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WO2025090753A1 - Exploitation de propriétés d'anticorps natifs pour guider une sélection bispécifique - Google Patents

Exploitation de propriétés d'anticorps natifs pour guider une sélection bispécifique Download PDF

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Publication number
WO2025090753A1
WO2025090753A1 PCT/US2024/052797 US2024052797W WO2025090753A1 WO 2025090753 A1 WO2025090753 A1 WO 2025090753A1 US 2024052797 W US2024052797 W US 2024052797W WO 2025090753 A1 WO2025090753 A1 WO 2025090753A1
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Prior art keywords
pool
molecule
antibody
multicomponent
building block
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Inventor
Alexander PARTIN
Evan Neal MIRTS
Danyang GONG
Timothy Patrick Riley
Fernando Garces
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Amgen Inc
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Amgen Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/10—Methods of screening libraries by measuring physical properties, e.g. mass
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8693—Models, e.g. prediction of retention times, method development and validation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange

Definitions

  • the disclosure relates to the generation and purification of multicomponent polypeptides, such as antibodies and particularly bispecific antibodies.
  • the disclosure relates to the use of methods utilizing physical properties of candidate components such as surface charge to select the appropriate components for integration into multicomponent polypeptides.
  • Bispecific antibodies are an exciting generation of biotherapeutics, enabling simultaneous or sequential targeting of two or more unique epitopes located on the same, or distinct, targets.
  • This dual-recognition capability enables diverse applications such as recruiting immune cells to kill tumor cells, crosslinking distinct cell surface receptors, or improving tissue specificity.
  • Bispecific T-cell Engager creates an artificial immune synapse between cytotoxic T cells and target tumor cells, by simultaneously binding a CD3 epitope on the surface of T cells and a tumor-associated antigen.
  • the first approach is to encode two or more unique fragment variable (Fv) sequences on the same polypeptide chain(s), as with formats such as BiTEs®, IgG-scFv, or DVD-Ig.
  • Fv fragment variable
  • This application relates to processes for screening and selecting potential components for inclusion in multicomponent polypeptides, and methods of constructing multicomponent peptides that incorporate the selected components.
  • those processes and methods involve measurement of a physical property such as conductivity.
  • the process of measuring the conductivity of a panel of potential components can be used as part of a method of generating multicomponent polypeptides.
  • the disclosed methods allow prediction of how well a panel of desired multicomponent species might be separated from undesired contaminating species, and prediction of which candidate components would best facilitate such separation.
  • the processes described herein into screening and production processes facilitates more efficient screening, and results in identifying multicomponent peptides that make better therapeutic candidates because they are able to more easily be purified away from contaminants.
  • Some embodiments described herein relate to a method of screening potential components for multicomponent polypeptides, wherein the multicomponent polypeptides each comprise at least one building block A and at least one building block B. and wherein the screening comprises multiple steps to identify preferred candidate components. Those steps include identifying and individually purify ing a group of molecules (pool A) wherein each molecule comprises one or more copies of a candidate building block A, as well as identifying and individually purifying a group of molecules (pool B) wherein each molecule comprises a one or more copies of a candidate building block B. Those steps also include determining the conductivity of each pool A molecule and of each pool B molecule.
  • the difference in conductivity (ACond or AConductivity) between each molecule of pool A and each molecule of pool B is determined.
  • the steps also include selecting a set of candidate multicomponent polypeptides that comprise a building block A-building block B pair, wherein the pool A and pool B molecules comprising the building blocks in the selected multicomponent peptides have a larger ACond than the ACond between the pool A and pool B molecules comprising the building block pairs in the non-selected multicomponent polypeptides.
  • the conductivity of each pool A molecule and of each pool B molecule is determined using cation-exchange chromatography (CIEX), anion-exchange chromatography (AIEX), hydrophobic interaction chromatography, or reversed phase chromatography.
  • CIEX cation-exchange chromatography
  • AIEX anion-exchange chromatography
  • hydrophobic interaction chromatography hydrophobic interaction chromatography
  • the selected building block A came from a pool A molecule measured to have a conductivity' within the top 10% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity within the bottom 10% of tested pool B molecules.
  • Particular embodiments relate to methods of screening in which each pool A molecule, each pool B molecule, or each pool A molecule and each pool B molecule comprises an immunoglobulin.
  • each pool A molecule, each pool B molecule, or each pool A molecule and each pool B molecule comprises an antibody.
  • building block A comprises all or part of an antibody heavy chain.
  • building block A comprises an antibody arm.
  • building block B comprises all or part of an antibody heavy chain. In some embodiments, building block B is an antibody arm. In some embodiments, building block A and building block B comprise all or part of an antibody heavy chain. In some embodiments, building block A and building block B comprise an antibody arm.
  • the multicomponent polypeptide of the method of screening may comprise a variety of different classes of polypeptides.
  • the multicomponent polypeptide is a bispecific antibody.
  • building block A comprises an antibody arm and building block B comprises an Fc-linked scFv.
  • the multicomponent polypeptide is an antibody drug conjugate or antibody fusion.
  • the multicomponent polypeptide comprises an antibody fusion, and wherein the antibody is fused to a cytokine.
  • the methods disclosed herein may combine analysis of ACond with further steps as part of the screening or design process.
  • the method further comprises estimating the absolute conductivity of the selected multicomponent polypeptides by summing the conductivity of the pool A molecule and pool B molecule that supply the building blocks for each multicomponent polypeptide, and keeping in the selected set only those multicomponent polypeptides within the upper 50% of the estimated summed conductivities.
  • the method comprises keeping only those multicomponent polypeptides with an absolute conductivity greater than 11 mS/cm.
  • the absolute conductivity is measured by summing the conductivity of each molecule as determined using cation-exchange chromatography (CIEX), anion-exchange chromatography (AIEX), hydrophobic interaction chromatography, or reversed phase chromatography.
  • CIEX cation-exchange chromatography
  • AIEX anion-exchange chromatography
  • hydrophobic interaction chromatography hydrophobic interaction chromatography
  • the method further comprises comparing the CIEX elution profile of each selected multicomponent polypeptide with the CIEX elution profiles of the pool A molecule and the pool B molecule from which its component parts were derived, and keeping only those multicomponent polypeptides whose principal elution peak have less than 25% overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule. In some embodiments, there is less than 10% overlap in peak area between the multicomponent peptide's principal peak and the principal peak of either the pool A molecule or the pool B molecule.
  • Some embodiments described herein relate to a method of constructing a multicomponent immunoglobulin molecule comprising the steps of (1) measuring the surface charge of each member of a pool A of immunoglobulins and measuring the surface charge of each member of a pool B of immunoglobulins; (2) selecting an immunoglobulin from pool A (immunoglobulin A) and an immunoglobulin from pool B (immunoglobulin B) that have a difference in conductivity (ACond) greater than 5 mS/cm. as measured by CIEX; and (3) combining a building block A that comprises part or all of immunoglobulin A with a building block B that comprises part or all of immunoglobulin B in the multicomponent immunoglobulin molecule.
  • the selected immunoglobulin A and immunoglobulin B have a ACond greater than 7 mS/cm, as measured by CIEX.
  • each pool A molecule and each pool B molecule comprises an immunoglobulin.
  • each pool A molecule, each pool B molecule, or each pool A molecule and each pool B molecule comprises an antibody.
  • building block A comprises all or part of an antibody heavy chain.
  • building block A comprises an antibody arm.
  • building block B comprises all or part of an antibody heavy chain.
  • building block B is an antibody arm.
  • building block A and building block B comprise all or part of an antibody heavy chain.
  • building block A and building block B comprise an antibody arm.
  • the multicomponent polypeptide of the method of constructing a multicomponent polypeptide may comprise a variety of different classes of polypeptides.
  • the multicomponent polypeptide is a bispecific antibody.
  • building block A comprises an antibody arm and building block B comprises an Fc-linked scFv.
  • the multicomponent polypeptide is an antibody drug conjugate or antibody fusion.
  • the multicomponent polypeptide comprises an antibody fusion, and wherein the antibody is fused to a cytokine.
  • the methods of constructing a multicomponent polypeptide disclosed herein may combine analysis of ACond with further steps as part of the screening or design process.
  • the method further comprises estimating the absolute conductivity of the selected multicomponent polypeptides by summing the conductivity of the pool A molecule and pool B molecule that supply the building blocks for each multicomponent polypeptide, and keeping in the selected set only those multicomponent polypeptides within the upper 50% of the estimated summed conductivities.
  • the method comprises keeping only those multicomponent polypeptides with an absolute conductivity greater than 11 mS/cm.
  • the absolute conductivity is measured by summing the conductivity of each molecule as determined using cation-exchange chromatography (CIEX), anion-exchange chromatography (AIEX), hydrophobic interaction chromatography, or reversed phase chromatography.
  • CIEX cation-exchange chromatography
  • AIEX anion-exchange chromatography
  • hydrophobic interaction chromatography hydrophobic interaction chromatography
  • FIGS. 1 A- IF are diagrams that show different antibodies and antibody-based structures.
  • FIG. 1 A is an example of a basic monospecific bivalent antibody, which contains two identical arms capable of binding antigen A (Building Block A) attached through their respective Fc regions.
  • FIG. IB shows an exemplary' bispecific antibody in which an arm capable of binding antigen A is linked to an arm capable of binding to antigen B (Building Block B) through their respective Fc regions, where the interaction's specificity is facilitated by complementary chargepair mutations in those Fc regions.
  • FIG. 1 A is an example of a basic monospecific bivalent antibody, which contains two identical arms capable of binding antigen A (Building Block A) attached through their respective Fc regions.
  • FIG. IB shows an exemplary' bispecific antibody in which an arm capable of binding antigen A is linked to an arm capable of binding to antigen B (Building Block B) through their respective Fc regions, where the interaction's specificity is facilitated by complementary chargepair mutations in those Fc regions.
  • FIG. 1C shows an exemplary AmAb, which is comprised of a standard antibody arm capable of binding antigen A (Building Block A) and an antibody arm capable of binding antigen B that has an scFv in place of the Fab region (Building Block B'), where the scFv optionally contains a stabilizing disulfide bond.
  • FIG. ID is a schematic breakdown of a bispecific antibody (as shown in FIG. IB) that shows the percent identity with each of the parent antibodies from which it was developed and the percentage of the bispecific antibody that is engineered to facilitate the bispecific antibody’s generation.
  • FIG. IE is a schematic breakdown of an AmAb (as shown in FIG.
  • FIG. IF is a diagram showing various species that may be formed during the creation of an AmAb from its constituent protein chains, including the desired AmAb (the “correct species”) and a range of different potential contaminants such as mispaired homodimers, single-arm species, and aggregates.
  • FIGS. 2A-2E show an overview of cation exchange chromatography (CIEX) purification profiles of different exemplary antibodies and antibody-based molecules.
  • FIG. 2A is an overlay of the CIEX elution profiles for a parental antibody capable of binding antigen A (Building Block A homodimer), a parental antibody capable of binding antigen B (Building Block B homodimer), and a hetero-IgG bispecific antibody generated from those parental antibodies that is capable of binding both antigen A and antigen B.
  • FIG. 2B is an overlay of the CIEX elution profiles for a parental antibody capable of binding antigen A (Building Block A homodimer), a parental antibody capable of binding antigen B (Building Block B homodimer), and an AmAb generated from those parental antibodies that is capable of binding both antigen A and antigen B.
  • FIG. 2C is an overlay of the CIEX elution profiles for a parental antibody capable of binding antigen A (Building Block A homodimer), a parental antibody that is an scFv-based homodimer capable of binding antigen B (Building Block B' homodimer), an scFv-linked antibody with the ClmAb format, and an scFv-linked antibody with the C2mAb format.
  • FIG. 1 is an overlay of the CIEX elution profiles for a parental antibody capable of binding antigen A (Building Block A homodimer), a parental antibody that is an scFv-based homodimer capable of binding antigen B (
  • FIG. 2D is an overlay of the CIEX elution profiles for a parental antibody capable of binding antigen A (Building Block A homodimer), a parental antibody capable of binding antigen B (Building Block B homodimer), an scFv-linked antibody with the C lmAb format, an scFv-linked antibody with the BlmAb format, and an scFv-linked antibody with the NlmAb format.
  • FIG. 2E is an overlay of the CIEX elution profiles for a parental antibody capable of binding antigen A (Building Block A homodimer), a cytokine, and an antibody-cytokine fusion comprising an antibody capable of binding antigen A and a cytokine receptor. [0019] FIG.
  • FIG. 3 is a schematic overview of purification-based Building Block Compatibility Screening (pBBCS)-aided CIEX separation.
  • the separation between parental mAbs (AConductivity) indicates the extent to which common contaminants such as homodimeric and single-arm species will separate from the AmAb species during CIEX.
  • FIGS. 4A and 4B show an overlay of the CIEX profiles for 8 unique mAbs against Target A (FIG. 4A) and an overlay of the CIEX profiles for 17 unique mAbs against Target B (FIG 4B).
  • FIGS. 5A-5C show representative CIEX profiles for AmAb designs, overlaid with the CIEX elution profiles of their respective pairs of parental antibodies with various AConductivities.
  • FIG. 5A shows the overlay of an AmAb’s elution profile along with its parent antibodies that have a AConductivity of 3.6 mS/cm.
  • FIG. 5B shows the overlay of an AmAb’s elution profile along with its parent antibodies that have a AConductivity of 6.3 mS/cm.
  • FIG. 5C shows the overlay of an AmAb’s elution profile along with its parent antibodies that have a AConductivity of 10.5 mS/cm.
  • FIG. 7 is a correlational analysis of predicted AmAb elution position (calculated as the mean of the two parental mAb elution positions) and observed AmAb elution position.
  • FIGS. 8A and 8B show correlational analyses of AConductivity 7 against the separation of AmAb from either superimposed Building Block A mAb (FIG. 8A) or superimposed Building Block B mAb (FIG. 8B) in CIEX. Data points are shaded to reflect qualitative separation behavior as either poor (grey squares) or good (black circles). The vertical dotted line represents a threshold AConductivity 7 of 7 mS/cm, above which good separation was consistently observed.
  • FIG. 8C is a breakdown of panel size after each step of pBBCS using the panels shown in FIGS 4A and 4B as potential combinations.
  • FIG 8C applies a AConductivity threshold of 7 mS/cm or greater, which was identified in FIGS. 8A and 8B as a threshold above-which good separation w ould be expected for resultant bispecific antibodies.
  • FIGS. 9A-9G show the characterization of a self-associating AmAb subspecies observed in CIEX.
  • FIG. 9A shows the CIEX elution profile of an AmAb antibody, with the two principal noted peaks highlighted.
  • FIGS. 9B and 9C are the SEC elution profiles of collected protein from Peak 1 and Peak 2 from FIG. 9A, respectively. As shown in FIGS. 9B and 9C, Peak 2 exhibited an additional and substantially earlier SEC elution position than Peak 1.
  • FIGS. 9D and 9E show intact mass spectrometry of collected protein from Peak 1 and Peak 2 from FIG.
  • FIGS. 9F and 9G are CIEX re-runs of collected protein from Peak 1 and Peak 2, respectively. The appearance of a peak identical in size to Peak 1 in Peak 2’s re-run provides further indication that Peak 2 was a self-associating multimer of AmAbs.
  • FIGS. 11 A-l 1C are an analysis of the affinity of the material in Peak 1 and Peak 2 from FIG. 10A for Target A and Target B.
  • FIGS. 11 A and 1 IB show in vitro analysis of the affinity of the material in Peak 1 and Peak 2.
  • FIG. 11C shows a cell-based functional assay to measure on and off-target activity.
  • FIGS. 12A and 12B show an analysis of the separation of monomer and multimer antibodies in relation to the conductivity of the monomer antibody.
  • FIG. 12A shows an exemplary CIEX elution profile for an AmAb, which notes the increased contact points with CIEX resin, potential intermol ecular basic patches, and increased elution conductivity of a multimeric species.
  • FIG12B shows a plot of the CIEX conductivity of a various AmAb monomers against the separation of Peak 1 (monomer) and Peak 2 (multimer) in CIEX elution profiles, with qualitatively poor separation noted when the conductivity of the monomer AmAb was below 11 mS/cm.
  • FIG12C shows a plot of the CIEX conductivity for Target B mAbs against separation of Peak 1 (monomer) and Peak 2 (multimer) in CIEX elution profiles, possibly due to a dominant role played by scFvs in defining how multimeric AmAbs interact with the CIEX column.
  • FIGS. 13A and 13B show analytical SEC of the Protein A eluates for two AmAbs, containing identical Fab arm sequences but different scFv arm sequences. Pairing the Target Al building block with the Target Bl building block produced the AlxBl AmAb, while pairing the Target Al building block with the Target B2 building block produced the AlxB2 AmAb. Both the AlxBl AmAb and the A1/B2 AmAb show the presence of aggregation in their respective SEC elution profdes in FIG. 13A and FIG. 13B.
  • FIGS. 15A-15F show an analysis of aggregation in a panel of AmAbs.
  • FIGS 15A- 15D show the SEC and CIEX elution profiles of four AmAbs, in which the proportion of aggregate peak in SEC correlated with the abundance of aggregate peak in CIEX.
  • FIG. 15E shows a plot of the average aggregate abundance with ten different Target B building blocks, with some scFv sequences having a higher propensity toward aggregation.
  • FIG. 15F shows a plot of the size of Peak 2 observed with CIEX against the percent aggregation measured in SEC, with a trendline for relation included.
  • FIG. 16 shows an overview' of pBBCS-based strategies for improving AmAb purification.
  • pBBCS improves the resolution between AmAbs and contaminants such as homodimers and single-arm species.
  • pBBCS aims to increase the overall conductivity of the AmAb molecule (which can be accurately predicted from the parental mAb elution profiles) to enable effective separation of aggregates due to the more pronounced avidity effects of high-conductivity AmAb molecules.
  • Bispecific antibodies engineered to recognize two targets simultaneously, demonstrate exceptional clinical potential for the therapeutic intervention of complex diseases.
  • These molecules are often composed of multiple polypeptide chains of differing sequences. To meet industrial scale productivity, enforcing the correct quaternary 7 assembly of these chains is critical. More broadly, the same demands on assembly are present in production of multicomponent polypeptides in general. The purification of multicomponent polypeptides resulting from a single cell expression is widely attractive from a manufacturing standpoint enabling lower costs and improve timelines. However, such approach may render cell extracts with undesirable species that must be separated from the molecule of interest.
  • the methods disclosed herein provide rational workflows for predicting success, based on investigating and identifying the native properties of component building blocks (e.g., antibodies) that can influence the purification of multicomponent polypeptides or their component building blocks.
  • component building blocks e.g., antibodies
  • This application describes Purification-Based Building Block Compatibility Screening (pBBCS), a method to predict the assembly of multicomponent polypeptides (e.g., bispecific antibodies) with the appropriate building blocks to enable superior purification properties.
  • pBBCS utilizes analysis of physical properties such as the surface charge of those components, and facilitates quickly focusing optimization efforts on only promising candidate components. Therefore, pBBCS can play a critical role in the next-generation workflows for multicomponent polypeptides to enable lower costs, higher speed, and an increase in overall success rates.
  • One property 7 that maybe applied in the context of pBBCS is elution conductivity of proteins.
  • Each multicomponent protein exhibits a unique surface charge that which impacts and defines the behavior of the that protein, just as the surface charge of contaminating unpaired or mispaired species impacts and defines their behavior.
  • These surface charge properties are important during certain types of chromatography 7 (e.g., cation exchange chromatography (CEX)), a critical step in multicomponent protein purification.
  • CEX cation exchange chromatography
  • the methods described herein use in silico prediction of bispecific CEX profiles to define compatibility requirements for component proteins.
  • the Examples below demonstrate the predictive power of pBBCS using a panel of Fab- scFv-Fc molecules by showing that distinct surface charge properties (as evidenced by differences in elution conductivities) of the two Fvs can improve separation of mispaired species.
  • the Examples also show that aggregates, a major contaminant in scFv-containing bispecifics, can be effectively separated through selection of Fv pairs predicted to generate a sufficiently high surface charge (i.e., a high CEX elution conductivity).
  • the terms “about” or “comprising essentially of’ refer to a value or composition that is within an acceptable error range for the value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of’ can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “comprising essentially of’ can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When values or compositions are provided in the application and claims, unless otherwise stated, the meaning of “about” or “comprising essentially of’ should be assumed to be within an acceptable error range for that value or composition.
  • antibody means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate. polynucleotide, lipid, or combinations of the foregoing.
  • a target such as a protein, polypeptide, peptide, carbohydrate. polynucleotide, lipid, or combinations of the foregoing.
  • antibody encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, recombinant antibodies, multispecific antibodies, and bispecific antibodies.
  • An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. For example, a common configuration for an antibody has two full length antibody heavy chains and two full length antibody light chains.
  • antibody heavy chain refers to an antibody heavy chain, consisting of a variable region and a constant region as defined for a full-length antibody.
  • a full-length antibody heavy chain is a polypeptide consisting in N-terminal to C- terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CHI), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR- CH2-CH3.
  • heavy chain when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (s), gamma (y), and mu (p), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG (e.g., IgGl, IgG2, IgG3, and IgG4) and subclasses of IgA (e.g., IgAl and IgA2). Heavy chain amino acid sequences are known in the art.
  • antibody light chain refers to an antibody light chain, consisting of a variable region and a constant region as defined for a full-length antibody.
  • a full- length antibody light chain is a polypeptide consisting in N-terminal to C- terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL.
  • VL antibody light chain variable domain
  • CL antibody light chain constant domain
  • the term light chain when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (X) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are known in the art.
  • antibody fragment refers to a portion of an intact antibody.
  • An "antigen-binding fragment,” “antigen-binding domain,” or “antigen-binding region,” refers to a portion of an intact antibody that binds to an antigen.
  • An antigen-binding fragment can contain the antigenic determining regions of an intact antibody (e.g., the complementarity determining regions (CDR)).
  • CDR complementarity determining regions
  • antigen-binding fragments of antibodies include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies.
  • An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat. or hamster) or humans, or can be artificially produced.
  • multispecific antibody means that an antigen binding protein is capable of specifically binding to two or more different antigens.
  • a subcategory of multispecific antibodies is "bispecific antibodies,” which are capable of specifically binding to two different antigens.
  • an antibody “‘specifically binds” to a target antigen when it has a significantly higher binding affinity for, and consequently is capable of distinguishing, that antigen, compared to its affinity for other unrelated proteins, under similar binding assay conditions.
  • Antigen binding proteins that specifically bind an antigen may have an equilibrium dissociation constant (KD) ⁇ 1 X 1 O' 6 M. The antigen binding protein specifically binds antigen with “high affinity” when the KD is ⁇ 1 x 10‘ 8 M.
  • an "isolated antibody” refers to an antibody population that comprises a single species of antibody.
  • a particular isolated antibody consists of an antibody population having a single heavy chain amino acid sequence and a single light chain amino acid sequence, which binds to a single epitope.
  • An isolated antibody can, however, have crossreactivity to other antigens, such as related molecules from different species.
  • a population of antibodies may still be an "isolated antibody” when contaminated by small amounts of other antibody species.
  • an isolated antibody may contain less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or no other antibody species.
  • a “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. Furthermore, “monoclonal” antibody refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.
  • variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
  • the variability' in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • VL and VL domain and VH region are used interchangeably to refer to the light chain variable region of an antibody.
  • VH and VH domain and VH region are used interchangeably to refer to the heavy chain variable region of an antibody.
  • the terms "constant region” and “constant domain” are interchangeable and have their common meaning in the art.
  • the constant region is an antibody portion, e g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen, but which can exhibit various effector functions, such as interaction with the Fc receptor.
  • the constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.
  • Fc region and “Fc domain” refer to a C-terminal region of an IgG heavy chain; in case of an IgGl antibody, the C-terminal region comprises - CH2-CH3 (see above).
  • an antibody arm refers to the combination of an antibody heavy chain and an antibody light chain.
  • an antibody comprises two antibody arms joined together by their respective heavy chain Fc regions.
  • the bispecific antibody comprises two distinct antibody arms joined together by their respective heavy chain Fc regions.
  • interface refers to the association surface that results from interaction one or more amino acids in a first antibody domain with one or more amino acids of a second antibody domain.
  • exemplary interfaces include the CHI /CL, VH/VL, CH2-CH2, and CH3/CH3 interfaces.
  • the interface includes, for example, hydrogen bonds, electrostatic interactions, or salt bridges between the amino acids forming an interface.
  • a “humanized antibody” refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human framework regions and constant regions.
  • a humanized antibody may comprise substantially all of at least one. and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, e.g., a non- human antibody refers to an antibody that has undergone humanization.
  • humanized antibodies are human immunoglobulins in which residues from the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability. Accordingly, humanized antibodies are also referred to as "CDR grafted" antibodies.
  • CDR grafted antibodies Early examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539; Roguska et al.. Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). Many additional examples and methods relating to humanization of antibodies have subsequently been published.
  • a "human antibody” refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term "human antibody,” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • ADC antibody drug conjugate
  • ADC antibody drug conjugate
  • ADC's "cargo” antibody drug conjugate
  • Such linkage is achieved via covalent linkage between the antibody or antibody fragment and its cargo, and such linkage is optionally facilitated by a linker.
  • the linked cargo is a drug or agent with an intended therapeutic effect.
  • antibody fusion refers to the combination of an antibody or antibody fragment with a non-antibody polypeptide into a single polypeptide.
  • an antibody fusion may comprise an antibody in which one or more heavy or light chains have been combined with a cytokine.
  • An antibody fusion can be generated by joining two or more genes that originally coded for the separate components of the fusion into a single new gene that, when translated, results in the combined polypeptide.
  • the term "host cell” can be any type of cell, e.g.. a primary’ cell, a cell in culture, or a cell from a cell line.
  • the term “host cell” refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, e.g., due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • the developed strategy for building block pair selection is called purification-based Building Block Compatibility Screening (pBBCS).
  • pBBCS purification-based Building Block Compatibility Screening
  • This is a 2-step assay that first requires empirically characterizing the physicochemical properties of building blocks (e.g., CIEX elution profiles) by expressing and purifying parental proteins from which the building blocks are derived.
  • CIEX elution profiles e.g., CIEX elution profiles
  • This first step may be adapted to a high-throughput process to enable the quick expression and purification of all candidate building blocks.
  • FIG. 3 is a schematic overview of pBBCS-aided CIEX separation that can be achieved by using AConductivity as an indicator of the extent to which common contaminant species will separate from the desired species during CIEX. Applying this in silico selection of compatible building blocks will reduce screening panel size and thereby save time and resources in the development of bispecifics.
  • the physiochemical property to be determined as part of pBBCS relates to the charge of proteins.
  • Properties that may be used include those that allow prediction of how the surface charge building blocks will contribute to the surface charge of a multicomponent polypeptide, and thus contribute to an understanding of how well undesired species can be separated from a desired multicomponent polypeptide.
  • Relevant charge-related properties that may be determined include the pH. pl, pKa, elution conductivity, surface residue charge, and number of charged residues of the proteins to be analyzed. The value or relative magnitude of charge-related properties can be determined by any range of methods.
  • Methods that may be used in the determination of the above charge-related properties of proteins include electrophoretic mobility assays (e.g., capillary electrophoresis, electrophoretic light scattering, or membrane confined electrophoresis), steady state electrophoresis, ion exchange chromatography (e.g., anion exchange chromatography (AIEX) or cation-exchange chromatography (CIEX)), hydrophobic interaction chromatography, reversed phase chromatography, and mass spectrometry.
  • the physiochemical property to be determined is the elution conductivity of the proteins in question, and the elution conductivity may be determined by chromatographic methods such as AIEX or CIEX.
  • one or more building blocks can be selected from a pool of candidates molecules (which may be defined as. e.g., "pool A”) that comprise candidate building blocks.
  • two or more building blocks can be selected from two pools of candidate molecules (which may be defined as, e.g., "pool A” and “pool B”) that comprise candidate building blocks A and B, respectively.
  • the physical property in question for each member of those pools may be analyzed.
  • the determined values of that physical property for a given pool of candidates may then be compared to the values of that physical property for another component, or to the values of candidates from another pool.
  • a physical property is measured for all members of a first pool (pool A) and of a second pool (pool B), and the those values are used to select two or more building blocks for a multicomponent polypeptide.
  • the physiochemical property to be determined in a pool A and a pool B is the elution conductivity of the proteins in question, and the elution conductivity may be determined by chromatographic methods such as AIEX or CIEX.
  • the values of the physical property' to be determined for candidate building blocks are used to predict the value of the physical property of potential multicomponent polypeptides.
  • the value of a physical property of a multicomponent polypeptide can be considered to be the average of the value of that physical property for each of its component parts. Therefore, when a priority is to be able to differentiate the multicomponent polypeptide from its component parts, building block selection may comprise comparing the property' values for candidate components and selecting those components with the greatest difference in magnitude. A large difference in component property magnitude results in the greatest chance that the multicomponent polypeptide's physical property' value will be distinguishable from the physical property 's values for the component parts.
  • the phy sical property to be determined for the candidate building blocks is conductivity. Therefore, in some embodiments, the difference in elution conductivities (z.e., AConductivity or ACond) of all possible building block combinations is determined.
  • the conductivity of candidate building blocks may' be determined by any method available in the art. In some embodiments, the conductivity' of each candidate building block is determined using cation-exchange chromatography (CIEX), anion-exchange chromatography (AIEX), hydrophobic interaction chromatography, or reversed phase chromatography.
  • the method of screening or method of constructing multicomponent polypeptides comprises selecting component building blocks (e.g., a building block A and a building block B) for a multicomponent polypeptide based on those building blocks have a larger AConductivity than other candidate pairs of building blocks.
  • component building blocks e.g., a building block A and a building block B
  • the selected building block A came from a pool A molecule measured to have a conductivity within the top 25% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity' within the bottom 25% of tested pool B molecules.
  • the selected building block A came from a pool A molecule measured to have a conductivity’ within the top 20% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity within the bottom 20% of tested pool B molecules.
  • the selected building block A came from a pool A molecule measured to have a conductivity within the top 15% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity within the bottom 15% of tested pool B molecules.
  • the selected building block A came from a pool A molecule measured to have a conductivity within the top 10% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity 7 w ithin the bottom 10% of tested pool B molecules.
  • the selected building block A came from a pool A molecule measured to have a conductivity within the top 5% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity within the bottom 5% of tested pool B molecules.
  • the selected building block A came from a pool A molecule measured to have a conductivity within the top 30% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity within the bottom 30% of tested pool B molecules.
  • the selected building block A came from a pool A molecule measured to have a conductivity' within the top 35% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity within the bottom 35% of tested pool B molecules.
  • the selected building block A came from a pool A molecule measured to have a conductivity within the top 40% of tested pool A molecules
  • the selected building block B came from a pool B molecule measured to have a conductivity within the bottom 40% of tested pool B molecules.
  • the method of screening or method of constructing multicomponent polypeptides comprises selecting component building blocks (e.g., a building block A and a building block B) for a multicomponent polypeptide based on those building blocks have at least a particular AConductivity.
  • the selected building blocks A and B respectively, come from pool A and B molecules that have a AConductivity greater than 4 mS/cm.
  • the selected building blocks A and B respectively, come from pool A and B molecules that have a AConductivity greater than 5 mS/cm.
  • the selected building blocks A and B come from pool A and B molecules that have a AConductivity' greater than 6 mS/cm. In some embodiments, the selected building blocks A and B, respectively, come from pool A and B molecules that have a AConductivity greater than 7 mS/cm. In some embodiments, the selected building blocks A and B, respectively, come from pool A and B molecules that have a AConductivity greater than 8 mS/cm. In some embodiments, the selected building blocks A and B, respectively, come from pool A and B molecules that have a AConductivity' greater than 9 mS/cm.
  • the pool molecules are immunoglobulins.
  • the pool molecules are antibodies or antibody fragments.
  • the building blocks to be incorporated as components may be part or all of the antibody or antibody fragment.
  • the building block may be part or all of an antibody heavy chain, an antibody light chain, an antibody arm (i.e., a combination of an antibody heavy chain and an antibody light chain), an antibody fragment (e.g., a Fab fragment, a VH domain, a VL domain, an Fc region), or a polypeptide comprising domains from the antibody (e.g., an scFv).
  • any multicomponent polypeptide may be constructed, and any components for such multicomponent polypeptides may be screened, using the methods described herein.
  • bispecific antibodies may be constructed using pools of antibodies that specifically bind to each of their two target antigens.
  • the bispecific antibodies to be constructed comprise two heavy chains and two light chains.
  • the bispecific antibodies to be constructed comprise two antibody arms, wherein each arm was taken from a separate pool of candidate antibodies.
  • the multicomponent polypeptide is comprises an antibody arm from a first pool and an Fc-linked scFv from a second pool.
  • the multicomponent polypeptide is a monovalent antibody comprising a first heavy chain from a first pool and a second heavy chain from a second pool.
  • the multicomponent polypeptide to be constructed or screened comprises a combination of an antibody-derived component and a non-antibody-derived component.
  • the multicomponent polypeptide comprises an antibody fusion with anon-antibody protein.
  • the multicomponent polypeptide comprises an antibody -cytokine fusion.
  • the multicomponent polypeptide comprises an antibody-drug conjugate.
  • the method of construction or screening comprises measuring the absolute conductivity of the components of the multicomponent polypeptide.
  • the method comprises estimating the absolute conductivity of the selected multicomponent polypeptides by summing the conductivity of the pool A molecule and pool B molecule that supply the building blocks for each multicomponent polypeptide, and keeping in the selected set only those multicomponent polypeptides within an upper range of the estimated summed conductivities.
  • the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 60% of the estimated summed conductivities.
  • the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 55% of the estimated summed conductivities.
  • the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 50% of the estimated summed conductivities. In some embodiments, the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 45% of the estimated summed conductivities. In some embodiments, the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 40% of the estimated summed conductivities. In some embodiments, the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 35% of the estimated summed conductivities. In some embodiments, the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 30% of the estimated summed conductivities.
  • the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 25% of the estimated summed conductivities. In some embodiments, the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 20% of the estimated summed conductivities. In some embodiments, the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 15% of the estimated summed conductivities. In some embodiments, the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 10% of the estimated summed conductivities. In some embodiments, the method comprises keeping in the selected set only those multicomponent polypeptides within the upper 5% of the estimated summed conductivities.
  • the method of construction or screening comprises selecting component building blocks (e.g., a building block A and a building block B) for a multicomponent polypeptides based on those building blocks have at least a particular absolute conductivity.
  • the absolute conductivity is determined by summing the conductivity of the component molecules, or summing the conductivity of the pool molecules from which the component molecules are derived.
  • the selected building blocks have an absolute conductivity greater than 4 mS/cm.
  • the selected building blocks have an absolute conductivity greater than 5 mS/cm.
  • the selected building blocks have an absolute conductivity greater than 6 mS/cm.
  • the selected building blocks have an absolute conductivity greater than 7 mS/cm. In some embodiments, the selected building blocks have an absolute conductivity greater than 8 mS/cm. In some embodiments, the selected building blocks have an absolute conductivity greater than 9 mS/cm. In some embodiments, the selected building blocks have an absolute conductivity greater than 10 mS/cm. In some embodiments, the selected building blocks have an absolute conductivity greater than 11 mS/cm. In some embodiments, the selected building blocks have an absolute conductivity greater than 12 mS/cm. In some embodiments, the selected building blocks have an absolute conductivity greater than 13 mS/cm. In some embodiments, the selected building blocks have an absolute conductivity greater than 14 mS/cm. In some embodiments, the selected building blocks have an absolute conductivity greater than 15 mS/cm.
  • the method of construction or screening comprises comparing the elution profile of each selected multicomponent polypeptide with the elution profiles of the pool A molecule and the pool B molecule from which its component parts were derived.
  • the elution profile is determined by CIEX chromatography.
  • the method comprises keeping, or proceeding with, only those multicomponent polypeptides whose principal elution peak has a small amount of overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule.
  • the multicomponent polypeptide's elution peak has less than 30% overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule. In some embodiments, the multicomponent polypeptide's elution peak has less than 25% overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule. In some embodiments, the multicomponent polypeptide's elution peak has less than 20% overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule.
  • the multicomponent polypeptide's elution peak has less than 15% overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule. In some embodiments, the multicomponent polypeptide's elution peak has less than 10% overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule. In some embodiments, the multicomponent polypeptide's elution peak has less than 5% overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule. In some embodiments, the multicomponent polypeptide's elution peak has no overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule.
  • a host cell when cultured under appropriate conditions, synthesizes heterodimeric antibody that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
  • the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
  • a host cell may be eukaryotic or prokaryotic.
  • Mammalian cell lines available as hosts for expression are known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC) and any cell lines used in an expression system known in the art can be used to make the recombinant polypeptides of the invention.
  • ATCC American Type Culture Collection
  • host cells are transformed with a recombinant expression vector that comprises DNA encoding a desired heterodimeric antibody.
  • the host cells that may be employed are prokaryotes, yeast, or higher eukaryotic cells.
  • Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli.
  • Higher eukary otic cells include insect cells and established cell lines of mammalian origin.
  • suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981. Cell 23: 175), L cells, 293 cells, C127 cells. 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (Rasmussen et al.. 1998, Cytotechnology 28: 31), HeLa cells, BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J.
  • human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells.
  • mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49, for example, can be used for expression of the polypeptide when it is desirable to use the polypeptide in various signal transduction or reporter assays.
  • it is possible to produce the polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria.
  • Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides.
  • Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides.
  • the antibody or antibody fragment is made in yeast or bacteria, it may be desirable to modify the product produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain a functional product. Such covalent attachments can be accomplished using known chemical or enzymatic methods.
  • a polypeptide can also be produced by operably linking the isolated nucleic acid of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif..
  • a host cell that comprises an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a “recombinant host celk’.
  • Cell lines may be selected through determining which cell lines have high expression levels and constitutively produce antigen binding proteins with the desired binding properties.
  • a cell line from the B cell lineage that does not make its own antibody but has a capacity to make and secrete a heterologous antibody can be selected.
  • a method of screening potential components for multicomponent polypeptides wherein the multicomponent polypeptides each comprise at least one building block A and at least one building block B, and wherein the screening comprises: a. identifying and individually purify ing a group of molecules (pool A) wherein each molecule comprises one or more copies of a candidate building block A; b. identifying and individually purifying a group of molecules (pool B) wherein each molecule comprises a one or more copies of a candidate building block B; c. determining the conductivity of each pool A molecule and of each pool B molecule; d. determining the difference in conductivity (ACond) between each molecule of pool A and each molecule of pool B; and e.
  • the screening comprises: a. identifying and individually purify ing a group of molecules (pool A) wherein each molecule comprises one or more copies of a candidate building block A; b. identifying and individually purifying a group of molecules (pool B) wherein each molecule comprises a one or more copies of
  • each pool A molecule and each pool B molecule comprises an antibody.
  • the multicomponent polypeptide is an antibody drug conjugate or antibody fusion.
  • the multicomponent polypeptide comprises an antibody fusion, and wherein the antibody is fused to a cytokine.
  • the method further comprises estimating the absolute conductivity of the selected multicomponent polypeptides by summing the conductivity of the pool A molecule and pool B molecule that supply the building blocks for each multicomponent polypeptide, and keeping in the selected set only those multicomponent polypeptides within the upper 50% of the estimated summed conductivities.
  • the method comprises keeping only those multicomponent polypeptides with an absolute conductivity greater than 11 mS/cm.
  • the method further comprises comparing the CIEX elution profile of each selected multicomponent polypeptide with the CIEX elution profiles of the pool A molecule and the pool B molecule from which its component parts were derived, and keeping only those multicomponent polypeptides whose principal elution peak have less than 25% overlap in peak area with the principal elution peak of either the pool A molecule or the pool B molecule.
  • a method of constructing a multicomponent immunoglobulin molecule comprising the steps of: a. measuring the surface charge of each member of a pool A of immunoglobulins and measuring the surface charge of each member of a pool B of immunoglobulins; b. selecting an immunoglobulin from pool A (immunoglobulin A) and an immunoglobulin from pool B (immunoglobulin B) that have a difference in conductivity' (ACond) greater than 5 mS/cm, as measured by CIEX; and c.
  • each pool A molecule and each pool B molecule comprises an immunoglobulin.
  • each pool A molecule and each pool B molecule comprises an antibody.
  • building block B comprises all or part of an antibody heavy chain.
  • the method of embodiment 28, wherein building block B is an antibody arm.
  • the method of any of embodiments 25-29, wherein the multicomponent polypeptide is a bispecific antibody.
  • the method of embodiment 32, wherein the multicomponent polypeptide comprises an antibody fusion, and wherein the antibody is fused to a cytokine. 34.
  • the method further comprises, prior to combining the building blocks, estimating the absolute conductivity of multicomponent polypeptides comprising the selected components by summing the conductivity of the pool A molecule and pool B molecule that supply the building blocks for each multicomponent polypeptide, and constructing only those multicomponent polypeptides within the upper 50% of the estimated summed conductivities.
  • Plasmid Construction genes to be expressed as polypeptides were synthesized by Twist Bioscience. Genes were cloned individually into vectors for mammalian transient expression using the Golden Gate assembly method. See Engler C et al.. PLoS One 3:e3647 (2008). which is incorporated herein by reference in its entirety) All chains containing Fc regions were constructed using a human IgGl scaffold (IgGl-SEFL2.2) carrying an aglycosylation mutation (N297G), as well as an engineered disulfide bond in the CH2 domain. ⁇ See Estes et al., iScience 24: 103447 (2021); and Jacobsen FW et al., J. Biol. Chem.
  • scFv sequences contained an engineered disulfide to increase stability of the Fv. After sequencing confirmation by Sanger, transfection-grade DNA was prepared using Maxi plasmid purification kits (Qiagen).
  • the purified plasmids were mixed at a ratio of 1 : 1 (HC:LC) for mAbs, 1 : 1 : 1 : 1 for Hetero-IgGs, 1 : 1 for IgG-scFvs, 1 : 1 : 1 for AmAbs and other 3-chain formats containing scFvs.
  • HC:LC Hetero-IgGs
  • IgG-scFvs 1 : 1 for AmAbs and other 3-chain formats containing scFvs.
  • 0.5 pig DNA was incubated with 1.5 pL PEImax reagent (Polysciences, catalog # 24765-2) in 100 pL FreeStyle F-17 medium for 10 minutes, then added to the cell culture.
  • CIEX cation-exchange chromatography
  • Protein A eluates were diluted with 20 ml of 20 mM MES, pH 6.2 and loaded onto a 1 ml cation ion-exchange column (HiTrap SP- HP, GE Life Sciences, catalog # GE29-0513-24) at 1 ml/min.
  • a 1 ml cation ion-exchange column HiTrap SP- HP, GE Life Sciences, catalog # GE29-0513-24
  • the samples were eluted with a linear 0-400 mM NaCl gradient over 40 column volumes at 0.4 ml/min.
  • Purity of purified samples were analyzed with non-reducing micro capillary electrophoresis (MCE) and analytical size exclusion chromatography (SEC).
  • MCE micro capillary electrophoresis
  • SEC analytical size exclusion chromatography
  • sample buffer 8.4 mM Tns-HCl pH 7.0, 7.98% Glycerol, 2.38 mM EDTA, 2.8% SDS and 2.4 mM lodoacetamide
  • sample buffer 8.4 mM Tns-HCl pH 7.0, 7.98% Glycerol, 2.38 mM EDTA, 2.8% SDS and 2.4 mM lodoacetamide
  • FIGS. 1A-1C are diagrams of a series of different antibody structures showing the Fc, Fv, Fab, CDR, and constant (VH, VL, CL, and CHI) regions identified on one of the arms of each antibody.
  • FIG. 1 A shows the monospecific and bivalent antibody structure used in most natural antibodies. Such an antibody contains two arms that can serve as a building block in constructing bispecific antibodies (labelled as “Building Block A”). Two bispecific antibodies constructed by combining Building Block A with other building blocks are shown in FIGS. IB and 1C.
  • FIG. 1A-1C are diagrams of a series of different antibody structures showing the Fc, Fv, Fab, CDR, and constant (VH, VL, CL, and CHI) regions identified on one of the arms of each antibody.
  • FIG. 1 A shows the monospecific and bivalent antibody structure used in most natural antibodies. Such an antibody contains two arms that can serve as a building block in constructing bispecific antibodies (labelled as “Building Block A”). Two bispecific antibodies constructed by combining Building Block
  • IB shows an exemplary bispecific antibody in which an arm capable of binding antigen A is linked to an arm capable of binding to antigen B (Building Block B) through their respective Fc regions, where the interaction's specificity is facilitated by complementary charge-pair mutations in those Fc regions.
  • FIG. 1C shows an exemplary AmAb. which is comprised of a standard antibody arm capable of binding antigen A (Building Block A) and an antibody arm capable of binding antigen B that has an scFv in place of the Fab region (Building Block B'). While the various building blocks can be combined to form desired AmAbs or other bispecific antibodies, those same building blocks may be present as. or form into, impurities that are difficult to purify away from the desired species. For example, FIG. IF shows exemplary impurities that may be present alongside the desired AmAb species when expressing the components of that AmAb.
  • Plasmids encoding the needed heavy and light chains of the Hetero-IgG’ s parental antibodies i.e., a Building Block A Homodimer and a Building Block B Homodimer, capable of binding antigen A and antigen B, respectively) were constructed using the same method and the same plasmid vector. Subsequently, each set of plasmids necessary for expressing a given bispecific antibody was transiently expressed in a population of suspension human embryonic kidney 293-6E cells (NRC- BRI) using the PEImax transfection reagent. Seven days following transfection, the conditioned medium from each population of transfected cells was harvested for antibody purification.
  • parental antibodies i.e., a Building Block A Homodimer and a Building Block B Homodimer, capable of binding antigen A and antigen B, respectively
  • each antibody solution was analyzed using CIEX chromatography to obtain a CIEX elution profile for those antibody solutions.
  • Raw chromatogram traces were exported (UNICORN 7, Cytiva) and CIEX profiles were generated with conductivity plotted against A280nm (Microsoft Excel). Elution positions, defined as the conductivity at which maximum A280nm signal was detected, were manually identified. Peak Separation was calculated as the difference in conductivities between the peaks indicated. Negative values were assigned as appropriate for Peak Separation when the AmAb elution position was earlier than both parental mAbs. For cases in which Peak 2 was directly overlapped with Peak 1, the peak separation was scored as zero. Separation was also qualitatively scored as either “good” or “bad” (as shown in FIGS. 8A-8B) based on the overlap observed between the AmAb main peak and the corresponding mAbs.
  • each antibody’s elution peak is labelled, illustrating that the Hetero-IgG eluted at a conductivity between those of the building block homodimers.
  • the CIEX elution profile can be used as a surrogate for surface charge, the Hetero-IgG has a surface charge between those of the building block homodimers.
  • the CIEX elution profile for an AmAb, Building Block A Homodimer, and Building Block B Homodimer were overlaid in a single chart, shown in FIG. 2B.
  • the AmAb had a surface charge between those of the building block homodimers.
  • 2D was a noncovalent homodimer that maintains a mAb-like quaternary structure due to HC/HC mispairing via the CH3 dimer, mimicking the CIEX profile of the corresponding parental mAh.
  • This contaminant may be the result of an imbalance in chain expression, yvith the scFv-containing polypeptide chain expressing at a low er level and the excess scFv-free chain driving homodimer formation.
  • the data in FIG. 2C also demonstrates that the stoichiometry of components (z.e., the number of scFvs) also impacts a bispecific antibody’s CIEX elution profile.
  • a bispecific antibody with two scFvs (C2mAb) displayed a greater apparent positive charge than a bispecific antibody yvith a single scFv (ClmAb) (FIG. 2C).
  • pBBCS Purification-Based Building Block Compatibility Screening
  • the second step calculates the difference in elution conductivities of two building blocks i.e., AConductivity or ACond) of all possible building block combinations for generating a bispecific.
  • FIG. 3 is a schematic overview of pBBCS-aided CIEX separation that can be achieved by using AConducti vity as an indicator of the extent to which common contaminants such as homodimeric and single-arm species will separate from the AmAb species during CIEX. Applying this in silico selection of compatible building blocks will reduce screening panel size and thereby save time and resources in the development of bispecifics.
  • FIG. 7 shows a plot of the measured conductivity and the predicted conductivity (z.e., the midpoint of the two parent mAbs).
  • FIGS. 5A-5C show exemplary overlays of AmAbs with their corresponding parental mAbs.
  • FIG. 5B shows an overlay with a AConductivity between parental mAbs of 6.3 mS/cm, which resulted in an AmAb with a peak that only slightly overlapped with the parental mAb peaks.
  • the soluble antigen binding affinity of the purified proteins from each peak were measured.
  • the antibodies were first captured onto streptavidin SAX biosensor tips with a biotinylated (1 biotin/molecule), polyclonal capture antibody (Jackson ImmunoResearch, catalog# 109-005-098) and then incubated with a dilution series of each soluble (monovalent) antigen.
  • This assay format was chosen so the bivalent antibodies would be immobilized and on the biosensor tips and tested versus the same serial dilution of each soluble antigen.
  • the measured quantitative KD affinities thus represent monovalent 1: 1 binding interaction and can be directly compared.
  • Experiments were run in a ForteBio Octet HTX instrument using the 96-tip mode with standard 5 Hz data acquisition rate at 27 °C and 1000 RPM.
  • Raw Octet binding data was processed with installed SPR kinetic curve fitting package and globally fit to a 1:1 binding model to determine the association rate constant (£ a ) and the dissociation rate constant (kd).
  • the equilibrium dissociation constant (A'D) was then calculated the as a ratio of kd'K.
  • the KD values are average ⁇ standard error from three independent experiments. As shown in FIGS. 11 A-l IB and Tables 2 and 3 below, the material in Peak 1 and Peak 2 show comparable binding affinities in vitro for both Target A and Target B.
  • Target A-expressing cells were harvested, counted and seeded to assay plates.
  • tested molecules were titrated in assay medium from >50nM 1:3 down for 12 points. Tested molecules were transferred to assay plates pre-seeded with Target A- expressing cells and incubated at room temperature for 30 minutes.
  • Cells stably expressing Target B (Target B binding indicates the crosslinking of the assayed molecules) were harvested, counted and resuspended to the desired concentration. Cells stably expressing Target B were then transferred to assay plates.
  • Target A- to Target B-expressing cells The ratio of Target A- to Target B-expressing cells was set to 1 : 1 based on assay development data. Assay plates were incubated at 37°C with 5% CO2. On Day 3, chemokine levels in culture supernatant were measured. The same experiment was performed using Target A-expressing cells co-cultured with parental cells which do not express Target B, to investigate the crosslinking-independent activities of the tested molecules. Chemokine levels were calculated and plotted against the antibody concentration as log
  • Peak 1 and Peak 2 maintained their CIEX elution positions after approximately 4 weeks at 4° C, indicating that the AmAb and AmAb-multimer subspecies show a stable conformation and can therefore likely be separated.
  • Some AmAbs showed separation of monomeric and multimeric species in CIEX, while others showed severe overlap.
  • the establishment of a method capable of predicting separation of this newly identified impurity in silico would increase the applicability of pBBCS. To this end, experiments were performed to gain a deeper understanding of the molecular basis for aggregate behavior during CIEX.
  • an AmAb with a high affinity for CIEX resin would generate multimers with a proportionally higher affinity for the column matrix, thereby increasing the species separation.
  • comparing main peak conductivities with the degree of separation between the AmAb and the aggregate peak showed that greater AmAb conductivity was correlated with greater separation from the aggregate.
  • AmAb conductivity can be accurately predicted from the building blocks conductivities (FIG. 7)
  • pBBCS can also inform the design of bispecifics with predictable separation from aggregate species. Analysis of the data showed that an AmAb conductivity of ⁇ 11 mS/cm or higher enabled effective separation of aggregates (FIG. 12B).
  • FIGS. 14A and 14B show examples in which two AmAbs, containing identical Fab arm sequences but different scFv arm sequences, generated aggregates with differing degrees of separation after CIEX.
  • Target Al building block with Target Bl building block (AlxBl) produced an AmAb with an elution conductivity of 5.6 mS/cm
  • pairing the same Target Al with Target B2 (AlxB2) generated an AmAb with an elution conductivity of 11.8 mS/cm.
  • Analytical SEC of the corresponding Protein A eluates shows the presence of aggregation in both molecules (FIGS. 13 A and 13B). However, the aggregate from the AlxBl AmAb fails to separate during SEC, as evidenced by the HMW species detected in each fraction by SEC (FIG. 14A, plotted as grey dots).

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Abstract

Des polypeptides à composants multiples tels que des anticorps bispécifiques comprennent de multiples chaînes polypeptidiques uniques, et garantissent que leur assemblage quaternaire approprié est essentiel à la fois pour l'efficacité et la sécurité. La présente invention concerne le criblage de compatibilité de blocs de construction (pBBCS) à base de purification, un nouveau procédé permettant de sélectionner des composants pour l'intégration réussie dans des polypeptides à composants multiples. Le pBBCS utilise l'analyse de propriétés physiques telles que la charge de surface de ces composants, et facilite la mise au point rapide d'efforts d'optimisation sur uniquement des composants candidats prometteurs. Par conséquent, le pBBCS peut jouer un rôle essentiel dans les flux de travail de nouvelle génération pour des polypeptides à composants multiples afin de garantir des coûts inférieurs, une vitesse supérieure et une augmentation des taux de réussite globaux.
PCT/US2024/052797 2023-10-26 2024-10-24 Exploitation de propriétés d'anticorps natifs pour guider une sélection bispécifique Pending WO2025090753A1 (fr)

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