Engineering monoclonal antibodies to improve stability and production titers

Abstract

This paper proposes to relate to engineered monoclonal antibodies and antibody variants to improve the method for stability and output thereof in cultivation. Specifically, these monoclonal antibodies can be engineered to glycine, alanine or serine at heavy chain residue 56 (AHo numbering), and / or engineered to hydrophobic residues such as alanine, isoleucine, phenylalanine, leucine, methionine or valine at position 80 (AHo numbering).

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 787,867, filed on January 3, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] The presented subject matter relates to the field of protein engineering. In particular, the presented subject matter relates to engineering antibodies, especially monoclonal antibodies and variants thereof, to improve their stability and yield. Background Art

[0004] Recombinantly produced monoclonal antibodies (mAbs) (and their active fragments) are important therapeutic tools. However, due to the complexity of these molecules, many challenges need to be faced in order to facilitate their production, storage and therapeutic administration.

[0005] Two challenges involve yield and stability. mAbs are produced in bioreactors by engineered cells (e.g., Chinese hamster ovary (CHO) cells). However, production levels can be low and can vary from mAb to mAb. Low production levels increase production costs, including production time, manpower, and consumed resources (e.g., components required to run the bioreactor). In addition, lack of stability can affect the "shelf life" of the mAb. Degraded mAbs may be less effective, while fragmented mAbs may present immunological risks.

[0006] Therefore, there is a need to improve the stability and production titer of mAbs. Summary of the invention

[0007] In a first aspect, the present invention provides a method for increasing the stability of a first antibody, the method comprising replacing glycine, alanine or serine at heavy chain position 56 (AHo numbering) to produce a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody. For example, at heavy chain position 56, glycine or serine can be substituted. For example, at heavy chain position 56, glycine or alanine can be substituted. For example, at heavy chain position 56, glycine can be substituted.

[0008] In a second aspect, there is provided herein a method for increasing the stability of a first antibody, the method comprising replacing a hydrophobic amino acid at position 80 (AHo numbering) of the heavy chain of the first antibody to produce 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 may include or be composed of: alanine, isoleucine, phenylalanine, leucine, methionine or valine. For example, the hydrophobic amino acid residue may include or be composed of: phenylalanine, leucine or valine.

[0009] In a third aspect, the present invention provides a method for increasing the stability of a first antibody, the method comprising replacing alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine at heavy chain position 80 (AHo numbering) of the first antibody to produce a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody. For example, at heavy chain position 80, phenylalanine, leucine or valine can be substituted. For example, at heavy chain position 80, isoleucine or methionine can be substituted. For example, at heavy chain position 80, isoleucine can be substituted. For example, at heavy chain position 80, methionine can be substituted.

[0010] In the sub-aspects of the first three aspects, the increase in stability of the second antibody is demonstrated by at least one selected from the group consisting of an increase in titer during cell culture, an increase in cell culture yield, an increase in purity after purification, a decrease in high molecular weight species, an increase in melting temperature, an increase in aggregation temperature, and an increase in melting onset temperature. In some sub-aspects, the increase in titer is measured by the rate of binding to the probe end coated with protein A using an Octet Forte Bio instrument; and / or the increase in yield is measured by capture by protein A or protein G; and / or the increase in 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 of each molecular weight; and / or the increase in melting 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 melting onset temperature is measured by DSF.

[0011] In some sub-aspects of the first aspect, the second antibody is further replaced by a hydrophobic amino acid residue at heavy chain position 80 (AHo numbering). For example, the hydrophobic amino acid residue may include or be composed of: alanine, isoleucine, phenylalanine, leucine, methionine or valine. For example, the hydrophobic amino acid residue may be selected from the group consisting of: phenylalanine, leucine and valine. In some sub-aspects of the first aspect, the second antibody is further replaced by methionine at position 80 (AHo numbering), or, alternatively, the second antibody is further replaced by isoleucine at position 80 (AHo numbering). In some sub-aspects of the first aspect, the second antibody is further replaced by alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine at position 80 (AHo numbering). In some sub-aspects of the first aspect, the second antibody is further replaced by phenylalanine, leucine or valine at position 80 (AHo numbering).

[0012] In some sub-aspects of the second and third aspects, the second antibody is further substituted with glycine, alanine or serine at position 56 (AHo numbering). In some sub-aspects of the second and third aspects, the second antibody is further substituted with glycine or alanine at position 56 (AHo numbering). In some sub-aspects of the second and third aspects, the second antibody is further substituted with glycine or serine at position 56 (AHo numbering). In some sub-aspects of the second and third aspects, the second antibody is further substituted with glycine at position 56 (AHo numbering).

[0013] In the first three aspects, the first antibody is a monoclonal antibody, such as a human antibody or a humanized antibody. In addition, the first antibody is an IgG antibody, such as an IgG antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody. 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.

[0014] In a fourth aspect, disclosed herein is a method for increasing the stability of a first antibody variant, the method comprising replacing glycine, alanine or serine at heavy chain position 56 (AHo numbering) to produce a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant. For example, at heavy chain position 56, glycine or serine can be substituted. For example, at heavy chain position 56, glycine or alanine can be substituted. For example, at heavy chain position 56, glycine can be substituted.

[0015] In a fifth aspect, disclosed herein is a method for increasing the stability of a first antibody variant, the method comprising replacing a hydrophobic amino acid residue (e.g., alanine, isoleucine, phenylalanine, leucine, methionine or valine) at heavy chain position 80 (AHo numbering) of the first antibody variant to produce 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 include or consist of: phenylalanine, leucine or valine. For example, the hydrophobic amino acid residue may include or consist of: methionine or isoleucine.

[0016] In a sixth aspect, disclosed herein is a method for increasing the stability of a first antibody variant, the method comprising replacing alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine at heavy chain position 80 (AHo numbering) of the first antibody variant to produce a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant. For example, at heavy chain position 80, phenylalanine, leucine or valine can be substituted. For example, at heavy chain position 80, methionine or isoleucine can be substituted. For example, at heavy chain position 80, methionine can be substituted. For example, at heavy chain position 80, isoleucine can be substituted.

[0017] In the sub-aspects of these fourth, fifth and sixth aspects, the increase in stability of the second antibody variant is demonstrated by at least one selected from the group consisting of: an increase in titer during cell culture, an increase in cell culture yield, an increase in purity after purification, a decrease in high molecular weight species, an increase in melting temperature, an increase in aggregation temperature, and an increase in melting onset temperature. In some sub-aspects, an Octet Forte Bio instrument is used to measure the increase in titer by the rate of binding to the probe end coated with protein A; and / or an increase in yield is measured by capture with protein A or protein G; and / or an increase in purity is measured by SEC of purified protein; and / or a decrease in high molecular weight species is measured by size exclusion chromatography (SEC) and the area under the curve of each peak of each molecular weight; and / or an increase in melting temperature is measured by differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC); and / or an increase in aggregation temperature is measured by DSF; and / or an increase in melting onset temperature is measured by DSF.

[0018] In some sub-aspects of the fourth aspect, the second antibody variant is further replaced by a hydrophobic amino acid residue at heavy chain position 80 (AHo numbering). For example, the hydrophobic amino acid residue can be selected from the group consisting of: alanine, isoleucine, phenylalanine, leucine, methionine and valine. For example, the hydrophobic amino acid residue can be selected from the group consisting of: phenylalanine, leucine and valine. In some sub-aspects of the fourth aspect, the second antibody variant is further replaced by methionine at position 80 (AHo numbering), or, alternatively, the second antibody is further replaced by isoleucine at position 80 (AHo numbering). In some sub-aspects of the fourth aspect, the second antibody variant is further replaced by alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine at position 80 (AHo numbering). In some sub-aspects of the fourth aspect, the second antibody variant is further replaced by phenylalanine, leucine or valine at position 80 (AHo numbering).

[0019] In some sub-aspects of the fifth and sixth aspects, the second antibody variant is further replaced by glycine, alanine or serine at position 56 (AHo numbering). For example, at position 56, the second antibody variant can be replaced by glycine or alanine. For example, at position 56, the second antibody variant can be replaced by glycine or serine. For example, at position 56, the second antibody variant can be replaced by glycine.

[0020] In some sub-aspects of these fourth, fifth and sixth aspects, the first antibody variant is a multispecific antibody, such as a bispecific or trispecific antibody. In some 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: Fab fragments, Fab' fragments, F'(ab)2 fragments, Fv fragments, single-chain antibodies, bifunctional antibodies), biparatopic peptides, domain antibodies (dAbs), CDR-grafted antibodies, single-chain antibodies (scFvs), single-chain antibody fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, mini antibodies, linear antibodies; chelated recombinant antibodies, triantibodies, biantibodies, intracellular antibodies, nanoantibodies, small modular immune drugs (SMIPs), antigen-binding domain immunoglobulin fusion proteins, single domain antibodies, and VHH-containing antibodies.

[0021] In addition, 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. In addition, the first antibody variant is an IgG antibody variant, such as an IgG antibody variant selected from the group consisting of an IgG1 antibody variant, an IgG2 antibody variant, an IgG3 antibody variant and an IgG4 antibody variant. That is, the IgG antibody variant can be an IgG1 antibody variant, the IgG antibody variant can be an IgG2 antibody variant, the IgG antibody variant can be an IgG3 antibody variant, and the IgG antibody variant can be an IgG4 antibody variant.

[0022] In a seventh aspect, disclosed herein is a method of increasing the stability of a first antibody or a first antibody variant, the method comprising

[0023] a. identifying the germline native amino acid sequence of the heavy chain of the antibody portion of the first antibody or the antibody variant;

[0024] 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 the antibody variant; and

[0025] c. substituting in the antibody portion of the first antibody or the antibody variant the identified residues from the germline original amino acid sequence at heavy chain positions 56 and 80, thereby producing a second antibody or a second antibody variant,

[0026] 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.

[0027] In a subaspect of this seventh aspect, the increase in stability of the second antibody or second antibody variant is demonstrated by at least one selected from the group consisting of: an increase in titer during cell culture, an increase in cell culture yield, an increase in purity after purification, a decrease in high molecular weight species, an increase in melting temperature, an increase in aggregation temperature, and an increase in melting onset temperature. In a subaspect of this seventh aspect, the increase in stability of the second antibody or second antibody variant is demonstrated by at least one selected from the group consisting of: an increase in titer during cell culture, an increase in cell culture yield, an increase in purity after purification, a decrease in high molecular weight species, an increase in melting temperature, an increase in aggregation temperature, and an increase in melting onset temperature. In some sub-aspects, using an Octet Forte Bio instrument, the increase in titer is measured by the rate of binding to a protein A coated probe end; 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 melting onset temperature is measured by DSF.

[0028] In this seventh aspect, the first antibody variant is a multispecific antibody, such as a bispecific or trispecific antibody. In some 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: Fab fragment, Fab' fragment, F'(ab)2 fragment, Fv fragment, single-chain antibody, bifunctional antibody), biparatope peptide, domain antibody (dAb), CDR-grafted antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, mini antibody, linear antibody; chelated recombinant antibody, tria antibody, biantibody, intracellular antibody, nano antibody, small modular immune drug (SMIP), antigen binding domain immunoglobulin fusion protein, single domain antibody and VHH-containing antibody.

[0029] In addition, in this seventh aspect, the first antibody variant is a monoclonal antibody variant, such as a human antibody variant or a humanized antibody variant. In addition, the first antibody variant is an IgG antibody variant, such as an IgG antibody variant selected from the group consisting of an IgG1 antibody variant, an IgG2 antibody variant, an IgG3 antibody variant, and an IgG4 antibody variant. That is, the IgG antibody variant can be an IgG1 antibody variant, the IgG antibody variant can be an IgG2 antibody variant, the IgG antibody variant can be an IgG3 antibody variant, and the IgG antibody variant can be an IgG4 antibody variant.

[0030] In addition, in this seventh aspect, the first antibody is a monoclonal antibody, such as a human antibody or a humanized antibody. In addition, the first antibody is an IgG antibody, such as an IgG antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody. 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.

[0031] In some sub-aspects of any one of the first to eighth aspects, the second antibody or second antibody variant is substituted by any one of the following residue pairs at heavy chain positions 56 and 80 (AHo numbering), respectively: GF, GI, GL, GT, GV, AF, AI, AL, AV, AA, AM, SA, SI or ST. For example, it should be noted that for this nomenclature, "GF" will refer to "G" at heavy chain position 56 and "F" at heavy chain position 80 (AHo numbering). In some sub-aspects of any one of the first to eighth aspects, the second antibody or second antibody variant is substituted by any one of the following residue pairs at heavy chain positions 56 and 80 (AHo numbering), respectively: GF, GL, GV, AF, AL or AV. Such substitutions may have higher titers and / or higher Tm compared to the first antibody (or first antibody variant). In some sub-aspects of any one of the first to eighth aspects, the second antibody or second antibody variant is substituted by any one of the following residue pairs at heavy chain positions 56 and 80 (AHo numbering): AA, AL, AM, AV, GF, GL, GT, SA or ST, respectively. Such substitutions may have higher titers than the first antibody (or first antibody variant). In some sub-aspects of any one of the first to eighth aspects, the second antibody or second antibody variant is substituted by any one of the following residue pairs at heavy chain positions 56 and 80 (AHo numbering): AI, AV, GI, SI or GV, respectively. Such substitutions may have higher Tm than the first antibody (or first antibody variant). In some sub-aspects of any one of the first to eighth aspects, the second antibody or second antibody variant is substituted by any one of the following residue pairs at heavy chain positions 56 and 80 (AHo numbering): GF, GL, GT, GV, AF, AL, AV, AA, AM, AV, SA and ST, respectively. Such substitutions may have higher titers than the first antibody (or first antibody variant). In some subaspects of any one of the first to eighth aspects, the second antibody or second antibody variant is substituted at heavy chain positions 56 and 80 (AHo numbering) by any one of the following residue pairs: 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).

[0032] In an eighth aspect, provided herein is a method of preparing a pharmaceutical composition formulated with a second antibody or second antibody variant produced by any of the preceding aspects.

[0033] In a ninth aspect, provided herein is an antibody or antibody variant prepared according to any one of the first seven aspects.

[0034] In a tenth aspect, provided herein is a pharmaceutical composition comprising an antibody or antibody variant prepared according to any one of the first seven aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figures 1A-1D is a series of graphs showing various characteristics of two monoclonal antibodies (engineered monoclonal antibody and its parent monoclonal antibody).

[0036] Figure 2A is a graph showing the following information: For mAbl, alanine and glycine have the highest titer at HC: 56. The HC80 residue is shown in the upper row of X-axis labels. The HC56 residue is shown in the lower row of X-axis labels.

[0037] Figure 2B is a graph showing the following information: For mAbl, phenylalanine, leucine, and valine have the highest titers at HC: 80. The HC56 residue is shown in the upper row of X-axis labels. The HC80 residue is shown in the lower row of X-axis labels.

[0038] Figure 3A is a graph showing the following information: For mAbl, the HC56 and HC80 variants with high titers also have high Tm. The HC80 residues are shown in the upper row labeled on the X-axis. The HC56 residues are shown in the lower row labeled on the X-axis.

[0039] Figure 3B is a graph showing the following information: for mAbl, molecules with phenylalanine, leucine or valine at HC80 have a Tm above 65°C. The HC56 residue is shown in the upper row of X-axis labels. The HC80 residue is shown in the lower row of X-axis labels.

[0040] Figure 4 is a graph showing the following information: As determined by SEC, for mAbl, the majority of high molecular weight (HMW) levels are below 5%. The HC80 residue is shown in the upper row of X-axis labels. The HC56 residue is shown in the lower row of X-axis labels.

[0041] Figure 5 is a graph showing the following information: mAb2 expresses well when it has residues, such as hydrophobic residues, at HC56 and HC80. The HC80 residues are shown in the upper row labeled on the X-axis. The HC56 residues are shown in the lower row labeled on the X-axis.

[0042] Figure 6 is a graph showing the following information: Tm is correlated with titer for substitutions at HC56 and HC80 of mAb2. HC80 residues are shown in the upper row labeled on the X-axis. HC56 residues are shown in the lower row labeled on the X-axis.

[0043] Figure 7is a graph showing high molecular weight species for substitutions at HC56 and HC80 of mAb2. The HC80 residue is shown in the upper row labeled on the X-axis. The HC56 residue is shown in the lower row labeled on the X-axis. DETAILED DESCRIPTION

[0044] Surprisingly, when the antibody heavy chain residue 56 (AHo numbering; residue 49 in Kabat numbering) of the mAb was modified to glycine, the antibody had higher titers and higher Tm in culture / production compared to molecules with the commonly observed alanine residue at this position. This effect has been observed across a large number of mAbs and germline comparisons, and in any case, it was independent of which residue was germline. This observation is in contrast to a study published by Mason et al. (Mason et al. 2012), who reported that alanine at residue 56 (AHo numbering) improved the expression titer of an IgG4 mAb. In addition, the use of methionine at heavy chain position 80 (AHo numbering) in a molecule-dependent manner compared to an isoleucine residue severely affected titers.

[0045] definition

[0046] 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.

[0047] "Antibody" or "immunoglobulin" means a tetrameric glycoprotein consisting of two heavy chains and two light chains, each of which comprises a variable domain (V) and a constant domain (C). "Heavy chain" and "light chain" mean substantially full-length standard immunoglobulin light and heavy chains; the variable domains (VL and VC) of the heavy and light chains constitute the V region of the antibody and contribute to antigen binding and specificity. "Antibodies" include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, and humanized antibodies. Light chains can be classified as kappa and lambda light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and the isotype of the antibody is 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 subdivided into subclasses including IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically connected by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 or more amino acids. The variable regions of each light / heavy chain pairing typically form an antigen binding site. "Monoclonal antibody" means an antibody obtained from a substantially homogeneous antibody population (i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts).

[0048] "Antibody variants" include antibody fragments and antibody-like proteins that are changed into the structure of standard tetrameric antibodies. Typical antibody variants include V regions that are changed into constant regions, or alternatively, V regions are optionally added to constant regions in a non-standard manner. Examples include multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), antigen-binding antibody fragments (e.g., Fab', F'(ab)2, Fv, single-chain antibodies, bifunctional antibodies), biparatopic peptides and recombinant peptides comprising the foregoing (as long as they exhibit the desired biological activity).

[0049] Multispecific antibodies target more than one antigen or epitope. For example, a "bispecific," "dual specific," or "bifunctional" antibody is a hybrid antibody with two different antigen binding sites. Bispecific antibodies can be produced by various methods, including fusion of hybridomas or linking Fab' fragments (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 preparing trispecific antibodies are known and are under further development (Wu & Demarest 2018, Wu et al. 2018)

[0050] "Antibody fragments" include antigen-binding portions of antibodies, including, for example, Fab, Fab', F(ab')2, Fv, domain antibodies (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv), single-chain antibody fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, minibodies, linear antibodies; chelated recombinant antibodies, triabodies or diabodies, intracellular antibodies, 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 containing at least a portion of an immunoglobulin (such as one, two, three, four, five or six CDR sequences) sufficient to confer antigen-specific binding to the polypeptide, as long as the antibody retains the desired binding activity.

[0051] Method Overview

[0052] The disclosed method comprises the following steps: identifying the residues at heavy chain positions 56 and 80 (AHo numbering) or the germline-derived amino acid residues at these positions in an antibody (or modified antibody), changing (mutating) the residue at position 56 to glycine, alanine or serine and / or changing (mutating) residue 80 to a hydrophobic residue (e.g., methionine or isoleucine) or changing (mutating) to any of alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine, and evaluating the stability of the modified antibody using any of the various techniques for measuring different features of the antibody. In some embodiments, the residue at heavy chain position 56 is changed to glycine.

[0053] The following discussion applies not only to antibodies, including IgG (IgG1, IgG2, IgG3, and IgG4), but also to antibody variants, such as those described in the "Definitions" section above.

[0054] Identify residues at positions 56 and / or 80 (AHo numbering)

[0055] In the first step, the amino acid residues at 56 and / or 80 (Aho numbering) in the heavy chain of Ab are identified. If position 56 is already glycine, no further identification is required because Ab does not need further engineering at this position. In most cases, the antibody polynucleotide sequence is cloned and sequenced, and then the sequence is translated into an amino acid sequence. Alternatively, the relevant amino acid sequence from an antibody or its region (e.g., variable region) can be determined by direct protein sequencing. In certain embodiments, if position 56 is already glycine, alanine or serine, no further identification is required because Ab does not need further engineering at this position. In certain embodiments, if position 56 is already glycine or serine, no further identification is required because Ab does not need further engineering at this position. In certain embodiments, if position 56 is already glycine or alanine, no further identification is required because Ab does not need further engineering at this position. In certain embodiments, if position 56 is already glycine or alanine, no further identification is required because Ab does not need further engineering at this position. In certain embodiments, if position 56 is already glycine, no further identification is required because Ab does not need further engineering at this position.

[0056] Alternatively, genomic DNA or cDNA encoding the monoclonal antibody of interest or binding fragment thereof can be isolated and sequenced from cells producing such antibodies using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).

[0057] DNA sequencing can be performed by any technique known in the art, such as that 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), ionic semiconductors (Rothberg et al. 2011), single-molecule real-time sequencing (Eid et al. 2009), sequencing by oligonucleotide ligation detection (SOLiD) (Valouev et al. 2008) and nanopore sequencing (discussed in Branton et al. (Branton et al. 2008)).

[0058] Once the polynucleotide sequence is obtained, the open reading frame is determined and the amino acid sequence is deduced from the genetic code. AHo numbering is used to determine position 56 and / or 80 and the amino acid residue is identified.

[0059] Direct protein sequencing of antibodies is also possible. Protein sequencing methods include, for example, using mass spectrometry, and Edman degradation approaches using a protein sequencer. In the case of Edman degradation, since the target antibody may be longer than 50-70 amino acids, the antibody may be digested using an endopeptidase such as trypsin or pepsin or chemically using cyanogen bromide, BNPS-skatole, formic acid, or chloramine T. The target fragment size for Edman degradation is 50-70 amino acids. Once the antibody is fragmented, the peptides can be analyzed in an automated manner using a protein sequencer, which performs the Edman degradation reaction and reads each released amino acid by a detection method such as high pressure liquid chromatography (HPLC). For mass spectrometry, the antibody can be fragmented with a protease (usually trypsin), the fragments separated by liquid chromatography (LC), and the fragments analyzed by a mass spectrometer using a de novo peptide sequencing algorithm; Medzihradszky and Chalkley discuss this approach (Medzihradszky & Chalkley 2015).

[0060] As described above, once the sequence is determined and AHo numbering is applied, the amino acid at position 56 and / or 80 can be determined.

[0061] Engineering one or more residues as target residues

[0062] Regardless of the method selected to identify the residues at AHo positions 56 and / or 80 of the antibody heavy chain, it is determined whether to mutate the residue at the polynucleotide level. In some embodiments, if the residue at position 56 is not glycine, then the residue is a candidate to be changed. For example, in the most common scenario, at position 56, when the residue is not glycine, it is alanine. Therefore, an A56G mutation can be formed. Similarly, for position 80, if the residue is not a hydrophobic residue (or not any of alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine), the residue is a candidate to be changed to a hydrophobic residue (or to be changed to any of alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine, such as methionine). For example, in some embodiments, in the case of this position (80), the residue is not methionine, and the residue is a candidate to be changed to methionine. For example, in some embodiments, in the case of this position (80), if the residue is not isoleucine, then the residue is a candidate to be changed to isoleucine. In the case of this position (80), even if the residue is a hydrophobic residue, such as methionine or isoleucine, the position is still a candidate to be changed to a different hydrophobic residue (e.g., isoleucine or methionine, respectively, because any of these amino acids (Met, Ile) can increase expression and stability in culture). In some embodiments, in the case of this position (80), the residue can be changed to any of alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine, or to a hydrophobic residue (e.g., alanine, phenylalanine, isoleucine, leucine, or methionine), even if the residue is already a different residue among alanine, phenylalanine, isoleucine, leucine, methionine, threonine, or valine, or a different hydrophobic residue. If the residue at position 56 is not glycine, then this residue can also be a candidate to be changed (e.g., 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.

[0063] Any known method can be used to modify the polynucleotide of encoding target antibody.After determining the amino acid residue at position 56 and / or 80, modify the nucleic acid sequence so that at position 56, it is coded as glycine (or alanine or serine), and / or at position 80, it is coded as alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine (or hydrophobic amino acid residue or methionine or isoleucine).In order to carry out this change, identify the codon of the amino acid encoding at position 56 and / or 80, and select one or more sudden changes according to Table 1, which shows the genetic code.

[0064] Table 1

[0065] Genetic code

[0066]

[0067] Most amino acids are encoded by more than one codon, as shown in Table 1. For example, alanine is encoded by four codons: GCU, GCC, GCA, and GCG; however, only Trp and Met are each encoded by a single codon (TGG and ATG, respectively). When selecting one or more mutations, considerations such as codon bias can be taken into account (Quax et al. 2015).

[0068] Mutations can be introduced into the nucleotide sequence encoding the antibodies of the present disclosure using standard techniques, including site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis to produce targeted amino acid substitutions. Commercial kits that can achieve the introduction of mutations into nucleic acids are also available, such as GeneArt TM system and Phusion kit (ThermoFisher Scientific; Waltham, MA); Site-Directed Mutagenesis Kit (New England BioLabs; Ipswich, MA); and customizable kits from Civic Bioscience (Montreal, Canada).

[0069] Alternatively, polynucleotide fragments can be synthesized using existing techniques and substitutions made within a polynucleotide comprising a complete coding sequence. In some cases, the entire coding sequence is synthesized with one or more targeted mutations.

[0070] In any case, the amino acid mutation method has no particular limitations as long as it can effectively achieve site mutation.

[0071] Cell selection and transfection of engineered polynucleotides

[0072] Recombinant DNA methods for producing antibodies are well known. DNA encoding an antibody (e.g., DNA encoding a VH domain, a VL domain, a single-chain variable fragment (scFv), or fragments and combinations thereof) (target polynucleotide) can be inserted into a suitable expression vector, which can then be transfected into a suitable host cell (e.g., an Escherichia coli cell, a COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell) (which otherwise would not produce an antibody) to obtain the desired antibody.

[0073] Suitable expression vectors are known in the art and include, for example, a polynucleotide encoding a target polypeptide connected to a promoter. 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 vectors to express a heavy chain, an entire light chain, or an entire heavy chain and a light chain (or a fragment thereof). The expression vector may be transferred to a host cell by conventional techniques, and the transfected cells may be cultured to produce antibodies.

[0074] Any cell line that can express or be engineered to express functional antibodies or antibody fragments can be used. For example, suitable mammalian cell lines include immortalized cell lines available from the American Type Culture Collection (Manassas, Virginia), including Chinese hamster ovary (CH)) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2) and human epithelial kidney 293 cells. In addition, cell lines or host systems can be selected to ensure the correct modification and processing of antibodies. Eukaryotic host cells with appropriate processing for primary transcripts, glycosylation of gene products and phosphorylated cell machinery can be used. These include CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7030 and HsS78Bst cells. Human cell lines generated by immortalizing human lymphocytes can also be used. Human cell lines (Janssen; Titusville, NJ) can be used to recombinantly produce monoclonal antibodies. 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., Saccharomyces cerevisiae, Pichia, etc.)), plant cells, or chicken cells.

[0075] Using conventional methods, antibodies can be stably expressed in cell lines. Stable expression can be used for long-term, high-yield production of recombinant proteins. For stable expression, host cells can be transformed with appropriately engineered vectors that include expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and selectable marker genes. Methods for producing high-yield stable cell lines are known in the art, and reagents are commercially available. Transient expression can also be achieved using conventional methods.

[0076] The cell line expressing the antibody can be maintained in a cell culture medium and maintained under culture conditions that result in expression and production of the antibody. The cell culture medium can be based on commercially available culture medium formulations, including, for example, DMEM or Ham's F12. In addition, the cell culture medium can be modified to support cell growth and increase in biological protein expression. Of course, for specific cell cultures, the cell culture medium can be optimized, including a cell culture growth medium formulated to promote cell growth or a cell culture production medium formulated to promote recombinant protein production.

[0077] Many cell culture media and cell culture nutrients and supplements are known. For example, suitable basal media include Dulbecco's Modified Eagle's Medium (DMEM), DME / F12, Minimum Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a-Minimum Essential Medium (a-MEM), Glasgow's Minimal Essential Medium (G-MEM), PF CHO and Iscove's Modified Dulbecco's Medium. Other examples of basal media include BME basal medium, Dulbecco's Modified Eagle's Medium.

[0078] The basal medium can be serum-free (which means that the medium does not contain serum (e.g., fetal bovine serum (FBS))) or an animal protein-free medium or a chemically defined medium. The basal medium can be modified to remove certain non-nutritional components found in the basal medium, such as various inorganic and organic buffers, one or more surfactants, and sodium chloride. The cell culture medium can contain a basal cell culture medium (modified or unmodified) and at least one of the following: an iron source, a recombinant growth factor; a buffer; a surfactant; an osmotic pressure regulator; an energy source; and a non-animal hydrolyzate. In addition, the modified basal cell culture medium can optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins. The modified basal medium can further contain glutamine, such as L-glutamine and / or methotrexate.

[0079] purification

[0080] Once the antibody has been produced, it can be purified by conventional methods, for example, by chromatography (e.g., ion exchange, affinity (particularly by affinity for the specific antigen, protein A, protein G) or fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the antibody can be fused to a heterologous polypeptide sequence ("tag") to facilitate purification.

[0081] Assessing stability

[0082] Titer in culture

[0083] You can, for example, use Supernatants from cell cultures expressing the engineered polynucleotides were analyzed using a Pall ForteBio platform instrument (Pall ForteBio; Fremont, CA). The platform provides biosensors for many different analytes, including biosensors for anti-human IgG quantification (AHQ), anti-mouse IgG quantification (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 using traditional ELISA formats, as well as HPLC and radioimmunoassay (RIA).

[0084] Melting Temperature (Differential Scanning Fluorescence (DSF) and Differential Scanning Calorimetry (DSC))

[0085] Thermal stability analysis by differential scanning fluorimetry (DSF) (also known as Protein Thermal Shift Assay; (Lo et al. 2004, Pantoliano et al. 2001, Semisotnov et al. 1991)) can be conveniently used to determine the melting temperature, Tm, of a protein. DSF utilizes a fluorescent dye that preferentially binds to unfolded (denatured) proteins. Heat-induced protein denaturation is typically monitored by measuring the fluorescence change of the dye using a real-time polymerase chain reaction (PCR) instrument. Examples of useful dyes include 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-(biscyanovinyl)julolidine (DCVJ). The method of Lo et al. was generally used, utilizing commonly available RE-PCR instruments and Orange (Lo et al. 2004).

[0086] Typically, the dye being analyzed and one or more proteins are mixed, a melting curve is determined, and the Tm is calculated from the melting curve.

[0087] In addition to DSF, any known technique can be used to determine the Tm of a protein. For example, techniques that utilize intrinsic fluorescence signals (e.g., from tryptophan) and techniques that utilize different ways of monitoring protein folding / unfolding (e.g., light scattering) can be used. Other techniques include rapid parallel proteolysis (Minde et al. 2012) and cellular thermal shift assays (Jafari et al. 2014).

[0088] Differential scanning calorimetry (DSC) can also be used to determine the Tm of a protein (Makhatadze 1998), although because DSC can also provide information about the unfolding pattern, multiple Tm values ​​can be obtained.

[0089] Typically, a spectrophotometer is used to determine the spectrum of a subject protein to quantify the protein (or alternatively, a different method is used to quantify the protein). The protein is then analyzed using the DSC program of the spectrophotometer.

[0090] Yield (as measured by antibody captured by Protein A)

[0091] A general method for purifying antibodies from clarified cell culture supernatants contains a capture step using protein A affinity chromatography; followed by a combination of anion and cation exchange chromatography (Fahrner et al. 2001, Kelley 2009)

[0092] Any method known in the art can be used for protein quantification. These methods include UV-Vis spectroscopy at 280nm (A280); based on the absorbance of tryptophan and tyrosine residues (or alternatively, the absorbance at 205nm (A205), detecting the protein backbone; Bradford assay (usually based on the use and absorbance of Coomassie Brilliant Blue G-250 dye); biuret test-derived assays, such as Lowry and bicinchoninic acid (BCA) assays; amino acid analysis (depending on the directional detection of modified amino acids); gel electrophoresis (as observed by gel band intensity) or dye-labeled proteins (thus correlating the detection of dye signals with the amount of protein), examples of dyes include fluorescamine and amido black 10B. In addition, HPLC and LC / MS methods can also be used.

[0093] To preserve samples and facilitate measurements, a NanoDrop spectrophotometer, such as that available from Thermo Fisher Scientific (Wilmington, DE), can be used. This device facilitates several methods of quantifying proteins.

[0094] High molecular weight (HMW) and main peak (MP) species as determined by size exclusion chromatography (SEC)

[0095] A general method for SEC was used to compare the species of Protein A eluted material, where the protein was passed through a column of dextran polymer porous beads to separate species according to size. The HMW percentage and MP percentage were determined by measuring the area under the SEC peak curve.

[0096] Aggregation (Taggregation) and melting initiation (Tmelt initiation or Tstartmelt)

[0097] Taggregate was measured by differential scanning fluorimetry (DSF) and using an excitation wavelength of 300 nm and emission wavelengths of 350 / 330 nm, the temperature at which 30 nm aggregated particles were formed was measured.

[0098] T onset is measured by DSF and fluorescence is measured at 350 / 330 nm. T onset is the temperature at which the first derivative of the emission wavelength at 350 / 330 nm rises above the basal level, which is also the time when the folded protein begins to unfold.

[0099] Pharmaceutical composition formulations and components

[0100] The antibodies and antibody variants prepared according to the methods disclosed herein can be formulated into pharmaceutical compositions suitable for administration to a patient.

[0101] Acceptable pharmaceutical ingredients are preferably non-toxic to patients at the dosages and concentrations used. Pharmaceutical compositions may contain agents for modifying, maintaining or preserving, for example, pH, osmotic concentration, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition.

[0102] In general, excipients can be classified based on the mechanism by which they stabilize proteins against various chemical and physical stressors. Some excipients mitigate the effects of specific stressors or modulate the specific sensitivity of a particular polypeptide. Other excipients generally affect the physical and covalent stability of proteins more.

[0103] Common excipients for liquid and lyophilized protein formulations are shown in Table 2 (see also (Kamerzell et al. 2011)).

[0104] Table 2

[0105] Examples of excipient components for peptide formulations

[0106]

[0107]

[0108] Other excipients are known in the art (e.g., see (Powell et al. 1998). One skilled in the art can determine what amount or range of excipients can be included in any particular formulation to obtain a biopharmaceutical composition that promotes retention of the stability of the biopharmaceutical. For example, the amount and type of salt to be included in the biopharmaceutical composition can be selected based on the desired osmolality of the final solution (i.e., isotonic, hypotonic, or hypertonic) and the amounts and osmolality of the other components to be included in the formulation.

[0109] Example

[0110] The following examples are presented as examples of the methods disclosed herein and are non-limiting. The Examples section follows this Examples section.

[0111] Embodiment 1. A method for increasing the stability of a first antibody, the method comprising replacing glycine, alanine or serine at heavy chain position 56 (AHo numbering) to produce a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody. For example, at heavy chain position 56, glycine can be substituted. For example, at heavy chain position 56, glycine or alanine can be substituted. For example, at position 56, glycine or serine can be substituted.

[0112] Embodiment 2. The method of embodiment 1, wherein at position 56 of the heavy chain, the glycine is substituted.

[0113] Embodiment 3. The method of any one of embodiments 1-2, wherein the second antibody is further substituted with a hydrophobic amino acid residue at position 80 (AHo numbering) of the heavy chain.

[0114] 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.

[0115] Embodiment 5. The method of embodiment 3, wherein the hydrophobic amino acid residue is selected from the group consisting of phenylalanine, leucine and valine.

[0116] Embodiment 6. The method of any one of embodiments 1-2, wherein the second antibody is further substituted with methionine at position 80 (AHo numbering).

[0117] Embodiment 7. The method of any one of embodiments 1 and 2, wherein the second antibody is further substituted with isoleucine at position 80 (AHo numbering).

[0118] Example 8. A method of increasing the stability of a first antibody, the method comprising substituting a hydrophobic amino acid residue at position 80 (AHo numbering) of the heavy chain of the first antibody to produce a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody.

[0119] 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.

[0120] Embodiment 10. The method of embodiment 8, wherein the hydrophobic amino acid residue is selected from the group consisting of phenylalanine, leucine and valine.

[0121] Example 11. A method for increasing the stability of a first antibody, the method comprising substituting alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine at position 80 (AHo numbering) of the heavy chain of the first antibody to produce a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody.

[0122] Embodiment 12. The method of embodiment 11, wherein the methionine at position 80 of the heavy chain of the first antibody is substituted.

[0123] Embodiment 13. The method of embodiment 11, wherein the isoleucine is substituted at position 80 of the heavy chain of the first antibody.

[0124] Embodiment 14. The method of any one of embodiments 8-13, wherein the second antibody is further substituted with alanine, glycine or serine at heavy chain position 56 (AHo numbering).

[0125] Embodiment 15. The method of any one of embodiments 8-13, wherein the second antibody is further substituted with alanine or glycine at position 56 (AHo numbering) of the heavy chain.

[0126] Embodiment 16. The method of any one of embodiments 8-13, wherein the second antibody is further substituted with glycine at position 56 (AHo numbering) of the heavy chain

[0127] Embodiment 17. The method as described in any of embodiments 1-16, wherein the increase in stability of the second antibody is demonstrated by at least one selected from the group consisting of: an increase in titer during cell culture, an increase in cell culture yield, an increase in purity after purification, a decrease in high molecular weight substances, an increase in melting point temperature, an increase in aggregation temperature and an increase in melting onset temperature.

[0128] Example 18. The method of Example 17, wherein the increase in titer is measured by the rate of binding to the protein A coated probe end using an Octet Forte Bio instrument.

[0129] Embodiment 19. The method of embodiment 17, wherein the increase in yield is measured by protein A or protein G capture.

[0130] Embodiment 20. The method of embodiment 17, wherein the increase in purity is measured by size exclusion chromatography (SEC) of the purified antibody.

[0131] Embodiment 21. The method of embodiment 17, wherein the reduction of high molecular weight species is measured by size exclusion chromatography (SEC) and the area under the curve of each peak at each molecular weight.

[0132] Embodiment 22. The method of embodiment 17, wherein the increase in melting point temperature is measured by differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC).

[0133] Embodiment 23. The method of embodiment 17, wherein the increase in aggregation temperature is measured by DSF.

[0134] Embodiment 24. The method of embodiment 17, wherein the increase in melting onset temperature is measured by DSF.

[0135] Embodiment 25. The method of any preceding embodiment, wherein the first antibody is a monoclonal antibody.

[0136] Embodiment 26. The method of any preceding embodiment, wherein the first antibody is a human monoclonal antibody or a humanized monoclonal antibody.

[0137] Embodiment 27. The method of any preceding embodiment, wherein the first antibody is an IgG antibody.

[0138] Embodiment 28. The method of embodiment 27, wherein the IgG antibody is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

[0139] Embodiment 29. The method of embodiment 27, wherein the IgG antibody is an IgG1 antibody.

[0140] Embodiment 30. The method of embodiment 27, wherein the IgG antibody is an IgG2 antibody.

[0141] Embodiment 31. The method of embodiment 27, wherein the IgG antibody is an IgG3 antibody.

[0142] Embodiment 32. The method of embodiment 27, wherein the IgG antibody is an IgG4 antibody.

[0143] Embodiment 33. A method for increasing the stability of a first antibody variant, the method comprising substituting glycine, alanine or serine at position 56 (AHo numbering) of the heavy chain to produce a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant.

[0144] Embodiment 34. The method of embodiment 33, wherein at position 56 of the heavy chain, the glycine is substituted.

[0145] Embodiment 35. The method of any one of embodiments 33 and 34, wherein the second antibody variant is further substituted with a hydrophobic amino acid residue at heavy chain position 80 (AHo numbering).

[0146] Embodiment 36. A method as described in embodiment 35, wherein the hydrophobic amino acid residue is selected from the group consisting of alanine, isoleucine, phenylalanine, leucine, methionine and valine.

[0147] Embodiment 37. The method of embodiment 35, wherein the hydrophobic amino acid residue is selected from the group consisting of phenylalanine, leucine and valine.

[0148] Embodiment 38. A method for increasing the stability of a first antibody variant, the method comprising substituting a hydrophobic amino acid residue at position 80 (AHo numbering) of the heavy chain of the first antibody variant to produce a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant.

[0149] 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.

[0150] Embodiment 40. The method of embodiment 38, wherein the hydrophobic amino acid residue is selected from the group consisting of phenylalanine, leucine and valine.

[0151] Example 41. A method for increasing the stability of a first antibody variant, the method comprising substituting alanine, phenylalanine, isoleucine, leucine, methionine, threonine or valine at position 80 (AHo numbering) of the heavy chain of the first antibody variant to produce a second antibody variant, wherein the second antibody variant is more stable than the unsubstituted first antibody variant.

[0152] Embodiment 42. The method of embodiment 41, wherein the methionine at heavy chain position 80 of the first antibody variant is substituted.

[0153] Embodiment 43. The method of embodiment 41, wherein the isoleucine is substituted at heavy chain position 80 of the first antibody variant.

[0154] Embodiment 44. The method of any one of embodiments 38-43, wherein the second antibody variant is further substituted with alanine, glycine or serine at heavy chain position 56 (AHo numbering).

[0155] Embodiment 45. The method of any one of embodiments 38-43, wherein the second antibody variant is further substituted with alanine or glycine at heavy chain position 56 (AHo numbering).

[0156] Embodiment 46. The method of any one of embodiments 38-43, wherein the second antibody variant is further substituted with glycine at heavy chain position 56 (AHo numbering)

[0157] Embodiment 47. The method of any one of embodiments 33-46, wherein the increase in stability of the second antibody variant is demonstrated by at least one selected from the group consisting of: an increase in titer during cell culture, an increase in cell culture yield, an increase in purity after purification, a decrease in high molecular weight substances, an increase in melting point temperature, an increase in aggregation temperature, and an increase in melting onset temperature.

[0158] Example 48. A method as described in Example 47, wherein the increase in titer is measured by the rate of binding to the protein A coated probe end using an Octet Forte Bio instrument.

[0159] Embodiment 49. The method of embodiment 47, wherein the increase in yield is measured by protein A or protein G capture.

[0160] Embodiment 50. The method of embodiment 47, wherein the increase in purity is measured by SEC of the purified antibody.

[0161] Embodiment 51. The method of embodiment 47, wherein the reduction of high molecular weight species is measured by SEC and the area under the curve of each peak at each molecular weight.

[0162] Embodiment 52. The method of embodiment 47, wherein the increase in melting point temperature is measured by DSF or DSC.

[0163] Embodiment 53. The method of embodiment 47, wherein the increase in aggregation temperature is measured by DSF.

[0164] Embodiment 54. The method of embodiment 47, wherein the increase in melting onset temperature is measured by DSF.

[0165] Embodiment 55. The method of any one of Embodiments 33-54, wherein the first antibody variant is a multispecific antibody.

[0166] Embodiment 56. The method of embodiment 55, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0167] Embodiment 57. The method of any one of embodiments 33-56, wherein the first antibody variant is an antibody fragment capable of binding to an antigen.

[0168] Example 58. A method as described in Example 57, wherein the antibody fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F'(ab)2 fragment, Fv fragment, single chain antibody, bifunctional antibody), biparatope peptide, domain antibody (dAb), CDR-grafted antibody, single chain antibody (scFv), single chain antibody fragment, chimeric antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, mini antibody, linear antibody; chelated recombinant antibody, tria antibody, diabody, intracellular antibody, nanobody, small modular immunopharmaceutical (SMIP), antigen binding domain immunoglobulin fusion protein, single domain antibody and VHH-containing antibody.

[0169] Embodiment 59. The method of any one of embodiments 33-58, wherein the first antibody variant is a human monoclonal antibody or a humanized monoclonal antibody variant.

[0170] Embodiment 60. The method of any one of embodiments 33-59, wherein the first antibody variant is an IgG antibody variant.

[0171] Embodiment 61. The method of embodiment 60, wherein the IgG antibody variant is selected from the group consisting of an IgG1 antibody variant, an IgG2 antibody variant, an IgG3 antibody variant, and an IgG4 antibody variant.

[0172] Embodiment 62. The method of embodiment 61, wherein the IgG antibody variant is an IgG1 antibody variant.

[0173] Embodiment 63. The method of embodiment 61, wherein the IgG antibody variant is an IgG2 antibody variant.

[0174] Embodiment 64. The method of embodiment 61, wherein the IgG antibody variant is an IgG3 antibody variant.

[0175] Embodiment 65. The method of embodiment 61, wherein the IgG antibody variant is an IgG4 antibody variant.

[0176] Example 66. A method for increasing the stability of a first antibody or a first antibody variant, the method comprising

[0177] a. identifying the germline native amino acid sequence of the heavy chain of the antibody portion of the first antibody or the antibody variant;

[0178] 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 the antibody variant; and

[0179] c. substituting in the antibody portion of the first antibody or the antibody variant the identified residues from the germline original amino acid sequence at heavy chain positions 56 and 80, thereby producing a second antibody or a second antibody variant,

[0180] 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.

[0181] Embodiment 67. The method as described in embodiment 66, wherein the increase in stability of the second antibody or second antibody variant is demonstrated by at least one selected from the group consisting of: an increase in titer during cell culture, an increase in cell culture yield, an increase in purity after purification, a decrease in high molecular weight substances, an increase in melting point temperature, an increase in aggregation temperature and an increase in melting onset temperature.

[0182] Embodiment 68. The method of embodiment 67, wherein the increase in titer is measured by the rate of binding to the protein A coated probe end using an Octet Forte Bio instrument.

[0183] Embodiment 69. The method of embodiment 67, wherein the increase in yield is measured by protein A or protein G capture.

[0184] Embodiment 70. The method of embodiment 67, wherein the increase in purity is measured by SEC of the purified protein.

[0185] Example 71. A method as described in Example 67, wherein the reduction of high molecular weight species is measured by SEC and the area under the curve of the peak at each molecular weight.

[0186] Embodiment 72. The method of embodiment 67, wherein the increase in melting point temperature is measured by DSF or DSC.

[0187] Embodiment 73. The method of embodiment 67, wherein the increase in aggregation temperature is measured by DSF.

[0188] Embodiment 74. The method of embodiment 67, wherein the increase in melting onset temperature is measured by DSF.

[0189] Embodiment 75. The method of any one of embodiments 66-74, wherein the first antibody is a monoclonal antibody.

[0190] Embodiment 76. The method of embodiment 75, wherein the first antibody is a human monoclonal antibody or a humanized monoclonal antibody.

[0191] Embodiment 77. The method of any one of Embodiments 66-76, wherein the first antibody is an IgG antibody.

[0192] Embodiment 78. The method of embodiment 77, wherein the IgG antibody is selected from the group consisting of: IgG1 antibody, IgG2 antibody, IgG3 antibody and IgG4 antibody.

[0193] Embodiment 79. The method of embodiment 78, wherein the IgG antibody is an IgG1 antibody.

[0194] Embodiment 80. The method of embodiment 78, wherein the IgG antibody is an IgG2 antibody.

[0195] Embodiment 81. The method of Embodiment 78, wherein the IgG antibody is an IgG3 antibody.

[0196] Embodiment 82. The method of Embodiment 78, wherein the IgG antibody is an IgG4 antibody.

[0197] Embodiment 83. The method of any one of Embodiments 66-82, wherein the first antibody variant is a multispecific antibody.

[0198] Embodiment 84. The method of embodiment 83, wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0199] Embodiment 85. The method of any one of Embodiments 66-84, wherein the first antibody variant is an antibody fragment capable of binding to an antigen.

[0200] Example 86. A method as described in Example 85, wherein the antibody fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F'(ab)2 fragment, Fv fragment, single chain antibody, bifunctional antibody), biparatope peptide, domain antibody (dAb), CDR-grafted antibody, single chain antibody (scFv), single chain antibody fragment, chimeric antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, mini antibody, linear antibody; chelated recombinant antibody, tria antibody, diabody, intracellular antibody, nanobody, small modular immunopharmaceutical (SMIP), antigen binding domain immunoglobulin fusion protein, single domain antibody and VHH-containing antibody.

[0201] Embodiment 87. The method of any one of Embodiments 66-86, wherein the first antibody variant is a human monoclonal antibody or a humanized monoclonal antibody variant.

[0202] Embodiment 88. The method of any one of Embodiments 66-87, wherein the first antibody variant is an IgG antibody variant.

[0203] Embodiment 89. The method of any one of embodiments 66-88, wherein the IgG antibody variant is selected from the group consisting of an IgG1 antibody variant, an IgG2 antibody variant, an IgG3 antibody variant, and an IgG4 antibody variant.

[0204] Embodiment 90. The method of embodiment 89, wherein the IgG antibody variant is an IgG1 antibody variant.

[0205] Embodiment 91. The method of Embodiment 89, wherein the IgG antibody variant is an IgG2 antibody variant.

[0206] Embodiment 92. The method of Embodiment 89, wherein the IgG antibody variant is an IgG3 antibody variant.

[0207] Embodiment 93. The method as described in Embodiment 89, wherein the IgG antibody variant is an IgG4 antibody variant.

[0208] Embodiment 94. The method of any one of embodiments 1-93, wherein the second antibody or second antibody variant is substituted at heavy chain positions 56 and 80 (AHo numbering) by any of the following residue pairs: GF, GI, GL, GT, GV, AF, AI, AL, AV, AA, AM, SA, SI or ST. For example, under this nomenclature, "GF" would refer to "G" at position 56 and "F" at position 80 (AHo numbering).

[0209] Embodiment 95. The method of any one of embodiments 1-94, further comprising formulating the second antibody or second antibody variant as a pharmaceutical composition.

[0210] Embodiment 96. An antibody or antibody variant prepared by the method as described in any one of embodiments 1-95.

[0211] Embodiment 97. A pharmaceutical composition comprising the antibody or antibody variant as described in Embodiment 96.

[0212] The following Examples section is given for illustrative purposes only and is not intended to limit the invention or the claims in any way.

[0213] Examples

[0214] Example 1 - Experimental Design

[0215] Antibody mutations were achieved by designing codon changes and having correspondingly synthesized nucleotides. The modified fragments were integrated into the open reading frame using the Golden Gate cloning method (Engler et al. 2009, Engler et al. 2008). Unless otherwise stated, antibodies were expressed in HEK 293-6E cells (a suspension cell line that expresses a truncated variant of Epstein Barr virus nuclear antigen (EBNA) 1 (Durocher et al. 2002). The antibodies produced were purified using protein A attached to a solid support.

[0216] The following germline families were examined: VH1 / VK4, VH4 / VL1, VH3 / VK3, VH6 / VK1, and VH2 / VL2. In the heavy chain, 56A and 56G, as well as 80I and 80M were tested.

[0217] The measurement parameters are:

[0218] Titer at harvest (mg / L)

[0219] Melting temperature (Tm; as measured by differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC), the latter giving two outputs: Tm1 and Tm2)

[0220] Yield (as measured by antibody captured by Protein A)

[0221] High molecular weight (HMW) species as determined by size exclusion chromatography (SEC) and peak molecular weight (Mp, defined as the molecular weight of the highest peak).

[0222] Example 2 - VH1 Germline

[0223] In this example, mutations in an antibody (mAb1 (IgG1), derived from the VH1 germline (VH1 / VK4)) were detected and the antibody was analyzed as described in Example 1.

[0224] Table 2.1

[0225] Observation on mAb1, a monoclonal antibody from the VH1 / VK4 germline

[0226]

[0227] + Indicates the amino acid at the specified position (AHo number)

[0228] * refers to the germline residue at the specified position

[0229] ∧mAb1 = "wild type" of mAb1. The suffixes refer to the residues found at positions 56 and 80, respectively.

[0230] ND, no data

[0231] ～ Variants of mAb1 with wild-type residues (G and L) at positions 56 and 80, respectively; data from a separate set of experiments

[0232] & Data are from DSC experiments and represent Tm1 and Tm2.

[0233] ** When measured by DSC, Tm1 was 76.9 and Tm2 was 84.4

[0234] In these experiments, when mAbl had an A at position 56 and an M at position 80, that antibody (mAb1AM) had the lowest titer of the antibodies tested observed. Similarly, mAblAI also had a low titer. However, mAblGM had the highest titer of the antibodies tested observed; mAblGI had the second highest titer.

[0235] Example 3 - VH3 germline

[0236] In this example, mutations in an antibody (mAb2 (IgG2 Ab), derived from the VH3 germline (VH3 / VK3)) were detected and the antibody was analyzed as described in Example 1.

[0237] Table 3.1

[0238] right Observation of mAb2, a monoclonal antibody from the VH3 / VK3 germline

[0239]

[0240] + Indicates the amino acid at the specified position (AHo number)

[0241] * refers to the germline residue at the specified position

[0242] ND, no data

[0243] In these experiments, when mAb2 had an A at position 56 and an M at position 80, this antibody (mAb2AM) had the lowest titer among the antibodies tested, as well as the lowest melting point (Tm), indicating that this antibody was less stable. However, mAb2AI and mAb2GM had higher titers and melting points among the antibodies tested.

[0244] Example 4 - VH4 germline

[0245] In this example, mutations in an antibody (mAb3 (IgG1 Ab), derived from the VH4 germline (VH4 / VL1)) were detected and the antibody was analyzed as described in Example 1.

[0246] Table 4.1

[0247] Observation on mAb3, a monoclonal antibody from the VH4 / VL1 germline

[0248]

[0249] + Indicates the amino acid at the specified position (AHo number)

[0250] * refers to the germline residue at the specified position

[0251] In these experiments, when mAb3 had A at position 56 and M or I at position 80, these antibodies (mAb3AM and mAb3AI) had the lowest titers among the antibodies tested, as well as the lowest melting points (Tm), indicating that these antibodies were less stable. However, mAb3(GI) and mAb3GM had higher titers and melting points among the antibodies tested.

[0252] Example 5 - VH6 germline

[0253] In this example, mutations in an antibody (mAb4 (IgG1 Ab), derived from the VH6 germline (VH6 / VK1)) were detected and the antibody was analyzed as described in Example 1.

[0254] Table 5.1

[0255] Observation on mAb4, a monoclonal antibody from the VH6 / VK1 germline

[0256]

[0257] + Indicates the amino acid at the specified position (AHo number)

[0258] * refers to the germline residue at the specified position

[0259] ND, no data

[0260] In these experiments, when mAb4 had an A at position 56 and an M at position 80, this antibody (mAb4AM) had the lowest titer among the antibodies tested observed; no melting point was determined. However, mAb4(GI) (germline) and mAb4GM had higher titers and melting points among the antibodies tested, with mAb4GI outperforming mAb4GM.

[0261] Example 6 - VH2 Germlines

[0262] In this example, mutations in an antibody (mAb5 (IgG1 Ab), derived from the VH2 germline (VH2 / VL2)) were detected and the antibody was analyzed as described in Example 1.

[0263] Table 6.1

[0264] Observation on mAb5, a monoclonal antibody from the VH2 / VL2 germline

[0265]

[0266] + Indicates the amino acid at the specified position (AHo number)

[0267] * refers to the germline residue at the specified position

[0268] In these experiments, both antibodies tested had high titers and good melting points.

[0269] When these antibodies were expressed by Chinese hamster ovary (CHO) cells, mAb5GM (“mutant” in Figure 1 ) had higher titers across clones and MTX levels compared to mAb5(AI) (germline; “WT” in Figure 1 ) antibody ( Figure 1A ), less HMW substances ( Figure 1B ) and higher MP purity ( Figure 1C ).

[0270] Example 7 - Comparison of Antibody Titers According to IgG Subtype and HC56 and HC80 Mutations

[0271] The harvest titers of mAb1 and mAb3 (the latter being a different subtype) were determined. Table 7.1 shows the results. In this experiment, the heavy chain variant HC:56G, HC:80M showed the most ideal final growth characteristics. The HC:56G, HC:80M variant also produced higher titers (day 6) in IgG1, IgG2 and IgG4 molecules, except for mAb3 (IgG4 form); in contrast, this molecule showed the highest titer in the case of HC:56A, HV:80M group substitution.

[0272] Table 7.1

[0273] Harvest titers of IgG subtypes with different HC56 and HC80 substitutions

[0274]

[0275]

[0276] Example 8 - Amino Acid Identification of IgG1 HC:56 and HC:80

[0277] In this example, additional choices for amino acids at HC:56 and HC:80 for IgG1 were identified using two approaches, one was an in-depth phylogenetic analysis and screening, and the other was based on the Rosetta model of all 400 H:56H:80 combinations. Notably, these experiments provided additional testing and analysis of possible choices at HC:56 and HC:80. Amino acid pairs with moderate results in one or more computational screens were mutated into model molecules and then compared to expression levels when harvested from 293-6E cell cultures and Tm obtained by DSF.

[0278] For phylogenetic analysis, blast searches of mAb1 and mAb2 were performed in protein NR (e=0.0001). CD-hit clustering was performed on the ~10,000 sequences identified for each molecule, and the most common residues were identified and ranked by frequency. The top amino acids were cloned into mAb1 and mAb2.

[0279] For the phylogenetic analysis of mAbl, the most common residue pairs are shown in Table 8.1 below (bold residue pairs were tested; asterisks indicate germline residues):

[0280] Table 8.1

[0281]

[0282]

[0283] For the phylogenetic analysis of mAb2, the most common residue pairs are shown in Table 8.2 below (residue pairs in bold were tested):

[0284] Table 8.2

[0285] H56 H80 A I A V G F G I G L G M G V S I M V T I V I

[0286] For Rosetta analysis, the energy scores of all amino acid combinations at HC:56 and HC:80 in mAb1 and mAb2 were measured using Rosetta software. Without being bound by theory, it can be assumed that under this method, Rosetta tried all 400 possible H56:H80 amino acid combinations using standard genetically encoded amino acids. In the Rosetta analysis, HC:56 and HC:80 amino acid combinations were compared and ranked by 1:1 weighted z scores based on the Rosetta "total score" and "p_aa_pp" scores. Variants with good energy scores that had not yet appeared in the phylogenetic analysis were cloned into mAb1 and mAb2 for further testing.

[0287] At HC:56, the results for mAbl indicated that the highest titers could be achieved with either HC:56G or HC:56A, followed by HC:56S (see, e.g., Figure 2A-2B ). The results for mAb2 indicate that the highest titers can be achieved with HC:56A, HC:56G, or HC:56S (see, e.g., Figure 5 ). Without being bound by theory, it is noteworthy that these residues are most similar in their relatively small size compared to the full class of amino acids.

[0288] At HC:80, the results for mAb1 indicate that the highest titers can be achieved with HC:80F, HC:80L, or HC:80V. The results for mAb2 indicate that the highest titers can be achieved with HC:80L, HC:80M, HC:80A, HC:80V, HC:80F, HC:80I. Additionally, in both cases, HC:80T resulted in relatively high expression. Without being bound by theory, a summary of these results is that using a hydrophobic residue at HC:80 will result in higher titers.

[0289] Among the engineered variants of mAb1, the six variants with the highest titers were associated with the six variants with the highest Tm (see Figure 2A-2B and Figure 3A-3B Among the variants of mAb2, the differences in Tm were less pronounced, although the low titer variant still had a lower Tm (see Figure 6 ). Additionally, the use of a hydrophobic residue at HC:80 was associated with a higher Tm.

[0290] Table 8.3 below summarizes the effects of residue combinations at heavy chain position 56 and heavy chain position 80 (AHo numbering) in each mAb1 and mAb2. In the nomenclature of Table 8, the first residue refers to heavy chain position 56 (AHo numbering), and the second residue refers to heavy chain position 80 (AHo numbering). Thus, for example, "GF" would refer to a "G" at heavy chain position 56 (AHo numbering) and an "F" at heavy chain position 80 (AHo numbering):

[0291] Table 8.3

[0292]

[0293] References

[0294] Bentley DR,Balasubramanian S,Swerdlow HP,Smith GP,Milton J,etal.2008.Accurate whole human genome sequencing using reversible terminatorchemistry.Nature 456:53-9

[0295] Branton D,Deamer DW,Marziali A,Bayley H,Benner SA,et al.2008.Thepotential and challenges of nanopore sequencing.Nat Biotechnol 26:1146-53

[0296] 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

[0297] Eid J, Fehr A, Gray J, Luong K, Lyle J, et al. 2009. Real-time DNAsequencing from single polymerase molecules. Science 323:133-8

[0298] Engler C,Gruetzner R,Kandzia R,Marillonnet S.2009.Golden gateshuffling:a one-pot DNA shuffling method based on type IIs restrictionenzymes.PLoS One 4:e5553

[0299] Engler C,Kandzia R,Marillonnet S.2008.A one pot,one step,precisioncloning method with high throughput capability.PLoS One 3:e3647

[0300] Fahrner RL,Knudsen HL,Basey CD,Galan W,Feuerhelm D,etal.2001.Industrial purification of pharmaceutical antibodies:development,operation,and validation of chromatography processes.Biotechnol Genet Eng Rev18:301-27

[0301] Honegger A,Pluckthun A.2001.Yet another numbering scheme forimmunoglobulin variable domains:an automatic modeling and analysis tool.J MolBiol 309:657-70

[0302] Jafari R,Almqvist H,Axelsson H,Ignatushchenko M,Lundback T,etal.2014.The cellular thermal shift assay for evaluating drug targetinteractions in cells.Nat Protoc 9:2100-22

[0303] Kamerzell TJ,Esfandiary R,Joshi SB,Middaugh CR,VolkinDB.2011.Protein-excipient interactions:mechanisms and biophysicalcharacterization applied to protein formulation development.Adv Drug DelivRev 63:1118-59

[0304] Kelley B.2009.Industrialization of mAb production technology:thebioprocessing industry at a crossroads.MAbs 1:443-52

[0305] Kostelny SA,Cole MS,Tso JY.1992.Formation of a bispecific antibody bythe use of leucine zippers.J Immunol 148:1547-53

[0306] Lo MC,Aulabaugh A,Jin G,Cowling R,Bard J,et al.2004.Evaluation offluorescence-based thermal shift assays for hit identification in drugdiscovery.Anal Biochem 332:153-9

[0307] Makhatadze G.1998.Measuring protein thermostability by differentialscanning calorimetry.Curr.Protocols Protein Sci.12:7.9.1-7.9.14

[0308] Margulies M,Egholm M,Altman WE,Attiya S,Bader JS,et al.2005.Genomesequencing in microfabricated high-density picolitre reactors.Nature 437:376-80

[0309] Mason M,Sweeney B,Cain K,Stephens P,Sharfstein ST.2012.Identifyingbottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in Chinese hamster ovary cells.Biotechnol Prog 28:846-55

[0310] Medzihradszky KF,Chalkley RJ.2015.Lessons in de novo peptidesequencing by tandem mass spectrometry.Mass Spectrom Rev 34:43-63

[0311] Minde DP,Maurice MM,Rudiger SG.2012.Determining biophysical proteinstability in lysates by a fast proteolysis assay,FASTpp.PLoS One 7:e46147

[0312] Nyren P,Lundin A.1985.Enzymatic method for continuous monitoring ofinorganic pyrophosphate synthesis.Anal Biochem 151:504-9

[0313] Pantoliano MW,Petrella EC,Kwasnoski JD,Lobanov VS,Myslik J,etal.2001.High-density miniaturized thermal shift assays as a general strategyfor drug discovery.J Biomol Screen 6:429-40

[0314] Powell MF,Nguyen T,Baloian L.1998.Compendium of excipients forparenteral formulations.PDA journal of pharmaceutical science and technology52:238-311

[0315] Quax TE,Claassens NJ,Soll D,van der Oost J.2015.Codon Bias as a Meansto Fine-Tune Gene Expression.Mol Cell 59:149-61

[0316] Ronaghi M,Uhlen M,Nyren P.1998.A sequencing method based on real-timepyrophosphate.Science 281:363,65

[0317] Rothberg JM,Hinz W,Rearick TM,Schultz J,Mileski W,et al.2011.Anintegrated semiconductor device enabling non-optical genome sequencing.Nature475:348-52

[0318] Sanger F,Nicklen S,Coulson AR.1977.DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci U S A 74:5463-7

[0319] Semisotnov GV,Rodionova NA,Razgulyaev OI,Uversky VN,Gripas AF,Gilmanshin RI.1991.Study of the"molten globule"intermediate state in proteinfolding by a hydrophobic fluorescent probe.Biopolymers 31:119-28

[0320] Songsivilai S,Lachmann PJ.1990.Bispecific antibody:a tool fordiagnosis and treatment of disease.Clin Exp Immunol 79:315-21

[0321] 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 universalsequence-dictated positioning.Genome Res 18:1051-63

[0322] Wu X,Demarest SJ.2018.Building blocks for bispecific and trispecificantibodies.Methods

[0323] Wu X,Yuan R,Bacica M,Demarest SJ.2018.Generation of orthogonal Fab-based trispecific antibody formats.Protein Eng Des Sel 31:249-56

[0324] The foregoing general description and the following detailed description are only exemplary and explanatory and non-restrictive. Unless otherwise specifically provided, the use of the singular includes the plural. Unless otherwise provided, the use of "or" means "and / or". The use of the term "including" and other forms (such as "includes" and "included") is not restrictive. Unless otherwise specifically provided, terms such as "element" or "component" cover elements and components comprising one unit and elements and components comprising more than one subunit. The use of the term "portion" may include a portion or the entire portion of a moiety. When referring to a numerical range (e.g., 1-5), all intermediate values, such as 1, 2, 3, 4 and 5, and fractions thereof, such as 1.5, 2.2, 3.4 and 4.1, are explicitly included.

[0325] "About" or "about (~)" means that when an amount is modified (e.g., "about" 3 mM), variations around the modified amount may occur. These variations may occur in a variety of ways, such as typical measurement and handling procedures, errors due to inadvertence, component purity, and the like.

[0326] "Comprising" and "comprises" are intended to mean that formulations and methods include the listed elements but do not exclude other unlisted elements. When used in the disclosed methods, the terms "consisting essentially of" and "consists essentially of" include the listed elements, exclude unlisted elements that change the basic properties of the formulation and / or method, but do not exclude other unlisted elements. A formulation consisting essentially of the elements will not exclude trace amounts of other elements, such as contaminants from any separation and purification methods or pharmaceutically acceptable carriers (e.g., phosphate buffered saline), preservatives, etc., but will exclude, for example, additional unspecified amino acids. When used to define formulations and methods, the terms "consisting of" and "consists of" exclude trace amounts of elements that are not just other ingredients and substantial method steps for administering the compositions described herein. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

Claims

1. A method for increasing the stability of a first antibody, the method comprising replacing the amino acid residue at heavy chain position 80 of the first antibody according to AHo numbering with a hydrophobic amino acid residue to produce a second antibody, wherein the second antibody is more stable than the unsubstituted first antibody, wherein the hydrophobic amino acid residue is selected from alanine, phenylalanine, leucine, methionine and valine, and wherein the second antibody is further substituted with alanine, glycine or serine at heavy chain position 56 according to AHo numbering.

2. The method of claim 1, wherein the hydrophobic amino acid residue is selected from phenylalanine, leucine and valine.

3. The method of claim 1, wherein position 80 of the heavy chain of the first antibody is substituted with methionine.

4. The method of any one of claims 1-3, wherein the second antibody is further substituted with alanine or glycine at position 56 of the heavy chain according to AHo numbering.

5. The method of any one of claims 1-3, wherein the second antibody is further substituted with glycine at position 56 of the heavy chain according to AHo numbering.

6. The method of any one of claims 1-3, wherein the first antibody is a monoclonal antibody.

7. The method of claim 6, wherein the monoclonal antibody is a human monoclonal antibody or a humanized monoclonal antibody.

8. The method of any one of claims 1-3, wherein the first antibody is an IgG antibody selected from an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

9. The method of claim 1, wherein the second antibody is substituted with any one of the following residue pairs: GF, GL, GV, AF, AL, AV, AA, AM or SA, respectively, wherein the first residue refers to heavy chain position 56 and the second residue refers to heavy chain position 80 according to AHo numbering.

10. The method of any one of claims 1-3, further comprising formulating the second antibody as a pharmaceutical composition.

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