Engineering monoclonal antibodies to improve stability and production titer

Abstract

To provide methods for improving monoclonal antibody (mAb) stability and production titer.SOLUTION: A method of increasing stability of a first antibody comprises substituting glycine, alanine or serine at heavy chain position 56 (AHo numbering) to create a second antibody. The second antibody is more stable than the unsubstituted first antibody.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 787,867, filed January 3, 2019, which is incorporated by reference herein in its entirety.

[0002] The subject matter presented herein relates to the field of protein engineering. In particular, the subject matter presented herein relates to antibody engineering, particularly monoclonal antibodies and their variant engineering, to improve their stability and production. [Background technology]

[0003] Recombinantly produced monoclonal antibodies (mAbs) (and their active fragments) are important therapeutic tools. However, due to the complexity of these molecules, there are numerous challenges that need to be met to facilitate their production, storage, and therapeutic administration.

[0004] Two challenges are involved in manufacturing and stability. mAbs are produced from engineered cells, such as Chinese hamster ovary (CHO) cells, in bioreactors. However, production levels are low and can vary between mAbs. Low production levels increase manufacturing costs, including resource requirements such as production time, labor, and components needed to run the bioreactor. Furthermore, lack of stability significantly impacts the "shelf-life" of mAbs. Degraded mAbs can be less potent, and fragmented mAbs can represent an immunological risk. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need to improve the stability and production titer of mAbs. [Means for solving the problem]

[0006] In a first aspect, provided herein is a method of increasing the stability of a first antibody, comprising substituting glycine, alanine, or serine at heavy chain position 56 (AHo numbering) to create a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody. For example, glycine or serine may be substituted at heavy chain position 56. For example, glycine or alanine may be substituted at heavy chain position 56. For example, glycine may be substituted at heavy chain position 56.

[0007] In a second aspect, provided herein is a method for increasing the stability of a first antibody, comprising substituting a hydrophobic amino acid at position 80 (AHo numbering) of the heavy chain of the first antibody to create a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody. Examples of hydrophobic amino acid residues include alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine. For example, the hydrophobic amino acid residue can comprise or consist of alanine, isoleucine, phenylalanine, leucine, methionine, or valine. For example, the hydrophobic amino acid residue can comprise or consist of phenylalanine, leucine, or valine.

[0008] In a third aspect, provided herein is a method of increasing the stability of a first antibody, comprising substituting alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine at position 80 (AHo numbering) of the heavy chain of the first antibody to create a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody. For example, phenylalanine, leucine, or valine may be substituted at position 80 of the heavy chain. For example, isoleucine or methionine may be substituted at position 80 of the heavy chain. For example, isoleucine may be substituted at position 80 of the heavy chain. For example, methionine may be substituted at position 80 of the heavy chain.

[0009] In subembodiments of these first three embodiments, the increased stability of the second antibody is evidenced by at least one selected from the group consisting of increased titer during cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature. In some subembodiments, the increased titer is measured by the rate of binding to a Protein A-coated probe tip using an Octet Forte Bio instrument; and / or the increased yield is measured by Protein A or Protein G capture; and / or the increased purity is measured by SEC of the purified protein; and / or the decrease in high molecular weight species is measured by size exclusion chromatography (SEC) and the area under the curve of each peak for each molecular weight; and / or the increased melting point temperature is measured by differential scanning fluorometry (DSF) or differential scanning calorimetry (DSC); and / or the increased aggregation temperature is measured by DSF; and / or the increased onset melting temperature is measured by DSF.

[0010] In some subembodiments of the first aspect, the second antibody is further substituted with a hydrophobic amino acid residue at position 80 (AHo numbering) of the heavy chain. For example, the hydrophobic amino acid residue can comprise or consist of: alanine, isoleucine, phenylalanine, leucine, methionine, or valine. For example, the hydrophobic amino acid residue can be selected from the group consisting of: phenylalanine, leucine, and valine. In some subembodiments of the first aspect, the second antibody is further substituted with a methionine at position 80 (AHo numbering), or alternatively, the second antibody is further substituted with an isoleucine at position 80 (AHo numbering). In some subembodiments of the first aspect, the second antibody is further substituted with an alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine at position 80 (AHo numbering). In some subembodiments of the first aspect, the second antibody further comprises a substitution at position 80 (AHo numbering) with phenylalanine, leucine, or valine.

[0011] In certain subembodiments of the second and third aspects, the second antibody is further substituted with glycine, alanine, or serine at position 56 (AHo numbering). In certain subembodiments of the second and third aspects, the second antibody is further substituted with glycine or alanine at position 56 (AHo numbering). In certain subembodiments of the second and third aspects, the second antibody is further substituted with glycine or serine at position 56 (AHo numbering). In certain subembodiments of the second and third aspects, the second antibody is further substituted with glycine at position 56 (AHo numbering).

[0012] In these first three aspects, the first antibody is a monoclonal antibody, such as a human antibody or a humanized antibody. Furthermore, the first antibody is an IgG antibody, such as an IgG antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 antibodies. That is, the IgG antibody can be an IgG1 antibody, the IgG antibody can be an IgG2 antibody, the IgG antibody can be an IgG3 antibody, and the IgG antibody can be an IgG4 antibody.

[0013] In a fourth aspect, provided herein is a method of increasing the stability of a first antibody variant, comprising substituting glycine, alanine, or serine at heavy chain position 56 (AHo numbering) to create a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant. For example, glycine or serine may be substituted at heavy chain position 56. For example, glycine or alanine may be substituted at heavy chain position 56. For example, glycine may be substituted at heavy chain position 56.

[0014] In a fifth aspect, provided herein is a method of increasing the stability of a first antibody variant, comprising substituting a hydrophobic amino acid residue (such as alanine, isoleucine, phenylalanine, leucine, methionine, or valine) at heavy chain position 80 (AHo numbering) of the first antibody variant to create a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant. For example, the hydrophobic amino acid residue may comprise or consist of: phenylalanine, leucine, or valine. For example, the hydrophobic amino acid residue may comprise or consist of methionine or isoleucine.

[0015] In a sixth aspect, provided herein is a method of increasing the stability of a first antibody variant, comprising substituting alanine, phenylalanine, isoleucine, leucine, methionine, or valine at position 80 of the heavy chain of the first antibody (AHo numbering) to create a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant. For example, phenylalanine, leucine, or valine may be substituted at position 80 of the heavy chain. For example, methionine may be substituted at position 80 of the heavy chain. For example, isoleucine may be substituted at position 80 of the heavy chain.

[0016] In sub-embodiments of these fourth, fifth, and sixth aspects, the increased stability of the second antibody variant is evidenced by at least one selected from the group consisting of increased titer during cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature. In some sub-embodiments, the increased titer is measured by the rate of binding to a Protein A-coated probe tip using an Octet Forte Bio instrument; and / or the increased yield is measured by Protein A or Protein G capture; and / or the increased purity is measured by SEC of the purified protein; and / or the decrease in high molecular weight species is measured by size exclusion chromatography (SEC) and the area under the curve of each peak for each molecular weight; and / or the increased melting point temperature is measured by differential scanning fluorometry (DSF) or differential scanning calorimetry (DSC); and / or the increased aggregation temperature is measured by DSF; and / or the increased melting temperature is measured by DSF.

[0017] In some sub-embodiments of the fourth aspect, the second antibody variant is further substituted with a hydrophobic amino acid residue at position 80 (AHo numbering) of the heavy chain. For example, the hydrophobic amino acid residue may be selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine. For example, the hydrophobic amino acid residue may be selected from the group consisting of: phenylalanine, leucine, and valine. In some sub-embodiments of the fourth aspect, the second antibody variant is further substituted with a methionine at position 80 (AHo numbering), or alternatively, the second antibody is further substituted with an isoleucine at position 80 (AHo numbering). In some sub-embodiments of the fourth aspect, the second antibody variant is further substituted with an alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine at position 80 (AHo numbering). In some sub-embodiments of the fourth aspect, the second antibody variant is further substituted with a phenylalanine, leucine, or valine at position 80 (AHo numbering).

[0018] In some sub-aspects of the fifth and sixth aspects, the second antibody variant is further substituted with glycine, alanine, or serine at position 56 (AHo numbering). For example, the second antibody variant may be substituted with glycine or alanine at position 56. For example, the second antibody variant may be substituted with glycine or serine at position 56. For example, the second antibody variant may be substituted with glycine at position 56.

[0019] In certain subembodiments of these fourth, fifth, and sixth aspects, the first antibody variant is a multispecific antibody, such as a bispecific or trispecific antibody. In certain subembodiments of these fourth, fifth, and sixth aspects, the first antibody variant is an antibody fragment capable of binding to an antigen; the antibody fragment may be selected from the group consisting of a Fab fragment, a Fab' fragment, an F'(ab)2 fragment, an Fv fragment, a single-chain antibody, a diabody, a biparatopic peptide, a domain antibody (dAb), a CDR-grafted antibody, a single-chain antibody (scFv), a single-chain antibody fragment, a chimeric antibody, a diabody, a triabody, a tetrabody, a minibody, a linear antibody; a chelating recombinant antibody, a tribody, a bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding domain immunoglobulin fusion protein, a single domain antibody, and a VHH-containing antibody.

[0020] Furthermore, in these fourth, fifth and sixth aspects, the first antibody variant is a monoclonal antibody variant, such as a human antibody variant or a humanized antibody variant. Furthermore, the first antibody variant is an IgG antibody variant, such as an IgG antibody variant selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibody variants. That is, the IgG antibody variant may be an IgG1 antibody variant, the IgG antibody variant may be an IgG2 antibody variant, the IgG antibody variant may be an IgG3 antibody variant, and the IgG antibody variant may be an IgG4 antibody variant.

[0021] In a seventh aspect, provided herein is a method of increasing the stability of a first antibody or first antibody variant, comprising: a. identifying the germline original amino acid sequence for the heavy chain of the antibody portion of the first antibody or antibody variant; b. identifying the amino acid residues at heavy chain position 56 (AHo numbering) and heavy chain position 80 (AHo numbering) of the antibody portion of the first antibody or antibody variant; and c. substituting residues identified from the germline original amino acid sequence of the antibody portion of the first antibody or antibody variant at heavy chain positions 56 and 80, thereby creating a second antibody or second antibody variant; Methods are provided wherein the second antibody is more stable than the unsubstituted first antibody; or wherein the second antibody variant is more stable than the unsubstituted first antibody variant.

[0022] In a subembodiment of this seventh aspect, the increased stability of the second antibody or second antibody variant is evidenced by at least one selected from the group consisting of increased titer during cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature. In a subembodiment of this seventh aspect, the increased stability of the second antibody or second antibody variant is evidenced by at least one selected from the group consisting of increased titer during cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature. In some sub-embodiments, the increase in titer is measured by the rate of binding to a Protein A coated probe tip using an Octet Forte Bio instrument; and / or the increase in yield is measured by Protein A or Protein G capture; and / or the increase in purity is measured by SEC of the purified protein; and / or the reduction in high molecular weight species is measured by size exclusion chromatography (SEC) and the area under the curve of each peak for each molecular weight; and / or the increase in melting point temperature is measured by differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC); and / or the increase in aggregation temperature is measured by DSF; and / or the increase in onset melting temperature is measured by DSF.

[0023] In this seventh aspect, the first antibody variant is a multispecific antibody, such as a bispecific or trispecific antibody. In certain sub-aspects of these fourth, fifth and sixth aspects, the first antibody variant is an antibody fragment capable of binding to an antigen; the antibody fragment may be selected from the group consisting of a Fab fragment, a Fab' fragment, an F'(ab)2 fragment, an Fv fragment, a single-chain antibody, a diabody, a biparatopic peptide, a domain antibody (dAb), a CDR-grafted antibody, a single-chain antibody (scFv), a single-chain antibody fragment, a chimeric antibody, a diabody, a triabody, a tetrabody, a minibody, a linear antibody; a chelating recombinant antibody, a tribody, a bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding domain immunoglobulin fusion protein, a single domain antibody and a VHH-containing antibody.

[0024] Furthermore, in this seventh aspect, the first antibody variant is a monoclonal antibody variant, such as a human antibody variant or a humanized antibody variant. Furthermore, the first antibody variant is an IgG antibody variant, such as an IgG antibody variant selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibody variants. That is, the IgG antibody variant may be an IgG1 antibody variant, the IgG antibody variant may be an IgG2 antibody variant, the IgG antibody variant may be an IgG3 antibody variant, and the IgG antibody variant may be an IgG4 antibody variant.

[0025] Further, in this seventh aspect, the first antibody is a monoclonal antibody, such as a human antibody or a humanized antibody. Furthermore, the first antibody is an IgG antibody, such as an IgG antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 antibodies. That is, the IgG antibody can be an IgG1 antibody, the IgG antibody can be an IgG2 antibody, the IgG antibody can be an IgG3 antibody, and the IgG antibody can be an IgG4 antibody.

[0026] In some subembodiments of any of the first through eighth aspects, the second antibody or second antibody variant has a substitution of any one of the following pairs of residues at positions 56 and 80 (AHo numbering) of the heavy chain: GF, GI, GL, GT, GV, AF, AI, AL, AV, AA, AM, SA, SI, or ST, respectively. By way of example, note that for this nomenclature, "GF" represents a "G" at position 56 in the heavy chain and an "F" at position 80 (AHo numbering) of the heavy chain. In some subembodiments of any of the first through eighth aspects, the second antibody or second antibody variant has a substitution of any one of the following pairs of residues at positions 56 and 80 (AHo numbering) of the heavy chain: GF, GL, GV, AF, AL, or AV, respectively. Such substitutions may have increased potency and / or a higher Tm compared to the first antibody (or first antibody variant). In some subembodiments of any of the first to eighth aspects, the second antibody or second antibody variant has a substitution of any one of the following pairs of residues at positions 56 and 80 (AHo numbering) of the heavy chain: AA, AL, AM, AV, GF, GL, GT, SA, or ST, respectively. Such substitutions may have higher potency compared to the first antibody (or first antibody variant). In some subembodiments of any of the first to eighth aspects, the second antibody or second antibody variant has a substitution of any one of the following pairs of residues at positions 56 and 80 (AHo numbering) of the heavy chain: AI, AV, GI, SI, or GV, respectively. Such substitutions may have higher Tm compared to the first antibody (or first antibody variant). In some subembodiments of any of the first to eighth aspects, the second antibody or second antibody variant has a substitution of any one of the following pairs of residues at positions 56 and 80 (AHo numbering) of the heavy chain: GF, GL, GT, GV, AF, AL, AV, AA, AM, AV, SA, and ST. Such substitutions may have higher potency compared to the first antibody (or first antibody variant). In some subembodiments of any of the first to eighth aspects, the second antibody or second antibody variant has a substitution of any one of the following pairs of residues at positions 56 and 80 (AHo numbering) of the heavy chain: GF, GI, GL, GV, AF, AL, AV, AI, or SI. Such substitutions may have a higher Tm compared to the first antibody (or first antibody variant).

[0027] In an eighth aspect, there is provided herein a method of producing a pharmaceutical composition formulated with a second antibody or second antibody variant produced by any of the preceding aspects.

[0028] In a ninth aspect, there is provided herein an antibody or antibody variant produced according to any of the first seven aspects.

[0029] In a tenth aspect, there is provided herein a pharmaceutical composition comprising an antibody or antibody variant produced according to any of the first seven aspects. [Brief explanation of the drawings]

[0030] [Figures 1A-1D] 1 is a series of graphs showing various characteristics of two monoclonal antibodies: an engineered monoclonal antibody and its parent monoclonal antibody. [Figure 2A] 1 is a graph showing that alanine and glycine at HC:56 had the highest potency for mAb1. The HC80 residue is shown in the row above the X-axis label. The HC56 residue is shown in the row below the X-axis label. [Figure 2B] 1 is a graph showing that for mAb1, phenylalanine, leucine, and valine at HC:80 had the highest potency. The HC56 residue is shown in the row above the X-axis label. The HC80 residue is shown in the row below the X-axis label. [Figure 3A] 1 is a graph showing that HC56 and HC80 mutants with high potency had high Tm for mAb1. HC80 residues are indicated in the column above the X-axis label. HC56 residues are indicated in the column below the X-axis label. [Figure 3B] 1 is a graph showing that for mAb1, molecules with phenylalanine, leucine, or valine at HC80 had a Tm greater than 65° C. The HC56 residue is shown in the column above the X-axis label. The HC80 residue is shown in the column below the X-axis label. [Figure 4]1 is a graph showing that for mAb1, the highest molecular weight (HMW) level, as determined by SEC, was less than 5%. The HC80 residue is shown in the column above the X-axis label. The HC56 residue is shown in the column below the X-axis label. [Figure 5] 1 is a graph showing that mAb2 is distinctly expressed with residues such as hydrophobic residues at HC56 and HC80. HC80 residues are shown in the column above the X-axis label. HC56 residues are shown in the column below the X-axis label. [Figure 6] 1 is a graph showing that Tm correlates with potency for substitutions at HC56 and HC80 of mAb2. HC80 residues are shown in the column above the X-axis label. HC56 residues are shown in the column below the X-axis label. [Figure 7]

[0023] Figure 1 is a graph showing high molecular weight species for substitutions at HC56 and HC80 of mAb2. HC80 residues are shown in the column above the X-axis label. HC56 residues are shown in the column below the X-axis label. DETAILED DESCRIPTION OF THE INVENTION

[0031] Surprisingly, when residue 56 (AHo numbering; residue 49 in Kabat numbering) of the antibody heavy chain of a mAb is altered to glycine, the antibody has higher potency and higher Tm in culture / production compared to molecules with the frequently observed alanine residue at that position. This effect is observed across multiple mAbs and germlines, and is independent of the germline identity of the residue. This observation contrasts with a study published by Mason et al. (Mason et al. 2012), which reported that alanine at residue 56 (AHo numbering) improves the expressed potency of an IgG4 mAb. Furthermore, potency is severely affected when a methionine residue is used instead of an isoleucine residue at heavy chain position 80 (AHo numbering) in a molecule-dependent manner.

[0032] definition The AHo numbering scheme is a structure-based numbering scheme that introduces gaps in the CDR regions to minimize deviations from the average structure of the aligned domains (Honegger & Pluckthun 2001). In the AHo numbering scheme, structurally equivalent positions in different antibodies will have the same residue number.

[0033] An "antibody" or "immunoglobulin" refers to a tetrameric glycoprotein consisting of two heavy chains and two light chains, each containing a variable domain (V) and a constant domain (C). "Heavy chain" and "light chain" refer to substantially full-length canonical immunoglobulin light and heavy chains; the variable domains of the heavy and light chains (VL and VC) constitute the V region of the antibody and contribute to antigen binding and specificity. "Antibodies" include monoclonal, polyclonal, chimeric, human, and humanized antibodies. Light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains are typically classified as μ (mu), δ (delta), γ (gamma), α (alpha), or ε (epsilon), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including IgM1 and IgM2. IgA is similarly further divided into subclasses including IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 additional amino acids. The variable regions of each light / heavy chain pair typically form the antigen-binding site. A "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0034] Antibody variants include antibody fragments and antibody-like proteins with alterations in the structure of a canonical tetrameric antibody. Exemplary antibody variants include V regions with alterations in the constant regions, or alternatively, V regions added to the constant regions, optionally in a non-canonical manner. Examples include multispecific antibodies (e.g., diabodies, bispecific antibodies), antibody fragments capable of binding to antigen (e.g., Fab', F'(ab)2, Fv, single-chain antibodies, diabodies, diabodies), biparatopic peptides and recombinant peptides containing the above, so long as they exhibit the desired biological activity.

[0035] Multispecific antibodies target two or more antigens or epitopes. For example, "bispecific," "dual-specific," or "bifunctional" antibodies are hybrid antibodies with two different antigen-binding sites. Bispecific antibodies can be generated by various methods, including hybridoma fusion or Fab' fragment linkage (Kostelny et al. 1992, Songsivilai & Lachmann 1990) (Kostelny et al. 1992, Songsivilai & Lachmann 1990, Wu & Demarest 2018). The two binding sites of a bispecific antibody each bind to a different epitope. Similarly, a trispecific antibody has three binding sites and binds to three epitopes. Several methods for generating trispecific antibodies are known and are under development (Wu & Demarest 2018, Wu et al. 2018).

[0036] Antibody fragments include, for example, Fab, Fab', F(ab'), Fv, domain antibodies (dAbs), complementarity-determining region (CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv), single-chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, linear antibodies; chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, single-domain antibodies (including camelized antibodies), VHH-containing antibodies, or variants or derivatives thereof, as well as polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding on the polypeptide, e.g., the antigen-binding portion of an antibody comprising one, two, three, four, five, or six CDR sequences, so long as the antibody retains the desired binding activity.

[0037] Overview of the method The methods disclosed herein include identifying residues at positions 56 and 80 (AHo numbering) of the heavy chain in an antibody (or modified antibody) or the germline native amino acid residues at these positions, altering (mutating) the residue at position 56 to glycine, alanine, or serine, and / or residue 80 to a hydrophobic residue (such as methionine or isoleucine) or to alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine, and assessing the stability of the modified antibody using any of a variety of techniques that measure various antibody properties. In some aspects, the residue at position 56 of the heavy chain is altered to glycine.

[0038] The following discussion is applicable not only to antibodies including IgG' (IgG1, IgG2, IgG3 and IgG4), but also to the antibody variants described in the "Definitions" section above.

[0039] Identifying residues at positions 56 and / or 80 (AHo numbering) In the first step, the amino acid residues at positions 56 and / or 80 (AHo numbering) in the heavy chain of the Ab are identified. If position 56 is already a glycine, no further identification is required, as the Ab does not require further manipulation at this position. In most cases, the antibody polynucleotide sequence is cloned and sequenced, and the sequence is then translated into an amino acid sequence. Alternatively, the relevant amino acid sequence from the antibody or a region thereof (e.g., a variable region) can be determined by direct protein sequencing. In some embodiments, if position 56 is already a glycine, alanine, or serine, no further identification is required, as the Ab does not require further manipulation at this position. In some embodiments, if position 56 is already a glycine or serine, no further identification is required, as the Ab does not require further manipulation at this position. In some embodiments, if position 56 is already a glycine or alanine, no further identification is required, as the Ab does not require further manipulation at this position. In some embodiments, if position 56 is already a glycine, no further identification is required, as the Ab does not require further manipulation at this position.

[0040] Alternatively, genomic or cDNA encoding a monoclonal antibody of interest or a binding fragment thereof can be isolated from cells that produce such an antibody (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody) and sequenced using conventional procedures.

[0041] DNA sequencing can be performed by any technique known in the art, such as the method described by Sanger et al. (Sanger et al. 1977) or high-throughput sequencing methods, such as pyrosequencing (Margulies et al. 2005, Nyren & Lundin 1985, Ronaghi et al. 1998), sequencing-by-synthesis (Bentley et al. 2008), ion semiconductor (Rothberg et al. 2011), single-molecule real-time sequencing (Eid et al. 2009), sequencing by oligo ligation detection (SOLiD) (Valouev et al. 2008) and nanopore sequencing (Branton et al. (Branton et al. 2008)).

[0042] Once the polynucleotide sequence is obtained, the open reading frame is determined and the amino acid sequence is deduced according to the genetic code. AHo numbering is applied to determine positions 56 and / or 80, and the amino acid residues are determined.

[0043] Direct protein sequencing of antibodies is also possible. Protein sequencing methods include, for example, by using mass spectrometry as well as the Edman degradation approach using a protein sequencer. In the case of Edman degradation, since the target antibody is likely to be longer than 50-70 amino acids, the antibody can be chemically digested using an endopeptidase (such as trypsin or pepsin) or using cyanogen bromide, BNPS-skatolet, formic acid, or chloramine T. The target fragment size for Edman degradation is 50-70 amino acids. Once the antibody has been fragmented, the peptides can be analyzed by automated methods using a protein sequenator that performs the Edman degradation reaction and reads each free amino acid by a detection method such as high-pressure liquid chromatography (HPLC). For mass spectrometry, antibodies are fragmented using a protease (commonly trypsin), the fragments are separated by liquid chromatography (LC), and the fragments are analyzed using mass spectrometry using a de novo peptide sequencing algorithm; this approach is discussed by Medzihradszky and Chalkley (Medzihradszky & Chalkley 2015).

[0044] As noted above, once the sequence is determined and AHo numbering is applied, the amino acids at positions 56 and / or 80 can be determined.

[0045] Manipulation of residues to target residues Regardless of the method selected to identify the residues at positions 56 and / or 80 of AHo in the antibody heavy chain, a determination is made at the polynucleotide level as to whether the residue should be mutated. In some embodiments, if the residue at position 56 is not a glycine, then the residue is a candidate for change. By way of example, in the most common scenario, the residue at position 56 is an alanine if it is not a glycine. Thus, an A56G mutation can be made. Similarly, for position 80, if the residue is not a hydrophobic residue (or is not alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine), then the residue is a candidate for change to a hydrophobic residue (or is not alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine, e.g., methionine). By way of example, in some subaspects, at this position (80), if the residue is not a methionine, then the residue is a candidate for change to methionine. As an example, in some embodiments, at position 80, if the residue is not isoleucine, then the residue is a candidate for change to isoleucine. At position 80, even if the residue is a hydrophobic residue, such as methionine or isoleucine, this position is still a candidate for change to a different hydrophobic residue (such as isoleucine or methionine, since either of these amino acids (Met, Ile) can increase expression and stability in culture, respectively). In some aspects, at position 80, the residue can be changed to alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine, or a hydrophobic residue (such as alanine, phenylalanine, isoleucine, leucine, or methionine), even if the residue is already alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine, or a different hydrophobic residue. If the residue at position 56 is not glycine, then the residue may also be a candidate for change, for example, to glycine. Examples of hydrophobic amino acid residues include alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and valine.In some embodiments, for any of the methods described herein, in the case of heavy chain position 80, the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

[0046] Any known method can be used to modify a polynucleotide encoding an antibody of interest. After determining the amino acid residues at positions 56 and / or 80, the nucleic acid sequence is modified so that glycine (or alanine or serine) is encoded at position 56, and / or alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine (or a hydrophobic amino acid residue, or methionine or isoleucine) is encoded at position 80. To make this change, the codons encoding the amino acids at positions 56 and / or 80 are identified, and one or more mutations are selected according to Table 1, which sets forth the genetic code.

[0047] [Table 1]

[0048] Most amino acids are coded for by more than one codon, as shown in Table 1. For example, alanine is coded for by four codons: GCU, GCC, GCA, and GCG; however, only Trp and Met are coded for by a single codon (TGG and ATG, respectively). When selecting one mutation or more than one mutation, considerations regarding, for example, codon bias can be explained by (Quax et al. 2015).

[0049] Mutations can be introduced into nucleotide sequences encoding the antibodies of this disclosure using standard techniques, including site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis, which result in targeted amino acid substitutions. Commercially available kits are also available for introducing mutations into nucleic acids, such as the GeneArt™ system and Phusion kit (ThermoFisher Scientific; Waltham, MA); Q5® Site-Directed Mutagenesis Kit (New England BioLabs; Ipswich, MA); and customized kits from Civic Bioscience (Montreal, Canada).

[0050] Alternatively, polynucleotide fragments can be synthesized using techniques known in the art and substituted into a polynucleotide comprising a full-length coding sequence. In some cases, the entire coding sequence with the targeted mutation is synthesized.

[0051] In any case, the amino acid mutation method is not particularly limited as long as it can effectively realize site mutation.

[0052] Cell Selection and Transfection with Engineered Polynucleotides Recombinant DNA methods for producing antibodies are well known. DNA encoding an antibody, for example, DNA encoding a VH domain, a VL domain, a single-chain variable fragment (scFv), or fragments thereof, and combinations thereof (target polynucleotide), can be inserted into a suitable expression vector, which can then be transfected into suitable host cells that do not otherwise produce antibodies, such as Escherichia coli cells, COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the desired antibody.

[0053] For example, suitable expression vectors containing a polynucleotide encoding a target polypeptide linked to a promoter are known in the art. Such vectors may include a nucleotide sequence encoding the constant region of an antibody molecule, and the variable domains of the antibody may be cloned into such a vector for expression of the heavy chain, the entire light chain, or both the entire heavy and light chains (or fragments thereof). The expression vector may be transferred to a host cell by conventional techniques, and the transfected cells may be cultured to produce the antibody.

[0054] Any cell line capable of expressing or engineered to express a functional antibody or antibody fragment can be used. For example, suitable mammalian cell lines include immortalized cell lines available from the American Type Culture Collection (Manassas, VA), including Chinese hamster ovary (CH) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human stem cell carcinoma cells (e.g., Hep G2), and human epithelial kidney 293 cells. Furthermore, cell lines or host systems can be selected to ensure correct modification and processing of the antibody. Eukaryotic host cells that possess the cellular machinery for proper processing of the primary product, glycosylation, and phosphorylation of the gene product can be used. These include CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7030, and HsS78Bst cells. Human cell lines developed by immortalizing human lymphocytes can also be used. For recombinant production of monoclonal antibodies, the human cell line PER.C6® (Janssen; Titusville, NJ) can be used. Examples of non-mammalian cells that can also be used include insect cells (e.g., Sf21 / Sf9, Trichoplusia ni Bti-Tn5bl-4), or yeast cells (e.g., Saccharomyces (e.g., S. cerevisiae, Pichia spp., etc.)), plant cells, or chicken cells.

[0055] Antibodies can be stably expressed in cell lines using conventional methods. Stable expression can be used for long-term, high-yield production of recombinant proteins. For stable expression, host cells can be transformed with an appropriate engineered vector containing expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and a selectable marker gene. Methods for generating high-yield, stable cell lines are known in the art, and reagents are commercially available. Transient expression can also be performed using conventional methods.

[0056] Antibody-expressing cell lines can be maintained in cell culture media and under culture conditions that allow antibody expression and production. Cell culture media can be based on commercially available media preparations, including, for example, DMEM or Ham's F12. Furthermore, cell culture media can be modified to support increases in both cell growth and biological protein expression. Of course, cell culture media can be optimized for specific cell cultures, including cell culture growth media formulated to promote cell growth or cell production media formulated to promote recombinant protein production.

[0057] Numerous cell culture media and cell culture nutrients and supplements are known. For example, suitable defined media include Dulbecco's Modified Eagle's Medium (DMEM), DME / F12, Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F-10, F-12, α-Minimum Essential Medium (α-MEM), Glasgow's Minimum Essential Medium (G-MEM), PF CHO, and Iscove's Modified Dulbecco's Medium. Other examples of basal media that can be used include BME Basal Medium and Dulbecco's Modified Eagle's Medium.

[0058] Basal media can be serum-free, meaning that the media does not contain serum (e.g., fetal bovine serum (FBS)) or is an animal protein-free medium or chemically defined. Basal media can be modified to remove certain non-nutritional components found in basal media, such as various inorganic and organic buffers, surfactants, and sodium chloride. Cell culture media can contain a basal cell culture medium (modified or unmodified) and at least one of the following: an iron source, recombinant growth factors, buffers, surfactants; an osmolality adjuster; an energy source; and non-animal hydrolysates. Additionally, modified basal cell culture media can optionally contain certain amino acids, vitamins, or a combination of both amino acids and vitamins. Modified basal media can further contain glutamine, e.g., L-glutamine, and / or methotrexate.

[0059] purification Once produced, the antibodies can be purified by conventional methods, such as by chromatography (e.g., by ion exchange, affinity, particularly affinity for the specific antigen, Protein A, Protein G, or size exclusion column chromatography), centrifugation, absorbance differential solubility, or any other standard technique for protein purification. Additionally, antibodies can be fused to heterologous polypeptide sequences ("tags") to facilitate purification.

[0060] Evaluate stability Titer in culture Supernatants from cell cultures expressing engineered polynucleotides can be analyzed using, for example, the Octet® platform instrument (Pall ForteBio; Fremont, CA). The Octet® platform offers biosensors for a number of different analytes, including biosensors for anti-human IgG quantitation (AHQ), anti-mouse IgG quantitation (AMQ), anti-FLAG (FLG), protein A (ProA), protein G (ProG), protein L (ProL), anti-Penta-His (HIS), streptavidin (SA), anti-human Fab-cH1 (FAb), anti-GST (GST), and Ni-NTA (NTA). Other options include traditional ELISA formats as well as HPLC and radioimmunoassays (RIA).

[0061] Melting temperature (differential scanning fluorescence (DSF) and differential scanning calorimetry (DSC)) Thermal stability by differential scanning fluorometry (DSF) (also known as protein thermal shift assay; (Lo et al. 2004, Pantoliano et al. 2001, Semisotnov et al. 1991)) can be conventionally determined using the protein's melting temperature, Tm. DSF utilizes a fluorescent dye that preferentially binds to unfolded (denatured) proteins. Real-time polymerase chain reaction (PCR) instruments are often used to monitor heat-induced protein denaturation by measuring changes in the dye's fluorescence. Examples of useful dyes include SYPRO® Orange (Thermo Fisher Scientific; Waltham, MA), 8-anilinonaphthalene-1-sulfonic acid (ANS), N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM), and 4-(dicyanovinyl)julolidine (DCVJ). The method of Lo et al. is often used with commonly used RE-PCR instruments and SYPRO® Orange (Lo et al 2004).

[0062] Generally, the dye and the protein to be analyzed are mixed, a melting curve is determined, and the Tm is calculated from the melting curve.

[0063] In addition to DSF, any known technique for determining the Tm of a protein can be used. For example, techniques that utilize intrinsic fluorescent signals (e.g., from tryptophan) as well as various modes of monitoring protein folding / unfolding, such as light scattering, can be used. Other techniques include rapid parallel proteolysis (Minde et al., 2012) and cellular thermal shift assays (Jafari et al., 2014).

[0064] Differential scanning calorimetry (DSC) can also be used to determine the Tm of a protein (Makhatadze 1998), but DSC can also provide information on the mode of unfolding, allowing multiple Tm values ​​to be obtained.

[0065] Generally, the spectrum of the subject protein is determined using a spectrophotometer to quantitate the protein (or alternatively, the protein is quantitated using a different method). The protein is then subjected to the DSC program of the spectrophotometer.

[0066] Yield (measured by antibody captured by Protein A) A common process for purifying antibodies from clarified cell culture supernatant involves protein A affinity chromatography followed by a capture step using a combination of anion and cation exchange chromatography (Fahrner et al 2001, Kelley 2009).

[0067] Protein quantification can be performed using any method known in the art. These include UV-Vis spectroscopy, which detects the protein backbone based on the absorbance of tryptophan and tyrosine residues at 280 nm (A280) (or alternatively, the absorbance at 205 nm (A205)); Bradford assay (usually based on the use and absorbance of Coomassie Brilliant Blue G-250 dye); Biuret test-derived assays, such as the Lowry and Bicinchoninic Acid (BCA) assays; amino acid analysis (which relies on the direct detection of modified amino acids); gel electrophoresis (observed by gel band intensity) or dye-labeling of proteins (thus correlating detection of the dye signal to protein amount), examples of dyes being fluorescamine and amido black 10B. In addition, HPLC and LC / MS methods can also be used.

[0068] To store samples and facilitate measurements, a NanoDrop spectrophotometer, available from Thermo Fisher Scientific (Wilmington, DE), for example, can be used. This device facilitates several approaches to protein quantitation.

[0069] High molecular weight (HMW) species and main peak (MP) species determined by size exclusion column chromatography (SEC) A common process for comparing species of Protein A eluted material for SEC is used, in which the protein is passed through a column of porous beads of dextran polymer to separate species based on size. The percentage of HMW to MP is determined by measuring the area under the curve of the SEC peak.

[0070] Coagulation (coagulation T) and onset of melting (onset of melting T or onset of melting T) The aggregation T is measured by differential scanning fluorometry (DSF), which measures the temperature at which 30 nm aggregate particles are formed using an excitation wavelength of 300 nm and emission of 350 / 330 nm.

[0071] The melting onset T is measured by DSF, measuring fluorescence at 350 / 330 nm. The onset T is the temperature at which the first derivative of 350 / 330 nm emission rises above the basal level, representing the level at which the folded protein begins to unfold.

[0072] Pharmaceutical composition formulations and ingredients The antibodies and antibody variants produced by the methods disclosed herein can be formulated into pharmaceutical compositions suitable for administration to a patient.

[0073] Acceptable pharmaceutical ingredients are preferably non-toxic to patients at the dosages and concentrations employed. Pharmaceutical compositions can include agents to alter, maintain, or protect, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition.

[0074] In general, excipients can be classified based on the mechanism by which they stabilize proteins against various chemical and physical stresses. Some excipients mitigate the effects of specific stresses or modulate the specific susceptibility of specific polypeptides. Other excipients have a more general effect on the physical and covalent stability of proteins.

[0075] Common excipients for liquid and lyophilized protein formulations are listed in Table 2 (see also (Kamerzell et al 2011)).

[0076] [Table 2]

[0077] [Table 3]

[0078] Other excipients are known in the art (see, e.g., (Powell et al 1998)). One of skill in the art can determine what amount or range of excipients may be included in any particular preparation to obtain a biopharmaceutical composition of the invention that promotes the retention of stability of the biopharmaceutical. For example, the amount and type of salt to include in a biopharmaceutical composition of the invention can be selected based on the desired osmolality (i.e., isotonic, hypotonic, or hypertonic) of the final solution and the amount and osmolality of other components to be included in the preparation.

[0079] Embodiment The following embodiments are presented as non-limiting examples of the methods disclosed herein. An Examples section follows this Embodiments section.

[0080] Embodiment 1. A method of increasing the stability of a first antibody, comprising substituting glycine, alanine, or serine at position 56 (AHo numbering) of the heavy chain to create a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody. For example, glycine may be substituted at position 56 of the heavy chain. For example, glycine or alanine may be substituted at position 56 of the heavy chain. For example, glycine or serine may be substituted at position 56 of the heavy chain.

[0081] Embodiment 2 The method of embodiment 1, wherein a glycine is substituted at heavy chain position 56.

[0082] Embodiment 3 The method of any one of embodiments 1 or 2, wherein the second antibody is further substituted with a hydrophobic amino acid residue at heavy chain position 80 (AHo numbering).

[0083] Embodiment 4. The method of embodiment 3, wherein the hydrophobic amino acid residue is selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine.

[0084] Embodiment 5. The method of embodiment 3, wherein the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

[0085] Embodiment 6 The method of any one of embodiments 1 or 2, wherein the second antibody is further substituted with a methionine at position 80 (AHo numbering).

[0086] Embodiment 7 The method of any one of embodiments 1 or 2, wherein the second antibody is further substituted with isoleucine at position 80 (AHo numbering).

[0087] Embodiment 8. A method of increasing the stability of a first antibody, comprising substituting a hydrophobic amino acid residue at position 80 (AHo numbering) of the heavy chain of the first antibody to create a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody.

[0088] Embodiment 9. The method of embodiment 8, wherein the hydrophobic amino acid residue is selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine.

[0089] Embodiment 10. The method of embodiment 8, wherein the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

[0090] Embodiment 11. A method of increasing the stability of a first antibody, comprising substituting alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine at position 80 of the heavy chain of the first antibody (AHo numbering) to create a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody.

[0091] Embodiment 12 The method of embodiment 11, wherein a methionine is substituted at position 80 of the heavy chain of the first antibody.

[0092] Embodiment 13 The method of embodiment 11, wherein an isoleucine is substituted at position 80 of the heavy chain of the first antibody.

[0093] Embodiment 14 The method of any one of embodiments 8 to 13, wherein the second antibody is further substituted with alanine, glycine, or serine at heavy chain position 56 (AHo numbering).

[0094] Embodiment 15. The method of any one of embodiments 8 to 13, wherein the second antibody is further substituted with alanine or glycine at heavy chain position 56 (AHo numbering).

[0095] Embodiment 16 The method of any one of embodiments 8 to 13, wherein the second antibody is further substituted with glycine at heavy chain position 56 (AHo numbering).

[0096] Embodiment 17. The method of any one of embodiments 1 to 16, wherein the increased stability of the second antibody is evidenced by at least one selected from the group consisting of increased titer during cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature.

[0097] Embodiment 18. The method of embodiment 17, wherein the increase in titer is measured by the rate of binding to a Protein A coated probe tip using an Octet Forte Bio instrument.

[0098] Embodiment 19. The method of embodiment 17, wherein the increase in yield is measured by Protein A or Protein G capture.

[0099] Embodiment 20. The method of embodiment 17, wherein the increase in purity is measured by size exclusion column chromatography (SEC) of the purified antibody.

[0100] Embodiment 21. The method of embodiment 17, wherein the reduction of high molecular weight species is measured by size exclusion column chromatography (SEC) and the area under the curve for each peak at each molecular weight.

[0101] Embodiment 22. The method of embodiment 17, wherein the increase in melting point temperature is measured by differential scanning fluorescence (DSF) or differential scanning calorimetry (DSC).

[0102] Embodiment 23. The method of embodiment 17, wherein the increase in aggregation temperature is measured by DSF.

[0103] Embodiment 24 The method of embodiment 17, wherein the increase in onset melting temperature is measured by DSF.

[0104] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the first antibody is a monoclonal antibody.

[0105] Embodiment 26 The method of any one of embodiments 1 to 25, wherein the first antibody is a human monoclonal antibody or a humanized monoclonal antibody.

[0106] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the first antibody is an IgG antibody.

[0107] Embodiment 28. The method of embodiment 27, wherein the IgG antibody is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibodies.

[0108] Embodiment 29. The method of embodiment 27, wherein the IgG antibody is an IgG1 antibody.

[0109] Embodiment 30. The method of embodiment 27, wherein the IgG antibody is an IgG2 antibody.

[0110] Embodiment 31 The method of embodiment 27, wherein the IgG antibody is an IgG3 antibody.

[0111] Embodiment 32 The method of embodiment 27, wherein the IgG antibody is an IgG4 antibody.

[0112] Embodiment 33. A method of increasing the stability of a first antibody variant, comprising substituting glycine, alanine, or serine at heavy chain position 56 (AHo numbering) to create a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant.

[0113] Embodiment 34 The method of embodiment 33, wherein a glycine is substituted at heavy chain position 56.

[0114] Embodiment 35 The method of any one of embodiments 33 or 34, wherein the second antibody is further substituted with a hydrophobic amino acid residue at heavy chain position 80 (AHo numbering).

[0115] Embodiment 36. The method of embodiment 35, wherein the hydrophobic amino acid residue is selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine.

[0116] Embodiment 37. The method of embodiment 35, wherein the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

[0117] Embodiment 38. A method of increasing the stability of a first antibody variant, comprising substituting a hydrophobic amino acid residue at heavy chain position 80 (AHo numbering) of the first antibody variant to create a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody.

[0118] Embodiment 39. The method of embodiment 38, wherein the hydrophobic amino acid residue is selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine.

[0119] Embodiment 40. The method of embodiment 38, wherein the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

[0120] Embodiment 41. A method of increasing the stability of a first antibody variant, comprising substituting alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine at heavy chain position 80 (AHo numbering) of the first antibody variant to create a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant.

[0121] Embodiment 42 The method of embodiment 41, wherein a methionine is substituted at heavy chain position 80 of the first antibody variant.

[0122] Embodiment 43 The method of embodiment 41, wherein an isoleucine is substituted at heavy chain position 80 of the first antibody variant.

[0123] Embodiment 44 The method of any one of embodiments 38 to 43, wherein the second antibody variant further comprises a substitution at heavy chain position 56 (AHo numbering) with alanine, glycine, or serine.

[0124] Embodiment 45 The method of any one of embodiments 38 to 43, wherein the second antibody variant is further substituted with alanine or glycine at heavy chain position 56 (AHo numbering).

[0125] Embodiment 46 The method of any one of embodiments 38 to 43, wherein the second antibody variant is further substituted with a glycine at heavy chain position 56 (AHo numbering).

[0126] Embodiment 47. The method of any one of embodiments 33 to 46, wherein the increased stability of the second antibody variant is evidenced by at least one selected from the group consisting of increased titer in cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature.

[0127] Embodiment 48. The method of embodiment 47, wherein the increase in titer is measured by the rate of binding to a Protein A coated probe tip using an Octet Forte Bio instrument.

[0128] Embodiment 49. The method of embodiment 47, wherein the increase in yield is measured by Protein A or Protein G capture.

[0129] Embodiment 50. The method of embodiment 47, wherein the increase in purity is measured by SEC of the purified antibody.

[0130] Embodiment 51. The method of embodiment 47, wherein the reduction in high molecular weight species is measured by SEC and the area under the curve for each peak at each molecular weight.

[0131] Embodiment 52 The method of embodiment 47, wherein the increase in melting point temperature is measured by DSF or DSC.

[0132] Embodiment 53. The method of embodiment 47, wherein the increase in aggregation temperature is measured by DSF.

[0133] Embodiment 54 The method of embodiment 47, wherein the increase in onset melting temperature is measured by DSF.

[0134] Embodiment 55. The method of any one of embodiments 33 to 54, wherein the first antibody variant is a multispecific antibody.

[0135] Embodiment 56 The method of embodiment 55, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0136] Embodiment 57. The method of any one of embodiments 33 to 56, wherein the first antibody variant is an antibody fragment capable of binding to the antigen.

[0137] Embodiment 58. The method of embodiment 57, wherein the antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, an F'(ab)2 fragment, an Fv fragment, a single-chain antibody, a diabody, a biparatopic peptide, a domain antibody (dAb), a CDR-grafted antibody, a single-chain antibody (scFv), a single-chain antibody fragment, a chimeric antibody, a diabody, a triabody, a tetrabody, a minibody, a linear antibody; a chelating recombinant antibody, a tribody, a bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding domain immunoglobulin fusion protein, a single-domain antibody, and a VHH-containing antibody.

[0138] Embodiment 59. The method of any one of embodiments 33 to 58, wherein the first antibody variant is a human monoclonal antibody or a humanized monoclonal antibody variant.

[0139] Embodiment 60. The method of any one of embodiments 33 to 59, wherein the first antibody mutant is an IgG antibody mutant.

[0140] Embodiment 61 The method of embodiment 60, wherein the IgG antibody variant is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibody variants.

[0141] Embodiment 62 The method of embodiment 61, wherein the IgG antibody variant is an IgG1 antibody variant.

[0142] Embodiment 63 The method of embodiment 61, wherein the IgG antibody variant is an IgG2 antibody variant.

[0143] Embodiment 64 The method of embodiment 61, wherein the IgG antibody variant is an IgG3 antibody variant.

[0144] Embodiment 65 The method of embodiment 61, wherein the IgG antibody variant is an IgG4 antibody variant.

[0145] Embodiment 66. A method for increasing the stability of a first antibody or first antibody variant, comprising: a. identifying the germline original amino acid sequence for the heavy chain of the antibody portion of the first antibody or antibody variant; b. identifying the amino acid residues at heavy chain position 56 (AHo numbering) and heavy chain position 80 (AHo numbering) of the antibody portion of the first antibody or antibody variant; and c. substituting residues identified from the germline original amino acid sequence of the antibody portion of the first antibody or antibody variant at heavy chain positions 56 and 80, thereby creating a second antibody or second antibody variant; Methods are provided wherein the second antibody is more stable than the unsubstituted first antibody; or wherein the second antibody variant is more stable than the unsubstituted first antibody variant.

[0146] Embodiment 67. The method of embodiment 66, wherein the increased stability of the second antibody variant or second antibody variant is evidenced by at least one selected from the group consisting of increased titer in cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature.

[0147] Embodiment 68. The method of embodiment 67, wherein the increase in titer is measured by the rate of binding to a Protein A coated probe tip using an Octet Forte Bio instrument.

[0148] Embodiment 69. The method of embodiment 67, wherein the increase in yield is measured by Protein A or Protein G capture.

[0149] Embodiment 70. The method of embodiment 67, wherein the increase in purity is measured by SEC of the purified protein.

[0150] Embodiment 71. The method of embodiment 67, wherein the reduction in high molecular weight species is measured by SEC and the area under the curve for each peak at each molecular weight.

[0151] Embodiment 72. The method of embodiment 67, wherein the increase in melting point temperature is measured by DSF or DSC.

[0152] Embodiment 73. The method of embodiment 67, wherein the increase in aggregation temperature is measured by DSF.

[0153] Embodiment 74 The method of embodiment 67, wherein the increase in onset melting temperature is measured by DSF.

[0154] Embodiment 75. The method of any one of embodiments 66 to 74, wherein the first antibody is a monoclonal antibody.

[0155] Embodiment 76 The method of embodiment 75, wherein the first antibody is a human monoclonal antibody or a humanized monoclonal antibody.

[0156] Embodiment 77. The method of any one of embodiments 66 to 76, wherein the first antibody is an IgG antibody.

[0157] Embodiment 78. The method of embodiment 77, wherein the IgG antibody is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibodies.

[0158] Embodiment 79. The method of embodiment 78, wherein the IgG antibody is an IgG1 antibody.

[0159] Embodiment 80. The method of embodiment 78, wherein the IgG antibody is an IgG2 antibody.

[0160] Embodiment 81 The method of embodiment 78, wherein the IgG antibody is an IgG3 antibody.

[0161] Embodiment 82. The method of embodiment 78, wherein the IgG antibody is an IgG4 antibody.

[0162] Embodiment 83 The method of any one of embodiments 66 to 82, wherein the first antibody variant is a multispecific antibody.

[0163] Embodiment 84 The method of embodiment 83, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0164] Embodiment 85. The method of any one of embodiments 66 to 84, wherein the first antibody variant is an antibody fragment capable of binding to the antigen.

[0165] Embodiment 86. The method of embodiment 85, wherein the antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, an F'(ab)2 fragment, an Fv fragment, a single-chain antibody, a diabody, a biparatopic peptide, a domain antibody (dAb), a CDR-grafted antibody, a single-chain antibody (scFv), a single-chain antibody fragment, a chimeric antibody, a diabody, a triabody, a tetrabody, a minibody, a linear antibody; a chelating recombinant antibody, a tribody, a bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding domain immunoglobulin fusion protein, a single-domain antibody, and a VHH-containing antibody.

[0166] Embodiment 87. The method of any one of embodiments 66 to 86, wherein the first antibody variant is a human monoclonal antibody or a humanized monoclonal antibody variant.

[0167] Embodiment 88. The method of any one of embodiments 66 to 87, wherein the first antibody mutant is an IgG antibody mutant.

[0168] Embodiment 89. The method of any one of embodiments 66 to 88, wherein the IgG antibody variant is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibody variants.

[0169] Embodiment 90. The method of embodiment 89, wherein the IgG antibody variant is an IgG1 antibody variant.

[0170] Embodiment 91 The method of embodiment 89, wherein the IgG antibody variant is an IgG2 antibody variant.

[0171] Embodiment 92 The method of embodiment 89, wherein the IgG antibody variant is an IgG3 antibody variant.

[0172] Embodiment 93 The method of embodiment 89, wherein the IgG antibody variant is an IgG4 antibody variant.

[0173] Embodiment 94. The method of any one of embodiments 1 to 93, wherein the second antibody or second antibody variant is substituted with any one of the following pairs of residues at positions 56 and 80 (AHo numbering) of the heavy chain: GF, GI, GL, GT, GV, AF, AI, AL, AV, AA, AM, SA, SI, or ST, respectively. By way of example, note that under this nomenclature, "GF" represents "G" at heavy chain position 56 and "F" at heavy chain position 80 (AHo numbering).

[0174] Embodiment 95 The method of any one of embodiments 1 to 94, further comprising formulating the second antibody or second antibody variant into a pharmaceutical composition.

[0175] Embodiment 96. An antibody or antibody variant produced by the method of any one of embodiments 1 to 95.

[0176] Embodiment 97. A pharmaceutical composition comprising the antibody or antibody variant of embodiment 96.

[0177] The Examples section below is offered by way of example only and is not intended to limit the disclosure or claims herein in any way. [Example]

[0178] Example 1 - Experimental Design Antibody mutations were performed by designing codon changes and synthesizing nucleotides accordingly. Modified fragments were integrated into the open reading frame using the Golden Gate cloning method (Engler et al., 2009, Engler et al., 2008). Antibodies were expressed in HEK 293-6E cells (a suspension cell line expressing a truncated mutant of Epstein-Barr virus nuclear antigen (EBNA) 1) unless otherwise noted (Durocher et al., 2002). The resulting antibodies were purified using Protein A attached to a solid support.

[0179] The following germline families were tested: VH1 / VK4, VH4 / VL1, VH3 / VK3, VH6 / VK1 and VH2 / VL2. Within the heavy chain, 56A and 56G were tested, as well as 80I and 80M.

[0180] The parameters measured were: Potency at time of collection (mg / L) Melting temperature (Tm; measured by differential scanning fluorometry (DSF) or differential scanning calorimetry (DSC), the latter yielding two outputs: Tm1 and Tm2) Yield (measured by antibody captured by Protein A) High molecular weight (HMW) species and peak molecular weight (defined as the molecular weight of the highest peak, Mp) determined by size exclusion column chromatography (SEC)

[0181] Example 2 – VH1 germline In this example, mAb1 (IgG1) derived from the VH1 germline (VH1 / VK4) mutations within the antibody were tested, and the antibody was assayed as described in Example 1.

[0182] [Table 4]

[0183] From these experiments, when mAb1 had an A at position 56 and an M at position 80, the antibody (mAb1AM) had the lowest potency observed of the antibodies tested. Similarly, mAb1AI had an even lower potency. However, mAb1GM had the highest potency observed of the antibodies tested; mAb1GI had the second-highest potency.

[0184] Example 3 – VH3 germline In this example, mutations within the antibody, mAb2 (IgG2 Ab) originating from the VH3 germline (VH3 / VK3), were tested and the antibody was assayed as described in Example 1.

[0185] [Table 5]

[0186] From these experiments, when mAb2 had an A at position 56 and an M at position 80, the antibody (mAb2AM) had the lowest potency and the lowest melting temperature (Tm) observed among the antibodies tested, suggesting that this antibody was less stable. However, mAb2AI and mAb2GM had higher potency and melting temperatures than the antibodies tested.

[0187] Example 4 – VH4 germline In this example, mutations within the antibody, mAb3 (IgG1 Ab) originating from the VH4 germline (VH4 / VL1), were tested and the antibody was assayed as described in Example 1.

[0188] [Table 6]

[0189] From these experiments, when mAb3 had an A at position 56 and an M at position 80, the antibody (mAb3AMmAb3AI) had the lowest potency and the lowest melting temperature (Tm) observed among the antibodies tested, suggesting that this antibody was less stable. However, mAb3 and mAb3GM had higher potency and melting temperatures than the antibodies tested.

[0190] Example 5 – VH6 germline In this example, mutations within the antibody, mAb4 (IgG1 Ab) originating from the VH6 germline (VH6 / VK1), were tested and the antibody was assayed as described in Example 1.

[0191] [Table 7]

[0192] From these experiments, when mAb4 had an A at position 56 and an M at position 80, the antibody (mAb4AM) had the lowest potency observed of the antibodies tested; the melting temperature was not determined. However, mAb4(GI) (germline) and mAb4GM had higher potency and melting temperatures of the antibodies tested, with mAb4GI being superior to mAb4GM.

[0193] Example 6 – VH2 germline In this example, mAb5 (IgG1 Ab) originating from the VH2 germline (VH2 / VL2), mutations within the antibody, were tested and the antibody was assayed as described in Example 1.

[0194] [Table 8]

[0195] From these experiments, the antibodies tested had both high potency and favorable melting temperatures.

[0196] When these antibodies were expressed in Chinese hamster ovary (CHO) cells, mAb5GM ("mutant" in Figure 1) had higher titers (Figure 1A), fewer HMW species (Figure 1B), and higher MP purity (Figure 1C) across clones and MTX levels compared to mAb5(AI) (germline; "WT" in Figure 1).

[0197] Example 7 – Comparison of antibody titers by IgG subtype and HC56 and HC80 mutations The harvest titers of mAb1 and mAb3 (the latter within a different subtype) were determined. Table 7.1 shows the results. In this experiment, the heavy chain variant HC:56G, HC:80M exhibited the most desirable final growth characteristics. The HC:56G, HC:80M variant also produced higher titers (day 6) in IgG1, IgG2, and IgG4 molecules, with the exception of mAb3 (IgG4 format); instead, this molecule exhibited the highest titer from the HC:56A, HV:80M set of substitutions.

[0198] [Table 9]

[0199] Example 8 – Amino acid identification for IgG1 HC:56 and HC:80 In this example, additional amino acid options for use in IgG1 HC:56 and HC:80 were identified using two methods: one based on precise phylogenetic analysis and screening, and the other based on Rosetta modeling of all 400 H:56 H:80 combinations. Note that these experiments provide additional testing and analysis of potential options in HC:56 and HC:80. Amino acid pairings with moderate results in one or more computational screens were mutated into model molecules, followed by comparison of expression levels after harvest from 293-6E cell culture with DSF and Tm.

[0200] For phylogenetic analysis, a blast search of mAb1 and mAb2 was performed in Protein NR (e = 0.0001). Approximately 10,000 sequences identified for each molecule were CD-hit clustered to identify the most common residues and ranked according to frequency. The top amino acids were cloned into mAb1 and mAb2.

[0201] For the phylogenetic analysis of mAb1, the most common residue pairs are shown in Table 8.1 below (residue pairs in bold were tested; asterisks indicate germline residues):

[0202] [Table 10]

[0203] For the phylogenetic analysis of mAb2, the most common residue pairs are shown in Table 8.2 below (residue pairs in bold were tested);

[0204] [Table 11]

[0205] For Rosetta analysis, Rosetta software was used to measure the energy scores of all HC:56 and HC:80 amino acid combinations in mAb1 and mAb2. Without being limited by theory, under this approach, Rosetta tried all 400 possible H56:H80 amino acid combinations using standard genetically encoded amino acids. From the Rosetta analysis, HC:56 and HC:80 amino acid combinations were compared according to Rosetta "total score" and "p_aa_pp" scores and ranked by a 1:1 weighted z-score. Variants with favorable energy scores not previously present in the phylogenetic analysis were cloned into mAb1 and mAb2 for further testing.

[0206] For HC:56, results from mAb1 indicate that the highest titers were achieved with HC:56G or HC:56A, followed by HC:56S (see, e.g., Figures 2A-2B). Results from mAb2 indicate that the highest titers were achieved with HC:56A, HC:56G, or HC:56S (see, e.g., Figure 5). Without being limited by theory, it is noted that these residues are most similar in terms of their relatively small size when compared to the full range of amino acids.

[0207] For HC:80, results from mAb1 indicate that the highest titers were achieved using HC:80F, HC:80L, or HC:80V. Results from mAb2 indicate that the highest titers were achieved using HC:80L, HC:80M, HC:80A, HC:80V, HC:80F, and HC:80I. Furthermore, in two cases, HC:80T produced significantly higher expression. Without being limited by theory, a generalization of these results is that the use of hydrophobic residues in HC:80 produces higher titers.

[0208] Among the engineered variants of mAb1, the six variants with the highest potency correlated with the six variants with the highest Tm (see, e.g., Figures 2A-2B and 3A-3B). Among the variants of mAb2, the differences in Tm were less pronounced, although the low-potency variants continued to have low Tm (see, e.g., Figure 6). Furthermore, the use of hydrophobic residues at HC:80 correlated with higher Tm.

[0209] The combined effects of residues at heavy chain positions 56 and 80 (AHo numbering) in mAb1 and mAb2, respectively, are summarized in Table 8.3 below. In the nomenclature of Table 8, the first residue represents heavy chain position 56 (AHo numbering) and the second residue represents heavy chain position 80 (AHo numbering). Thus, as an example, note that "GF" represents "G" at heavy chain position 56 (AHo numbering) and "F" at heavy chain position 80 (AHo numbering):

[0210] [Table 12]

[0211] References Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, et al. 2008. Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53-9 Branton D, Deamer DW, Marziali A, Bayley H, Benner SA, et al. 2008. The potential and challenges of nanopore sequencing. Nat Biotechnol 26:1146-53 Durocher Y,Perret S,Kamen A.2002.High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.Nucleic Acids Res 30:E9 Eid J,Fehr A,Gray J,Luong K,Lyle J,et al.2009.Real-time DNA sequencing from single polymerase molecules.Science 323:133-8 Engler C,Gruetzner R,Kandzia R,Marillonnet S.2009.Golden gate shuffling:a one-pot DNA shuffling method based on type IIs restriction enzymes.PLoS One 4:e5553 Engler C,Kandzia R,Marillonnet S.2008.A one pot,one step,precision cloning method with high throughput capability.PLoS One 3:e3647 Fahrner RL,Knudsen HL,Basey CD,Galan W,Feuerhelm D,et al.2001.Industrial purification of pharmaceutical antibodies:development,operation,and validation of chromatography processes.Biotechnol Genet Eng Rev 18:301-27 Honegger A,Pluckthun A.2001.Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool.J Mol Biol 309:657-70 Jafari R,Almqvist H,Axelsson H,Ignatushchenko M,Lundback T,et al.2014.The cellular thermal shift assay for evaluating drug target interactions in cells.Nat Protoc 9:2100-22 Kamerzell TJ,Esfandiary R,Joshi SB,Middaugh CR,Volkin DB.2011.Protein-excipient interactions:mechanisms and biophysical characterization applied to protein formulation development.Adv Drug Deliv Rev 63:1118-59 Kelley B.2009.Industrialization of mAb production technology:the bioprocessing industry at a crossroads.MAbs 1:443-52 Kostelny SA,Cole MS,Tso JY.1992.Formation of a bispecific antibody by the use of leucine zippers.J Immunol 148:1547-53 Lo MC,Aulabaugh A,Jin G,Cowling R,Bard J,et al.2004.Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.Anal Biochem 332:153-9 Makhatadze G.1998.Measuring protein thermostability by differential scanning calorimetry.Curr.Protocols Protein Sci.12:7.9.1-7.9.14 Margulies M,Egholm M,Altman WE,Attiya S,Bader JS,et al.2005.Genome sequencing in microfabricated high-density picolitre reactors.Nature 437:376-80 Mason M,Sweeney B,Cain K,Stephens P,Sharfstein ST.2012.Identifying bottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in Chinese hamster ovary cells.Biotechnol Prog 28:846-55 Medzihradszky KF,Chalkley RJ.2015.Lessons in de novo peptide sequencing by tandem mass spectrometry.Mass Spectrom Rev 34:43-63 Minde DP,Maurice MM,Rudiger SG.2012.Determining biophysical protein stability in lysates by a fast proteolysis assay,FASTpp.PLoS One 7:e46147 Nyren P,Lundin A.1985.Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis.Anal Biochem 151:504-9 Pantoliano MW,Petrella EC,Kwasnoski JD,Lobanov VS,Myslik J,et al.2001.High-density miniaturized thermal shift assays as a general strategy for drug discovery.J Biomol Screen 6:429-40 Powell MF,Nguyen T,Baloian L.1998.Compendium of excipients for parenteral formulations.PDA journal of pharmaceutical science and technology 52:238-311 Quax TE,Claassens NJ,Soll D,van der Oost J.2015.Codon Bias as a Means to Fine-Tune Gene Expression.Mol Cell 59:149-61 Ronaghi M,Uhlen M,Nyren P.1998.A sequencing method based on real-time pyrophosphate.Science 281:363,65 Rothberg JM,Hinz W,Rearick TM,Schultz J,Mileski W,et al.2011.An integrated semiconductor device enabling non-optical genome sequencing.Nature 475:348-52 Sanger F,Nicklen S,Coulson AR.1977.DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci U S A 74:5463-7 Semisotnov GV,Rodionova NA,Razgulyaev OI,Uversky VN,Gripas AF,Gilmanshin RI.1991.Study of the “molten globule” intermediate state in protein folding by a hydrophobic fluorescent probe.Biopolymers 31:119-28 Songsivilai S,Lachmann PJ.1990.Bispecific antibody:a tool for diagnosis and treatment of disease.Clin Exp Immunol 79:315-21 Valouev A,Ichikawa J,Tonthat T,Stuart J,Ranade S,et al.2008.A high-resolution,nucleosome position map of C.elegans reveals a lack of universal sequence-dictated positioning.Genome Res 18:1051-63 Wu X,Demarest SJ.2018.Building blocks for bispecific and trispecific antibodies.Methods Wu X,Yuan R,Bacica M,Demarest SJ.2018.Generation of orthogonal Fab-based trispecific antibody formats.Protein Eng Des Sel 31:249-56

[0212] Both the general description above and the detailed description below are exemplary and explanatory only and are not limiting. The use of the singular includes the plural unless specifically stated otherwise. The use of "or" means "and / or" unless specifically stated otherwise. The use of the term "comprising" and other forms such as "comprises" and "comprises" is not limiting. Terms such as "element" or "component" include both elements and components containing one unit and elements and components containing two or more subunits unless specifically stated otherwise. The use of the term "moiety" can include part of the moiety or the entire moiety. When a numerical range, such as 1 to 5, is mentioned, all intervening values, such as 1, 2, 3, 4, and 5, and fractions thereof, such as 1.5, 2.2, 3.4, and 4.1, are expressly included.

[0213] "About" or "to" when modifying an amount (e.g., "about" 3 mM) means that variation around the modified amount may occur. These variations may occur due to various means, such as typical measuring and processing procedures, inadvertent error, purity of components, etc.

[0214] "Comprising" and "comprises" are intended to mean that the preparations and methods include the recited elements, but do not exclude other unrecited elements. The terms "consisting essentially of" and "consists essentially of," when used in reference to disclosed methods containing recited elements, exclude unrecited elements that alter the basic nature of the preparation and / or method, but do not exclude other unrecited elements. Thus, a preparation consisting essentially of elements does not exclude contaminants from any separation and purification methods, or trace amounts of other elements, such as pharmaceutically acceptable carriers (e.g., phosphate-buffered saline), preservatives, etc., but does exclude, for example, additional unspecified amino acids, etc. The terms "consisting of" and "consists of," when used to define preparations and methods, exclude more than trace amounts of other components and substantial method steps for administering the compositions described herein. Embodiments defined by each of these transitional terms are included within the scope of this disclosure.

Claims

1. 1. A method for increasing the stability of a first antibody, comprising substituting glycine, alanine, or serine at heavy chain position 56 (AHo numbering) to create a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody.

2. 2. The method of claim 1, wherein the glycine is substituted at heavy chain position 56.

3. 3. The method of claim 1 or 2, wherein the second antibody further comprises a substitution at heavy chain position 80 (AHo numbering) with a hydrophobic amino acid residue.

4. 4. The method of claim 3, wherein the hydrophobic amino acid residue is selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine.

5. 4. The method of claim 3, wherein the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

6. 3. The method of claim 1 or 2, wherein the second antibody is further substituted with a methionine at position 80 (AHo numbering).

7. 3. The method of claim 1 or 2, wherein the second antibody is further substituted with isoleucine at position 80 (AHo numbering).

8. 1. A method for increasing the stability of a first antibody, comprising substituting a hydrophobic amino acid residue at position 80 (AHo numbering) of the heavy chain of said first antibody to create a second antibody, wherein said second antibody is more stable than the unsubstituted first antibody.

9. 9. The method of claim 8, wherein the hydrophobic amino acid residue is selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine.

10. 9. The method of claim 8, wherein the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

11. 1. A method for increasing the stability of a first antibody, comprising substituting alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine at position 80 of the heavy chain of said first antibody (AHo numbering) to create a second antibody, wherein said second antibody is more stable than the unsubstituted first antibody.

12. 12. The method of claim 11, wherein the methionine is substituted at position 80 of the heavy chain of the first antibody.

13. 12. The method of claim 11, wherein the isoleucine is substituted at position 80 of the heavy chain of the first antibody.

14. 14. The method of any one of claims 8 to 13, wherein the second antibody further comprises a substitution at heavy chain position 56 (AHo numbering) with alanine, glycine, or serine.

15. 14. The method of any one of claims 8 to 13, wherein the second antibody further comprises a substitution with alanine or glycine at position 56 of the heavy chain (AHo numbering).

16. 14. The method of any one of claims 8 to 13, wherein the second antibody is further substituted with glycine at position 56 of the heavy chain (AHo numbering).

17. 17. The method of any one of claims 1 to 16, wherein the increased stability of the second antibody is evidenced by at least one selected from the group consisting of increased titer during cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature.

18. 18. The method of claim 17, wherein the increase in titer is measured by the rate of binding to a Protein A coated probe tip using an Octet Forte Bio instrument.

19. 18. The method of claim 17, wherein the increase in yield is measured by protein A or protein G capture.

20. 18. The method of claim 17, wherein the increase in purity is measured by size exclusion column chromatography (SEC) of the purified antibody.

21. 18. The method of claim 17, wherein the reduction in high molecular weight species is measured by size exclusion column chromatography (SEC) and the area under the curve for each peak at each molecular weight.

22. 18. The method of claim 17, wherein the increase in melting point temperature is measured by differential scanning fluorescence (DSF) or differential scanning calorimetry (DSC).

23. 18. The method of claim 17, wherein the increase in aggregation temperature is measured by DSF.

24. 18. The method of claim 17, wherein the increase in onset melting temperature is measured by DSF.

25. The method of any one of claims 1 to 24, wherein the first antibody is a monoclonal antibody.

26. The method of any one of claims 1 to 25, wherein the first antibody is a human monoclonal antibody or a humanized monoclonal antibody.

27. The method of any one of claims 1 to 26, wherein the first antibody is an IgG antibody.

28. 28. The method of claim 27, wherein the IgG antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 antibodies.

29. 28. The method of claim 27, wherein the IgG antibody is an IgG1 antibody.

30. 28. The method of claim 27, wherein the IgG antibody is an IgG2 antibody.

31. 28. The method of claim 27, wherein the IgG antibody is an IgG3 antibody.

32. 28. The method of claim 27, wherein the IgG antibody is an IgG4 antibody.

33. A method for increasing the stability of a first antibody variant, comprising substituting glycine, alanine or serine at heavy chain position 56 (AHo numbering) to create a second antibody variant, wherein said second antibody variant is more stable than said unsubstituted first antibody variant.

34. 34. The method of claim 33, wherein the glycine is substituted at heavy chain position 56.

35. 35. The method of claim 33 or 34, wherein the second antibody mutant further comprises a substitution at heavy chain position 80 (AHo numbering) with a hydrophobic amino acid residue.

36. 36. The method of claim 35, wherein the hydrophobic amino acid residue is selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine.

37. 36. The method of claim 35, wherein the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

38. A method for increasing the stability of a first antibody variant, comprising substituting a hydrophobic amino acid residue at position 80 of the heavy chain (AHo numbering) of the first antibody variant to create a second antibody, wherein the second antibody variant is more stable than the unsubstituted first antibody variant.

39. 39. The method of claim 38, wherein the hydrophobic amino acid residue is selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine, and valine.

40. 39. The method of claim 38, wherein the hydrophobic amino acid residue is selected from the group consisting of: phenylalanine, leucine, and valine.

41. 1. A method for increasing the stability of a first antibody variant, comprising substituting alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine at position 80 of the heavy chain of said first antibody (AHo numbering) to create a second antibody variant, wherein said second antibody variant is more stable than the unsubstituted first antibody variant.

42. 42. The method of claim 41 , wherein the methionine is substituted at heavy chain position 80 of the first antibody mutant.

43. 42. The method of claim 41 , wherein the isoleucine is substituted at heavy chain position 80 of the first antibody mutant.

44. 44. The method of any one of claims 38 to 43, wherein the second antibody mutant further comprises a substitution at heavy chain position 56 (AHo numbering) with alanine, glycine, or serine.

45. 44. The method of any one of claims 38 to 43, wherein the second antibody mutant further comprises a substitution with alanine or glycine at heavy chain position 56 (AHo numbering).

46. 44. The method of any one of claims 38 to 43, wherein the second antibody mutant further comprises a substitution with glycine at heavy chain position 56 (AHo numbering).

47. 47. The method of any one of claims 33 to 46, wherein the increased stability of the second antibody variant is evidenced by at least one selected from the group consisting of increased titer in cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature.

48. 48. The method of claim 47, wherein said increase in titer is measured by the rate of binding to a Protein A coated probe tip using an Octet Forte Bio instrument.

49. 48. The method of claim 47, wherein the increase in yield is measured by protein A or protein G capture.

50. 48. The method of claim 47, wherein the increase in purity is measured by SEC of the purified antibody.

51. 48. The method of claim 47, wherein the reduction in high molecular weight species is measured by SEC and area under the curve for each peak at each molecular weight.

52. 48. The method of claim 47, wherein the increase in melting point temperature is measured by DSF or DSC.

53. 48. The method of claim 47, wherein the increase in aggregation temperature is measured by DSF.

54. 48. The method of claim 47, wherein the increase in onset melting temperature is measured by DSF.

55. 55. The method of any one of claims 33 to 54, wherein the first antibody mutant is a multispecific antibody.

56. 56. The method of claim 55, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

57. 57. The method of any one of claims 33 to 56, wherein the first antibody mutant is an antibody fragment capable of binding to an antigen.

58. 58. The method of claim 57, wherein the antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, an F'(ab)2 fragment, an Fv fragment, a single chain antibody, a diabody, a biparatopic peptide, a domain antibody (dAb), a CDR-grafted antibody, a single chain antibody (scFv), a single chain antibody fragment, a chimeric antibody, a diabody, a triabody, a tetrabody, a minibody, a linear antibody; a chelating recombinant antibody, a tribody, a bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding domain immunoglobulin fusion protein, a single domain antibody, and a VHH-containing antibody.

59. 59. The method of any one of claims 33 to 58, wherein the first antibody variant is a human monoclonal antibody or a humanized monoclonal antibody variant.

60. 60. The method of any one of claims 33 to 59, wherein the first antibody mutant is an IgG antibody mutant.

61. 61. The method of claim 60, wherein the IgG antibody variant is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibody variants.

62. 62. The method of claim 61, wherein the IgG antibody variant is an IgG1 antibody variant.

63. 62. The method of claim 61, wherein the IgG antibody variant is an IgG2 antibody variant.

64. 62. The method of claim 61, wherein the IgG antibody variant is an IgG3 antibody variant.

65. 62. The method of claim 61, wherein the IgG antibody variant is an IgG4 antibody variant.

66. 1. A method for increasing the stability of a first antibody or first antibody variant, comprising: a. identifying the germline amino acid sequence for the heavy chain of the antibody portion of the first antibody or the antibody variant; b. identifying the amino acid residues at heavy chain position 56 (AHo numbering) and heavy chain position 80 (AHo numbering) of the antibody portion of the first antibody or antibody variant; and c. substituting the identified residues at heavy chain positions 56 and 80 from the germline amino acid sequence of the antibody portion of the first antibody or antibody variant, thereby creating a second antibody or second antibody variant; The method, wherein the second antibody is more stable than the unsubstituted first antibody; or the second antibody variant is more stable than the unsubstituted first antibody variant.

67. 67. The method of claim 66, wherein the increased stability of the second antibody or second antibody variant is evidenced by at least one selected from the group consisting of increased titer in cell culture, increased yield from cell culture, increased purity after purification, a decrease in high molecular weight species, an increased melting point temperature, an increased aggregation temperature, and an increased onset melting temperature.

68. 68. The method of claim 67, wherein said increase in titer is measured by the rate of binding to a Protein A coated probe tip using an Octet Forte Bio instrument.

69. 68. The method of claim 67, wherein the increase in yield is measured by protein A or protein G capture.

70. 68. The method of claim 67, wherein the increase in purity is measured by SEC of the purified protein.

71. 68. The method of claim 67, wherein the reduction in high molecular weight species is measured by SEC and area under the curve for each peak at each molecular weight.

72. 68. The method of claim 67, wherein the increase in melting point temperature is measured by DSF or DSC.

73. 68. The method of claim 67, wherein the increase in aggregation temperature is measured by DSF.

74. 68. The method of claim 67, wherein the increase in onset melting temperature is measured by DSF.

75. 75. The method of any one of claims 66 to 74, wherein the first antibody is a monoclonal antibody.

76. 76. The method of claim 75, wherein the first antibody is a human or humanized monoclonal antibody.

77. 77. The method of any one of claims 66 to 76, wherein the first antibody is an IgG antibody.

78. 78. The method of claim 77, wherein the IgG antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 antibodies.

79. 79. The method of claim 78, wherein the IgG antibody is an IgG1 antibody.

80. 79. The method of claim 78, wherein the IgG antibody is an IgG2 antibody.

81. 79. The method of claim 78, wherein the IgG antibody is an IgG3 antibody.

82. 79. The method of claim 78, wherein the IgG antibody is an IgG4 antibody.

83. 83. The method of any one of claims 66 to 82, wherein the first antibody mutant is a multispecific antibody.

84. 84. The method of claim 83, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

85. 85. The method of any one of claims 66 to 84, wherein the first antibody mutant is an antibody fragment capable of binding to an antigen.

86. 86. The method of claim 85, wherein the antibody fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, an F'(ab)2 fragment, an Fv fragment, a single chain antibody, a diabody, a biparatopic peptide, a domain antibody (dAb), a CDR-grafted antibody, a single chain antibody (scFv), a single chain antibody fragment, a chimeric antibody, a diabody, a triabody, a tetrabody, a minibody, a linear antibody; a chelating recombinant antibody, a tribody, a bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding domain immunoglobulin fusion protein, a single domain antibody, and a VHH-containing antibody.

87. 87. The method of any one of claims 66 to 86, wherein the first antibody variant is a human monoclonal antibody or a humanized monoclonal antibody variant.

88. 88. The method of any one of claims 66 to 87, wherein the first antibody mutant is an IgG antibody mutant.

89. 89. The method of any one of claims 66 to 88, wherein the IgG antibody variant is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibody variants.

90. 90. The method of claim 89, wherein the IgG antibody variant is an IgG1 antibody variant.

91. 90. The method of claim 89, wherein the IgG antibody variant is an IGg2 antibody variant.

92. 90. The method of claim 89, wherein the IgG antibody variant is an IgG3 antibody variant.

93. 90. The method of claim 89, wherein the IgG antibody variant is an IgG4 antibody variant.

94. 94. The method of any one of claims 1 to 93, wherein the second antibody or second antibody variant comprises a substitution of any one of the following pairs of residues at positions 56 and 80 (AHo numbering) of the heavy chain: GF, GI, GL, GT, GV, AF, AI, AL, AV, AA, AM, SA, SI or ST.

95. 95. The method of any one of claims 1 to 94, further comprising formulating said second antibody or second antibody variant into a pharmaceutical composition.

96. 96. An antibody or antibody variant produced by the method of any one of claims 1 to 95.

97. 97. A pharmaceutical composition comprising the antibody or antibody variant of claim 96.

98. 94. The method of any one of claims 1 to 93, further comprising formulating said second antibody or second antibody variant into a pharmaceutical composition.

99. 94. An antibody or antibody variant produced by the method of any one of claims 1 to 93.

100. 100. A pharmaceutical composition comprising the antibody or antibody variant of claim 99.

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