, CH2, CH1, and VH domain. The CH3 domain allows heterodimerization of heavy chain antibodies and contains either DE or KK residues for two different heavy chains. The CH2 domain is a human CH2 domain. The VH determines the specificity of the antibody, whereby DE heavy chains target tetanus toxin (TT) (MF1516) and KK heavy chains target cMET (MF3462). The sequences are shown in Tables 10 and 11 below.

[0226] The CH1 region of the heavy chain is either wild-type or a separation domain described herein that creates a charge difference from the wild-type domain. Heavy chains with DE arms are mutants of the human wild-type CH3 domain to promote heterodimerization. Heavy chains with KK arms are mutants of the human wild-type CH3 to promote heterodimerization. The DE arm is linked to a separation domain that has a negative charge difference compared to the wild-type domain, and the KK arm is linked to a separation arm that has a positive charge difference compared to the wild-type domain.

[0227] [Table 11]

[0228] [Table 12]

[0229] The amino acid sequence of the antibody identified by the number that targets tetanus toxoid has the amino acid sequence of MF1337. MF1337 EVQLVETGAEVKKPGASVKVSCKASDYIFTKYDINWVRQAPGQGLEWMGWMSANTGNTGYAQKFQGRVTMTRDTSINTAYMELSSLTSGDTAVYFCARSSLFKTETAPYYHFALDVWGQGTTVTVSS

[0230] Bispecific antibodies were produced by transfecting HEK293 cells with an IgG1 heavy chain construct containing a light chain construct as follows: 293 cells in suspension were transfected at 3.0 x 10 6 The cells were cultured in a T125 flask on a shaker plateau until a density of 0.3–0.5 × 10 cells / mL was reached in each well of a 24-well deep-well plate. 6Cells were seeded at a density of 1000 viable cells / mL. Cells were transiently transfected with the respective sterile DNA:PEI mixtures according to standardized procedures and further cultured. Seven days after transfection, the supernatant was harvested and filtered through a 0.22 μM filter. The sterile supernatant was stored at 4°C until the antibody was purified by protein A affinity chromatography. Subsequently, the antibody was expressed in HEK293 cells by transient transfection and purified from the culture supernatant using protein A affinity chromatography according to standard procedures.

[0231] Purification of IgG for functional screening Protein A affinity chromatography was used to purify IgG at small scale (<500 μg), medium scale (<10 mg), and large scale (>10 mg). Small-scale purification was performed using filtration in 24-well filter plates under sterile conditions. First, the pH of the medium was adjusted to pH 8.0. Subsequently, the IgG-containing supernatant was incubated with Protein A Sepharose CL-4B beads (50% v / v) (Pierce) for 2 hours at 25°C at 600 rpm on a shaking platform. The beads were then collected by filtration. The beads were washed twice with PBS pH 7.4. Bound IgG was then eluted with 0.1 M citrate buffer at pH 3.0, and the eluate was immediately neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using a Multiscreen Ultracel 10 multiplate (Millipore). Samples were finally collected in PBS pH 7.4. IgG concentrations were measured using Octet (ForteBio). Protein samples were stored at 4°C.

[0232] The following constructs were made and used in the experiments. Constructs were sequence verified before the experiments were performed. The encoded heavy chains were produced and analyzed using SDS-PAGE under reducing and non-reducing conditions. All heavy chains generated bispecific monovalent antibodies and half antibodies of the expected size.

[0233] [Table 13]

[0234] To generate bispecific antibodies, various DE heavy chains were combined with various KK heavy chains. The products were analyzed by CIEX as described in Example 2. Both combinations of DE / KK antibodies and the generation of one arm with either DE or KK were analyzed. The generation of one arm with DE gave DE / DE homodimers, and the generation of one arm with KK gave KK half antibodies. The generation of KK / KK homodimers was not observed.

[0235] The table below lists the retention times for various antibody species and single arm formations. The relative difference in retention time between bispecific antibodies (DE / KK) and homodimers (DE / DE) or KK half antibodies indicates the distance between the peaks in the CIEX spectrum. A larger difference makes it easier to separate the fractions that form homodimers and half antibodies in bispecific antibodies.

[0236] [Table 14]

[0237] The retention times of the various antibody species listed in Table 13 are shown in Figures 5 to 10. As shown in Figure 5, the CIEX retention times of the wild-type DE / DE homodimer and the KK half antibody are relatively close. In contrast, the use of a mutant CH1 separation domain along with the DE and KK CH3 heterodimerization domains of these heavy chains alters the CIEX retention times of the heavy chains, increasing the difference in retention times between the homodimer, bispecific heterodimer, and half antibody. Figure 6 shows the CH1 region of the DE heavy chain with mutations T197D and K213Q. The CH1 region of the KK heavy chain with mutations N159K and hinge residue E216K is also shown. Therefore, the CIEX retention times of the homodimer (DE / DE) and half antibody (KK) have a large difference in retention time, as shown in Figure 6. The retention time of the bispecific antibody (DE / KK) is further separated from the other species, allowing for better separation of the different species.

[0238] The effect of mutations in the CH1 separation domain on the CIEX retention time of bispecific antibodies is shown in Figures 7-10.

[0239] [Table 15-1]

[0240] [Table 15-2]

[0241] [Table 15-3]

[0242] [Table 15-4]

[0243] [Table 15-5]

[0244] Example 6 Further analysis of CH1 variants from Examples 1 to 5 and new CH1 variants The antibody is produced as described in Example 1, with the proviso that the antibody in this example is a monospecific, bivalent antibody with two identical heavy chains and two identical light chains. The antibody is not a bispecific antibody and therefore has a wild-type CH3 domain.

[0245] ELISA to evaluate binding of various CH1 variants to fibrinogen-coated plates. The antibodies were IgG1 antibodies containing the indicated CH1 variants. All antibodies were bivalent monospecific antibodies with variable domains containing the VH of MF1122 and the common light chain of Figure 13A (designated as PG1122). As a negative control, the same antibodies, but now containing the VH of the PD-PL1 antibody (designated as PG PD-PL1), were tested on the same fibrinogen-coated plates (see Figure 16: fibrinogen plate-positive sample set 1, fibrinogen plate-positive sample set 2, and fibrinogen plate-negative sample set, respectively). The same antibodies were evaluated for binding to PD-PL1-coated plates (see Figure 16: PD-PL1 plate-positive sample set and PD-PL1 plate-negative sample set).

[0246] Fibrinogen ELISA plates were coated with 10 μg / mL human fibrinogen (Sigma Aldrich; Cat. No. F4753). Antibodies were incubated at a range of 10-fold dilutions starting at 5 μg / mL to a final concentration of 0.005 μg / mL.

[0247] PD-L1 ELISA plates were coated with 2.5μg / mL human PD-PL1 Fc (R&D systems; catalog no. 156-B7). The antibody was incubated at a range of 10-fold dilutions starting at 5μg / mL for a final concentration of 0.005μg / mL. Bound antibody was detected with a 1:1000 dilution of an HRP-conjugated Protein L-based secondary antibody (Pierce, catalog no. 32420) that binds to the kappa light chain.

[0248] The PD-L1 binding antibodies were tested together as negative controls in a fibrinogen ELISA, and the fibrinogen binding antibodies were tested together as negative controls in a PD-L1 ELISA. The negative controls, with opposite binding specificities, however, had the same CH2, CH3, and CH1 variant sequences as the test antibodies. The amino acid sequence of the VH variable region of MF1122 is shown in Table 4. The sequences of each CH1 variant are shown in Table 14. Thus, these data demonstrate that the separation residues do not affect the binding of the designated heavy chain variable region to its target antigen.

[0249] The conclusion of the ELISA assays is that all antibodies tested bind to the targets specified by the variable domain sequences and, importantly, do not bind to non-specific targets (Figure 16). In other words, fibrinogen-specific antibodies bind to fibrinogen in the fibrinogen ELISA and do not bind to PD-L1 in the PD-L1 ELISA, and PD-PL1-specific antibodies bind to PD-L1 in the PD-L1 ELISA and do not bind to fibrinogen in the fibrinogen ELISA.

[0250] [Table 16]

[0251] The indicated mutants were analyzed in a wild-type IgG1 background. Also shown is whether the CH1 mutants are associated with heavy chains that have either DE or KK arms. The heavy chain variable region (VH) of the heavy chain has the sequence of MF1122 (see Table 4). CH1 mutants that increase charge (first 7 entries in the figure) were tested with the PD-L1 heavy chain variable region (RT ∼11 min and FAB Tm ∼76°C).

[0252] Various CH1 variants were combined with other wild-type IgG1s to generate IgG1 antibodies with the indicated variable domains and CH1 variants. The antibodies are monospecific, bivalent antibodies with identical heavy and light chains. Each antibody has two identical CH1 domains and two identical variable domains. The products were analyzed by CIEX as described in Example 2. The CIEX retention times (RT) of each antibody and the relative difference from the retention time of an IgG1 antibody with the same variable domains and wild-type CH1 (WT) are shown in Table 16. A larger difference makes it easier to separate the fractions that form homodimers and half antibodies in bispecific antibodies.

[0253] [Table 17]

[0254] In conclusion, amino acid mutations predictably increase ΔRT (defined as the difference between the RT of the IgG mutant and wild-type IgG). Some mutants exhibit larger ΔRT than others. Both VH sequences tested (VH1122 and PD) are affected to the same extent by the mutations.

[0255] Analysis of the stability of antibodies incorporating separation moieties Example 3 describes the Uncle stability assay. The data shown below is obtained with the Uncle instrument using the method described in Example 3.

[0256] The monospecific bivalent antibodies shown in Table 16 were tested for various stability parameters, and the results are shown in Table 17.

[0257] [Table 18]

[0258] [Table 19]

[0259] In all cases, some mutation combinations reduce TAGG, but the reduction is well within acceptable levels. Overall thermal stability is affected to a similar extent in both VHs, demonstrating that it is independent of the specific VH sequence in the associated variable domains. The N159 K modification, which contains the anti-PD-L1-containing variable domain, is associated with an early melting event of approximately 66°C in this analysis. This is likely a measurement issue, as the values for this variant, which contains the MF1122-containing variable domain, do not show the same difference as the wild-type. This trend is also seen in various combinations with N159K, which typically do not show any difference from the wild-type.

[0260] [Table 20]

[0261] Compared to the other CH1 variants listed in Table 18, the single amino acid variant N201K appears to produce the largest shift in CIEX (2.5-3.4 min) while maintaining high thermal stability (TAGG is reduced by 0-2°C). This is true for both VH sequences tested, regardless of their antigen binding specificity and the germline V region from which they are derived. The other listed single amino acid variants also show good stability and useful shifts in CIEX retention time.

[0262] [Table 21]

[0263] All tested mutants have similar Tm1 values, and Tm2 is within the favorable range. The single mutation K213Q causes a large CIEX shift (-0.8 min) while maintaining good thermal stability (TAGG is reduced by 0.7 °C). The double mutant N201K + N159K has a significant effect on CIEX retention time while showing a limited effect on thermal stability. In this case, the tested triple mutant showed the largest CIEX retention time shift.

[0264] Example 7a: Identification of non-surface residues for separation design Based on the structural information of the IgG1 CH2 region with a separate CH2 region surface, the positions of non-surface-exposed and buried amino acid residues within the CH2 region were identified using the program GETAREA 1.0 with default parameters. (Negi et al., "Solvent Accessible Surface Areas, Atomic Solvation Energies, and Their Gradients for Macromolecules," Last modified Wednesday, April 17, 3:00 PM, 2015.) A model of the CH2 region with the sequence in Table 20 was entered into the Swiss-model website (Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structural homology modeling. Bioinformatics. 2006 Jan 15;22(2):195-201). The same procedure was performed for the IgG1 CH3 region.

[0265] High-quality homology models of CH2 and CH3 were obtained using Swiss-Model version 1.3.0 from the Swiss-Model web server essentially as described above. Several suitable crystal structures exist (these are high-quality structures, with alignments showing >95% sequence identity with the CH2 domain used as the "original" or template sequence in many embodiments herein). Many other CH2 regions in the PDB could provide high-quality starting structures (with >95% sequence identity and high-quality structures with the CH2 region used here). Many are readily identified using commonly used homology modeling tools, as in Example 1. A structural model of the CH2 domain was obtained with 98.2% sequence identity at full length starting from the PDB template 5vu0 of the CH2 query sequence (note that mismatches occur in the engineered and terminal / linker regions, and the model obtains a Swiss-Model GMQE score of 0.99 in version 1.3.0).

[0266] The pdb file generated by Swiss-Model was uploaded and this structure was processed by GETAREA1.0β. Based on the default parameters of GETAREA1.0β, amino acids with more than 50% surface exposure are referred to as "Out" or surface, while non-OUT or non-surface exposed residues with more than 50% to 20% accessibility and less than 20% accessibility are referred to as "in" by GETAREA1.0β or are referred to herein as buried amino acids.

[0267] CH3 Similarly, the "original" or any engineered CH3 domain embodied herein can be modeled as a homodimer (two interacting CH3 chains) or as a monomer using homology modeling. Several suitable crystal structures exist (these are high-quality structures, with alignments showing >92% sequence identity with the DE or KK-CH3 domains used as the "original" sequences in many of the embodiments herein). Many other CH3 regions in the PDB could provide high-quality starting structures (with >92% sequence identity and high-quality structures with the CH3 regions used here). Many are readily identified using commonly used homology modeling tools, as in Example 1. For example, starting from the PDB template 5w38 of the DE-CH3 query sequence, we obtain a structural model of the CH3 domain (DE-CH3) with a full-length sequence identity of 93.46 (note that mismatches occur in the engineered region and the linker / domain end region, and the model obtains a Swiss-Model GMQE score of 0.99 in version 1.3.0). The PDB file generated by Swiss-Model was uploaded, and this structure was processed with GETAREA1.0β. Based on the default parameters of GETAREA1.0β, amino acids with surface exposure greater than 50% are referred to as "Out" or surface, while non-OUT or non-surface-exposed residues with accessibility greater than 50% to 20% and less than 20% are referred to as "In" by GETAREA1.0β or as buried amino acids herein (see Tables 20-22).

[0268] The CH2 region modeled is a human CH2 modified to silence positions 235 and 236 according to EU numbering. The CH3 region modeled is a human CH3 modified to include the L351D and L368E mutations in Table 21 and the T366K and L351K mutations in Table 22 according to EU numbering, thereby modeling the CH3 chain of the CH3 DEKK heterodimerization domain.

[0269] [Table 22-1]

[0270] [Table 22-2]

[0271] [Table 22-3]

[0272] Sequence and modeling information for CH2. Positions are indicated by arbitrary numbers. Residue ALA numbered 2 corresponds to EU number 231, residue PRO numbered 3 corresponds to EU number 232, etc., up to residue LYS numbered 111, which has position number 340 according to EU numbering (see IMGT table shown in Figure 17).

[0273] The sequence of the CH2 region contains Fc silent mutations (L235G and G236R mutations) at positions 235 and 236. The column In / Out indicates whether the amino acid is considered buried (i) or surface-exposed (o). A blank indicates the value of an amino acid that is neither surface-exposed nor buried.

[0274] [Table 23-1]

[0275] [Table 23-2]

[0276] [Table 23-3]

[0277] Sequence and modeling information for CH3. Positions are indicated by arbitrary numbers. Residue numbered 1 GLY corresponds to EU number 341, residue numbered 2 GLN corresponds to EU number 342, etc., up to residue numbered 103 LEU, which has position number 443 according to EU numbering (see IMGT table shown in Figure 17).

[0278] The sequence used for the CH3 region contains DE heterodimerization mutations at positions 351 and 368 (mutations L351D and L368E are positions 11 and 28 in the above numbering, respectively). The column In / Out indicates whether the amino acid is considered buried (i) or surface-exposed (o). A blank indicates the value of an amino acid that is neither surface-exposed nor buried.

[0279] [Table 24-1]

[0280] [Table 24-2]

[0281] [Table 24-3]

[0282] Sequence and modeling information for CH3. Positions are indicated by arbitrary numbers. Residue numbered 1 GLY corresponds to EU number 341, residue numbered 2 GLN corresponds to EU number 342, etc., up to residue numbered 103 LEU, which has position number 443 according to EU numbering (see IMGT table shown in Figure 17).

[0283] The sequence used for the CH3 region contains KK heterodimerization mutations at positions 351 and 366 (the L351K and T366K mutations are positions 11 and 26 in the above numbering, respectively). The column In / Out indicates whether the amino acid is considered buried (i) or surface-exposed (o). A blank indicates the value for an amino acid that is not surface-exposed.

[0284] Example 7b: Construct Design Non-surface and buried positions in CH2 and CH3 are altered to alter the charge of the multimerized proteins incorporating these immunoglobulin regions. A total of nine exemplary CH2 and CH3 region mutations are generated and incorporated into monospecific and multispecific antibodies for comparison with monospecific and multispecific antibodies with wild-type CH2 and CH3 regions. Constructs expressing heavy chain molecules containing these separate CH2 / CH3 regions are prepared similarly to the methods detailed in Example 1.

[0285] The amino acid mutations of the tested mutants are shown in Table 23.

[0286] [Table 25]

[0287] All mutants contain Fc silent mutations in the CH2 region as shown in Table 20. The mutants also contain a CH3 heterodimerization domain as shown in Table 21 for the DE mutants and Table 22 for the KK mutants. Mutations that provided an increased negative charge were incorporated into the DE CH3 backbone. For those that provided an increased positive charge, they were incorporated into the KK CH3 backbone.

[0288] Each heavy chain was engineered with a heavy chain variable region that, together with the common light chain in Figure 13a, forms a variable domain that binds to tetanus toxoid (TT) or the extracellular portion of c-MET. The TT variable domain has a heavy chain variable region comprising the amino acid sequence of MF1516. The c-MET variable domain has a heavy chain variable region comprising the amino acid sequence of MF3462. The amino acid sequences of VH MF1516 and MF3462 are shown above. The production of heavy chains containing interchangeable heterodimerization regions allows for the preferential formation of bispecific antibodies. Heavy chains containing VH MF1516 contain a DE mutant CH3 domain, while heavy chains containing VH3462 have a KK mutant CH3 domain.

[0289] The identity of the final construct was confirmed by sequencing. For the production of bispecific antibodies, one heavy chain contained the variable region of MF1516, a wild-type CH1 region and hinge region, an Fc silent CH2 region, and a DE CH3 region. The other heavy chain contained the variable region of MF3462, a wild-type CH1 region and hinge region, an Fc silent CH2 region, and a KK CH3 region. As described above, a mutation shown in Table 23 that results in an increased negative charge was incorporated into the heavy chain with a DE CH3 region. A mutation that results in an increased positive charge was incorporated into the heavy chain containing a KK CH3 region. Hereinafter, when referring to the wild type, a bispecific antibody refers to one having the above heavy and light chains but without one of the mutations listed in Table 23.

[0290] Bispecific antibodies were produced by combining two heavy chains. The antibodies were expressed and purified using methods essentially as described in Example 1c. Briefly, constructs expressing two heavy chains and a common light chain having the sequences of Figure 13a were transfected into HEK293 cells. Six days after transfection, the cell culture medium was harvested. Antibodies were then purified from this medium as described in Example 1. The antibodies produced by this method are listed in Figure 19.

[0291] Monospecific bivalent antibodies with the variants listed in Table 23 were produced using heavy chains containing a CH3 domain that does not contain an interchangeable heterodimerization CH3 domain. The heavy chains contained either a VH containing the amino acid sequence of MF1516 or MF3462, a wild-type CH1 region and hinge region, an Fc silent CH2 region, and a wild-type CH3 region. The amino acid mutations listed in Table 23 were introduced into either the Fc silent CH2 region or the wild-type CH3 region. Mutations listed in Table 23 that result in an increased negative charge were incorporated into a heavy chain containing the VH region of MF1516. Mutations that result in an increased positive charge were incorporated into a heavy chain containing the MF3462 region. When referring to the wild-type antibody hereafter, this refers to an antibody having the heavy and light chains listed above but without one of the variants listed in Table 23. Briefly, constructs expressing the indicated heavy and common light chains containing the sequences of Figure 13a were transfected into HEK293 cells. Six days after transfection, the cell culture medium was harvested, and antibodies were then purified from this medium as described in Example 1. The antibodies produced in this manner are listed in Figure 19.

[0292] ELISA To assess the binding of various antibodies, ELISA plates were coated with c-MET, tetanus toxoid, or thyroglobulin (c-MET (R&D systems, catalog no. 358-MT / CF) 2.5 μg / mL, tetanus toxoid (Statens Institute, catalog no. T162-2) 2 μg / mL, and thyroglobulin (Sigma Aldrich, catalog no. T1126-500MG) 10 μg / mL). Antibodies were incubated at 10, 1, 0.1, or 0.01 μg / mL. Bound antibodies were detected with a 1:2000 dilution of an HRP-conjugated protein L-based secondary antibody (Pierce, catalog no. 32420) that binds to the kappa light chain.

[0293] The ELISA results are summarized in FIG. Antibody PG1337 is a monospecific bivalent TT IgG1 antibody. Antibody PG1025 is a monospecific bivalent tyrosine globulin IgG1 antibody. Antibody PG2994 is a monospecific bivalent cMET IgG1 antibody. It is concluded that all bispecific antibodies bind to c-MET and tetanus toxoid in a dose-dependent manner. The bispecific antibodies do not bind to the negative control antigen (thyroglobulin). Binding does not appear to differ between antibodies with wild-type CH2 / CH3 regions or their mutants.

[0294] Example 8: CIEX profile of each bispecific antibody. CIEX experiments were performed as described in Example 2. The results for each antibody are shown in Figures 20 and 21 and summarized in Table 24.

[0295] [Table 26]

[0296] Example 9: Melting Temperatures of Antibodies Containing Each CH2 CH3 Separating Domain Thermal stability was measured by UNCLE as described in Example 6.

[0297] [Table 27]

[0298] Most bispecific antibodies containing isolation variants show only a small decrease in melting temperature (approximately 2-3°C).

[0299] TM1: Half IgG An early TM was observed in the half IgG and one PB, which may be due to the half IgG in the preparation of that PB. TM1 was similar for all half IgGs (lower for KK compared to DE half antibody transfections, and lowest for V303K).

[0300] Melting of TM2:Fc PBs with KK mutations show a decrease in TM2 (2-3 degrees). Mutation of V303 (on both the DE and KK sides) reduces TM2 of the PB.

[0301] TM3: Fab melting As expected from the wild-type IgG1 control, all PBs were detected at approximately 78-79°C (see Figures 20 and 21), indicating that PB stability was not significantly affected by the Fc mutations.

[0302] TAGG: Same in all PBs, higher in KK half IgG. The half IgG of E388T has high TAGG.

[0303] summary

[0304] [Table 28]

[0305] All CH2 and CH3 mutants tested favorably affect the separation of bispecific antibodies from DEDE and KK molecules, with some mutants having a greater effect. Thermal stability is only slightly affected by the separation mutation. The proportion of half antibody in these relatively crude preparations is also relatively constant across each separation mutation and similar to wild-type, with the exception of E388T, which effectively has 0% half antibody.

[0306] The present invention provides the following aspects as part of the present invention.

[0307] Aspects Aspect 1 is a CH1 region of an immunoglobulin, comprising a mutation of an amino acid that is not surface-exposed in the immunoglobulin, wherein the mutation Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and CH1 region of an immunoglobulin selected from mutations of negatively charged amino acids to positively charged amino acids.

[0308] Embodiment 2 The immunoglobulin region of embodiment 1, comprising two or more mutations of non-surface exposed amino acids in the immunoglobulin.

[0309] Embodiment 3. The immunoglobulin region of embodiment 1 or 2, which is a human immunoglobulin region.

[0310] Aspect 4 The immunoglobulin region of any one of aspects 1 to 3, wherein non-surface exposed amino acids are buried.

[0311] Aspect 5 An immunoglobulin region according to any one of Aspects 1 to 4, which is an IgG region, preferably an IgG1 region.

[0312] Aspect 6. A CH1 region of an immunoglobulin, comprising an amino acid mutation selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, and K213 (EU numbering).

[0313] Embodiment 7. The CH1 region of the immunoglobulin according to embodiment 6, which comprises an amino acid mutation selected from D148, Y149, V154, N159, A172, S190 and N201.

[0314] Embodiment 8. The CH1 region of the immunoglobulin according to embodiment 6 or 7, which comprises an amino acid mutation selected from N159 and / or N201.

[0315] Aspect 9: An antibody comprising the CH1 region according to any one of aspects 1 to 8.

[0316] Embodiment 10: The antibody according to embodiment 9, comprising two or more CH1 regions according to any one of embodiments 1 to 8.

[0317] Embodiment 11. The antibody of embodiment 9 or 10, wherein the antibody comprises different heavy chains.

[0318] Embodiment 12. The antibody of embodiment 11, which is a multispecific antibody.

[0319] Embodiment 13. The multispecific antibody according to embodiment 11 or 12, wherein the heavy chains comprise interchangeable heterodimerization regions.

[0320] Embodiment 14. The multispecific antibody according to embodiment 13, wherein the antibody comprises interchangeable heterodimerized CH3 regions.

[0321] Aspect 15. The multispecific antibody according to any one of aspects 12 to 14, wherein one of the heavy chains comprises the CH3 mutations L351D and L368E, and the other heavy chain comprises the CH3 mutations T366K and L351K.

[0322] Aspect 16 The antibody according to any one of aspects 9 to 15, which is an IgG1 antibody.

[0323] Embodiment 17. The antibody of any one of embodiments 9 to 16, comprising one or more antibody light chains.

[0324] Aspect 18: The antibody of any one of claims 9 to 17, comprising a common light chain.

[0325] Aspect 19. A composition comprising an immunoglobulin region according to any one of aspects 1 to 8 or an antibody according to any one of aspects 9 to 18.

[0326] Aspect 20. A pharmaceutical composition comprising an immunoglobulin domain according to any one of aspects 1 to 8 or an antibody according to any one of aspects 9 to 18.

[0327] Aspect 21: A nucleic acid encoding the CH1 region of any one of Aspects 1 to 8 or the antibody of any one of Aspects 9 to 18.

[0328] Aspect 22 A nucleic acid encoding the antibody of any one of aspects 9 to 18.

[0329] Embodiment 23. A recombinant host cell comprising nucleic acid according to embodiment 21 or 22.

[0330] Aspect 24: A method for producing an antibody according to any one of aspects 9 to 18, the method comprising: providing a nucleic acid encoding a first heavy chain comprising a CH1 region according to any one of aspects 1 to 8; providing a nucleic acid encoding a second heavy chain, wherein the first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and harvesting the antibody from the host cell culture; Clarifying the harvest; capturing the protein; performing anion exchange chromatography; and performing cation exchange chromatography to separate the antibody from other antibodies or antibody fragments; The method comprises producing an antibody by performing at least one of the following steps.

[0331] Aspect 25: A method for producing an antibody according to any one of aspects 9 to 18, the method comprising: providing a nucleic acid encoding a first heavy chain comprising a CH1 region according to any one of aspects 1 to 8; providing a nucleic acid encoding a second heavy chain, wherein the first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and harvesting the antibody from the host cell culture; and A method comprising, in the separating step, separating the antibody from other antibodies or antibody fragments by isoelectric focusing on a gel.

[0332] Embodiment 26. The method of embodiment 24 or 25, wherein the first and second heavy chains comprise interchangeable heterodimerization regions, preferably interchangeable CH3 heterodimerization regions.

[0333] Aspect 27. A method for producing a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: providing a nucleic acid encoding a CH1 region of a first heavy chain and a nucleic acid encoding a CH1 region of a second heavy chain such that the isoelectric points of the first encoded heavy chain and the second encoded heavy chain are different, wherein at least one of the CH1 regions comprises an amino acid mutation at a position selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201, and K213 (EU numbering); Culturing the host cells to express the nucleic acid; and harvesting the multispecific antibodies from the host cell culture using differences in isoelectric points; harvesting the antibody from the host cell culture; Clarifying the harvest; capturing the protein; performing anion exchange chromatography; and The method further comprises performing cation exchange chromatography to separate the antibody from other antibodies or antibody fragments.

[0334] Aspect 28. A method for purifying a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: providing a nucleic acid encoding a CH1 region of a first heavy chain and / or a nucleic acid encoding a CH1 region of a second heavy chain, such that the isoelectric points of the first encoded heavy chain and the second encoded heavy chain are different, wherein at least one of the CH1 regions comprises an amino acid mutation at a position selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201 and K213 (EU numbering); and Culturing the host cells to express the nucleic acid; and purifying the multispecific antibody from the host cell culture by isoelectric focusing to separate the multispecific antibody from other antibodies or antibody fragments.

[0335] Embodiment 29. The method of embodiment 27 or 28, wherein the nucleic acids encoding the homomultimer of the first heavy chain, the homomultimer of the second heavy chain, and the heteromultimer of the first and second heavy chains are expressed as proteins with different isoelectric points, resulting in different retention times in ion exchange chromatography.

[0336] Aspect 30: wherein the mutation of the one or more amino acids at the positions is Neutral to negatively charged amino acids, Positively charged amino acids to neutral amino acids, positively charged amino acids to negatively charged amino acids, Neutral amino acids to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and 30. The method according to any one of aspects 27 to 29, wherein the mutation is selected from a negatively charged amino acid to a positively charged amino acid.

[0337] Embodiment 31 The method of any one of embodiments 27 to 30, wherein the first heavy chain and the second heavy chain comprise compatible CH3 heterodimerization regions.

[0338] Embodiment 32. The method of embodiment 31, wherein one of the compatible CH3 heterodimerization regions comprises the L351D and L368E mutations, and the other comprises the T366K and L351K mutations.

[0339] Aspect 33. A CH1-containing immunoglobulin polypeptide comprising a first charged amino acid residue at position 120, 147, 148, 149, 154, 159, 172, 175, 190, 201, or 213.

[0340] Embodiment 34. The CH1-containing immunoglobulin polypeptide according to embodiment 33, which comprises, in addition to the charged residue according to embodiment 33, a second charged amino acid residue at a different position selected from: 120, 147, 148, 149, 154, 159, 172, 175, 190, 201 or 213.

[0341] Embodiment 35. A CH1-containing immunoglobulin polypeptide comprising a neutral or negatively charged amino acid residue at position 197 and / or 213.

[0342] Embodiment 36. A CH1-containing immunoglobulin polypeptide comprising a neutral or positively charged amino acid residue at position 159 and a positively charged amino acid residue at hinge position 216.

[0343] Aspect 37 An immunoglobulin protein comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the first and / or second CH1-containing immunoglobulin polypeptide comprises one or more mutations of one or more amino acids selected from amino acids in the CH1 region that are not surface exposed, such that the isoelectric point of the immunoglobulin protein comprising the first CH1-containing immunoglobulin polypeptide and the second CH1-containing immunoglobulin polypeptide is different from the isoelectric point of an immunoglobulin protein containing only the first CH1 immunoglobulin polypeptide or the isoelectric point of a protein containing only the second CH1 immunoglobulin polypeptide.

[0344] Embodiment 38. The immunoglobulin protein of embodiment 37, wherein the one or more mutations of one or more amino acids selected from amino acids in the CH1 region are buried.

[0345] Aspect 39. A composition comprising an immunoglobulin region or an antibody according to any one of aspects 1 to 18, further comprising an amino acid selected from T197 and a mutation at hinge position E216.

[0346] EMBODIMENT 40: An immunoglobulin protein comprising a first CH1 region-containing immunoglobulin polypeptide and a second CH1 region-containing immunoglobulin polypeptide, wherein one CH1 region comprises one or more mutations of non-surface exposed amino acids, and wherein the one or more mutations of amino acids are Neutral to negatively charged amino acids, Positively charged to neutral amino acids, and a positively charged amino acid to a negatively charged amino acid, or Neutral amino acids to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and An immunoglobulin protein selected from mutations of negatively charged amino acids to positively charged amino acids.

[0347] 41. An immunoglobulin protein comprising a first CH1 region-containing immunoglobulin polypeptide and a second CH1 region-containing immunoglobulin polypeptide, wherein one CH1 region comprises one or more mutations of non-surface exposed amino acids, and wherein the one or more mutations of amino acids are Neutral to negatively charged amino acids, Positively charged to neutral amino acids, and A mutation from a positively charged amino acid to a negatively charged amino acid, The other CH1 region contains one or more mutations of amino acids that are not surface exposed, and one or more mutations of amino acids Neutral amino acids to positively charged amino acids, Negatively charged amino acids to neutral amino acids, and Immunoglobulin proteins, which are mutations of negatively charged amino acids to positively charged amino acids.

[0348] Aspect 42: An immunoglobulin protein comprising a first CH1 region-containing immunoglobulin polypeptide and a second CH1 region-containing immunoglobulin polypeptide, wherein the first and / or second CH1 region-containing immunoglobulin polypeptide comprises one or more mutations of one or more amino acids selected from amino acids in the CH1 region that are not surface-exposed, such that the isoelectric point of the immunoglobulin protein comprising the first CH1 region-containing immunoglobulin polypeptide and the second CH1 region-containing immunoglobulin polypeptide is different from the isoelectric point of an immunoglobulin protein containing only the first CH1 region immunoglobulin polypeptide and the isoelectric point of an immunoglobulin protein containing only the second CH1 region immunoglobulin polypeptide.

[0349] Embodiment 43. The immunoglobulin protein of any one of embodiments 40 to 42, which comprises a human CH1 region.

[0350] Embodiment 44. The immunoglobulin protein according to any one of embodiments 40 to 43, which is an IgG.

[0351] Embodiment 45. The immunoglobulin protein of any one of embodiments 40 to 44, wherein non-surface exposed amino acids are buried.

[0352] Aspect 46. The immunoglobulin protein of any one of aspects 40 to 44, which comprises an amino acid mutation in the CH1 region of an amino acid selected from T120, K147, D148, Y149, V154, N159, A172, Q175, S190, N201 and K213.

[0353] Embodiment 47. The immunoglobulin protein according to embodiment 46, which comprises an amino acid mutation selected from D148, Y149, V154, N159, A172, S190 and N201.

[0354] Embodiment 48. The immunoglobulin protein according to embodiment 47, which comprises an amino acid mutation at N159 and / or N201.

[0355] Aspect 49 The immunoglobulin protein of any one of aspects 40 to 48, wherein the first CH1 region-containing immunoglobulin polypeptide and the second CH1 region-containing immunoglobulin polypeptide are heavy chains.

[0356] Embodiment 50. The immunoglobulin protein of any one of embodiments 40 to 49, which is an antibody.

[0357] Embodiment 51. The antibody according to embodiment 50, which is a bispecific antibody.

[0358] Embodiment 52. The antibody according to embodiment 50, which is a multispecific antibody.

[0359] Embodiment 53. The immunoglobulin protein according to any one of embodiments 40 to 52, further comprising an amino acid selected from T197 and a mutation at hinge position E216.

[0360] Embodiment 54. A composition comprising an immunoglobulin region according to any one of embodiments 1 to 8 or an antibody according to any one of embodiments 9 to 18, wherein the immunoglobulin region further comprises one or more of the following mutations: G122P, I199V, N203I, S207T, and V211I.

Claims

1. A CH1 region of an immunoglobulin comprising at least one amino acid mutation, wherein the mutation is - N201K; - N201D; - A172P, S190A and N201K; - A172P, S190A and N201D; - N201D and K213Q; - T120K and N201K; - N159K and N201K; - T120K, N201K and N159K; - N201D and N159D; or - N201D, K213Q and N159D (EU numbering) A CH1 region of an immunoglobulin selected from:

2. The CH1 region of the immunoglobulin described in claim 1, which is the CH1 region of a human immunoglobulin.

3. A CH1 region of the immunoglobulin described in claim 1, which is an IgG region, preferably an IgG1 region.

4. An immunoglobulin CH1 / CL domain comprising an immunoglobulin region described in any one of claims 1 to 3.

5. A protein comprising the immunoglobulin domain described in claim 4.

6. A CH1-containing immunoglobulin polypeptide comprising the CH1 region of an immunoglobulin defined in any one of claims 1 to 5.

7. An antibody, preferably a multispecific antibody, comprising the immunoglobulin domain described in claim 4.

8. An immunoglobulin protein comprising a first CH1-containing immunoglobulin polypeptide and a second CH1-containing immunoglobulin polypeptide, wherein the first and / or second CH1-containing immunoglobulin polypeptide is: - N201K; - N201D; - A172P, S190A and N201K; - A172P, S190A and N201D; - N201D and K213Q; - T120K and N201K; - N159K and N201K; - T120K, N201K and N159K; - N201D and N159D; or - N201D, K213Q (EU numbering) and one or more mutations of one or more amino acids selected from Thus, the isoelectric point of the immunoglobulin protein comprising the first CH1-containing immunoglobulin polypeptide and the second CH1-containing immunoglobulin polypeptide is different from the isoelectric point of an immunoglobulin protein containing only the first CH1 immunoglobulin polypeptide and a protein containing only the second CH1 immunoglobulin polypeptide.

9. An immunoglobulin protein described in claim 8, comprising a human CH1 region.

10. The immunoglobulin protein of claim 8, comprising a human IgG CH1 region, preferably a human IgG1 CH1 region.

11. The immunoglobulin protein described in claim 8, wherein the first CH1 region-containing immunoglobulin polypeptide and the second CH1 region-containing immunoglobulin polypeptide are heavy chains.

12. The immunoglobulin protein of claim 8, which is an antibody.

13. An antibody described in claim 12, comprising different heavy chains.

14. An antibody comprising a first heavy chain and a second heavy chain, wherein one of the heavy chains comprises at least one amino acid mutation, and wherein the mutation is: - N201K; - N201D; - A172P, S190A and N201K; - A172P, S190A and N201D; - N201D and K213Q; - T120K and N201K; - N159K and N201K; - T120K, N201K and N159K; - N201D and N159D; or - N201D, K213Q (EU numbering) An antibody selected from:

15. An antibody described in any one of claims 12 to 14, which is a bispecific or multispecific antibody.

16. An antibody described in any one of claims 12 to 15, wherein the heavy chain comprises an interchangeable heterodimerization region.

17. An antibody described in claim 16, comprising a compatible CH3 heterodimerization region.

18. An antibody described in claim 17, wherein one of the heavy chains contains CH3 mutations L351D and L368E and the other heavy chain contains CH3 mutations T366K and L351K.

19. An antibody described in any one of claims 12 to 18, which is an IgG antibody, preferably an IgG1 antibody.

20. An antibody described in any one of claims 12 to 19, comprising one or more antibody light chains.

21. A composition comprising an immunoglobulin region, domain, protein, or antibody described in any one of claims 1 to 20.

22. A pharmaceutical composition comprising an immunoglobulin region, domain, protein, or antibody according to any one of claims 1 to 20, and preferably a pharmaceutically acceptable excipient.

23. A nucleic acid encoding an immunoglobulin region, domain, protein, or antibody described in any one of claims 1 to 20.

24. A nucleic acid encoding an antibody described in any one of claims 12 to 20.

25. A recombinant host cell comprising the nucleic acid described in claim 23 or 24.

26. A method for producing the antibody of any one of claims 12 to 20, comprising: Providing a nucleic acid encoding a first heavy chain comprising a CH1 region according to any one of claims 1 to 3 or a domain according to claim 4, and CH2 and CH3 regions; providing a nucleic acid encoding a second heavy chain, wherein said first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; harvesting the antibody from the host cell culture; Clarifying the harvest; capturing the protein; performing anion exchange chromatography; and performing cation exchange chromatography to separate the antibody from other antibodies or antibody fragments; The method comprises producing the antibody by performing at least one of the following steps.

27. A method for producing the antibody of any one of claims 12 to 20, comprising: Providing a nucleic acid encoding a first heavy chain comprising a CH1 region according to any one of claims 1 to 3 or a domain according to claim 4, and CH2 and CH3 regions; providing a nucleic acid encoding a second heavy chain, wherein said first and second heavy chains can be the same or different; providing a nucleic acid encoding a light chain; introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and harvesting the antibody from the host cell culture; and A method comprising, in the separating step, separating the antibody from other antibodies or antibody fragments by isoelectric focusing on a gel.

28. A method for producing a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, said method comprising: providing nucleic acids encoding CH1, CH2, and CH3 regions of a first heavy chain and nucleic acids encoding CH1, CH2, and CH3 regions of a second heavy chain such that the isoelectric points of the first encoded heavy chain and the second encoded heavy chain are different, wherein at least one of the CH1 regions is - N201K; - N201D; - A172P, S190A and N201K; - A172P, S190A and N201D; - N201D and K213Q; - T120K and N201K; - N159K and N201K; - T120K, N201K and N159K; - N201D and N159D; or - N201D, K213Q (EU numbering) and Culturing the host cell to express the nucleic acid; and harvesting the multispecific antibody from the host cell culture using differences in isoelectric points; harvesting the antibody from the host cell culture; Clarifying the harvest; capturing the protein; performing anion exchange chromatography; and The method further comprises the step of performing cation exchange chromatography to separate said multispecific antibody from other antibodies or antibody fragments.

29. A method for purifying a multispecific antibody comprising a first heavy chain and a second heavy chain having different isoelectric points, the method comprising: providing nucleic acids encoding CH1, CH2, and CH3 regions of a first heavy chain and nucleic acids encoding CH1, CH2, and CH3 regions of a second heavy chain such that the isoelectric points of the first and second encoded heavy chains are different, wherein at least one of the regions is: - N201K; - N201D; - A172P, S190A and N201K; - A172P, S190A and N201D; - N201D and K213Q; - T120K and N201K; - N159K and N201K; - T120K, N201K and N159K; - N201D and N159D; or - N201D, K213Q (EU numbering) and Culturing the host cell to express the nucleic acid; and purifying said multispecific antibody from said host cell culture by isoelectric focusing to separate said multispecific antibody from other antibodies or antibody fragments.

30. The method described in claim 28 or 29, wherein the nucleic acids encoding the homomultimer of the first heavy chain, the homomultimer of the second heavy chain, and the heteromultimer of the first and second heavy chains are expressed as proteins with different isoelectric points, resulting in different retention times in ion exchange chromatography.

31. The method of any one of claims 28 to 30, wherein the first heavy chain and the second heavy chain comprise compatible CH3 heterodimerization regions.

32. The method described in claim 31, wherein one of the compatible CH3 heterodimerization regions contains the mutations L351D and L368E, and the other contains the mutations T366K and L351K.

33. The method of claim 31 or 32, wherein the first heavy chain comprises CH3 mutations L351D and L368E, and the second heavy chain comprises CH3 mutations T366K and L351K.

34. A method described in any one of claims 28 to 33, wherein the CH1, CH2 and CH3 regions are human CH1, CH2 and CH3 regions.

35. The method of any one of claims 28 to 33, wherein the CH1, CH2 and CH3 regions are human IgG CH1, CH2 and CH3 regions, preferably human IgG1 CH1, CH2 and CH3 regions.

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