CN117794949A - Antibody optimisation - Google Patents

Antibody optimisation Download PDF

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CN117794949A
CN117794949A CN202280054863.XA CN202280054863A CN117794949A CN 117794949 A CN117794949 A CN 117794949A CN 202280054863 A CN202280054863 A CN 202280054863A CN 117794949 A CN117794949 A CN 117794949A
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amino acid
domain
numbering
acid residue
antibody
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B·D·鲍斯
D·J·斯托克尔
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Eli Lilly and Co
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Eli Lilly and Co
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Priority claimed from PCT/US2022/074581 external-priority patent/WO2023015280A1/en
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Abstract

The present disclosure relates to methods of developing, designing and producing therapeutic antibodies to improve the developability of antibodies or antibody drug conjugates.

Description

Antibody optimisation
Technical Field
The invention belongs to the field of medicine. More specifically, the present invention provides methods for developing, designing and producing therapeutic antibodies and conjugates thereof. In certain embodiments, the methods disclosed herein provide for improvements in the developability of antibodies or antibody drug conjugates, e.g., in viscosity and/or aggregation reduction.
Background
Therapeutic antibodies have been developed to treat a variety of diseases. Common challenges faced in the development of therapeutic antibodies include, for example, high viscosity, aggregation, low solubility, half-life extension, and immunogenicity. Some of these challenges can affect the stability of therapeutic antibodies, which can be further exacerbated when formulated for delivery. While therapeutic antibodies are typically administered via Intravenous (IV) infusion, subcutaneous administration provides several advantages over IV, such as a more effective pharmacokinetic profile, options for self-administration, which are important for chronic diseases requiring frequent chronic dosing. In addition, subcutaneous administration enables a ready-to-use pre-filled delivery device, provides greater patient comfort, reduces treatment time, potentially improves compliance, treatment outcome, and reduces cost.
However, the development of therapeutic antibody formulations suitable for subcutaneous administration presents multiple challenges, in particular, the limited volumes available for subcutaneous administration require highly concentrated antibody solutions. Highly concentrated antibody solutions can result in increased viscosity and/or aggregation. High viscosity can increase injection time and pain at the injection site, affect patient compliance, and can also destabilize the drug substance during bioprocessing, affect pharmacokinetic profiles, bioactivity, increase manufacturing costs, and potentially hinder development and progression of the drug. Similarly, aggregation is also a major problem, as the trend towards high concentration solutions increases the probability of protein-protein interactions, which in turn facilitate aggregation.
Methods of reducing the viscosity of antibody therapeutics have been investigated. Such methods include changing formulation conditions, such as adjusting pH, buffer conditions, ionic strength, or adding other excipients. However, these methods have proven to be limited in addressing the viscosity issues associated with a broader range of antibody therapeutics. For example, in some cases, changing formulation conditions may require additional manufacturing process steps that affect stability, aggregation, immunogenicity, and/or pharmacokinetic profiles, all of which can be costly and compromise development of antibody therapeutics. In addition, attempts to optimize the amino acid sequence in the variable region of IgG1 antibodies to reduce viscosity have been reported (TOmar et al, mabs 2016,8 (2): 216-228). However, such methods are limited to the specific antibody variable region to be optimized. Thus, there remains a need for additional methods of reducing the viscosity of therapeutic antibodies at high concentrations, wherein the methods are applicable across a broad spectrum of antibody therapeutics, such as IgG1, igG2, igG3, or IgG4 antibodies, and are not limited to a particular therapeutic of interest, and wherein such methods do not negatively affect affinity, stability, aggregation, immunogenicity, or biological function, and do not require expensive process or formulation changes.
Methods have been studied to improve the half-life of peptides and small proteins, for example, fusing peptides to the Fc portion of antibodies for extended time. In some cases, such methods involve the expression of smaller proteins or peptides on the N-or C-terminus of an antibody Fc consisting of hinge, CH2 and CH3 domains. This type of fusion can improve half-life by, for example, reducing renal clearance. However, such fusion may result in an increase in viscosity or a decrease in pH stability of the fusion protein, which may require significant formulation optimization or further engineering of the peptide or protein sequence.
Disclosure of Invention
Accordingly, the present disclosure addresses one or more of the above needs by providing alternative compositions and methods for improving the developability of antibody therapeutics, for example by reducing the viscosity of antibodies or antibody drug conjugates at high concentrations and/or improving half-life. In particular, the present disclosure provides compositions and methods that include modification of the constant regions of antibodies, wherein the methods do not negatively impact antibody affinity, aggregation, and/or stability, or biological functions such as effector functions, or require expensive alterations in formulation and downstream manufacturing processes. More particularly, the present disclosure provides compositions and methods comprising amino acid substitutions in the IgG heavy chain constant domain of antibodies, which may be useful across a wide range of antibody therapeutic platforms, such as IgG2, igG3, or IgG4 antibody therapeutics.
Accordingly, in certain embodiments, the present disclosure provides antibodies having a modified human IgG Heavy Chain (HC) constant region comprising one or more of the following amino acid substitutions compared to a wild-type human IgG heavy chain constant region: e137G, D203N, Q274K, Q355R, E419Q (all positions numbered according to EU numbering). Accordingly, in certain embodiments, the present disclosure provides antibodies comprising a human IgG HC constant region comprising a constant heavy chain 1 (CH 1) domain, a constant heavy chain 2 (CH 2) domain, and a constant heavy chain 3 (CH 3) domain, and a human IgG Light Chain (LC) constant region, wherein the human IgG HC constant region comprises: lysine at amino acid residue 274 (EU numbering) of the CH2 domain; arginine at amino acid residue 355 (EU numbering) of the CH3 domain; glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; glycine at amino acid residue 137 (EU numbering) of the CH1 domain; asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and arginine at amino acid residue 355 (EU numbering) of the CH3 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glutamine at amino acid residue 419 of the CH3 domain (EU numbering); lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glycine at amino acid residue 137 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glycine at amino acid residue 137 of the CH1 domain (EU numbering); arginine at amino acid residue 355 of the CH3 domain (EU numbering) and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); glutamine at amino acid residue 419 of the CH3 domain (EU numbering) and glycine at amino acid residue 137 of the CH1 domain (EU numbering); glutamine at amino acid residue 419 of the CH3 domain (EU numbering) and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); glycine at amino acid residue 137 (EU numbering) of the CH1 domain and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering); arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), asparagine at amino acid residue 203 of the CH1 domain (EU numbering); arginine at amino acid residue 355 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); glutamine at amino acid residue 419 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering); glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; or lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CHl domain.
In certain embodiments, the disclosure provides antibodies having a modified human IgG Heavy Chain (HC) constant region comprising two or more of the following amino acid substitutions compared to a wild-type human IgG heavy chain constant region: e137G, D203N, Q274K, Q355R, E419Q, R409K (all positions numbered according to EU numbering). Accordingly, in certain embodiments, the present disclosure provides antibodies having a modified human IgG Heavy Chain (HC) constant region comprising three or more of the following amino acid substitutions compared to a wild-type human IgG heavy chain constant region: e137G, D203N, Q274K, Q355R, E419Q, R409K (all positions numbered according to EU numbering).
According to some embodiments, the present disclosure provides antibodies comprising a human IgG HC constant region comprising CH1, CH2, and CH3 domains, and a human IgG light chain constant region, wherein the human IgG HC constant region comprises: lysine at amino acid residue 274 (EU numbering) of the CH2 domain; arginine at amino acid residue 355 (EU numbering) of the CH3 domain; glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and arginine at amino acid residue 355 (EU numbering) of the CH3 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glutamine at amino acid residue 419 of the CH3 domain (EU numbering); or lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.
According to a further embodiment, the present disclosure provides an antibody comprising a human IgG HC constant region comprising CH1, CH2 and CH3 domains, and a human IgG light chain constant region, wherein the human IgG heavy chain constant region comprises lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.
In a further embodiment, an antibody of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3.
In some embodiments, the antibodies of the disclosure have a human IgG2, human IgG3, or human IgG4 isotype. In some embodiments, the antibodies of the disclosure have a human IgG4 isotype. In some embodiments, antibodies as described herein comprise a modified human IgG4 hinge region comprising an S228P mutation (EU numbering) that reduces IgG4 Fab arm exchange in vivo (see Labrijn et al, nat. Biotechnol.2009, 27 (8): 767). In some embodiments, the antibodies described herein have a modified IgG4 Fc region with reduced or eliminated Fc effector function (i.e., igG4 Fc effector is not effective). Such antibodies comprise, for example, amino acid residue modifications F234A and/or L235A in the IgG4 Fc region to reduce binding to fcγr (all residues are numbered according to EU numbering). In some embodiments, the antibodies described herein comprise an IgG4 hinge region comprising proline at residue 228, and an IgG4 Fc region comprising alanine at residues 234 and 235 (all residues numbered according to EU numbering).
In some embodiments, the antibodies of the disclosure comprise a human IgG heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 8. 9, 10, 12, 13 or 16.
In some embodiments, the disclosure also provides antibody drug conjugates comprising the antibodies disclosed herein.
In some embodiments, the antibodies of the present disclosure have modified human IgG HC constant regions that reduce the viscosity and/or improve the stability of the antibodies. In some embodiments, the antibodies or antibody drug conjugates of the disclosure have a reduced viscosity when compared to a wild-type antibody comprising a wild-type human IgG heavy chain constant region. In a further embodiment of the present disclosure, the viscosity of the antibody or antibody drug conjugate is reduced by about 25% to about 80% when compared to the wild-type antibody. In some embodiments, the antibodies of the present disclosure have a modified human IgG4HC constant region comprising one or more of the following amino acid substitutions compared to the wild-type human IgG4HC constant region: e137G, D203N, Q274K, Q355R, E419Q (all positions numbered according to EU numbering). In some embodiments, the antibodies of the present disclosure have a modified human IgG4HC constant region comprising one or more of the following amino acid substitutions compared to the wild-type human IgG4HC constant region: Q274K, Q355R, E419Q. In some embodiments, an antibody or antibody drug conjugate of the disclosure has a modified human IgG4HC constant region that reduces the viscosity of the antibody by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, or about 100%. In some embodiments, the antibodies of the disclosure have a therapeutically acceptable viscosity for delivery to a patient. In such embodiments, the viscosity is below 14cP, below 12cP, or below 10cP. In some embodiments, the antibody is administered by subcutaneous administration or intravenous administration.
In some embodiments, the antibodies of the present disclosure have a modified human IgG2 HC constant region comprising one or more of the following amino acid residues: E137G, D203N, Q274K (all positions are numbered according to EU numbering). In some embodiments, the antibodies of the present disclosure have a modified human IgG3 HC constant region comprising amino acid residue Q274K (numbered according to EU numbering). In such embodiments, the human IgG2 and/or human IgG3 antibodies have reduced viscosity when compared to a wild-type antibody comprising a wild-type human IgG2 or human IgG3 heavy chain constant region, respectively.
In some embodiments, the present disclosure provides methods of reducing the viscosity of a human IgG4 antibody comprising generating a variant of a human IgG4 antibody comprising a modified human IgG4 heavy chain constant region comprising CH1, CH2, and CH3 domains, and a human IgG light chain constant region, wherein the modified human IgG4 heavy chain constant region comprises the following amino acid residues: lysine at amino acid residue 274 (EU numbering) of the CH2 domain; arginine at amino acid residue 355 (EU numbering) of the CH3 domain; glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; glycine at amino acid residue 137 (EU numbering) of the CH1 domain; asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and arginine at amino acid residue 355 (EU numbering) of the CH3 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glutamine at amino acid residue 419 of the CH3 domain (EU numbering); lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glycine at amino acid residue 137 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glycine at amino acid residue 137 of the CH1 domain (EU numbering); arginine at amino acid residue 355 of the CH3 domain (EU numbering) and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); glutamine at amino acid residue 419 of the CH3 domain (EU numbering) and glycine at amino acid residue 137 of the CH1 domain (EU numbering); glutamine at amino acid residue 419 of the CH3 domain (EU numbering) and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); glycine at amino acid residue 137 (EU numbering) of the CH1 domain and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering); arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), asparagine at amino acid residue 203 of the CH1 domain (EU numbering); arginine at amino acid residue 355 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); glutamine at amino acid residue 419 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering); glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain; or lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain.
In some embodiments, the present disclosure provides methods of reducing the viscosity of a human IgG4 antibody comprising generating a variant of a human IgG4 antibody comprising a modified human IgG4 heavy chain constant region comprising CH1, CH2, and CH3 domains, and a human IgG light chain constant region, wherein the modified human IgG4 heavy chain constant region comprises the following amino acid residues: lysine at amino acid residue 274 (EU numbering) of the CH2 domain; arginine at amino acid residue 355 (EU numbering) of the CH3 domain; glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and arginine at amino acid residue 355 (EU numbering) of the CH3 domain; lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain; arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glutamine at amino acid residue 419 of the CH3 domain (EU numbering); or lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.
In some embodiments, the present disclosure provides methods of reducing the viscosity of a human IgG4 antibody comprising generating a variant of a human IgG4 antibody comprising a modified human IgG4 heavy chain constant region comprising CH1, CH2, and CH3 domains, and a human IgG light chain constant region, wherein the modified human IgG4 heavy chain constant region comprises the following amino acid residues: lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.
In some embodiments, nucleic acid sequences encoding antibodies of the present disclosure are provided. In some embodiments of the disclosure, nucleic acids encoding HC or LC of antibodies are provided. In some embodiments of the disclosure, nucleic acids encoding VH or VL of an antibody are provided. Some embodiments of the present disclosure provide vectors comprising nucleic acid sequences encoding antibodies HC or LC. Some embodiments of the present disclosure provide vectors comprising nucleic acid sequences encoding antibody VH or VL.
The nucleic acids of the present disclosure may be expressed in a host cell, for example, after the nucleic acid has been operably linked to an expression control sequence. Expression control sequences capable of expressing nucleic acids to which they are operably linked are well known in the art. Expression vectors may include sequences encoding one or more signal peptides that facilitate secretion of the polypeptide from the host cell. Expression vectors containing a nucleic acid of interest (e.g., a nucleic acid encoding an antibody heavy or light chain) can be transferred into a host cell by well known methods, such as stable or transient transfection, transformation, transduction, or infection. In addition, the expression vector may contain one or more selectable markers, such as tetracycline, neomycin, and dihydrofolate reductase, to aid in the detection of host cells transformed with the desired nucleic acid sequences.
In another aspect, provided herein are cells, e.g., host cells, comprising a nucleic acid, vector, or nucleic acid composition described herein. The host cell may be a cell stably or transiently transfected, transformed, transduced or infected with one or more expression vectors which express all or a portion of the antibodies described herein. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced or infected with an expression vector that expresses the HC and LC polypeptides of the antibodies of the disclosure. In some embodiments, the host cell may be stably or transiently transfected, transformed, transduced or infected with a first vector that expresses the HC polypeptide of the antibodies described herein and a second vector that expresses the LC polypeptide. Such host cells, e.g., mammalian host cells, can express antibodies that specifically bind human IL-4 ra as described herein. Mammalian host cells capable of expressing antibodies are known to include CHO cells, HEK293 cells, COS cells and NS0 cells.
In some embodiments, a cell, e.g., a host cell, comprises a first vector and a second vector comprising a nucleic acid sequence encoding an antibody as provided herein. In a further embodiment, the host cell is a mammalian cell.
The present disclosure further provides methods for producing an antibody or antibody binding fragment thereof as described herein by: culturing the above-described host cell, e.g., a mammalian host cell, under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium. The medium into which the antibody has been secreted may be purified by conventional techniques. Various protein purification methods can be employed, and such methods are known in the art and are described, for example, in Deutscher, methods in Enzymology 182:83-89 (1990) scenes, protein Purification: principles and Practice, 3 rd edition, springer, N.Y. (1994).
The disclosure further provides antibodies or antibody-binding fragments thereof produced by any of the methods described herein.
In another aspect, provided herein are pharmaceutical compositions comprising an antibody, nucleic acid, or vector described herein. Such pharmaceutical compositions may also comprise one or more pharmaceutically acceptable excipients, diluents or carriers. Pharmaceutical compositions may be prepared by methods well known in the art (e.g., remington: the Science and Practice of Pharmacy, 22 nd edition (2012), A.Loyd et al, pharmaceutical Press).
In some embodiments, the present disclosure provides antibodies and antibody drug conjugates for use in therapy. In some embodiments, the present disclosure provides antibodies and antibody drug conjugates for treating medical conditions. In some embodiments, the medical condition is cancer, cardiovascular disease, autoimmune disease, or neurodegenerative disease.
In a further embodiment, the present disclosure provides the use of an antibody or antibody drug conjugate in the manufacture of a medicament for the treatment of cancer, cardiovascular disease, autoimmune disease or neurodegenerative disease.
In a further embodiment, the present disclosure provides a method of administering a therapeutically effective amount of an antibody or antibody drug conjugate as disclosed herein, wherein the antibody or antibody drug conjugate is administered intravenously. In other embodiments, the present disclosure provides methods of administering a therapeutically effective amount of an antibody or antibody drug conjugate as disclosed herein, wherein the antibody or antibody drug conjugate is administered subcutaneously.
As used herein, the term "antibody" refers to an immunoglobulin molecule that binds an antigen. Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. Antibodies can be of any class (e.g., igG, igE, igM, igD, igA) and any subclass (e.g., igG1, igG2, igG3, igG 4).
As used interchangeably herein, the term "antibody conjugate" or "conjugated antibody" or "antibody drug conjugate" refers to a complex of an antibody or fragment thereof and a non-antibody molecule, which may comprise an antigen binding fragment of an antibody or a constant domain fragment of an antibody thereof. In one embodiment, the antibody and non-antibody molecules are linked by a linker.
As used herein, the term "non-antibody molecule" refers to a molecule that is not an immunoglobulin molecule. In one embodiment, the non-antibody molecule is a peptide comprising 2 or more amino acid residues. In a further embodiment, the non-antibody molecule is a small molecule drug. In a further embodiment, the non-antibody molecule is an oligonucleotide.
In a further embodiment, the oligonucleotide is an RNA oligonucleotide. In a further embodiment, the oligonucleotide is an RNAi oligonucleotide. In a further embodiment, the oligonucleotide is a double stranded oligonucleotide. In a further embodiment, the oligonucleotide is chemically modified.
Exemplary antibodies are immunoglobulin G (IgG) type antibodies that are composed of four polypeptide chains: two Heavy Chains (HC) and two Light Chains (LC) crosslinked via inter-chain disulfide bonds. The amino terminal portion of each of the four polypeptide chains includes a variable region of about 100-125 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. The constant region refers to the region of the antibody comprising the Fc region and CH1 domain of the antibody heavy chain. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., igG1, igG2, igG3, and IgG 4). Amino acid residue numbering in the constant regions is based on the EU index as in Kabat. Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, bethesda, MD: U.S. Dept.of Health and Human Services, public Health Service, national Institutes of Health (1991). The terms EU index number or EU numbering are used interchangeably herein.
VH and VL regions can be further subdivided into regions of high variability termed Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved termed Framework Regions (FR). CDRs are exposed on the surface of proteins and are important regions of antibodies for antigen binding specificity. Each VH and VL is composed of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, three CDRs of a heavy chain are referred to as "HCDR1, HCDR2, and HCDR3", and three CDRs of a light chain are referred to as "LCDR1, LCDR2, and LCDR3". CDRs contain most of the residues that interact specifically with antigen formation. The assignment of amino acid residues to CDRs can be accomplished according to well known protocols, including those described in the following: kabat (Kabat et Al, "Sequences of Proteins of Immunological Interest," National Institutes of Health, bethesda, md. (1991)), chothia (Chothia et Al, "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology,196, 901-917 (1987)), al-Lazikani et Al, "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology,273, 927-948 (1997)), north (North et Al, "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology,406, 228-256 (2011)), or IMGT (International ImmunoGenetics database available at www.imgt.org; see Lefranc et Al, nucleic Acids Res.1999; 27:209-212). North CDR definition for this paper describes specific binding to human IL-4Rα antibody.
Embodiments of the disclosure also include antibody fragments or antigen-binding fragments, such as Fab, fab ', F (ab') 2, fv fragments, scFv, scFab, disulfide-linked Fv (sdFv), fd fragments, which may be fused, for example, to an Fc region or an IgG heavy chain constant region.
As used herein, the term "Fc region" refers to an antibody region comprising the CH2 and CH3 domains of an antibody heavy chain. Optionally, the Fc region may comprise a portion of the hinge region or the entire hinge region of the heavy chain of the antibody. Biological activity, such as effector function, can be attributed to the Fc region, which varies with antibody isotype. Examples of antibody effector functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), C1q binding, complement-dependent cytotoxicity (CDC), phagocytosis, down-regulation of cell surface receptors (e.g., B-cell receptors); and B cell activation.
As referred to herein, a "wild-type" antibody is an antibody comprising the natural heavy chain constant region (including natural Fc) and the natural light chain constant region of a human IgG1, igG2, igG3, or IgG4 antibody.
The terms "bind" and "binding" as used herein are intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in the proximity of two proteins or molecules as determined by common methods known in the art, unless otherwise indicated.
As used interchangeably herein, the term "nucleic acid" or "polynucleotide" refers to a polymer of nucleotides, including single-and/or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, that incorporate natural nucleotides, modified nucleotides, and/or analogs of nucleotides. Polynucleotides of the present disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or synthetic reactions.
As used herein, the term "subject" refers to a mammal, including but not limited to humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Preferably, the subject is a human.
As used herein, the term "inhibit" refers to, for example, a decrease, slow down, decrease, stop, destroy, cancel, antagonize, or block of a biological response or activity, but does not necessarily indicate a complete elimination of the biological response.
As used herein, the term "treatment" or "treatment" refers to all processes in which there may be a slowing, controlling, delaying, or stopping of the progression of a disorder or disease disclosed herein, or ameliorating symptoms of a disorder or disease, but does not necessarily indicate a complete elimination of all of the disorder or disease symptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for treating a disease or condition in a patient, particularly a human.
As used herein, the term "about" means within 5%.
As used herein, the terms "a," "an," "the," and similar terms used in the context of the present disclosure (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
Drawings
FIG. 1 shows amino acid sequence alignment for human IgG1, igG2, igG3, and IgG4 heavy chain CH1-CH2-CH3 domains highlighting GNKRQ amino acid residues.
Figure 2 compares the Differential Scanning Calorimetry (DSC) thermograms of human IgG4P antibody variants with and without KRQ amino acid substitutions in the HC CH1 and CH2 domains.
Figure 3 shows that KRQ amino acid substitutions in the heavy chain CH1 and CH2 domains of IgG4P antibodies do not affect C1q Elisa binding affinity relative to IgG4P antibodies lacking KRQ amino acid substitutions.
Figure 4 shows that KRQ amino acid substitutions in the heavy chain CH1 and CH2 domains of IgG4P antibodies do not affect ADCC activity relative to IgG4P antibodies lacking KRQ amino acid substitutions.
Fig. 5 shows that KRQ amino acid substitutions in the heavy chain CH1 and CH2 domains of IgG4P antibodies do not affect CDC activity relative to IgG4P antibodies lacking KRQ amino acid substitutions.
Figures 6A-6B show that KRQ amino acid substitutions in the heavy chain CH1 and CH2 domains of IgG4P antibodies do not affect cell binding in B cells (6A) or monocytes (6B) relative to IgG4P antibodies lacking KRQ amino acid substitutions.
Examples
Example 1: antibody production and engineering
Antibody engineering, expression and purification ： Exemplary antibody molecules 1, 2, 3, 4, 5, 6 and 7 were engineered in the Heavy Chain (HC) constant domain to improve viscosity by alleviating the charge balance of potential electrostatic interactions between Fab and Fc. The HC CH1, CH2 and CH3 domains of human IgG4 antibodies have lower isoelectric points (pI) when compared to the HC constant domains of human IgG1 (table 1) due to uneven charge distribution. Five amino acid residues in the CH1, CH2 and CH3 domains of IgG4 were identified as affecting charge balance: 1) E137 (CH 1 domain), 2) D203 (CH 1 domain), 3) Q274 (CH 2 domain), 4) Q355 (CH 3 domain), and 5) E419 (CH 3 domain). Alignment of the human IgG4 HC constant region with the human IgG1 HC constant region (fig. 1) shows that similar positions of these amino acid residues are different in the human IgG1 HC constant region and are found to affect the overall pI of each domain. Alignment of human IgG1, igG2, igG3, and IgG4 HC constant regions (FIG. 1) also shows that similar positions of the GNKRQ amino acids are at certain positions The differences are different.
In order to match pI for both the CH2 and CH3 domains of an IgG4 antibody to an IgG1 antibody and minimize potential introduction of immunogenic peptides, residues at three of the five identified positions in an IgG4 Fc were converted to the corresponding residues found in an IgG1 wild-type Fc. Amino acid residue substitutions include: positively charged lysines for neutral charged glutamine at position 274 (Q274K), positively charged arginines for neutral charged glutamine at position 355 (Q355R), and neutral charged glutamines for negatively charged glutamates at position 419 (E419Q). The resulting IgG4 Fc was designated "KRQ". In order to match the pI for the CH1 domain of IgG4 to that of IgG1, the residues at positions 137 and 203 were converted to the corresponding residues found in the IgG1 wild-type CH1 domain. Amino acid substitutions include: neutral charged glycine replaces the negatively charged glutamic acid at position 137 (E137G), and polar asparagine replaces the negatively charged aspartic acid at position 204 (D203N). The resulting IgG4 Fc with all 5 amino acid substitutions was referred to as "GNKRQ".
In addition, double mutants called "RQ", comprising Q355R and E419Q amino acid substitutions concentrated only in the CH3 domain, and single mutants comprising Q274K amino acid substitutions concentrated in the CH2 domain were also evaluated.
To generate variants for molecules 1 to 6, two forms of IgG4 Fc were utilized: 1) IgG4 Fc with S228P amino acid residue substitutions (which stabilize the hinge and prevent arm exchange) are referred to as "IgG4P", or 2) IgG4 Fc with S228P amino acid residue substitutions (which are known to minimize effector function) in combination with F234A and L235A amino acid substitutions are referred to as "IgG4PAA". The variable domains of each of molecules 1 to 6 were cloned into IgG4PAA Fc, igG4PAA KRQ Fc, igG4PAA GNKRQ Fc and wild-type IgG1 Fc to generate separate antibody variants for each of the 6 molecules. For molecule 7, the variable domains were cloned into IgG4P Fc, igG4P KRQ Fc, and IgG4PAAKRQ Fc. Molecule 7 variants also include engineered cysteines (referred to herein as "eCys") at positions 124 and 378 of the heavy chain that serve as attachment points for small molecules in the generation of Antibody Drug Conjugates (ADCs). For each of molecules 1 to 7, the wild-type CH1 domains of human IgG4 and IgG1 were used in wild-type and KRQ variants, respectively, while the light chain variable domain was cloned onto the human K constant region for all molecules and matched pairs were expressed together to generate the complete antibodies required for viscosity and biophysical evaluation (see fig. 1-IgG4P GNKRQeCys).
Molecule 1 to 7 antibody variants are synthesized, expressed and purified by methods well known in the art, e.g., using predetermined HCs: LC vector ratio (if two vectors are used), or a single vector system encoding both heavy and light chains, a suitable host cell, such as chinese hamster ovary Cells (CHO), is transiently or stably transfected with the expression system for secreting antibodies. The clarified medium into which the antibodies are secreted is purified using generally known techniques. The HC constant domains and hinge sequences for the antibody variants of molecules 1 to 7 are shown in table 2. Theoretical pI (pI is the pH at which the net charge of the molecule is neutral) was calculated for each heavy chain domain with and without amino acid substitutions and compared using software analysis methods well known in the art (all without C-terminal lysines, which often undergo post-translational cleavage).
The results as demonstrated in table 1 show that theoretical pIs of the individual heavy chain CH1, CH2 and CH3 domains in the hig 4 antibody variants increased to values similar to the pI of the respective IgG1 domains when compared to wild type IgG4 due to the amino acid substitutions introduced within the IgG4 HC constant region.
Table 1: effect of constant domain amino acid substitutions on theoretical pI
Table 2: antibody sequences with respect to HC constant regions of molecules 1 to 7
Example 2: biophysical properties of exemplary antibody variants
Biophysical properties of exemplary antibody variants for molecules 1-7 were evaluated.
Intrinsic viscosity: the exemplified molecule 1 to 6 antibody variants were concentrated to 130mg/mL in 5mM histidine, 280mM mannitol, pH 6 buffer (H6M), and the molecule 7 variants were concentrated to 125mg/mL. Protein concentration was prepared and quantified using a variable optical path SoloVPE instrument (CTech). Average of 9 replicates was used, usinginitium (RheoSense) the intrinsic viscosity of each antibody was measured at 15 ℃. A minimum of 35 μl was used, with 27 μl of sample drawn and injected onto the B05 chip with the system set to an automatic test mode that adjusts the flow rate to achieve a 50% full scale pressure reading.
The results as demonstrated in table 3 show that IgG4PAA KRQ antibody variants with respect to molecules 1, 3, 4 and 6 have a significant viscosity reduction ranging from about 74% to about 30% when compared to the respective IgG4PAA variants lacking the KRQ amino acid substitution. Furthermore, surprisingly, 5 of the 6 IgG4PAA KRQ antibody variants (with respect to molecules 2, 3, 4, 5, 6) demonstrated a reduction in viscosity when compared to the respective IgG1 variants. In particular, igG4PAA KRQ variants with respect to molecules 2, 3 and 4 showed a viscosity reduction ranging from about 82% to about 48% when compared to the respective IgG1 variants. Further viscosity improvements were observed for molecules 1, 3, 4 and 6 for the addition of the E137G and D204N mutations in the CH1 domain when compared to the separate IgG4PAA variants lacking the KRQ amino acid substitution. For molecules 2 and 5, where the initial viscosity was lower with respect to IgG4PAA prior to substitution, no benefit was observed with respect to CH1 substitution addition when compared to KRQ substitution. Surprisingly, however, 5 of the 6 IgG4PAA GNKRQ antibody variants (molecules 1, 2, 3, 4, 6) demonstrated a decrease in viscosity when compared to the respective IgG1 variants. In particular, igG4PAAKRQ variants with respect to molecules 3, 4 and 6 showed a viscosity reduction ranging from about 83% to about 55% when compared to the respective IgG1 variants.
In addition, the results as demonstrated in table 4 show that the molecule 7 IgG4P GNKRQ eCys (9.6 cP), igG4P KRQ eCys (11.6 cP), igG4P RQ eCys (14.9 cP) and IgG4P K eCys (21.0 cP) antibody variants, respectively, have a viscosity reduction from about 78% to about 51%, thus showing the additive effect of the combination of mutations when compared to the molecule 7 iggg 4P eCys (43 cP) variants lacking any of the GNKRQ amino acid substitutions.
Table 3: intrinsic viscosity of antibody variants of molecules 1 to 6
Table 4: intrinsic viscosity of molecular 7 IgG4P antibody variants
cp=centipoise
Intrinsic viscosity of the antibody variant in the form of an antibody drug conjugate as molecule 7: molecular 7 antibody variants IgG4P KRQ eCys, igG4P GNKRQ, and IgG4P eCys were tested for their intrinsic viscosities as antibody drug conjugates. Briefly, engineered cysteines at positions 124 and 378 in the heavy chains of the molecules 7 IgG4P eCys, igG4P KRQ eCys, and IgG4P GNKRQ antibody variants were used to generate antibody drug conjugates using lipophilic small molecules with short linkers. Conjugation is accomplished by a short reduction in the presence of the reducing agent dithiothreitol, followed by a desalting step to remove the reducing agent and a short oxidation step in the presence of dehydroascorbic acid. The reduction step and subsequent desalting steps remove any cysteines (cysteines) on the engineered cysteines at positions 124 and 378 of the heavy chain, which occur during cell culture expression. The oxidation step reforms the native disulfide bond, which includes two pairs of disulfide bonds in the hinge and 1 pair of disulfide bonds between the heavy and light chains. The small molecule is then conjugated to an antibody using maleimide-based chemistry that reacts with the free thiol of the engineered cysteine residue. The viscosity is measured substantially as described above.
The results as demonstrated in table 5 show that molecule 7 IgG4P KRQ eCys ADC (12.2 cP) and molecule 7 IgG4P GNKRQ eCys ADC (10.2 cP) have a viscosity reduction of 1/48 and 1/58, respectively, when compared to molecule 7 iggg 4p eCys ADC (592 cP) lacking GNKRQ amino acid residue substitution, thus showing the additive effect of the combination of mutations.
Table 5: intrinsic viscosity of molecular 7 igg4p KRQ antibody drug conjugate
Intrinsic viscosity (cP)
Molecular 7-IgG4P eCys-ADC 592
Molecular 7-IgG4P KRQ eCys-ADC 12.2
Molecular 7-IgG4P GNKRQ eCys-ADC 10.2
cp=centipoise
Thermal stability ： Differential Scanning Calorimetry (DSC) was used to evaluate the stability of exemplified variants 1 to 7 against thermal denaturation. Run DSC using Malvern MircoCal VP-DSC instrument. The sample was heated from 20 ℃ to 110 ℃ at a constant rate of 60 ℃/hour. The analytical method was performed using the MicroCal VP-Capillary DSC Automated Analysis program. Baseline correction is performed and tstart and TM1 are determined. Although the transition temperature for unfolding of 3 domains (including CH2, CH3 and Fab) was not adequately resolved for IgG4P eCys or IgG4P KRQ eCys, the thermal melting temperatures of the molecule 7 IgG4P eCys and IgG4P KRQ eCys antibody variants were measured in PBS, pH 7.2 buffer.
The results as demonstrated in table 6 and fig. 2 show that the introduction of amino acid residue KRQ into CH2 and CH3 of the IgG4 constant domain of the antibody does not negatively affect the thermostability of the resulting antibody.
Thermal aggregation (Tagg) initiation: internal Fluorescence (IF) and light scattering measurements were performed using a promethaus Panta (Nano Temper Technologies) equipped with a high sensitivity capillary. Samples at 0.5mg/mL were prepared in PBS, pH 7.2 buffer, and aggregation (Tagg) initiation was determined using a thermal gradient from 20-95 ℃ at a constant rate of 1 ℃/min. Using PR Panta Analysis (X64) software (V1.0.2), the onset of aggregation was determined using a 2-state fit based on the retro-reflected signal at 385nm, with a threshold of 0.5% increase from baseline.
The results as demonstrated in table 6 show that the onset of aggregation is comparable between the molecule 7 IgG4P KRQ eCys and IgG4P eCys antibody variants and thus that the introduction of amino acid residue KRQ into CH2 and CH3 of the IgG4 constant domain of the antibody does not negatively affect the thermostability of the resulting antibody.
TABLE 6 thermal aggregation of KRQ eCys antibody variants for molecule 7 IgG4P
All values are in degrees Celsius (C.)
Aggregation at temperature stress pairs: exemplary molecular variants were evaluated for solution stability over time at approximately 100mg/mL in a common 5mM histidine pH 6.0 buffer with excipients. The concentrated samples were incubated at 5℃and 35℃for a period of 4 weeks, respectively. After incubation, samples were analyzed for the percentage of high molecular weight species (% HMW) using Size Exclusion Chromatography (SEC).
As demonstrated in table 7, the KRQ amino acid residue substitutions did not significantly affect aggregation over a 4 week period at 5 ℃ or 35 ℃; in particular, the results show that high concentration solution stability of IgG4P KRQ eCys antibodies is comparable to IgG4P eCys antibody variants.
The freeze/thaw stability was evaluated using 3 repeated slow, controlled temperature cycles that simulate freeze/thaw conditions for a large number of bulk drug substances placed at-70 ℃. Samples were evaluated in both 5mM histidine, 280mM mannitol, 0.05% PS80, pH 6 (H6 MT) and 5mM histidine, 280mM sucrose, 0.05% PS80, pH 6 (H6 ST). After incubation, samples were analyzed for changes in the percentage of high molecular weight species (Δhmw) using analytical size exclusion chromatography (aec). The samples were also visually inspected for phase separation or precipitate formation.
As demonstrated in table 7, the introduction of amino acid residue KRQ into CH2 and CH3 of the IgG4 constant domain of the antibody did not affect the freeze/thaw stability of the resulting antibodies. In particular, the molecule 7 IgG4P KRQ eCys antibody variant showed Δ3.4% hmw in H6MT and Δ0.5% hmw in H6ST, which was comparable to IgG4P eCys antibodies, which showed Δ2.5% hmw in H6MT and Δ0.3% hmw in H6ST, as measured by aSEC after 3 freeze-thaw cycles. Visual observations did not show any phase separation or precipitation with respect to IgG4P KRQ eCys or IgG4P eCys antibody variants.
Solubility: solubility was assessed by concentrating 100mg of antibody variant to a volume of about 0.5mL using a 30kDa molecular weight cut-off centrifuge filter (e.g., amicon u.c. filter, millipore, catalog #ufc 903024). The final concentration of the samples was measured by UV absorbance at 280nm using a Solo VPE spectrophotometer (C Technologies, inc). After incubation, the samples were analyzed for the percentage of high molecular weight species (% HMW) using analytical size exclusion chromatography (aec). The samples were also visually inspected for phase separation or precipitate formation.
The results as demonstrated in table 7 show that the molecular 7 igg4p KRQ eCys antibody variant exhibiting Δ0.6% hmw is comparable to the molecular 7 igg4p eCvs antibody exhibiting Δ0.4% hmw as measured by aeec. Visual observations did not show any phase separation or precipitation with respect to IgG4P KRQ eCys or IgG4P eCys.
TABLE 7 molecular parameters for the variants of the molecular 7 IgG4P and IgG1 antibodies
Biophysical analysis demonstrated that the introduction of amino acid residue KRQ into the CH2 and CH3 domains of IgG4 significantly reduced viscosity when compared to IgG4P antibodies lacking the KRQ amino acid substitution without negatively affecting the aggregation, solubility, or thermal stability of the antibody.
Example 3 effector function Activity
In vitro fcγ receptor binding, ADCC, and CDC assays were performed to assess whether amino acid substitutions in the Fc region of the antibody altered Fc function.
Human fcγ receptor binding: the binding affinity of the molecular 7 IgG4 antibody variants to human fcγ receptor was assessed by Surface Plasmon Resonance (SPR) analysis. Biacore T100 (Cytiva), biacore reagent and Scubber 2Biacore Evaluation Software (Biologics 2008) were used for SPR analysis. A series of S CM5 chips (Cytiva P/N BR 100530) were prepared using the manufacturer' S EDC/NHS amine coupling method (Cytiva P/N BR 100050). Briefly, the surface of all 4 Flow Cells (FC) was activated by injecting a 1:1 mixture of EDC/NHS at 10. Mu.L/min for 7 min. Protein A (Calbiochem P/N539202) was diluted to 100 μg/mL in 10mM acetate, pH 4.5 buffer, and approximately 4000RU was fixed to all 4 FCs by 7 minute injection at a flow rate of 10 μl/min. Unreacted sites were blocked with a 7 minute injection of 10 μl/min ethanolamine. Injection of 2X 10. Mu.L glycine, pH 1.5 was used to remove any non-covalently bound protein. The running buffer was 1 XHBS-EP+ (TEKNOVA, P/N H8022). Fcγr extracellular domain (ECD) -fcγri (CD 64), fcγriia_131R and fcγriia_131H (CD 32 a), fcγriiia_158V, fc γriiia_158F (CD 16 a) and fcγriib (CD 32 b) were produced from stable CHO cell expression and purified using IgG sepharose and size exclusion chromatography. For fcγri binding, the antibodies were diluted to 2.5 μg/mL in running buffer, and approximately 150RU of each antibody was captured in FCs 2 to 4 (RU capture). FC1 is a reference FC, so there is no capture antibody in FC 1. Fcyri ECD was diluted to 200nM in running buffer and then serially twice diluted to 0.78nM in running buffer. Duplicate injections of each concentration were injected at 40 μl/min onto all FCs for 120 seconds followed by a dissociation phase of 1200 seconds. Regeneration was performed by injecting 15 μl of 10mM glycine, pH 1.5, at 30 μl/min on all FCs. The reference-subtracted data were collected as FC2 FC1, FC3-FC1 and FC4-FC1 and measurements were obtained at 25 ℃. Using "1" in steady state equilibrium analysis or BIA evaluation by scanner 2Biacore Evaluation Software: 1 (Langmuir) bind "model to calculate affinity (KD). For fcyriia, fcyriib, and FcyRIIIa binding, the antibodies are diluted to 5 μg/mL in running buffer and about 500RU of each antibody is captured in fc2 to 4 (RU capture). FC1 becomes the reference FC again. Fcγ receptor ECD was diluted to 10 μm in running buffer and then serially diluted 2-fold to 39nM in running buffer. Duplicate injections of each concentration were injected at 40 μl/min onto all FCs for 60 seconds followed by a dissociation phase of 120 seconds. Regeneration was performed by injecting 15 μl of 10mM glycine, pH 1.5, at 30 μl/min on all FCs. The reference-subtracted data were collected as FC2-FC1, FC3-FC1 and FC4-FC1 and measurements were obtained at 25 ℃. Affinity (KD) was calculated using steady state equilibrium analysis by scanner 2Biacore Evaluation Software. Each receptor was assayed at least twice.
The results as demonstrated in table 8 show that the molecular 7 IgG4P KRQ eCys and IgG4P eCys antibody variants have comparable binding affinities for each fcγ receptor, thus showing that the introduction of amino acid residue KRQ into CH2 and CH3 of the IgG4 constant domain of the antibody does not affect the Fc binding activity of the resulting antibody.
Table 8: binding affinity of molecular 7 IgG4P antibody variants to human Fc gamma receptor
C1q binding: binding of the molecular 7 IgG4 antibody variant to human C1q was assessed by Elisa. 96-well microplates were coated with 100. Mu.L/well of molecular 7 IgG4 KRQ eCys antibody variants diluted from 10. Mu.g/mL to 0.19. Mu.g/mL in DPBS (Dulbecco's HyClone) and incubated overnight at 4 ℃. The coated reagent was removed and the plate was blocked with 200. Mu.L/Kong Lao protein blocking buffer (Thermo) and incubated for 2 hours at Room Temperature (RT). Plates were washed 3 times with wash buffer (1 XTBE with 0.05% Tween 20) and 10. Mu.g/mL human C1q (MS Biomedical) diluted in casein blocking reagent was added at 100. Mu.L/well and incubated for 3 hours at RT. Humanized IgG1 and humanized IgG4P isotype control antibodies were used as positive and negative controls, respectively. The plates were then washed 3 times with wash buffer and 100 μl/well of sheep anti-human C1q-HRP (abcam#ab 46191) diluted 1:800 in casein blocking agent was added and incubated for 1 hour at RT. Plates were then washed 6 times with wash buffer and 100 μl/well of TMB substrate (Pierce) was added to each well and incubated for 7 minutes. 100. Mu.L/well of 1N HCl was added to terminate the reaction. The optical density at 450nm was measured immediately on a colorimetric microplate reader. Data were analyzed using SoftMax Pro 7.1Data Acquisition and Analysis Software.
As demonstrated in figure 3, the molecular 7 IgG4P KRQ eCys antibody variant has comparable C1q binding to the IgG4P control, thus showing that the introduction of amino acid residue KRQ into CH2 and CH3 of the IgG4 constant domain of the antibody does not affect complement binding of the resulting antibody.
Antibody Dependent Cellular Cytotoxicity (ADCC): an in vitro ADCC assay for the molecule 7 antibody variant was evaluated using a reporter-based ADCC assay.
Briefly, daudi cells (ATCC, # CCL-213) and human CD20 target cell lines were used as effector cell lines, and Jurkat cells (Eli Lilly and Company) expressing functional FcgammaRIIIa (V158) -NFAT-Luc. All test antibody variants and cells were diluted in assay medium containing RPMI-1640 (phenol red free) with 0.1mM non-essential amino acid (NEAA), 1mM sodium pyruvate, 2mM L-glutamine, 500U/mL penicillin-streptomycin and 0.1% w/v BSA. The test antibodies were first diluted to a 3X concentration of 3.3. Mu.g/mL and then serially diluted 7-fold at a 1:4 ratio. 50 μl/well of each antibody was aliquoted into white opaque bottom 96-well plates (Costar, # 3917). CD20 antibody was used as positive control. Daudi target cells were then plated at 5X 10 4 A 50 μl aliquot of each cell/well was added to the plate and incubated for 1 hour at 37 ℃. Next, jurkat V158 cells were added to the wells in 50. Mu.L aliquots of 150,000 cells/well, and incubated at 37℃for 4 hours, followed by the addition of 100. Mu.L/well of One-Glo luciferase substrate (Promega, # E8130). The contents of the plates were mixed at low speed using a plate shaker, incubated at room temperature for 5 minutes, and the luminescence signal read using 0.2cps integration on a BioTek microplate reader (BioTek Instruments). Data were analyzed using GraphPad Prism 9 and Relative Luminescence Units (RLUs) for each antibody concentration were plotted in a scatter format of antibody concentration versus RLU. The results are representative of two independent experiments.
As demonstrated in fig. 4, the molecular 7 antibody variants IgG4Pe Cys and IgG4P KRQ ecs have comparable ADCC activity (i.e., do not induce ADCC activity) in the reporter-based ADCC assay, thus indicating that the introduction of amino acid residue KRQ into CH2 and CH3 of the IgG4 constant domain of the antibody does not affect the effector function activity of the resulting antibody. The positive control CD20 antibody showed potent ADCC activity.
Complement Dependent Cytotoxicity (CDC): in vitro CDC assays of the variant of the molecular 7 IgG4 antibody were performed using Daudi cells (ATCC, # CCL-213). All test antibodies, complements and cells were diluted in assay medium consisting of RPMI-1640 (phenol red free) with 0.1mM nonessential amino acid (NEAA), 1mM sodium pyruvate, 2mM L-glutamine, 500U/mL penicillin-streptomycin and 0.1% w/v BSA. The test antibodies were first diluted to a 3X concentration of 100. Mu.g/mL and then serially diluted 7-fold at a 1:4 ratio. 50 μl/well of each antibody was aliquoted into white opaque bottom 96-well plates (Costar, # 3917). Daudi target cells were then added in 50. Mu.L aliquots of 50,000 cells/well along with the exemplified antibodies, and the CD20 positive control antibodies were incubated for 1 hour at 37 ℃. Next, human serum complement (Quidel, #A113) thawed rapidly in a 37℃water bath was diluted 1:6 in assay medium and added to assay plates at 50. Mu.L/well. Plates were incubated at 37℃for 2 hours, followed by the addition of 100. Mu.L/well CellTiter Glo substrate (Promega, #G7571) to each well. The contents of the plates were mixed at low speed using a plate shaker, incubated at room temperature for 5 minutes, and the luminescence signal read using 0.2cps integration on a BioTek microplate reader (BioTek Instruments). Data were analyzed using GraphPad Prism v9 and Relative Luminescence Units (RLUs) for each antibody concentration were plotted in a scatter format of antibody concentration versus RLU.
As demonstrated in figure 5, the molecule 7 IgG4P eCys and IgG4P KRQ eCys antibody variants have comparable CDC activity in Daudi cells, thus showing that the introduction of amino acid residue KRQ into CH2 and CH3 of the IgG4 constant domain of the antibody does not affect the CDC activity of the resulting antibody. CDC assay positive control CD20 antibodies showed potent CDC activity.
The results of fcγ, C1q binding assays and cell-based effector function ADCC and CDC assays showed that the introduction of KRQ amino acid residues into the constant domain of the antibody variant did not affect the Fc function of the antibody.
EXAMPLE 4 cell binding of variants
Binding to B cells and monocytes: exemplary molecular 7 igg4p antibody variants were tested for binding to B cells and monocytes in a Fluorescence Activated Cell Sorting (FACS) assay. By standard Ficoll-Paque TM plus(GE HEALTHCARE) density gradient centrifugation, human PBMCs were isolated from human blood samples. Freshly isolated cellular PBMC were resuspended at 200 ten thousand cells/mL and allowed to stand at room temperature for 15 minutes before plating at 100. Mu.L/well to a round bottom 96 well plateInside, and with FACS buffer (containing from +.>PBS of 2% fetal bovine serum). According to the manufacturer's protocol (Thermo Fisher Scientific) and Alexa- >647 conjugated exemplified IgG4P antibody variants were added to the wells at 66.67nM and 4-fold dilutions were performed in duplicate. An equal volume of 2X antibody mixture containing the following was then added to the wells: human TruStain FcX TM FITC anti-human CD3 antibody, alexa->700 anti-human CD4 antibodies (all from +.>) CD20 monoclonal antibody (2H 7), perCP-cyanine5.5 (Thermo Fisher Scientific) and CD14 PE-Cy TM 7 mice were anti-human CD14 (BD Biosciences). Cells were incubated at 4 ℃ for 30 min, then washed twice with FACS buffer, and resuspended in a final volume of 100 μl FACS buffer. Adding a vital dye Sytox TM blue (Thermo Fisher Scientific), and via a flow cytometer (LSRFortessa) TM X-20; BD BIOSCIENCES), samples were analyzed. Data analysis was performed using FlowJo software and statistical analysis was performed using GraphPad Prism 9. Data represent mean ± SEM of positive cell percentages of CD 20B cells and CD3/CD20 negative, CD14 positive monocyte populations from two donors. By fitting log (Ab concentration) relative to the percentage of Ab positive expressing cells from each cell populationThe S-shaped curve of the percentages generates a curve.
As shown in table 9 and the results confirmed in fig. 6A and 6B, the exemplified molecular 7 antibody variants IgG4P KRQ eCys, igG4P RQ eCys, and IgG4P eCys separated B cells from PBMCs with comparable affinities (ECs of 0.20nM, 0.18nM, and 0.17nM, respectively 50 ) And monocytes (EC of 1.04nM, 0.90nM and 1.00nM, respectively 50 ) Binding, therefore, shows that the introduction of amino acid residues KRQ or RQ into CH2 and CH3 of the IgG4 constant domain of an antibody does not affect the binding of the variable region of the resulting antibody.
Table 9: binding of molecular 7 IgG4P variant antibodies to B cells and monocytes
Antibodies to B cell EC 50 (nM) Monocytes EC 50 (nM)
Molecule 7-IgG4P KRQ eCys 0.20 1.04
Molecular 7-IgG4P RQ eCys 0.18 0.90
Molecule 7-IgG4P eCys 0.17 1.00
Sequence listing
SEQ ID NO:1 human kappa constant (for molecules 1, 2, 3, 4, 5, 6, 7)
SEQ ID NO:2 human IgG1 CH1 (for molecules 12, 3, 4, 5, 6)
SEQ ID NO:3 human IgG1 hinge (for molecules 1, 2, 3, 4, 5, 6)
SEQ ID NO:4 human IgG1 CH 1-hinge-CH 2-CH3 (for molecules 1, 2, 3, 4, 5, 6)
SEQ ID NO:5 human IgG4 CH1 (for molecules 1, 2, 3, 4, 5, 6)
SEQ ID NO:6 human IgG4 (S228P) hinge (for molecules 1, 2, 3, 4, 5, 6, 7)
SEQ ID NO:7 human IgG4PAA (S228P, F234A, L A) CH 1-hinge-CH 2-CH3 (for molecules 1, 2, 3, 4, 5, 6)
SEQ ID NO:8 human IgG4PAA KRQ (S228P, Q274K, F234A, L235A, Q355R, E419Q) CH 1-hinge-CH 2-CH3 (for molecules 1, 2, 3, 4, 5, 6)
SEQ ID NO:9 human IgG4P KRQ eCys (S124C, S228P, Q274K, Q355R, A378C, E419Q) CH 1-hinge-CH 2-CH3 (for molecule 7)
SEQ ID NO:10 human IgG4P RQ eCys (S124C, S228P, Q355R, A378C, E419Q) CH 1-hinge-CH 2-CH3 (for molecule 7)
SEQ ID NO:11 human IgG4P eCys (S124C, S P, A378C) CH 1-hinge-CH 2-CH3 (for molecule 7)
SEQ ID NO:12 human IgG4P GNKRQ eCys (S124C, E137G, D203N, S P, Q274K, Q355R, A378C, E419Q) CH 1-hinge-CH 2-CH3 (for molecule 7)
SEQ ID NO:13 human IgG4P K eCys (S124C, S228P, Q274K, A378C) CH 1-hinge-CH 2-CH3 (for molecule 7)
SEQ ID NO:14 human IgG2 CH 1-hinge-CH 2-CH3
SEQ ID NO:15 human IgG3 CH 1-hinge-CH 2-CH3
SEQ ID NO:16 human IgG4PAA GNKRQ (S228P, E137G, D203N, Q274K, F234A, L235A, Q355R, E419Q) CH 1-hinge-CH 2-CH3 (for molecules 1, 2, 3, 4, 5, 6)
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Claims (25)

1. An antibody comprising a human IgG heavy chain constant region comprising CH1, CH2, and CH3 domains and a human IgG light chain constant region, wherein the human IgG heavy chain constant region comprises:
lysine at amino acid residue 274 (EU numbering) of the CH2 domain;
arginine at amino acid residue 355 (EU numbering) of the CH3 domain;
glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
Asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and arginine at amino acid residue 355 (EU numbering) of the CH3 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glutamine at amino acid residue 419 of the CH3 domain (EU numbering);
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glycine at amino acid residue 137 of the CH1 domain (EU numbering);
arginine at amino acid residue 355 of the CH3 domain (EU numbering) and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
glutamine at amino acid residue 419 of the CH3 domain (EU numbering) and glycine at amino acid residue 137 of the CH1 domain (EU numbering);
Glutamine at amino acid residue 419 of the CH3 domain (EU numbering) and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
glycine at amino acid residue 137 (EU numbering) of the CH1 domain and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), and glycine at amino acid residue 137 of the CH1 domain (EU numbering);
arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
arginine at amino acid residue 355 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
glutamine at amino acid residue 419 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
Arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); or (b)
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain.
2. The antibody of claim 1, comprising a human IgG heavy chain constant region comprising CH1, CH2, and CH3 domains and a human IgG light chain constant region, wherein the human IgG heavy chain constant region comprises:
lysine at amino acid residue 274 (EU numbering) of the CH2 domain;
arginine at amino acid residue 355 (EU numbering) of the CH3 domain;
glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and arginine at amino acid residue 355 (EU numbering) of the CH3 domain;
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glutamine at amino acid residue 419 of the CH3 domain (EU numbering); or (b)
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.
3. The antibody of claim 2, wherein the human IgG heavy chain constant region comprises:
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.
4. The antibody of any one of claims 1 to 3, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3.
5. The antibody of any one of claims 1 to 4, wherein the antibody is a human IgG2, human IgG3 or human IgG4 antibody.
6. The antibody of claim 5, wherein the antibody is a human IgG4 antibody.
7. An antibody comprising a human IgG heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 8. 9, 10, 12, 13 or 16.
8. An antibody drug conjugate comprising the antibody of any one of claims 1-7.
9. The antibody of any one of claims 1-7, or the antibody drug conjugate of claim 8, wherein the antibody or antibody drug conjugate has a reduced viscosity compared to a wild-type antibody comprising a wild-type human IgG heavy chain constant region.
10. The antibody or antibody drug conjugate of claim 9, wherein the viscosity is reduced by about 25% to about 80% when compared to the wild-type antibody.
11. A method of reducing the viscosity of a human IgG4 antibody comprising generating a variant of a human IgG4 antibody, said variant comprising a modified human IgG4 heavy chain constant region and a human IgG light chain constant region, said modified human IgG4 heavy chain constant region comprising CH1, CH2 and CH3 domains, wherein said modified human IgG4 heavy chain constant region comprises the amino acid residues:
lysine at amino acid residue 274 (EU numbering) of the CH2 domain;
arginine at amino acid residue 355 (EU numbering) of the CH3 domain;
Glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and arginine at amino acid residue 355 (EU numbering) of the CH3 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glutamine at amino acid residue 419 of the CH3 domain (EU numbering);
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glycine at amino acid residue 137 of the CH1 domain (EU numbering);
arginine at amino acid residue 355 of the CH3 domain (EU numbering) and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
Glutamine at amino acid residue 419 of the CH3 domain (EU numbering) and glycine at amino acid residue 137 of the CH1 domain (EU numbering);
glutamine at amino acid residue 419 of the CH3 domain (EU numbering) and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
glycine at amino acid residue 137 (EU numbering) of the CH1 domain and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), and glycine at amino acid residue 137 of the CH1 domain (EU numbering);
arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
arginine at amino acid residue 355 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
glutamine at amino acid residue 419 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering);
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and glycine at amino acid residue 137 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain;
Arginine at amino acid residue 355 of the CH3 domain (EU numbering), glutamine at amino acid residue 419 of the CH3 domain (EU numbering), glycine at amino acid residue 137 of the CH1 domain (EU numbering), and asparagine at amino acid residue 203 of the CH1 domain (EU numbering); or (b)
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, glutamine at amino acid residue 419 (EU numbering) of the CH3 domain, glycine at amino acid residue 137 (EU numbering) of the CH1 domain, and asparagine at amino acid residue 203 (EU numbering) of the CH1 domain.
12. The method of claim 11, wherein the modified human IgG4 heavy chain constant region comprises the amino acid residues:
lysine at amino acid residue 274 (EU numbering) of the CH2 domain;
arginine at amino acid residue 355 (EU numbering) of the CH3 domain;
glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
lysine at amino acid residue 274 (EU numbering) of the CH2 domain and arginine at amino acid residue 355 (EU numbering) of the CH3 domain;
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain;
arginine at amino acid residue 355 of the CH3 domain (EU numbering) and glutamine at amino acid residue 419 of the CH3 domain (EU numbering); or (b)
Lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.
13. The method of claim 11 or 12, wherein the modified human IgG4 heavy chain constant region comprises the amino acid residues:
lysine at amino acid residue 274 (EU numbering) of the CH2 domain, arginine at amino acid residue 355 (EU numbering) of the CH3 domain, and glutamine at amino acid residue 419 (EU numbering) of the CH3 domain.
14. The method of any one of claims 11 to 13, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the VL comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3.
15. The method of any one of claims 11 to 14, further comprising generating an antibody drug conjugate comprising the variant human IgG4 antibody.
16. The method of claims 11 to 15, wherein the antibody has a reduced viscosity compared to a wild-type human IgG4 antibody.
17. The method of claim 16, wherein the viscosity is reduced by about 25% to about 80% when compared to a wild-type human IgG4 antibody.
18. A nucleic acid comprising a sequence encoding the heavy or light chain of the antibody of any one of claims 1-7.
19. A vector comprising the nucleic acid of claim 18.
20. A cell comprising the vector of claim 19.
21. The cell of claim 20, wherein the cell is a mammalian cell.
22. A method of producing an antibody comprising culturing the cell of claim 21 under conditions such that the antibody is expressed, and recovering the expressed antibody from the culture medium.
23. An antibody produced by the method of claim 22.
24. A pharmaceutical composition comprising the antibody or antibody drug conjugate of any one of claims 1-10 or 23, and a pharmaceutically acceptable excipient, diluent or carrier.
25. A method of administering the antibody or antibody drug conjugate of any one of claims 1-10 or 23 to a subject in need thereof, wherein the antibody is administered subcutaneously to the subject.
CN202280054863.XA 2021-08-05 2022-08-05 Antibody optimisation Pending CN117794949A (en)

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