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WO2024221187A1 - Polypeptides hétéromultimères - Google Patents

Polypeptides hétéromultimères Download PDF

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Publication number
WO2024221187A1
WO2024221187A1 PCT/CN2023/090376 CN2023090376W WO2024221187A1 WO 2024221187 A1 WO2024221187 A1 WO 2024221187A1 CN 2023090376 W CN2023090376 W CN 2023090376W WO 2024221187 A1 WO2024221187 A1 WO 2024221187A1
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Prior art keywords
domain
polypeptide
modified immunoglobulin
heteromultimer
sequence
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Inventor
Tong Zhang
Wei Jiang
Xiaoyun Guo
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Biomap Intelligence Technology Sg PteLtd
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Biomap Intelligence Technology Sg PteLtd
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Priority to PCT/CN2023/090376 priority Critical patent/WO2024221187A1/fr
Priority to CN202380097505.1A priority patent/CN121100128A/zh
Publication of WO2024221187A1 publication Critical patent/WO2024221187A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to heteromultimer polypeptides comprising a heterodimeric polypeptide complex and methods of production favoring heterodimeric polypeptide complex formation.
  • Conventional antibodies generally comprise two antigen binding domains able to bind to a particular epitope.
  • the binding domains are provided heavy chain variable regions or a combination of heavy and light chain variable regions.
  • Naturally occurring human antibodies are a multimer generally comprising two identical heavy chains and two identical light chains. Each heavy chain and light chain provide the same variable regions, resulting in a monospecific antibody. Certain animals such as lamas and camels can produce heavy chain only antibodies.
  • Each of the two heavy chains in a naturally occurring antibody comprise two identical CH3-CH2-hinge-CH1-variable regions.
  • a dimer formed between two CH3-CH2-hinge regions provides for an Fc.
  • Antibody production involving Fc homodimerization is primarily mediated by a large and compact interface between the two CH3 domains followed by disulfide bond formation in the hinge region (Schroeder et al. J Allergy Clin Immunol. 2010. 125 (2 Suppl 2) : S41–52; Ha et al. Front. Immunol. 2016. 7: 394) .
  • bispecific antibodies Compared with conventional monospecific antibodies, bispecific antibodies (BsAbs) , bind to two epitopes, which may be present on the same or different molecules.
  • Bispecific IgG antibodies are a multimer made up of two heavy chain polypeptides and two light chain polypeptides. Expressing the different polypeptides in a cell can result in the production of different antibodies, including monospecific antibodies resulting from Fc homodimerization and bispecific antibodies resulting from Fc heterodimerization (Klein et al. MAbs. 2012.4 (6) : 653-63. ) .
  • a variety of different types of multispecific binding molecules can be produced using different binding domains, including antigen binding domains.
  • the use of different CH3 regions can facilitate production of multispecific binding molecules. Publications mentioning techniques for facilitating heterodimerization mediated by CH3 regions include: Spiess et al. Molecular Immunology. 2015. 67 (2 Pt A) : 95-106; Klein et al. MAbs. 2012.4 (6) : 653-63; Carter. J Immunol Methods. 2001.248 (1-2) : 7-15; Brinkmann et al. MAbs. 2017.
  • the present invention relates to heteromultimer polypeptides comprising a heterodimeric polypeptide complex, wherein the heterodimeric polypeptide comprises two modified immunoglobulin CH3 domains.
  • the modified immunoglobulin CH3 domains comprise substitutions favoring heterodimer formation.
  • the different polypeptides making up the heterodimeric polypeptide complex can be combined with different binding domains to provide molecules able to bind different targets.
  • heteromultimer polypeptides include, for example, bispecific antibodies and different types of molecules containing two or more antibody variable regions providing for multispecificity. Additional examples include, for example, heteromultimer polypeptides comprising different types of binding agents providing for specificity, such as polypeptide receptors and ligands.
  • a first aspect of the present invention features a heteromultimer comprising a heterodimeric polypeptide complex, wherein the heterodimeric polypeptide complex comprises:
  • a second polypeptide comprising a second modified immunoglobulin CH3 domain having at least one amino acid substitution relative to a wildtype immunoglobulin CH3 domain at positions 366 and 360, wherein the substitution is selected from the group consisting of 366Y, 366W, 366F, 366L, 360Y, 360W, 360F, and 360L, wherein numbering is according to the EU index as in Kabat.
  • Another aspect of the present invention is directed to a heteromultimer comprising a heterodimeric polypeptide complex, wherein the heterodimeric polypeptide complex comprises:
  • a second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W or 366Y; wherein numbering is according to the EU index as in Kabat
  • modified immunoglobulin CH3 domain indicates a CH3 derived from a human Ig CH3 region and/or a polypeptide region having a sequence identity of at least 85%to SEQ ID NO: 3.
  • a derived sequence is produced by altering one or more amino acids from a reference sequence. Alterations can include amino acid insertions, deletions, and/or substitutions. The amino acid reference number is with respect to a reference sequence prior to any alterations.
  • the CH3 is derived from a human IgG. Corresponding changes can be made in IgM, IgA, IgD, or IgE CH3, where such changes may involve different numbering than IgG, but corresponding locations.
  • a wildtype immunoglobulin CH3 domain is a naturally occurring human Ig CH3 domain.
  • the wildtype Ig CH3 is a human IgG; or is IgM, IgA, IgD, and IgE.
  • amino acid reference number provides the location in the corresponding wildtype sequence. In some circumstances, such as amino acid addition or deletion, the numbering from the wildtype sequence can be shifted, however the corresponding location would remain the same.
  • Another aspect of the present invention is directed to a heteromultimer comprising a heterodimeric polypeptide complex, wherein the dimeric polypeptide complex comprises a first polypeptide comprising a first modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, and a second polypeptide comprising a second modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, wherein:
  • the first modified immunoglobulin CH3 domain comprises the substitution 405V, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;
  • the first modified immunoglobulin CH3 domain comprises the substitution 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366Y;
  • the first modified immunoglobulin CH3 domain comprises the substitution 349T, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T and 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366W;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A
  • the second modified immunoglobulin CH3 domain comprises the substitutions 360W and 366Y
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A
  • the second modified immunoglobulin CH3 domain comprises the substitutions 360Y, 366W, 435R and 436F;
  • Another aspect is directed to a nucleic acid combination comprising a first nucleic acid encoding the first polypeptide of a heterodimeric complex and a second nucleic acid encoding the second polypeptide of a heterodimeric complex.
  • Another aspect is directed to a nucleic acid comprising a first nucleic acid encoding the first polypeptide of a heterodimeric complex and a second nucleic acid encoding the second polypeptide of a heterodimeric complex.
  • Another aspect is directed to a vector combination comprising a vector encoding the first polypeptide of a heterodimeric complex and a second vector encoding the second polypeptide of a heterodimeric complex.
  • Another aspect is directed to a host cell comprising one or more recombinant nucleic acids encoding for a heteromultimer.
  • references to “recombinant” nucleic acid indicates an association of elements different than occurring in nature.
  • examples of recombinant nucleic acid include heterologous regulatory elements such as a promoter and/or polyadenylation signal; the presence, association, and/or location of coding regions; and/or the of presence, association, and/or location of non-coding regions.
  • Recombinant nucleic acid can be provided, for example, through modification of a host cell genome and/or extrachromosomal elements such as vectors.
  • Additional aspects include method of producing heteromultimers using a recombinant cell comprising recombinant nucleic acid encoding the multimer; and methods of obtaining heteromultimers (e.g., recovering the heteromultimers from such recombinant cells) .
  • FIGs. 1A-1C illustrates a “Fab 2 ⁇ Fc” , where the Fc is homodimeric.
  • FIG. 1B depicts a “Fab ⁇ Fc” monospecific antibody.
  • FIG. 1C illustrates a “Fc” homodimer.
  • FIG. 2 shows the calculation of the percentage of correctly paired bispecific antibodies.
  • the box around the curve indicates 3 peaks representing 3 fractions of 3 formats, which are Fab 2 ⁇ Fc, Fab ⁇ Fc, and Fc from left to right.
  • the present invention includes modified CH3 domains comprising specific amino acid substitutions to promote heterodimer formation.
  • substitutions are noted with respect to IgG and/or a reference sequence.
  • IgG subclasses e.g., IgG 1 , IgG 2 , IgG 3 , and IgG 4
  • allotypes e.g., for IgG1: G1m (z, a) , G1m (f) , and G1m (f, a)
  • Vidarsson et al. Front Immunol. 2014: 5: 520 which is herein incorporated by reference in its entirety.
  • Amino acid modifications that may be present in a modified CH3 domain include, but are not limited to, one or more amino acid insertions, deletions, and/or substitutions compared to the IgG CH3 domain.
  • the modifications of the CH3 domain and the modified CH3 domains are referred to herein collectively as “CH3 modifications” , “modified CH3 domains” , “modified CH3” , “variant CH3 domains” , “CH3 variants” , “CH3 mutations” , or “mutated CH3 domains” .
  • Modified CH3 domains may be incorporated into molecules of choice to facilitate dimerization.
  • the modified CH3 domains are present as a heterodimer joining two polypeptides.
  • heterodimeric polypeptide complex may be part of a multimer.
  • Favoring formation of the heterodimeric complex can be useful, for example, in producing multispecific binding molecules, wherein the different polypeptides making up the heterodimeric complex have different binding domains.
  • multispecific binding molecules can have a variety of uses including diagnosis, prophylaxis, and therapeutic.
  • therapeutic uses include cancer treatment, inflammatory diseases, autoimmunity, neurodegeneration, bleeding disorders, psoriasis, wet macular degeneration, and infections (Spiess et al. Molecular Immunology. 2015. 67 (2 Pt A) : 95-106; and Brinkmann et al. MAbs. 2017. 9 (2) : 182-212. ) .
  • heterodimers As used herein, terms such as “heterodimers” , “heterodimeric complex” and “heterodimeric” indicate the dimer region comprises two polypeptides having a different amino acid sequence.
  • homodimers As used herein, terms such as “homodimers” , “homodimeric complex” and “homodimeric” indicate the dimer region comprises two polypeptides having the same amino acid sequence.
  • Multimers comprising a heterodimeric polypeptide complex may comprise one or more additional domains positioned outside the heterodimeric polypeptide complex, such as binding domains, antibody constant domains (or parts thereof) , and antibody hinges; and/or additional polypeptides such as light chain antibodies or binding fragments thereof.
  • An antibody binding fragment comprises one or more variable regions.
  • Multimers preferably comprise two or more different binding domains.
  • a “binding domain” refers to a polypeptide able to specifically bind to another molecule.
  • polypeptide, ” “protein” and “peptide” can be used interchangeably to refer to an amino acid sequence without regard to function.
  • Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acid acids.
  • Amino acids include naturally occurring amino acids and amino acids provided by cellular modification.
  • a first option refers to the applicability of the first element without the second.
  • a second option refers to the applicability of the second element without the first.
  • a third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore, satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore, satisfy the requirement of the term “and/or. ”
  • references to “comprise” , and variations such as “comprises” and “comprising” , used with respect to an element or group of elements is open-ended and does not exclude additional unrecited elements or method steps. Terms such as “including” , “containing” and “characterized by” are synonymous with comprising. In the different aspects and embodiments described herein reference to an open-ended term such as “comprising” can be replaced by “consisting” or “consisting essentially of” .
  • RNA and the corresponding DNA are considered the same unless provided otherwise by the employed context, for example, providing reference to the polynucleotide being RNA or DNA.
  • Corresponding RNA and DNA include uracil for thymine and replacement of the ribose backbone for the deoxyribose backbone.
  • sequence “identical” or “identity” SEQ ID NO: 1 can be calculated by determining the number of identical amino acids in aligned sequences, dividing by the total number of amino acids in SEQ ID NO: 1 (125 amino acids) and multiplying by 100. Percent “identical” or “identity” for nucleic acid sequences can be determined in an analogous manner where nucleotides to the reference sequence are aligned to achieve maximal alignment taking into account nucleotide differences and gaps, dividing by the total number of nucleotides in the reference sequence and multiplying by 100.
  • any variable of one component may be used with any variable on another component.
  • SEQ ID NO: X and SEQ ID NO: Y refer to two different sequences
  • reference to a first modified IgG CH3 domain with a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO: X in combination with a second modified immunoglobulin CH3 with a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO: Y include embodiments where the first modified immunoglobulin CH3 domain can have a sequence identity of at least 95%to SEQ ID NO: X, and the second modified immunoglobulin CH3 domain can have a sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%to SEQ ID NO:
  • the term “about” refers to a value within 10%of the underlying parameter (i.e., plus or minus 10%) .
  • “about 1: 10” includes 1.1: 10.1 or 0.9: 9.9
  • “about 5 hours” includes 4.5 hours or 5.5 hours.
  • the term “about” at the beginning of a string of values modifies each of the values by 10%. In certain embodiments, about refers to plus or minus 5%of an indicated value.
  • the instant invention is generally disclosed herein using affirmative language to describe the numerous embodiments of the instant invention.
  • the instant invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.
  • materials and/or method steps are excluded.
  • the present invention provides for CH3 substitution combinations on different polypeptides favoring formation of a heterodimeric polypeptide complex.
  • Reference to “substitution” , “mutation” and “modification” of an amino acid provides for a different amino acid than in the wildtype sequence, and is not a description concerning production.
  • Heterodimer formation can be favored in different ways such as knobs-into-holes (KIH or knob-in-hole) , electrostatic, and/or cysteine bond formation (Wei et al. Oncotarget. 2017. 8 (31) : 51037-51049; Klein et al. MAbs. 2012.4 (6) : 653-63; and Brinkmann et al. MAbs. 2017. 9 (2) : 182-212) .
  • one or more amino acids in the provided amino acid combinations of the first and second polypeptide making up the heterodimer complex may favor heterodimerization through a knob-in-hole mechanism.
  • the heterodimeric polypeptide complex comprises:
  • a second polypeptide comprising a second modified immunoglobulin CH3 domain having at least one amino acid substitution relative to a wildtype immunoglobulin CH3 domain at positions 366 and 360, wherein the substitution is selected from the group consisting of 366Y, 366W, 366F, 366L, 360Y, 360W, 360F, and 360L, wherein numbering is according to the EU index as in Kabat.
  • the dimeric polypeptide complex comprises a first polypeptide comprising a first modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, and a second polypeptide comprising a second modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, wherein:
  • the first modified immunoglobulin CH3 domain comprises the substitution 405V such as F405V
  • the second modified immunoglobulin CH3 domain comprises the substitution 366W such as T366W
  • the first modified immunoglobulin CH3 domain comprises the substitutions 405V and 407A such as F405V and F407A
  • the second modified immunoglobulin CH3 domain comprises the substitution 366W such as T366W
  • the first modified immunoglobulin CH3 domain comprises the substitution 407A such as Y407A
  • the second modified immunoglobulin CH3 domain comprises the substitution 366Y such as T66Y;
  • the first modified immunoglobulin CH3 domain comprises the substitution 349T such as Y349T, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y such as K360Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T and 407A such as Y349T and Y407A
  • the second modified immunoglobulin CH3 domain comprises the substitution 360Y such as K360Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A such as Y349T, F405V, and Y407A
  • the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366W such a K360Y and T366W;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A such as Y349T, F405V, and Y407A
  • the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366Y such as K360Y and T366Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A such as Y349T, F405V, and Y407A
  • the second modified immunoglobulin CH3 domain comprises the substitutions 360W and 366Y such as K360W and T366Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A such as Y349T, F405V, and Y407A
  • the second modified immunoglobulin CH3 domain comprises the substitutions 360Y, 366W, 435R and 436F such as K360Y, T366W, H435R, and Y436F;
  • reference to a modified immunoglobulin CH3 domain indicates a CH3 derived from a human Ig CH3 region by providing for the indicated substitutions and up to 10 additional modifications.
  • 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 amino acids modification are provided.
  • the alterations are with respect to human IgG, IgM, IgA, IgD, and IgE; human IgG; human IgG 1 ; or human IgG 4 .
  • Human immunoglobulins can be assigned to five major isotypes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG 1 , IgG 2 , IgG 3 and IgG 4 .
  • Antibody light chains of vertebrate species may be assigned to one of two clearly distinct types, namely kappa ( ⁇ ) and lambda ( ⁇ ) , based on the amino acid sequences of their constant domains.
  • EU index Kabat
  • references to amino acid modification indicates a deletion, substitution or addition. When more than one amino acid modification is present, each modification can independently be either a deletion, substitution or addition.
  • reference to a modified immunoglobulin CH3 domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG 1 CH3.
  • the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 3; or in addition to the indicated substitutions can differ from SEQ ID NO: 3 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • reference to a modified immunoglobulin CH3 domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG 2 CH3.
  • the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 19; or in addition to the indicated substitutions can differ from SEQ ID NO: 19 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • reference to a modified immunoglobulin CH3 domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG 3 CH3.
  • the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 20; or in addition to the indicated substitutions can differ from SEQ ID NO: 20 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • reference to a modified immunoglobulin CH3 domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG 4 CH3.
  • the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 21; or in addition to the indicated substitutions can differ from SEQ ID NO: 21 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • additional modifications that can be present include, for example, modifications favoring heterodimer formation, modifications altering one or more Fc effector activity, and/or modifications not significantly decreasing (e.g., less than 20%or less than 10%impact) heterodimer formation.
  • the first polypeptide further comprises a first CH2 immunoglobulin domain joined to the amino terminus of the first CH3 immunoglobulin domain and the second polypeptide further comprises a second CH2 immunoglobulin domain joined to the amino terminus of the second CH3 amino terminus.
  • the first and second CH2 immunoglobulin domains may be the same or different.
  • the first CH3-CH2 hinge domain and the second CH3-CH2 hinge domain are derived from a human Ig CH3-CH2 region by, in addition to providing for the indicated substitutions, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modification are provided or any subcombination thereof, for example, 0 to 15, 0 to 10, 0 to 5, or 0 to 3 additional amino acids are modified.
  • the modifications are with respect to any of human IgG, IgM, IgA, IgD, and IgE; human IgG; or human IgG 1 or IgG 4 .
  • the first polypeptide further comprises a first immunoglobulin hinge region joined to the amino terminus of the first CH2 domain providing a first CH3-CH2-hinge domain and the second polypeptide further comprises a second CH2 immunoglobulin hinge region joined to the amino terminus of the second CH2 amino terminus providing a second CH3-CH2-hinge domain, wherein the hinge domains are joined by one or more disulfide bonds.
  • the heterodimeric complex formed between the first and the second CH3-CH2-hinge domain corresponds to an Fc region.
  • the Fc may also provide for different effector functions such as antibody-dependent cellular cytotoxicity (ADCC) , antibody-dependent cellular phagocytosis (ADCP) , and complement-dependent cytotoxicity (CDC) .
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • Additional possible advantages include potential for increased half-life (e.g., binding to FcRn) and purification (e.g., using protein A or protein G affinity columns) .
  • the first CH3-CH2-hinge domain and the second CH3-CH2 hinge domain are derived from a human Ig CH3-CH2-hinge domain by, in addition to providing for the indicated substitutions, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or any subcombination thereof, of example 0-15, 0 to 10, 0 to 5, 0-3 amino acids are modified.
  • the modifications are with respect to any of human IgG, IgM, IgA, IgD, and IgE; human IgG; or human IgG 1 or IgG 4 .
  • reference to a modified immunoglobulin CH3-CH2-hinge domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to a IgG 1 CH3-CH2-hinge domain.
  • the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 12; or in addition to the indicated substitutions can differ from SEQ ID NO: 12 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • IgG 2 , IgG 3 and IgG 4 CH2 domains are highly homologous with IgG 1 CH2.
  • SEQ ID NOs: 22, 23 and 24 provide examples of CH3-CH2-hinge domains from IgG 2 , IgG 3 and IgG 4 .
  • reference to a modified immunoglobulin CH3-CH2-hinge domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG 2 CH3.
  • the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 22; or in addition to the indicated substitutions can differ from SEQ ID NO: 22 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • reference to a modified immunoglobulin CH3-CH2-hinge domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG 3 CH3.
  • the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 23; or in addition to the indicated substitutions can differ from SEQ ID NO: 23 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • reference to a modified immunoglobulin CH3-CH2-hinge domain indicates a polypeptide having the indicated substitutions and a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to IgG 4 CH3.
  • the polypeptide comprising a sequence having the indicated substitutions has a sequence identity of at least 85%, at least 90%, at least 95%, or at least 99%to SEQ ID NO: 24; or in addition to the indicated substitutions can differ from SEQ ID NO: 24 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • the heterodimeric polypeptide may comprise not only, substitutions described herein facilitating heterodimer formation, but also comprise one or more Fc modifications increasing stability, increasing half-life, and/or altering effector functions.
  • Fc modifications increasing cellular cytotoxic effector functions include:
  • the Fc can prolong half-life in circulation through its interaction with FcRn.
  • Half-life extending modifications include:
  • the heterodimeric polypeptide may comprise Fc modifications that abolish or reduce immune effector functions. Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (Fc ⁇ R) can be useful limiting unwanted inflammatory responses to therapeutic antibodies. Fc variants that abolish or reduce immune effector functions include:
  • Antibodies like all polypeptides have an isoelectric point (pI) , which is generally defined as the pH at which a polypeptide carries no net charge. Protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pI) of the protein. It is possible to optimize solubility by modifying the number and location of ionizable residues in the antibody to adjust the pI (see U.S. Patent No. 10,457,742, which is herein incorporated by reference in its entirety) .
  • the first and second polypeptide comprise CH3 modifications provided for in Table 1:
  • heteromultimers are produced (e.g., in a cell) , or provided in preparation, wherein of the total multimers, the heteromultimers are about 90%or greater, about 93%or greater, about 95%or greater, or about 97%or greater of the total multimers. Percentage of heteromultimers can be assessed as provided in the Examples below (e.g., CE-SDS or HPLC) .
  • binding domains can be provided to different polypeptide making up a heteromultimer.
  • a particular binding domain can specifically bind to another molecule.
  • Reference to “specifically” or “specific” with respect to binding does not require absolute specificity. Rather the specificity is provided by the binding domain able to distinguish the targeted molecule from most or all other molecule that are present.
  • the binding domain is able to distinguish the targeted molecule from at least 75%, 85%, 95, or 99%from other molecules present in a biological sample or subject containing the targeted molecules.
  • Biological molecules containing targeted molecules may include, for example, material obtained from an animal (e.g., human) and/or present in an animal (e.g., human) .
  • heterodimeric polypeptide complex can be combined with different binding domains to provide for molecules that are able to bind different targets.
  • Different types of heteromultimer polypeptides can be produced, including, for example, bispecific antibodies and different types of molecules containing two or more antibody variable regions, providing for multispecificity, polypeptide receptors, and ligands.
  • the binding domain is an antigen binding domain.
  • An “antigen binding domain” refers to one or more immunoglobulin variable regions able to bind to an epitope present on an antigen.
  • An immunoglobulin variable region contains three complementary determining regions (CDRs) interspaced within a framework (FR) .
  • CDRs complementary determining regions
  • FR framework
  • Different types and sources of immunoglobulin variable regions can be used, such as variable regions obtained from primate (e.g., humans, camels, monkeys) antibodies and variable regions obtained from other animals such as sharks, camels, llamas, mice, and rats.
  • framework regions for human immunoglobulins use humanized or human framework regions.
  • Antigen binding domains comprise at least one variable region.
  • two variable regions are provided, for example, domains provided by a heavy and a light chain variable region contribute to binding.
  • the heavy chain can be viewed as an extension from the CH3 domain, while the light chain variable region provides an additional polypeptide.
  • Certain animals provide for naturally occurring antibodies with only a heavy chain, for example, camel, shark, and llama single domain heavy chain antibodies (see Flajnik et al. PLoS Biol. 2011. 9 (8) : e1001120, which is herein incorporated by reference in its entirety) .
  • the heteromultimer contains two or more different antigen binding domains.
  • Each antigen binding domain can be a single domain binding site provided by one variable region or a two-domain binding site provided by two immunoglobulin variable regions.
  • An antigen binding domain can be provided for by a variety of different antigen binding fragments comprising such a domain.
  • antibody binding fragments include VH, VL, VHH (sdAb or dAb) , Fab, Fab’, F (ab’) 2 , scFab, scFv, (scFv) 2 , Fv, taFv, DART and (bispecific T-cell engager) .
  • Multispecificity can be provided for by a variety of different formats (see Spiess et al. Molecular Immunology. 2015. 67 (2 Pt A) : 95-106 and Brinkmann et al. MAbs. 2017. 9 (2) : 182-212, both of which are incorporated by reference herein in their entirety) .
  • Examples of such formats include scFv-CH3, scFv-Fc, scFab (single chain Fab) , Fab-scFab, orthogonal Fab, DuetMab, CrossMAb, Ig-Fv, scFab-Fc-scFv2, scFab-Fc-scFv, DVD-Ig TM (dual variable domain immunoglobulin) , taFv (tandem scFv) -Fc, scFv-Fc-Fv, Fab-Fc-scFv, Fab-scFv-Fc, DART-Fc, scFv-CH3, and TriFabs.
  • DuetMab refers to the replacement of the native interchain disulfide bond with one of the 2 CH1-CL interfaces with an engineered interchain disulfide bond using knobs-into-holes technology (see Mazor et al. MAbs. 2015. 7 (2) : 377-389) .
  • CrossMAb is a type of knobs-into-holes technology in which either the light chain of one Fab arm is exchanged by the Fd of the corresponding heavy chain, or only one pair of the variable or constant domain of one Fab is swapped between the light and heavy chain (see Brinkmann et al. MAbs. 2017. 9 (2) : 182-212) .
  • Canonical molecules are comprised of two scFvs from an anti-TAA (tumor-associated antigen) and an anti-CD3 monoclonal antibody with a short linker connecting them in tandem (see Zhou et al. Biomarker Research. 2021. 9 (38) : hyperlink/doi. org/10.1186/s40364-021-00294-9) .
  • TriFabs indicate two regular Fabs fused through flexible 20-residue peptide linkers, (G 4 S) 4 , to an asymmetric Fab-like entity as heterodimerization module or stem region.
  • the overall structure of TriFabs resembles that of an IgG with the Fc region substituted by the stem region (see Brinkmann et al. MAbs. 2017. 9 (2) : 182-212) .
  • DVD-Ig TM indicates a tetra-specific antibody format (four-in-one) that is constructed by fusing two dual variable domain immunoglobulin antibodies (see DiGiammarino et al. Methods Mol Biol. 2012. 899: 145-56, which is herein incorporated by reference in its entirety) .
  • antibody includes both single domain antibodies containing variable regions provided by an antibody heavy chain (e.g., VHH) and two domain antibodies containing variable regions provided by antibody heavy and light chains.
  • Antibodies can be provided in different preparations, including polyclonal antibodies or monoclonal antibodies.
  • Monoclonal antibodies refer to a population of antibodies having substantially the same structure, where any structural variation is provided by a hybridoma expressing the antibody or cell lines designed to express only a particular antibody (e.g., recombinant cell comprising antibody encoding nucleic acid) .
  • the base subunit for a full-length human antibody is two heavy chains and two light chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region and a heavy chain constant region (comprised of at least domains CH1, hinge, CH2 and CH3) .
  • Each light chain is comprised of a light chain variable region and a light chain constant region.
  • the VH and the VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions, interspersed with framework regions.
  • Each VH and VL is composed of three complementarity determining regions and four framework segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • multispecific antibody refers to an antibody that comprises multiple immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein) .
  • the first and second epitopes overlap or substantially overlap.
  • the first and second epitopes do not overlap or do not substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein) .
  • a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain.
  • a multispecific antibody is a bispecific antibody molecule, a trispecific antibody, or a tetraspecific antibody molecule.
  • antigen binding domains can be from any animal origin including birds and mammals (e.g., human, primate, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken) .
  • mammals e.g., human, primate, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken
  • the antibody variable regions are of human origin.
  • the antigen binding domains are selected from the group consisting of CD19 (B4, CVID3, B-lymphocyte antigen CD19) , CD20 (MS41, Bp35, B-lymphocyte antigen CD20) , EpCAM (epithelial cell adhesion molecule, CD326, GA733-2, M1S2, M4S1, MIC18, TACSTD1, TROP1) , CEA (Carcinoembryonic antigen-related cell adhesion molecule) , PSMA (Prostate-specific membrane antigen, FOLH1, NAALAD1, PSM, GIG27) , GD2 Ganglioside, CD30 (TNFRSF8, Tumor necrosis factor receptor superfamily member 8) , CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1, T10) , CD47 (Leukocyte surface antigen CD47, MER6) , CD52 (CAMPATH-1 antigen, CDW52, HE5) , c-Met
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.
  • the term “antigen binding fragment” refers to one or more polypeptides comprising an immunoglobulin variable region (e.g., single and two domain binding regions) such as, a diabody, a Fab, a Fab′, a F (ab′) 2 , scFab, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv′) , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , a single domain antibody (sdAb) an scFv dimer (bivalent diabody) , taFv, a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody or a bivalent domain
  • the antigen binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment of the heavy chain.
  • the Fd segment is approximately the first 220 amino acids from the N-terminus of the heavy chain, contained within the heavy chain of the Fab.
  • the antigen binding fragment comprises Fab and F (ab′) .
  • single-domain antibody refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.
  • Fab or “Fab fragment” refer to an antibody fragment composed of VH, CH1, VL and CL domains.
  • Fv refers to the minimum antibody fragment that contains a complete antigen-recognition and antigen binding site. This region may be made of a heavy chain variable region or a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association.
  • Single-chain Fv, ” “single-chain antibody, ” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • specific binding refers to preferential binding of the two proteins with typically an equilibrium dissociation constant (K D ) of about 1 ⁇ 10 -8 M or less, for example about 1 ⁇ 10 -9 M or less, about 1 ⁇ 10 -10 M or less, about 1 ⁇ 10 -11 M or less, or about 1 ⁇ 10 -12 M or less, typically with the K D that is at least one hundred-fold less than its K D for binding to a non-specific antigen (e.g., BSA, casein) .
  • K D equilibrium dissociation constant
  • antibody derivative refers to a molecule comprising a full-length antibody or an antigen binding fragment thereof, wherein one or more amino acids are chemically modified or substituted.
  • Chemical modifications that can be used in antibody derivatives include, e.g., alkylation, PEGylation, acylation, ester formation or amide formation or the like, e.g., for linking the antibody to a second molecule.
  • Exemplary modifications include PEGylation (e.g., cysteine-PEGylation) , biotinylation, radiolabeling, and conjugation with a second agent (such as a cytotoxic agent) .
  • Additional binding domains include a receptor able to bind to a ligand, and a ligand able to bind to a receptor.
  • Fusion proteins providing for specific binding can be produced by appending a receptor or ligand onto one or both polypeptides making a heterodimeric immunoglobulin complex comprising a CH3 dimer.
  • Multispecificity can be provided using different formats such as appending different receptors to each polypeptide; appending different ligands to each polypeptide; appending a receptor to one polypeptide and a ligand to another polypeptide; and attaching a receptor or ligand to one polypeptide and providing an antigen domain to another polypeptide.
  • Ligand and receptor combinations present on a particular multimer are selected to not bind to each other.
  • one or more antigen binding domains are provided as described in II. A. supra.
  • the heteromultimer comprises a heterodimeric immunoglobulin complex comprising a CH3-CH2 heterodimer as provided for in II. A. supra.
  • the heterodimeric immunoglobulin complex comprises a Fc as provided for example in II. A. supra.
  • the heterodimeric multimer is an immunoadhesin or an antibody-immunoadhesin chimera.
  • An immunoadhesin comprises an Fc region and receptor binding domains made up of receptors and/or ligands.
  • An antibody-immunoadhesin chimera comprises an Fc region at least one binding domain joined to a receptor or ligand and at least one binding domain joined to an antigen binding domain (e.g., antigen binding fragment) .
  • antigen binding domain e.g., antigen binding fragment
  • Heteromultimers comprising a heterodimeric polypeptide complex can be produced by expressing the individual polypeptides and allowing for the formation of the heterodimeric polypeptide complex.
  • the multimers may also comprise additional polypeptides, for example, antibody light chains or binding fragments thereof.
  • the produced heteromultimer can be purified from other material, including homodimers as described in the Examples below or use alternative purification techniques. (see U.S. Patent No. 10,875,931 and Yao et al. Nature Communications. 2022. 13: 1539; each of which is herein incorporated by reference in its entirety. )
  • Polypeptides can be produced from a single nucleic acid encoding for each of the polypeptides or through the use of multiple nucleic acids encoding for the different polypeptides.
  • the different nucleic acids can exist inside a host cell as extrachromosomal elements and/or be integrated into a host genome.
  • encoding sequences can be provided for additional polypeptides associated with heterodimers, for example, in the case of an antibody, antibody light chains, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies.
  • the heteromultimer is produced in a host cell comprising a first recombinant nucleic acid segment encoding for a first polypeptide of the heterodimer and a second recombinant nucleic acid segment encoding for a second polypeptide.
  • the first and second recombinant nucleic acid segments can be provided on the same or on different nucleic acids.
  • Reference to “recombinant nucleic acid segment” refers to a combination of different nucleic acids than those found in nature.
  • the recombinant nucleic acid segment can exist in a vector along with vector elements and/or be integrated inside a host genome.
  • nucleic acid encoding the first and second polypeptides are provided on one or more nucleic acid vectors.
  • Vectors can be produced by different methods including, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • the vector comprises a nucleotide sequence encoding a heterodimeric polypeptide, operably linked to a promoter.
  • Vector refers to an extrachromosomal polynucleotide capable of being duplicated within a biological system or that can be moved between such systems.
  • Vector polynucleotides typically contain elements, such as origins of replication, a polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, virus, animal, plant, and reconstituted biological systems.
  • “Host cell” refers to a cell able to express nucleic acid encoding for multimer polypeptides.
  • the host cell is bacterial host; a mammalian cell line, such as a human cell line (e.g., HeLa) ; a hamster cell line (e.g., CHO) ; or a murine cell line.
  • An expression vector can be used to facilitate delivery and expression of an encoding nucleic acid to a host cell. The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce a heterodimeric polypeptide of the invention.
  • heterodimeric polypeptides described herein e.g., antibodies
  • host-expression vector systems may be utilized to express the heterodimeric polypeptides described herein (e.g., antibodies) (see, e.g., U.S. Pat. No. 5,807,715, which is herein incorporated by reference in its entirety) .
  • Heterodimeric polypeptides may be purified by different methods such as chromatography (e.g., ion exchange, affinity, Protein A, and/or sizing column chromatography) , centrifugation, and differential solubility.
  • chromatography e.g., ion exchange, affinity, Protein A, and/or sizing column chromatography
  • centrifugation e.g., centrifugation, and differential solubility.
  • Polypeptides expressed with a signal sequence can generally be recovered from the culture medium as a secreted polypeptide, while polypeptides expressed without a signal sequence may be recovered from host cell lysate. If the heterodimeric polypeptide is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton TM X-100) .
  • a suitable detergent solution e.g., Triton TM X-100
  • heteromultimers can be facilitated using signal peptides (see, for example, Haryadi et al. PLOS ONE. 2015. 10 (2) : e0116878. doi: 10.1371/journal, describing optimization of heavy chain and light chain signal peptides for increase expression, which is herein incorporated by reference in its entirety) .
  • Multimers can be purified, for example, by methods including ion-exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, size-exclusion chromatography, dialysis, or affinity chromatography (see U.S. Patent No. 10,457, 742, which is herein incorporated by reference in its entirety) .
  • Embodiment 1 A heteromultimer comprising a heterodimeric polypeptide complex, wherein the heterodimeric polypeptide complex comprises:
  • a second polypeptide comprising a second modified immunoglobulin CH3 domain having at least one amino acid substitution relative to a wildtype immunoglobulin CH3 domain at positions 366 and 360, wherein the substitution is selected from the group consisting of 366Y, 366W, 366F, 366L, 360Y, 360W, 360F, and 360L, wherein numbering is according to the EU index as in Kabat.
  • Embodiment 2 The heteromultimer of Embodiment 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 405 and 407; and the second modified immunoglobulin CH3 domain comprises a substitution at position 366.
  • Embodiment 3 The heteromultimer of Embodiment 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 349 and 407; and the second modified immunoglobulin CH3 domain comprises a substitution at position 360.
  • Embodiment 4 The heteromultimer of Embodiment 1, wherein the first modified immunoglobulin CH3 domain comprises substitutions at positions 349, 405 and 407; and the second modified immunoglobulin CH3 domain comprises substitutions at positions 360 and 366.
  • Embodiment 5 The heteromultimer of any one of Embodiments 1-4, wherein the substitutions are selected from 349T, 405V, 407A, 366W, 366Y, 360W, and 360Y.
  • Embodiment 6 A heteromultimer comprising a heterodimeric polypeptide complex, wherein the dimeric polypeptide complex comprises a first polypeptide comprising a first modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, and a second polypeptide comprising a second modified immunoglobulin CH3 domain having one or more substitutions relative to a wildtype immunoglobulin CH3 domain, wherein:
  • the first modified immunoglobulin CH3 domain comprises the substitutions 405V, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366W;
  • the first modified immunoglobulin CH3 domain comprises the substitution 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 366Y;
  • the first modified immunoglobulin CH3 domain comprises the substitution 349T, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T and 407A, and the second modified immunoglobulin CH3 domain comprises the substitution 360Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366W;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A, and the second modified immunoglobulin CH3 domain comprises the substitutions 360Y and 366Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A
  • the second modified immunoglobulin CH3 domain comprises the substitutions 360W and 366Y;
  • the first modified immunoglobulin CH3 domain comprises the substitutions 349T, 405V and 407A
  • the second modified immunoglobulin CH3 domain comprises the substitutions 360Y, 366W, 435R and 436F;
  • Embodiment 7 The heteromultimer of Embodiment 1, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitutions 405V and 407A and the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W or 366Y.
  • Embodiment 8 The heteromultimer of Embodiment 7, wherein the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitution 366W.
  • Embodiment 9 The heteromultimer of Embodiment 8, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 5; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 6.
  • Embodiment 10 The heteromultimer of Embodiment 8, wherein the second modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 360Y.
  • Embodiment 11 The heteromultimer of Embodiment 10, where in the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T.
  • Embodiment 12 The heteromultimer of Embodiment 10, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 8.
  • Embodiment 13 The heteromultimer of Embodiment 11, wherein the second modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitutions 435R and 436F.
  • Embodiment 14 The heteromultimer of Embodiment 13, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 11.
  • Embodiment 15 The heteromultimer of Embodiment 7, wherein the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T and second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitutions 366Y and 360Y.
  • Embodiment 16 The heteromultimer of Embodiment 15, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 9.
  • Embodiment 17 The heteromultimer of Embodiment 7, wherein the first modified immunoglobulin CH3 domain polypeptide further comprises the amino acid substitution 349T and the second modified immunoglobulin CH3 domain polypeptide comprises the amino acid substitutions 366Y and 360W.
  • Embodiment 18 The heteromultimer of Embodiment 17, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 7; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 10.
  • Embodiment 19 The heteromultimer of Embodiment 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitution 349T and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 360Y.
  • Embodiment 20 The heteromultimer of Embodiment 19, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 13; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 14.
  • Embodiment 21 The heteromultimer of Embodiment 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitutions 349T and 405V and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 360Y.
  • Embodiment 22 The heteromultimer of Embodiment 21, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 15; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 14.
  • Embodiment 23 The heteromultimer of Embodiment 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitutions 349T and 407A and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 360Y.
  • Embodiment 24 The heteromultimer of Embodiment 23, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 16; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 14.
  • Embodiment 25 The heteromultimer of Embodiment 6, wherein the first modified immunoglobulin CH3 domain comprises the amino acid substitution 405V and the second modified immunoglobulin CH3 domain comprises the amino acid substitution 366W.
  • Embodiment 26 The heteromultimer of Embodiment 25, wherein the first modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 17; and the second modified immunoglobulin CH3 domain polypeptide comprises a sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or 100%to SEQ ID NO: 6.
  • Embodiment 27 The heteromultimer of any one of Embodiments 1-26, wherein the first modified immunoglobulin CH3 domain and the second first modified immunoglobulin CH3 domain are derived from a human IgG.
  • Embodiment 27a The heteromultimer of Embodiment 27, wherein the human IgG is selected from IgG 1 , IgG 2 , IgG 3 , or IgG 4 .
  • Embodiment 27b The heteromultimer of Embodiment 27a, wherein the human IgG is most preferably IgG 1 .
  • Embodiment 28 The heteromultimer of any one of Embodiments 1-27a, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least99%with SEQ ID NO: 3 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 3.
  • Embodiment 29 The heteromultimer of any one of Embodiments 1-27a, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 19 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 19.
  • Embodiment 30 The heteromultimer of any one of Embodiments 1-27a, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 20 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 20.
  • Embodiment 31 The heteromultimer of any one of Embodiments 1-27a, wherein the first modified immunoglobulin CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 21 and the second modified CH3 domain comprises a sequence having a sequence identity of at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%with SEQ ID NO: 21.
  • Embodiment 32 The heteromultimer of any one of Embodiments 1-27a, wherein the first polypeptide further comprises a first CH2 immunoglobulin domain joined to the amino terminus of the first CH3 immunoglobulin domain and the second polypeptide further comprises a second CH2 immunoglobulin domain joined to the amino terminus of the second CH3 amino terminus, wherein the first and second CH2 immunoglobulin domains may be the same or different.
  • Embodiment 33 The heteromultimer of Embodiment 32, wherein the first CH3-CH2-hinge domain is an IgG 1 or IgG 4 and the second CH3-CH2-hinge domain is an IgG 1 or IgG 4 .
  • Embodiment 34 The heteromultimer of any one of Embodiments 1-33, wherein the first polypeptide further comprises a first immunoglobulin hinge region joined to the amino terminus of the first CH2 domain providing a first CH3-CH2-hinge domain and the second polypeptide further comprises a second immunoglobulin hinge region joined to the amino terminus of the second CH2 amino terminus providing a second CH3-CH2-hinge domain, wherein the first and second hinge regions are joined by one or more disulfide bonds, and wherein the first CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at least 90%to SEQ ID NOs: 12, 22, 23 or 24 and the second CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at 85%to SEQ ID NOs: 12, 22, 23 or 24.
  • Embodiment 35 The heteromultimer of Embodiment 34, wherein the first CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or at least 99%to any of SEQ ID NO: 12, 22, 23, or 24 and the second CH3-CH2-hinge domain comprises an amino acid sequence with a sequence identity of at least 90%, at least 95%, at least 97%, or at least 99%to SEQ ID NO: 12, 22, 23, or 24; or in addition to the indicated substitutions can differ from any of SEQ ID NO: 12, 22, 23, or 24 by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modifications, or any subcombination thereof, for example, 0 to 10, 0 to 5, or 0 to 3 additional modifications.
  • Embodiment 36 The heteromultimer of Embodiments 1 or 6, wherein either the first polypeptide comprises the sequence of SEQ ID NO: 4 and the second polypeptide comprises the sequence of SEQ ID NO: 6; the first polypeptide comprises the sequence of SEQ ID NO: 5 and the second polypeptide comprises the sequence of SEQ ID NO: 6; the first polypeptide comprises the sequence of SEQ ID NO: 13 and the second polypeptide comprises the sequence of SEQ ID NO: 14; the first polypeptide comprises the sequence of SEQ ID NO: 15 and the second polypeptide comprises the sequence of SEQ ID NO: 14; the first polypeptide comprises the sequence of SEQ ID NO: 16 and the second polypeptide comprises the sequence of SEQ ID NO: 14; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 8; the first polypeptide comprises the sequence of SEQ ID NO: 7 and the second polypeptide comprises the sequence of SEQ ID NO: 9; the first polypeptide comprises the sequence of S
  • Embodiment 37 The heteromultimer of any one of Embodiments 1-36, wherein the first polypeptide further comprises a first binding domain and the second polypeptide further comprises a second binding domain, wherein the first and second binding domains are different from each other.
  • Embodiment 38 The heteromultimer of Embodiment 37, wherein the first and second binding domains are independently selected from a receptor or a ligand.
  • Embodiment 39 The heteromultimer of Embodiment 37, wherein the first binding domain is a first antigen binding domain and the second binding domain is a second antigen binding domain, wherein the first and second antigen binding domains bind to different epitopes.
  • Embodiment 40 The heteromultimer of Embodiment 39, wherein the first and second antigen binding domains bind to different antigens.
  • Embodiment 41 The heteromultimer of any one of Embodiments 39 or 40, wherein at least one of the first and second polypeptides further comprises a third antigen binding domain such as a third antigen binding domain comprising a third Fab, a third scFv, or a third VHH.
  • a third antigen binding domain such as a third antigen binding domain comprising a third Fab, a third scFv, or a third VHH.
  • Embodiment 42 The heteromultimer of Embodiment 41, wherein the first polypeptide further comprises a third antigen binding domain, and the second polypeptide further comprises a fourth antigen binding domain.
  • Embodiment 43 The heteromultimer of Embodiment 42, wherein the first polypeptide further comprises a fourth antigen binding domain, such as a fourth antigen binding domain comprising a fourth Fab, a fourth scFv, or a fourth VHH.
  • a fourth antigen binding domain such as a fourth antigen binding domain comprising a fourth Fab, a fourth scFv, or a fourth VHH.
  • Embodiment 44 The heteromultimer of any one of claims 39-43, wherein each antigen binding domain is selected from the group consisting of a VH, VL, VHH (sdAb or dAb) , Fab, Fab’, F (ab’) 2 , scFab, scFv, (scFv) 2 , Fv, taFv, DART and bispecific T-cell engager.
  • each antigen binding domain is selected from the group consisting of a VH, VL, VHH (sdAb or dAb) , Fab, Fab’, F (ab’) 2 , scFab, scFv, (scFv) 2 , Fv, taFv, DART and bispecific T-cell engager.
  • Embodiment 45 The heteromultimer of Embodiments 34 or 35, wherein the heteromultimer is selected from the group comprising a DART-Fc, CrossMAb, DuetMab, TriFabs, (controlled Fab arm exchange generated bispecific antibodies) , scFv-CH3, scFv-Fc, scFab (single chain Fab) , Fab-scFab, orthogonal Fab, DuetMab, CrossMAb, Ig-Fv, scFab-Fc-scFv2, scFab-Fc-scFv, DVD-Ig TM (dual variable domain immunoglobulin) , taFv (tandem scFv) -Fc, scFv-Fc-Fv, Fab-Fc-scFv, Fab-scFv-Fc, and scFv-CH3.
  • a DART-Fc CrossMAb
  • DuetMab TriFab
  • Embodiment 46 The heteromultimer of any one of Embodiments 39-45, wherein the heteromultimer binds to one, two, three, four or more antigenic polypeptides.
  • Embodiment 47 The heteromultimer of Embodiment 46, wherein the antigenic polypeptides are selected from the group consisting of CD19, CD20, EpCAM, CEA, PSMA, GD2, CD30, CD38, CD47, CD52, c-Met, CA19-9, CA72-4, CAM 17.1, CD1a, CD5, CD11A, CD40, CD44, B7-H3, B7-H4, B7-H6, PD-L1 or antigenic fragments thereof.
  • Embodiment 48 The heteromultimer of any one of Embodiments 1-35 or 37-47, wherein the first and/or second polypeptide further comprises one or more Fc mutations selected from the group comprising:
  • G236A, A330L, and I332E ( “GAALIE” ) ;
  • G236A, S239D, A330L, and I332E ( “GASDALIE” ) ;
  • Embodiment 49 A nucleic acid combination comprising a first nucleic acid encoding the first polypeptide and a second nucleic acid encoding the second polypeptide of any one of Embodiments 1-48.
  • Embodiment 50 A nucleic acid encoding both the first polypeptide and the second polypeptide of any one of Embodiments 1-48.
  • Embodiment 51 A vector combination comprising a first vector comprising a nucleic acid encoding the first polypeptide and a second vector comprising a second nucleic acid encoding the second polypeptide of any one of Embodiments 1-48.
  • Embodiment 52 A vector comprising nucleic acid encoding the first and second polypeptides of any one of Embodiments 1-48.
  • Embodiment 53 A host cell comprising one or more recombinant nucleic acid encoding the first polypeptide and second polypeptide of any one of Embodiments 1-48.
  • Embodiment 54 A method of producing a heteromultimer comprising culturing the host cell of Embodiment 53 under conditions to produce the heteromultimer.
  • Embodiment 55 The method of Embodiment 54, further comprising recovering the heteromultimer from the cell or cell culture.
  • Embodiment 56 A pharmaceutical composition comprising the composition of any one of Embodiments 1-48 and a pharmaceutically acceptable carrier.
  • Nucleic acids encoding for a first and second polypeptide comprising different CH3 IgG 1 substitutions were produced to evaluate heterodimer formation.
  • the different substitutions are shown in Table 3.
  • HD-41 is wildtype CH3 and was used as a negative control. Multiple substitutions are separated by slash marks ( "/" ) .
  • CH3A and CH3B, as illustrated in FIG. 1, provide an IgG 1 heavy chain polypeptides (CH3A) and a half Fc (CH3B) .
  • CH3A and CH3B comprise the substitutions shown in Table 3.
  • CH3A CH3A (homodimer)
  • CH3A CH3B heterodimer
  • CH3B CH3B (homodimer)
  • Component 1 comprises a CH3A: CH3A (homodimer) and is a Fab 2 x Fc.
  • Component 2 comprises CH3A: CH3B heterodimer and is a Fab ⁇ Fc.
  • Component 3 comprises CH3B: CH3B (homodimer) and is a Fc.
  • the different components have significantly different molecular weights.
  • the relative molecular weights of the different components are: Component 1 > Component 2 > Component 3.
  • the different Components are illustrated in FIGs. 1A-1C.
  • Table 4 provides the particular amino acid sequences for the Fab heavy and light chains used in the evaluation. (Carter et al. J Immunol Methods. 2001. 248 (1–2) : 7–15) .
  • the amino acid sequence of the Fab heavy chain polypeptide is provided by SEQ ID NO: 1 and the amino acid sequence of the Fab light chain polypeptide is provided by SEQ ID NO: 2.
  • Nucleic encoding for polypeptides described in Example 2 were designed starting with nucleic acid encoding for SP34 (Carter et al. J Immunol Methods. 2001. 248 (1–2) : 7–15) . Encoding sequence sequences were codon-optimized (Biointron) . PCR primers were designed for each fragment and synthesized by Genewiz. Target genes encoding heavy chains, light chains, a heavy chain constant region with a Kozak sequence, an N-terminal signal peptide, and a C-terminal stop codon were synthesized and subcloned into digested mammalian expression vector-pcDNA3.4 using the Gibson method (Gibson, D., Young, L., Chuang, RY. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009. 6: 343–345) .
  • Expression vectors encoding the polypeptide sequences were transformed simultaneously into chemically competent E. coli DH5-alpha cells (NEB or similar) for plasmid preparation. After validation by Sanger sequencing (Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec. 74 (12) : 5463-7) , positive colonies were inoculated in LB (Luria Broth) medium culture with appropriate antibiotic. Desired amounts of plasmids were purified from cell pellets using a plasmid preparation kit DNA concentration was determined by UV-Vis (ultraviolet-visible) absorbance measurement at 260 nm.
  • ExpiCHO-S TM cells were used for transient expression with a commercially available transfection kit and culture medium (ThermoFisher Scientific) .
  • a high-throughput transfection protocol was used on a Hamilton STAR liquid handling station.
  • plasmids were mixed and incubated with ExpiFectamine TM CHO reagent and OptiPRO TM medium, and the mixture was added to the cells (viable cell density at 4-6E6 cells/mL) in a 24-deep-well plate. The final concentration of the plasmid mixture was 1 ⁇ g/mL.
  • Transfected cells were shaken at 37°C with 80%relative humidity and 8%CO 2 .
  • ExpiFectamine TM enhancer and feed were supplemented and the temperature was shifted to 32°C. Cells were harvested on day 7 post-transfection.
  • High-throughput purification was performed on a Hamilton STAR liquid handling station, using Tip columns containing Protein A resin (MicroSep Biological Science) .
  • Cell culture was clarified by plate centrifugation. Clarified supernatant was loaded into a Tip column 10-20 times by repeated aspirating and dispensing. The columns were washed with neutral pH buffer (PBS (phosphate-buffered saline) ) .
  • PBS phosphate-buffered saline
  • Polypeptides were then eluted by 5-10 times by repeated aspirating and dispensing into a clean deep-well plate with low pH buffer (50 mM NaAc, pH 3.5) . Eluted polypeptides were then titrated to neutral pH by the addition of high pH buffer (1 M Arg, pH 9.0) . Protein concentration was quantified by UV-Vis absorbance measurement at 280 nm.
  • SEC-UPLC (Vanquish TM Flex TM , Thermo Fisher Scientific) was performed for aggregation measurement. Specifically, 50 ⁇ L purified antibody sample was loaded to a size exclusion column (Unix TM -C SEC-300) , with PBS used as mobile phase and flow rate set at 0.3 mL/min Total run time for each sample was set at 10 min. The correctly paired BsAbs peaks were identified based on retention time and purity of the BsAbs was quantified as relative peak area (%) .
  • CE-SDS GXII Touch TM HT, Perkin Elmer
  • the non-reduced samples were prepared by mixing 2 ⁇ L purified antibody with 7 ⁇ L SDS loading solution (HT Protein Express Sample Buffer, Perkin Elmer, with 1%SDS (sodium dodecyl sulfate) and 12.5 mM NEM (N-ethylmaleimide) ) .
  • SDS loading solution HT Protein Express Sample Buffer, Perkin Elmer, with 1%SDS (sodium dodecyl sulfate) and 12.5 mM NEM (N-ethylmaleimide)
  • Non-reduced samples were heated at 70°C for 10 min and then mixed with an additional amount of 35 ⁇ L H 2 O.
  • Prepared polypeptide samples were transferred to a 96-well plate and loaded into the Protein Express Chips were prepared for the sample runs according to the user manual (Protein Express Assay Quick Guide, Perkin Elmer) .
  • Purity of the different components can be discerned by the percentage of main peak.
  • the purity of the heterodimeric format can be determined by integrating the main and side peaks observed, and calculating the percentage of main peak's AUC (area under curve) relative to the total AUC of all the three formats’ AUC.
  • the relative peak area (59.170%) was quantified as the percentage of the main peak area to the sum of all peak areas, which can be used to represent the percentage of correctly paired BsAb (heterodimer) . As summarized in Table 5, the percentage of the correctly paired BsAb was 59.170%.
  • Table 6 summarizes the results for different constructs, analyzed using CE-SDS and SEC-UPLC. Wildtype (HD-41) was used as a negative control.
  • substitutions F405V/Y407A introduced into CH3A and substitution T366W introduced into CH3B can significantly improve the correct pairing rate of CH3A and CH3B as compared to wildtype (without any mutations in the CH3 domain) .
  • further replacements of Y349T into CH3A and K360Y/H435R/Y436F into CH3B i.e., HD-18
  • Table 7 provides results of another set of experiments, evaluating heterodimer formation for different constructs. Again, substitutions of residues F405V and Y407A in one CH3 domain and T366W in the other CH3 domain significantly improved the correct pairing of CH3A and CH3B. The impact of additional modifications at different positions are also illustrated in the Table 7.

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Abstract

L'invention concerne des polypeptides hétéromultimères comprenant un complexe polypeptidique hétérodimère et des procédés de production favorisant la formation d'un complexe polypeptidique hétérodimère. Les complexes polypeptidiques hétérodimères comprennent deux domaines CH3 d'immunoglobuline polypeptidique associés ensemble, chaque région contenant une ou plusieurs mutations favorisant la formation d'hétérodimères. Des complexes hétéromultimères peuvent être utilisés, par exemple, pour permettre la production de protéines de liaison multispécifiques.
PCT/CN2023/090376 2023-04-24 2023-04-24 Polypeptides hétéromultimères Pending WO2024221187A1 (fr)

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WO2020014429A2 (fr) * 2018-07-11 2020-01-16 Kurtagic Elma Compositions et méthodes associées à des constructions de domaine de liaison à un antigène fc ciblées sur ccr4
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WO2013166594A1 (fr) * 2012-05-10 2013-11-14 Zymeworks Inc. Constructions hétéromultimères de chaînes lourdes d'immunoglobulines comprenant des mutations dans le domaine fc
WO2020014429A2 (fr) * 2018-07-11 2020-01-16 Kurtagic Elma Compositions et méthodes associées à des constructions de domaine de liaison à un antigène fc ciblées sur ccr4
WO2021034890A1 (fr) * 2019-08-19 2021-02-25 Pandion Therapeutics, Inc. Immunotolérance ciblée avec un agoniste de pd-1
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