WO2019183406A1 - Purification d'anticorps multispécifiques à l'aide d'une résine ch1 - Google Patents

Purification d'anticorps multispécifiques à l'aide d'une résine ch1 Download PDF

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Publication number
WO2019183406A1
WO2019183406A1 PCT/US2019/023447 US2019023447W WO2019183406A1 WO 2019183406 A1 WO2019183406 A1 WO 2019183406A1 US 2019023447 W US2019023447 W US 2019023447W WO 2019183406 A1 WO2019183406 A1 WO 2019183406A1
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domain
amino acid
domains
acid sequence
antigen
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PCT/US2019/023447
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English (en)
Inventor
Qufei LI
Lucas Bailey
Bryan Glaser
Roland Green
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Invenra Inc.
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Publication of WO2019183406A1 publication Critical patent/WO2019183406A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • 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
    • C07K2317/522—CH1 domain

Definitions

  • Antibodies are an invaluable tool in the medical field.
  • the importance of monoclonal antibodies, including their roles in scientific research and medical diagnostics, have been widely recognized for several decades.
  • the full potential of antibodies, especially their successful use as therapeutic agents has only more recently been demonstrated, as demonstrated by the successful therapies adalimumab (Humira), rituximab (Rituxan), infliximab (Remicade), bevacizumab (Avastin), trastuzumab
  • An area of active research in the antibody therapeutic field is the design and use of multispecific antibodies, i.e. a single antibody engineered to recognize multiple targets. These antibodies offer the promise of greater therapeutic control. For example, a need exists to improve target specificity in order to reduce the off-target effects associated with many antibody therapies, particularly in the case of antibody-based immunotherapies.
  • multispecific antibodies offer new therapeutic strategies, such as synergistic targeting of multiple cell receptors, especially in an immunotherapy context.
  • a method of purifying an antigen binding CH1 -substituted protein comprising the steps of: i) contacting a sample comprising the antigen-binding CH1 -substituted protein with a CH1 binding reagent, wherein the antigen-binding CH1- substituted protein comprises at least a first, a second, a third, and a fourth polypeptide chain associated in a complex, wherein the complex comprises at least one CH1 domain, or portion thereof, and wherein the number of CH1 domains in the complex is at least one fewer than the valency of the complex, and wherein the contacting is performed under conditions sufficient for the CH1 binding reagent to bind the CH1 domain, or portion thereof; and ii) purifying the complex away from one or more incomplete complexes, wherein the incomplete complexes do not comprise the first, the second, the third, and the fourth polypeptide chain.
  • the antigen-binding CH1- substituted protein comprises at least a first, a second, a third
  • the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C- terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence;
  • the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence;
  • the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein domain H has a variable region
  • the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C- terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence;
  • the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence;
  • the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein domain I is the single CH1
  • domain B and domain G have a CH3 amino acid sequence.
  • amino acid sequences of the B and the G domains are identical, wherein the sequence is an endogenous CH3 sequence
  • the amino acid sequences of the B and the G domains are different and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the B domain interacts with the G domain, and wherein neither the B domain nor the G domain significantly interacts with a CH3 domain lacking the orthogonal modification
  • the orthogonal modifications of the B and the G domains comprise mutations that generate engineered disulfide bridges between domain B and G.
  • the mutations of the B and the G domains that generate engineered disulfide bridges are a S354C mutation in one of the B domain and G domains, and a 349C in the other domain.
  • the orthogonal modifications of the B and the G domains comprise knob-in-hole mutations.
  • the knob-in hole mutations of the B and the G domains are a T366W mutation in one of the B domain and G domain, and a T366S, L368A, and aY407V mutation in the other domain.
  • the orthogonal modifications of the B and the G domains comprise charge- pair mutations.
  • the charge-pair mutations of the B and the G domains are a T366K mutation in one of the B domain and G domain, and a L351D mutation in the other domain.
  • domain B and domain G have an IgM CH2 amino acid sequence or an IgE CH2 amino acid sequence.
  • the IgM CH2 amino acid sequence or the IgE CH2 amino acid sequence comprise orthogonal modifications.
  • domain E and domain K have a CH3 amino acid sequence.
  • the amino acid sequences of the E and K domains are identical, wherein the sequence is an
  • the amino acid sequences of the E and the K domains are different.
  • the different sequences of the E and the K domains separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain significantly interacts with a CH3 domain lacking the orthogonal modification.
  • the orthogonal modifications of the E and the K domains comprise mutations that generate engineered disulfide bridges between domain E and K.
  • the mutations of the E and the K domains that generate engineered disulfide bridges are a S354C mutation in one of the E domain and K domain, and a 349C in the other domain.
  • the orthogonal modifications in the E and K domains comprise knob-in-hole mutations.
  • the knob-in hole mutations of the E and the K domains are a T366W mutation in one of the E domain or K domain and a T366S, L368A, and aY407V mutation in the other domain.
  • the orthogonal modifications of the E and the K domains comprise charge-pair mutations.
  • the charge-pair mutations of the E and the K domains are a T366K mutation in one of the E domain or K domain and a
  • the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C- terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, wherein domain B and domain D have a constant region domain amino acid sequence, and wherein domain E is the single CH1 domain, or portion thereof;
  • the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence;
  • the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C- tenninus, in a H-I-J
  • the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C- terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B and domain D have a constant region domain amino acid sequence, and wherein domain E has a CL amino acid sequence
  • the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence
  • the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C- terminus, in a H-I-J-K orientation, and
  • domain A has a VL amino acid sequence and domain F has a VH amino acid sequence. In certain aspects, domain A has a VH amino acid sequence and domain F has a VL amino acid sequence. In certain aspects, domain H has a VL amino acid sequence and domain L has a VH amino acid sequence. In certain aspects, domain H has a VH amino acid sequence and domain L has a VL amino acid sequence.
  • domain D and domain J have a CH2 amino acid sequence.
  • the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen
  • the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen
  • the first polypeptide chain or the third polypeptide chain further comprises a domain N and a domain O, wherein domain N has a variable region domain amino acid sequence, wherein domain O has a constant region amino acid sequence, wherein domains N and O are arranged, from N-terminus to C-terminus, in a N-0 orientation, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain or to the N-terminus of domain H of the third polypeptide chain; the binding molecule further comprises a fifth polypeptide chain, comprising: a domain P and a domain Q, wherein the domains are arranged, from N- terminus to C-terminus, in a P-Q orientation, and domain P has a variable region domain amino acid sequence and domain Q has a constant region amino acid sequence; and either the first or third polypeptide chain is associated with the fifth polypeptide chain through an interaction between the N and the P domains and an interaction between
  • the first polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain.
  • the third polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain H of the third polypeptide chain.
  • the amino acid sequences of domain N and domain A are identical, the amino acid sequences of domain H is different from domains N and A, the amino acid sequences of domain O and domain B are identical, the amino acid sequences of domain I is different from domains O and B, the amino acid sequences of domain P and domain F are identical, the amino acid sequences of domain L is different from domains P and F, the amino acid sequences of domain Q and domain G are identical, the amino acid sequences of domain M is different from domains Q and G; and (b) wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the first antigen.
  • the amino acid sequences of domain N, domain A, and domain H are different, the amino acid sequences of domain O, domain B, and domain I are different, the amino acid sequences of domain P, domain F, and domain L are different, and the amino acid sequences of domain Q, domain G, and domain M are different; and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for a third antigen.
  • domain N, domain A, and domain H each comprise a VL amino acid sequence
  • domain P, domain F, and domain L each comprise a VH amino acid sequence
  • domain O and domain Q each comprise a CH3 amino acid sequence
  • domain B and domain I each comprise a CL amino acid sequence
  • domain G and domain M each comprise a CH1 amino acid sequence.
  • domain N, domain A, and domain H are VL domains
  • domain P, domain F, and domain L are VH domains
  • domain O and domain Q are CH3 domains
  • domain B and domain I are CL domains
  • domain G and domain M are CH1 domains.
  • domain N, domain A, and domain H each comprise a VL amino acid sequence
  • domain P, domain F, and domain L each comprise a VH amino acid sequence
  • domain O and domain Q each comprise a CH3 amino acid sequence
  • domain B and domain G each comprise a CH3 amino acid sequence
  • domain I comprises a CL amino acid sequence
  • domain M comprises a CH1 amino acid sequence.
  • domain N, domain A, and domain H are VL domains
  • domain P, domain F, and domain L are VH domains; domain O and domain Q are CH3 domains; domain B and domain G are CH3 domains; domain l is a CL domain; and domain M is a CH1 domain.
  • amino acid sequences of the O and the Q domains are identical, and the sequences of the O and the Q domains are endogenous CH3 sequences.
  • the amino acid sequences of the O and the Q domains are different and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, the O domain interacts with the Q domain, and neither the O domain nor the Q domain significantly interacts with a CH3 domain lacking the orthogonal modification.
  • the orthogonal modifications of the O and the Q domains comprise mutations that generate engineered disulfide bridges between domain O and G.
  • the mutations of the O and the Q domains that generate engineered disulfide bridges are a S354C mutation in one of the O domain and Q domains, and a 349C in the other domain.
  • the orthogonal modifications of the O and the Q domains comprise knob-in-hole mutations.
  • the knob-in hole mutations of the O and the Q domains are a T366W mutation in one of the O domain and Q domain, and a T366S, L368A, and aY407V mutation in the other domain.
  • the orthogonal modifications of the O and the Q domains comprise charge-pair mutations.
  • the charge-pair mutations of the O and the Q domains are a T366K mutation in one of the O domain and Q domain, and a L351D mutation in the other domain.
  • the antigen-binding CH1- substituted protein further comprises: a sixth polypeptide chain, wherein: (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C- terminus, in a R-S-H-I-J-K orientation, and wherein domain R has a variable region domain amino acid sequence and domain S has a constant domain amino acid sequence; (b) the binding molecule further comprises a sixth polypeptide chain, comprising: a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T- U orientation, and wherein domain T has a variable region domain amino acid sequence and domain U has a constant domain amino acid sequence; and (c) the third and the sixth polypeptides are associated through an interaction between the R and the T domains and an interaction between the S and the U domains to form the binding molecule.
  • the amino acid sequences of domain R and domain A are identical, the amino acid sequences of domain H is different from domain R and A, the amino acid sequences of domain S and domain B are identical, the amino acid sequences of domain I is different from domain S and B, the amino acid sequences of domain T and domain F are identical, the amino acid sequences of domain L is different from domain T and F, the amino acid sequences of domain U and domain G are identical, the amino acid sequences of domain M is different from domain U and G and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the first antigen.
  • the antigen-binding CH1- substituted protein further comprises a second CH1 domain, or portion thereof.
  • the amino acid sequences of domain R and domain H are identical, the amino acid sequences of domain A is different from domain R and H, the amino acid sequences of domain S and domain I are identical, the amino acid sequences of domain B is different from domain S and I, the amino acid sequences of domain T and domain L are identical, the amino acid sequences of domain F is different from domain T and L, the amino acid sequences of domain U and domain M are identical, the amino acid sequences of domain G is different from domain U and M and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the second antigen.
  • the amino acid sequences of domain S and domain I are CH1 sequences.
  • the amino acid sequences of domain U and domain M are CH1 sequences.
  • the amino acid sequences of domain R, domain A, and domain H are different, the amino acid sequences of domain S, domain B, and domain I are different, the amino acid sequences of domain T, domain F, and domain L are different, and the amino acid sequences of domain U, domain G, and domain M are different; and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for a third antigen.
  • the antigen binding CH1 -substituted protein further comprises a second CH1 domain, or portion thereof.
  • the amino acid sequences of domain S and domain I are CH1 sequences.
  • the amino acid sequences of domain U and domain M are CH1 sequences.
  • sequence that forms the junction between the A domain and the B domain is IKRTPREP or IKRTVREP.
  • sequence that forms the junction between the F domain and the G domain is SSASPREP.
  • At least one CH3 amino acid sequence has a C-terminal tripeptide insertion connecting the CH3 amino acid sequence to a hinge amino acid sequence, wherein the tripeptide insertion is selected from the group consisting of PGK, KSC, and GEC.
  • sequences are human sequences.
  • At least one CH3 amino acid sequence is an IgG sequence.
  • the IgG sequences are IgGl sequences.
  • At least one CH3 amino acid sequence has one or more isoallotype mutations.
  • the isoallotype mutations are D356E and L358M.
  • At least one of the at least one CH1 domain comprises a human CH1 amino acid sequence, and wherein the CH1 binding reagent binds to a human CH1 epitope.
  • at least one of the at least one CH1 domain comprises an CH1 amino acid sequence selected from the group consisting of: an IgG CH1, an IgA CH1, an IgE CH1, an IgM CH1, and an IgD CH1.
  • at least one of the at least one CH1 domain comprises an IgG CH1 amino acid sequence.
  • the IgG CH1 amino acid sequence comprises an IgGl CH1 amino acid sequence.
  • At least one of the at least one CH1 domain comprises an IgA CH1 amino acid sequence.
  • At least one of the at least one CH1 domain comprises SEQ ID NO:23.
  • At least one of the at least one CH1 domain comprises one or more orthogonal modifications.
  • the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, the mutations selected from the group consisting of: an engineered cysteine at position 138 of the CH1 sequence and position 116 of the CL sequence; an engineered cysteine at position 128 of the CH1 sequence and position 119 of the CL sequence, and an engineered cysteine at position 129 of the CH1 sequence and position 210 of the CL sequence.
  • the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, wherein the mutations comprise and engineered cysteines at position 128 of the CH1 sequence and position 118 of a CL Kappa sequence.
  • the orthogonal modifications comprise mutations that generate engineered disulfide bridges between the at least one CH1 domain and a CL domain, the mutations selected from the group consisting of: a Fl 18C mutation in the CL sequence with a corresponding A141C in the CH1 sequence; a Fl 18C mutation in the CL sequence with a corresponding L128C in the CH1 sequence; and a S162C mutations in the CL sequence with a corresponding P171C mutation in the CH1 sequence.
  • the orthogonal modifications comprise charge-pair mutations between the at least one CH1 domain and a CL domain, the charge-pair mutations selected from the group consisting of: a Fl 18S mutation in the CL sequence with a corresponding A141L in the CH1 sequence; a Fl 18A mutation in the CL sequence with a corresponding A141L in the CH1 sequence; a Fl 18V mutation in the CL sequence with a corresponding A141L in the CH1 sequence; and a T129R mutation in the CL sequence with a
  • modifications comprise charge-pair mutations between the at least one CH1 domain and a CL domain, the charge-pair mutations selected from the group consisting of: a N138K mutation in the CL sequence with a corresponding G166D in the CH1 sequence,; and a N138D mutation in the CL sequence with a corresponding G166K in the CH1 sequence.
  • the CH1 binding reagent comprises an anti-CHl antigen binding site. In certain aspects, the CH1 binding reagent comprises an anti-CHl antibody. In certain aspects, the anti-CHl antibody comprises a single-domain antibody. In certain aspects, the single-domain antibody comprises a Camelid-derived antibody.
  • the CH1 binding reagent is attached to a surface of a solid support.
  • the solid support is selected from the group consisting of: an agarose bead, a magnetic bead, and a resin.
  • the CH1 binding reagent is attached to the surface prior to step (ii).
  • the CH1 binding reagent is attached to the surface subsequent to step (ii).
  • the purifying step is selected from the group consisting of:
  • the method further comprises an elution step following step (ii) generating an eluate comprising antigen-binding CH1 -substituted protein.
  • the elution step comprises contacting the antigen-binding CH1- substituted protein bound to the CH1 binding reagent with a low-pH solution.
  • the low-pH solution comprises 0.1 M acetic acid pH 4.0.
  • the method further comprises an additional purification step following the elution step.
  • the additional purification step comprises an ion exchange chromatography purification.
  • the ion exchange chromatography purification comprises an ion exchange chromatography purification.
  • chromatography purification comprises cation exchange chromatography.
  • greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is the antigen-binding CH1- substituted protein.
  • the greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is obtained following a single iteration of steps (i)-(iii).
  • the greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% (w/w) of the total protein in the eluate is obtained using any of the above methods wherein the purifying step does not comprise use of a binding reagent other than the CH1 binding reagent of the methods disclosed or described herein.
  • the less than 5% of the first and the second polypeptides are unassociated in the eluate. In certain aspects, less than 5% of the third and the fourth polypeptides are unassociated in the eluate. In certain aspects, the less than less than 5% of the first and the third polypeptides, the less than 5% of the first and the second polypeptides, or the less than 5% of the third and the fourth polypeptides unassociated in the eluate is obtained following a single iteration of steps (i)-(iii).
  • the less than less than 5% of the first and the third polypeptides, the less than 5% of the first and the second polypeptides, or the less than 5% of the third and the fourth polypeptides unassociated in the eluate is obtained using any of the above methods wherein the purifying step does not comprise use of a binding reagent other than the CH1 binding reagent of the methods disclosed or described herein.
  • the sample is a supernatant or a lysate of an expression system.
  • the expression system is selected from the group consisting of: a cell free expression system, a mammalian cell culture, a bacterial cell culture, a yeast cell culture.
  • the mammalian cell culture comprises an immortalized cell line.
  • the immortalized cell line is a chine hamster ovary (CHO) or human 293 derived cell line.
  • the expression system stably expresses the polypeptide chains of the antigen-binding CH1 -substituted protein.
  • the expression system is a serum-free expression system.
  • an antigen-binding CH1 -substituted protein purified by any of the methods disclosed or described herein.
  • composition comprising the antigen binding CH1 -substituted protein purified by any of the methods disclosed or described herein.
  • Also disclosed herein is a method of treatment, comprising administering to a subject in need of treatment the pharmaceutical composition comprising any of the antigen binding CH1 -substituted protein purified by any of the methods disclosed or described herein and any of the pharmaceutically acceptable carriers disclosed or described herein.
  • Fig. 1 shows SDS-PAGE analysis of bispecific antibodies comprising standard knob-hole orthogonal mutations introduced into CH3 domains found in their native positions within the Fc portion of the bispecific antibody that have been purified using a single-step CH1 affinity purification step (Capture SelectTM CH1 affinity resin).
  • FIG. 2 presents schematic architectures, with respective naming conventions, for various antigen-binding CH1 -substituted proteins (also called antibody constructs) described herein.
  • FIG. 3 presents a higher resolution schematic of polypeptide chains and their domains, with respective naming conventions, for the bivalent lxl antibody constructs described herein.
  • FIG. 4 illustrates features of an exemplary bivalent lxl bispecific antigen-binding CH1- substituted protein,“BC1”.
  • FIG. 5A shows size exclusion chromatography (SEC) analysis of“BC1”, demonstrating that a single-step CH1 affinity purification step (CaptureSelectTM CH1 affinity resin) yields a single, monodisperse peak via gel filtration in which >98% is unaggregated bivalent protein.
  • FIG. 5B shows comparative literature data of SEC analysis of a CrossMab bivalent antibody construct [data from Schaefer el al. ( Proc Natl Acad Sci USA. 2011 Jul 5; 108(27): 11187-92)].
  • FIG. 6A is a cation exchange chromatography elution profile of“BC1” following one-step purification using the CaptureSelectTM CH1 affinity resin, showing a single tight peak.
  • FIG. 6B is a cation exchange chromatography elution profile of“BC1” following purification using standard Protein A purification.
  • FIG. 7 shows non-reducing SDS-PAGE gels of“BC1” at various stages of purification.
  • FIGS. 8A and 8B compare SDS-PAGE gels of“BC1” after single-step CH1- affmity purification under both non-reducing and reducing conditions (FIG. 8A) with SDS- PAGE gels of a CrossMab bispecific antibody under non-reducing and reducing conditions as published in the referenced literature (FIG. 8B).
  • FIGS. 9A and 9B show mass spec analysis of“BC1”, demonstrating two distinct heavy chains (FIG. 9A) and two distinct light chains (FIG. 9B) under reducing conditions.
  • FIG. 10 presents a mass spectrometry analysis of purified“BC1” under non reducing conditions, confirming the absence of incomplete pairing after purification.
  • FIG. 11 illustrates features of an exemplary bivalent lxl bispecific antigen-binding CH1- substituted protein,“BC6”, further described in Example 3.
  • FIG. 12A presents size exclusion chromatography (SEC) analysis of“BC6” following one-step purification using the CaptureSelectTM CH1 affinity resin,
  • FIG. 12B shows a SDS-PAGE gel of “BC6” under non-reducing conditions.
  • FIG. 13 illustrates features of an exemplary bivalent bispecific antigen-binding CH1- substituted protein,“BC28”, further described in Example 4.
  • FIG. 14 shows SEC analysis of“BC28” and“BC30”, each following one-step purification using the CaptureSelectTM CH1 affinity resin.
  • FIG. 15 presents a schematic of polypeptide chains and their domains, with respective naming conventions, for the trivalent 2x1 antibody constructs described herein.
  • FIG. 16 illustrates features of an exemplary trivalent 2x1 bispecific antigen-binding CH1- substituted protein,“BCl-2xl”, further described in Example 7.
  • FIG. 17 shows non-reducing SDS-PAGE of“BC1” and“BCl-2xl” protein expressed using the ThermoFisher Expi293 transient transfection system, at various stages of purification.
  • FIG. 18 presents a schematic of polypeptide chains and their domains, with respective naming conventions, for the trivalent 1x2 antibody constructs described herein.
  • FIG. 19 illustrates features of an exemplary trivalent 1x2 trispecific construct, “BC28-lxlxla”, further described in Example 11.
  • FIG. 20 shows size exclusion chromatography of“BC28-lxlxla” following transient expression and single step CH1 affinity resin purification, demonstrating a single well-defined peak.
  • FIG. 21 presents a schematic of polypeptide chains and their domains, with respective naming conventions, for certain tetravalent 2x2 constructs described herein.
  • FIG. 22 illustrates certain salient features of the exemplary tetravalent 2x2 construct,“BC22-2x2” further described in Example 14.
  • FIG. 23 is a non-reducing SDS-PAGE gel comparing the 2x2 tetravalent“BC22- 2x2” construct to a 1x2 trivalent construct“BC 12-1x2” and a 2x1 trivalent construct “BC21-2x1” at different stages of purification.
  • FIG. 24 shows SDS-PAGE results with bivalent and trivalent constructs, each after transient expression and one-step purification using the CaptureSelectTM CH1 affinity resin, under non-reducing and reducing conditions, as further described in Example 17.
  • FIG. 25 shows supernatant of the Expi293 Expression system transiently transfected with different ratios of vectors encoding the four polypeptide chains of a BC28 antibody and run directly on an native SDS-PAGE gel.
  • FIG. 26A shows the architecture of trivalent, bispecific constructs that benefit from CH1 purification.
  • FIG. 26B shows SDS-PAGE gel of the purification products of the constructs depicted in FIG. 26A, following expression using the Expi293 system and one- step purification using the CaptureSelectTM CH1 affinity resin.
  • FIG. 27A the architecture of trivalent bispecific constructs that were expressed using the ExpiCHO system.
  • FIG. 27B shows an SDS-PAGE gel of the resulting purification products of the constructs depicted in FIG. 27A, following expression using the ExpiCHO system and one-step purification using the CaptureSelectTM CH1 affinity resin.
  • FIG. 27C shows size exclusion chromatography results following ExpiCHO expression and one-step purification of the constructs depicted in FIG. 27A.
  • antigen binding site is meant a region of an antigen-binding CH1 -substituted protein that specifically recognizes or binds to a given antigen or epitope.
  • “B-Body” means any of the antigen-binding CH1- substituted protein constructs described herein.
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of multiple sclerosis, arthritis, or cancer.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • the term“sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.
  • the term“therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease.
  • a therapeutically effective amount can be a
  • prophylaxis can be considered therapy.
  • antibody constant region residue numbering is according to the Eu index as described at
  • Ranges provided herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of
  • the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • a method of purifying an antigen-binding CH1- substituted protein is provided.
  • the method comprises the steps of: i) contacting a sample comprising the antigen-binding CH1- substituted protein with a CH1 binding reagent, wherein the antigen-binding CH1- substituted protein comprises at least a first, a second, a third, and a fourth polypeptide chain associated in a complex, wherein the complex comprises at least one CH1 domain, or portion thereof, and wherein the number of CH1 domains in the complex is at least one fewer than the valency of the complex, and wherein the contacting is performed under conditions sufficient for the CH1 binding reagent to bind the CH1 domain, or portion thereof; and ii) purifying the complex from one or more incomplete complexes, wherein the incomplete complexes do not comprise the first, the second, the third, and the fourth polypeptide chain.
  • a typical antibody has two CH1 domains.
  • CH1 domains are described in more detail in Section 6.4.1.
  • the CH1 domain of a typical antibody can be substituted with a CH3 domain, generating an antigen-binding protein having only a single CH1 domain.
  • Antigen-binding proteins can also refer to molecules based on antibody architectures that have been engineered such that they no longer possess a typical antibody architecture.
  • an antibody can be extended at its N or C terminus to increase the valency (described in more detail in Section 6.4.14.1) of the antigen-binding protein, and in certain instances the number of CH1 domains is also increased beyond the typical two CH1 domains.
  • Such molecules can also have one or more of their CH1 domains substituted, such that the number of CH1 domains in the protein is at least one fewer than the valency of the antigen-binding protein.
  • the number of CH1 domains that are substituted by other domains generates an antigen-binding CH1- substituted protein having only a single CH1 domain. In other embodiments, the number of CH1 domains substituted by another domain generates an antigen-binding CH1 -substituted protein having two or more CH1 domains, but at least one fewer than the valency of the antigen-binding protein. In particular embodiments, where an antigen-binding CH1- substituted protein has two or more CH1 domains, the multiple CH1 domains can all be in the same polypeptide chain. In other particular embodiments, where an antigen-binding CH1- substituted protein has two or more CH1 domains, the multiple CH1 domains can be a single CH1 domain in multiple copies of the same polypeptide chain present in the complete complex.
  • CH1 binding reagents can be any molecule that specifically binds a CH1 epitope.
  • CH1 sequences that provide the CH1 epitope are described in more detail in Section 6.4.1, and specific binding is described in more detail in Section 6.4.14.1.
  • CH1 binding reagents are derived from
  • the CH1 binding reagent is an antibody, also referred to as an“anti-CHl antibody.”
  • the anti-CHl antibody can be derived from a variety of species.
  • the anti-CHl antibody is a mammalian antibody, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human antibodies.
  • the anti-CHl antibody is a single-domain antibody. Single-domain antibodies, as described herein, have a single variable domain that forms the ABS and specifically binds the CH1 epitope.
  • Exemplary single-domain antibodies include, but are not limited to, heavy chain antibodies derived from camels and sharks, as described in more detail in international application WO 2009/011572, herein incorporated by reference for all it teaches.
  • the anti-CHl antibody is a camel derived antibody (also referred to as a“camelid antibody”).
  • Exemplary camelid antibodies include, but are not limited to, human IgG-CHl CaptureSelectTM (Therm oFisher,
  • the anti-CHl antibody is a monoclonal antibody. Monoclonal antibodies are typically produced from cultured antibody-producing cell lines. In other embodiments, the anti-CHl antibody is a polyclonal antibody, i.e., a collection of different anti-CHl antibodies that each recognize the CH1 epitope. Polyclonal antibodies are typically produced by collecting the antibody containing serum of an animal immunized with the antigen of interest, or fragment thereof, here CH1.
  • CH1 binding reagents are molecules not derived from immunoglobulin proteins.
  • examples of such molecules include, but are not limited to, aptamers, peptoids, and affibodies, as described in more detail in Perret and Boschetti ( Biochimie , Feb. 2018, Vol 145:98-112).
  • the CH1 binding reagent can be attached to a solid support in various embodiments of the invention.
  • Solid supports as described herein, refers to a material to which other entities can be attached or immobilized, e.g ., the CH1 binding reagent.
  • Solid supports also referred to as “carriers,” are described in more detail in international application WO 2009/011572.
  • the solid support comprises a bead or nanoparticle.
  • beads and nanoparticles include, but are not limited to, agarose beads, polystyrene beads, magnetic nanoparticles (e.g., DynabeadsTM, ThermoFisher), polymers (e.g, dextran), synthetic polymers (e.g, SepharoseTM), or any other material suitable for attaching the CH1 binding reagent.
  • the solid support is modified to enable attachment of the CH1 binding reagent.
  • Example of solid support modifications include, but are not limited to, chemical modifications that form covalent bonds with proteins (e.g, activated aldehyde groups) and modifications that specifically pair with a cognate modification of a CH1 binding reagent (e.g, biotin-streptavidin pairs, disulfide linkages, polyhistidine-nickel, or“click-chemistry” modifications such as azido-alkynyl pairs).
  • chemical modifications that form covalent bonds with proteins e.g, activated aldehyde groups
  • modifications that specifically pair with a cognate modification of a CH1 binding reagent e.g, biotin-streptavidin pairs, disulfide linkages, polyhistidine-nickel, or“click-chemistry” modifications such as azido-alkynyl pairs.
  • the CH1 binding reagent is attached to the solid support prior to the CH1 binding reagent contacting the antigen-binding CH1- substituted proteins, herein also referred to as an“anti-CHl resin.”
  • anti-CHl resins are dispersed in a solution.
  • anti-CHl resins are“packed” into a column. The anti-CHl resin is then contacted with the antigen-binding CH1 -substituted proteins and the CH1 binding reagents specifically bind the antigen-binding CH1- substituted proteins.
  • the CH1 binding reagent is attached to the solid support after the CH1 binding reagent contacts the antigen-binding CH1 -substituted proteins.
  • a CH1 binding reagent with a biotin modification can be contacted with the antigen-binding CH1- substituted proteins, and subsequently the CH1 binding reagent/antigen-binding CH1- substituted protein mixture can be contacted with streptavidin modified solid support to attach the CH1 binding reagent to the solid support, including CH1 binding reagents specifically bound to the antigen-binding CH1 -substituted proteins.
  • the bound antigen-binding CH1- substituted proteins are released, or“eluted,” from the solid support forming an eluate having the antigen-binding CH1- substituted proteins.
  • the bound antigen-binding CH1- substituted proteins are released through reversing the paired modifications (e.g ., reduction of the disulfide linkage), adding a reagent to compete off the antigen-binding CH1- substituted proteins (e.g., adding imidazole that competes with a polyhistidine for binding to nickel), cleaving off the antigen-binding CH1 -substituted proteins (e.g, a cleavable moiety can be included in the modification), or otherwise interfering with the specific binding of the CH1 binding reagent for the antigen-binding CH1- substituted protein.
  • a reagent to compete off the antigen-binding CH1- substituted proteins e.g., adding imidazole that competes with a polyhistidine for binding to nickel
  • cleaving off the antigen-binding CH1 -substituted proteins e.g, a cleavable moiety can be included in the modification
  • Methods that interfere with specific binding include, but are not limited to, contacting antigen-binding CH1- substituted proteins bound to CH1 binding reagents with a low-pH solution.
  • the low-pH solution comprises 0.1 M acetic acid pH 4.0.
  • the bound antigen-binding CH1 -substituted proteins can be contacted with a range of low-pH solutions, i.e., a“gradient.”
  • a single iteration of the method using the steps of contacting the antigen-binding CH1 -substituted proteins with the CH1 binding reagents, followed by eluting the antigen-binding CH1- substituted proteins is used to purify the antigen-binding CH1- substituted proteins from the one or more incomplete complexes.
  • no other purifying step is performed.
  • one or more additional purification steps are performed to further purify the antigen-binding CH1 -substituted proteins from the one or more incomplete complexes.
  • the one or more additional purification steps include, but are not limited to, purifying the antigen-binding CH1- substituted proteins based on other protein characteristics, such as size (e.g, size exclusion chromatography), charge (e.g, ion exchange chromatography), or hydrophobicity (e.g ., hydrophobicity interaction chromatography).
  • size e.g, size exclusion chromatography
  • charge e.g, ion exchange chromatography
  • hydrophobicity e.g ., hydrophobicity interaction chromatography
  • an additional cation exchange chromatograph is performed.
  • the antigen-binding CH1- substituted proteins can be further purified repeating contacting the antigen-binding CH1- substituted proteins with the CH1 binding reagents as described above, as well as modifying the CH1 purification method between iterations, e.g., using a step elution for the first iteration and a gradient elution for a subsequent elution.
  • At least four distinct polypeptide chains associate together to form a complete complex, i.e., the antigen-binding CH1- substituted protein.
  • incomplete complexes can also form that do not contain the at least four distinct polypeptide chains.
  • incomplete complexes may form that only have one, two, or three of the polypeptide chains.
  • an incomplete complex may contain more than three polypeptide chains, but does not contain the at least four distinct polypeptide chains, e.g, the incomplete complex inappropriately associates with more than one copy of a distinct polypeptide chain.
  • the method of the invention purifies the complex, i.e., the completely assembled antigen-binding CH1- substituted protein, from incomplete complexes.
  • Methods to assess the efficacy and efficiency of the purification steps are well known to those skilled in the art and include, but are not limited to, SDS-PAGE analysis, ion exchange chromatography, size exclusion chromatography, and mass spectrometry. Purity can also be assessed according to a variety of criteria.
  • criterion examples include, but are not limited to: 1) assessing the percentage of the total protein in an eluate that is provided by the completely assembled antigen-binding CH1 -substituted protein, 2) assessing the fold enrichment or percent increase of the method for purifying the desired products, e.g., comparing the total protein provided by the completely assembled antigen binding CH1 -substituted protein in the eluate to that in a starting sample, 3) assessing the percentage of the total protein or the percent decrease of undesired products, e.g, the incomplete complexes described above, including determining the percent or the percent decrease of specific undesired products (e.g, unassociated single polypeptide chains, dimers of any combination of the polypeptide chains, or trimers of any combination of the polypeptide chains).
  • specific undesired products e.g, unassociated single polypeptide chains, dimers of any combination of the polypeptide chains, or trimers of any combination of the polypeptide chains.
  • Purity can be assessed after any combination of methods described herein. For example, purity can be assessed after a single iteration of using the anti-CHl binding reagent, as described herein, or after additional purification steps, as described in more detail in Section 6.3.3. The efficacy and efficiency of the purification steps may also be used to compare the methods described using the anti-CHl binding reagent to other purification methods known to those skilled in the art, such as Protein A purification.
  • the antigen-binding CH1- substituted proteins comprise a first and a second polypeptide chain, wherein: (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence; (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the
  • the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, wherein domain A has a variable region domain amino acid sequence, and wherein domain B, domain D, and domain E have a constant region domain amino acid sequence;
  • the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a variable region domain amino acid sequence and domain G has a constant region domain amino acid sequence;
  • the third polypeptide chain comprises a domain H, a domain I, a domain J, and a domain K, wherein the domains are arranged, from N-terminus to C-terminus, in a H-I-J-K orientation, and wherein the third
  • CH1 amino acid sequences are antibody heavy chain constant domain sequences.
  • CH1 sequences are sequences of the second domain of a native IgG antibody heavy chain, with reference from the N-terminus to C-terminus.
  • the CH1 sequences are endogenous sequences.
  • the CH1 sequences are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences.
  • the CH1 sequences are human sequences.
  • the CH1 sequences are from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM isotype. In a preferred embodiment, the CH1 sequences are from an IgGl isotype. In preferred embodiments, the CH1 sequence is ETniProt accession number P01857 amino acids 1-98. [00121]
  • the CL amino acid sequences useful in the antigen-binding CH1- substituted proteins described herein are light chain constant domain sequences. In some
  • Cl sequences are sequences of the second domain of a native IgG antibody light chain.
  • the CL sequences are endogenous sequences.
  • the CL sequences are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, CL sequences are human sequences.
  • the CL amino acid sequences are lambda (l) light chain constant domain sequences.
  • the CL amino acid sequences are human lambda light chain constant domain sequences.
  • the lambda (l) light chain sequence is UniProt accession number P0CG04.
  • the CL amino acid sequences are kappa (K) light chain constant domain sequences.
  • the CL amino acid sequences are human kappa (K) light chain constant domain sequences.
  • the kappa light chain sequence is UniProt accession number P01834.
  • the CH1 sequence and the CL sequence are both endogenous sequences.
  • the CH1 sequence, the CL sequence, or both the CH1 and Cl sequence are modified sequences.
  • the CH1 sequence and the CL sequences may separately comprise respectively orthogonal modifications in endogenous CH1 and CL sequences, as discussed below in greater detail in Section 6.4.1.1. It is to be understood that orthogonal mutations in the CH1 sequence do not eliminate the specific binding interaction between the CH1 binding reagent and the CH1 domain.
  • the orthogonal mutations may reduce, though not eliminate, the specific binding interaction.
  • CH1 and CL sequences can also be portions thereof, either of an endogenous or modified sequence, such that a domain having the CH1 sequence, or portion thereof, can associate with a domain having the CH1 sequence, or portion thereof.
  • the antigen-binding CH1- substituted protein having a portion of the CH1 sequences described above can be bound by the CH1 binding reagent.
  • the CH1 domain is also unique in that it’s folding is typically the rate limiting step in the secretion of IgG (Feige et al. Mol Cell. 2009 Jun l2;34(5):569-79; herein incorporated by reference in its entirety).
  • purifying the antigen-binding CH1- substituted proteins based on the rate limiting component of CH1 comprising polypeptide chains can provide a means to purify complete complexes from incomplete chains, i.e., purifying complexes have the limiting CH1 domain from complexes only having the one or more non-CHl comprising chains.
  • the CH1 limiting expression may be a benefit in some aspects, as discussed, there is the potential for CH1 to limit overall expression of the complete antigen binding CH1 -substituted proteins.
  • the expression of the polypeptide chain comprising the CH1 sequence(s) is adjusted to improve the efficiency of the antigen-binding CH1- substituted proteins forming complete complexes.
  • the ratio of a plasmid vector constructed to express the polypeptide chain comprising the CH1 sequence(s) can be increased relative to the plasmid vectors constructed to express the other polypeptide chains.
  • polypeptide chain comprising the CH1 sequence(s) when compared to the polypeptide chain comprising the CL sequence(s) can be the smaller of the two polypeptide chains.
  • expression of the polypeptide chain comprising the CH1 sequence(s) can be adjusted by controlling which polypeptide chain has the CH1 sequence(s).
  • engineering the antigen-binding CH1 -substituted protein such that the CH1 domain is present in a two-domain polypeptide chain e.g ., the 4 th polypeptide chain described herein
  • the CH1 sequence instead of the CH1 sequence’s native position in a four-domain polypeptide chain (e.g., the 3 rd polypeptide chain described herein)
  • a relative expression level of CH1 containing chains that is too high compared to the other chains can result in incomplete complexes the have the CH1 chain, but not each of the other chains.
  • the expression of the polypeptide chain comprising the CH1 sequence(s) is adjusted to both reduce the formation incomplete complexes without the CH1 containing chain, and to reduce the formation incomplete complexes with the CH1 containing chain but without the other chains present in a complete complex.
  • the CH1 sequence and the CL sequences separately comprise respectively orthogonal modifications in endogenous CH1 and CL sequences. Orthogonal mutations, in general, are described in more detail below in Sections 6.4.15.1- 6.4.15.3. [00128]
  • the orthogonal modifications in endogenous CH1 and CL sequences are an engineered disulfide bridge selected from engineered cysteines at position 138 of the CH1 sequence and position 116 of the CL sequence, at position 128 of the CH1 sequence and position 119 of the CL sequence, or at position 129 of the CH1 sequence and position 210 of the CL sequence, as numbered and discussed in more detail in U.S. Pat. No. 8,053,562 and U.S. Pat. No. 9,527,927, each incorporated herein by reference in its entirety.
  • the engineered cysteines are at position 128 of the CH1 sequence and position 118 of the CL Kappa sequence, as numbered by the Eu index.
  • the mutations that provide non- endogenous cysteine amino acids are a Fl 18C mutation in the CL sequence with a corresponding A141C in the CH1 sequence, or a Fl 18C mutation in the CL sequence with a corresponding L128C in the CH1 sequence, or a S162C mutations in the CL sequence with a corresponding P171C mutation in the CH1 sequence, as numbered by the Eu index.
  • the orthogonal mutations in the CL sequence and the CH1 sequence are charge-pair mutations.
  • the charge-pair mutations are a Fl 18S, Fl 18A or Fl 18V mutation in the CL sequence with a corresponding A141L in the CH1 sequence, or a T129R mutation in the CL sequence with a corresponding K147D in the CH1 sequence, as numbered by the Eu index and described in greater detail in Bonisch et al. (Protein Engineering, Design & Selection , 2017, pp. 1-12), herein incorporated by reference for all that it teaches.
  • the charge-pair mutations are a N138K mutation in the CL sequence with a corresponding G166D in the CH1 sequence, or a N138D mutation in the CL sequence with a
  • domain A has a variable region domain amino acid sequence.
  • Variable region domain amino acid sequences as described herein, are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences. VL and VH sequences are described in greater detail below in Sections 6.4.2.1 and 6.4.2.4, respectively.
  • domain A has a VL antibody domain sequence and domain F has a VH antibody domain sequence.
  • 6.4.2.1.VL Regions are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences.
  • VL amino acid sequences useful in the antigen-binding CH1- substituted proteins described herein are antibody light chain variable domain sequences.
  • a specific VL amino acid sequence associates with a specific VH amino acid sequence to form an antigen-binding site.
  • the VL amino acid sequences are mammalian sequences, including human sequences, synthesized sequences, or
  • VL amino acid sequences are mutated sequences of naturally occurring sequences.
  • the VL amino acid sequences are lambda (l) light chain variable domain sequences.
  • the VL amino acid sequences are kappa (K) light chain variable domain sequences.
  • the VL amino acid sequences are kappa (K) light chain variable domain sequences.
  • the C- terminus of domain A is connected to the N-terminus of domain B.
  • domain A has a VL amino acid sequence that is mutated at its C-terminus at the junction between domain A and domain B, as described in greater detail below in Section 6.4.20.1 and in Example 6.
  • the VL amino acid sequences comprise highly variable sequences termed “complementarity determining regions” (CDRs), typically three CDRs (CDR1, CD2, and CDR3).
  • CDRs complementarity determining regions
  • the CDRs are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences.
  • the CDRs are human sequences.
  • the CDRs are naturally occurring sequences.
  • the CDRs are naturally occurring sequences that have been mutated to alter the binding affinity of the antigen binding site for a particular antigen or epitope.
  • the naturally occurring CDRs have been mutated in an in vivo host through affinity maturation and somatic hypermutation.
  • the CDRs have been mutated in vitro through methods including, but not limited to, PCR-mutagenesis and chemical mutagenesis.
  • the CDRs are synthesized sequences including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries.
  • the VL amino acid sequences comprise“framework region” (FR) sequences.
  • FRs are generally conserved sequence regions that act as a scaffold for interspersed CDRs (see Section 6.4.2.2.), typically in a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 arrangement (from N-terminus to C-terminus).
  • the FRs are mammalian sequences, including, but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences.
  • the FRs are human sequences.
  • the FRs are naturally occurring sequences.
  • the FRs are synthesized sequences including, but not limited, rationally designed sequences.
  • the FRs and the CDRs are both from the same naturally occurring variable domain sequence.
  • the FRs and the CDRs are from different variable domain sequences, wherein the CDRs are grafted onto the FR scaffold with the CDRs providing specificity for a particular antigen.
  • the grafted CDRs are all derived from the same naturally occurring variable domain sequence.
  • the grafted CDRs are derived from different variable domain sequences.
  • the grafted CDRs are synthesized sequences including, but not limited to, CDRs obtained from random sequence CDR libraries and rationally designed CDR libraries.
  • the grafted CDRs and the FRs are from the same species. In certain embodiments, the grafted CDRs and the FRs are from different species.
  • an antibody is “humanized”, wherein the grafted CDRs are non-human mammalian sequences including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, and goat sequences, and the FRs are human sequences. Humanized antibodies are discussed in more detail in U.S. Pat. No. 6,407,213, the entirety of which is hereby incorporated by reference for all it teaches.
  • portions or specific sequences of FRs from one species are used to replace portions or specific sequences of another species’ FRs.
  • VH amino acid sequences in the antigen-binding CH1 -substituted proteins described herein are antibody heavy chain variable domain sequences.
  • a specific VH amino acid sequence associates with a specific VL amino acid sequence to form an antigen-binding site.
  • VH amino acid sequences are mammalian sequences, including human sequences, synthesized sequences, or combinations of non-human mammalian, mammalian, and/or synthesized sequences, as described in further detail above in Sections 6.4.2.2 and 6.4.2.3.
  • VH amino acid sequences are mutated sequences of naturally occurring sequences.
  • domain B has a constant region domain sequence. In some embodiments, domain B has a constant region domain sequence that is not a CH1 sequence. Constant region domain amino acid sequences, as described herein, are sequences of a constant region domain of an antibody.
  • the constant region sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the constant region sequences are human sequences. In certain embodiments, the constant region sequences are from an antibody light chain. In particular embodiments, the constant region sequences are from a lambda or kappa light chain. In certain embodiments, the constant region sequences are from an antibody heavy chain, except for the CH1 region of a heavy chain.
  • the constant region sequences are an antibody heavy chain sequence that is an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM isotype.
  • IgAl IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM isotype.
  • the constant region sequences are from an IgG isotype. In a preferred embodiment, the constant region sequences are from an IgGl isotype. In preferred specific embodiments, the constant region sequence is a CH3 sequence. CH3 sequences are described in greater detail below in Section 6.4.3.1. In other preferred embodiments, the constant region sequence is an orthologous CH2 sequence. Orthologous CH2 sequences are described in greater detail below in Section 6.4.3.2.
  • the constant region sequence is a CH1 or Cl sequence.
  • the constant region sequence is a Cl sequence.
  • CH1 and Cl sequences are described herein.
  • the CH1 or Cl sequence comprises one or more CH1 or Cl orthogonal modifications described herein.
  • the constant region sequence has been mutated to include one or more orthogonal mutations.
  • domain B has a constant region sequence that is a CH3 sequence comprising knob-hole (synonymously, “knob-in-hole,”“KIH”) orthogonal mutations, as described in greater detail below in Section 6.4.15.2, and either a S354C or a Y349C mutation that forms an engineered disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in in greater detail below in Section 6.4.15.1.
  • the knob-hole orthogonal mutation is a T366W mutation.
  • CH3 amino acid sequences are sequences of the C- terminal domain of an antibody heavy chain.
  • the CH3 sequences are mammalian sequences, including, but not limited to, mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences. In a preferred embodiment, the CH3 sequences are human sequences. In certain embodiments, the CH3 sequences are from an IgAl, IgA2, IgD, IgE, IgM, IgGl, IgG2, IgG3, IgG4 isotype or CH4 sequences from an IgE or IgM isotype. In a specific
  • the CH3 sequences are from an IgG isotype. In a preferred embodiment, the CH3 sequences are from an IgGl isotype.
  • the CH3 sequences are endogenous sequences.
  • the CH3 sequence is ETniProt accession number P01857 amino acids 224-330.
  • a CH3 sequence is a segment of an endogenous CH3 sequence.
  • a CH3 sequence has an endogenous CH3 sequence that lacks the N-terminal amino acids G224 and Q225.
  • a CH3 sequence has an endogenous CH3 sequence that lacks the C-terminal amino acids P328, G329, and K330.
  • a CH3 sequence has an endogenous CH3 sequence that lacks both the N-terminal amino acids G224 and Q225 and the C-terminal amino acids P328, G329, and K330.
  • an antigen binding CH1 -substituted protein has multiple domains that have CH3 sequences, wherein a CH3 sequence can refer to both a full endogenous CH3 sequence as well as a CH3 sequence that lacks N-terminal amino acids, C-terminal amino acids, or both.
  • the CH3 sequences are endogenous sequences that have one or more mutations.
  • the mutations are one or more orthogonal mutations that are introduced into an endogenous CH3 sequence to guide specific pairing of specific CH3 sequences, as described in more detail below in Sections 6.4.15.1-6.4.15.3.
  • the CH3 sequences are engineered to reduce immunogenicity of the antibody by replacing specific amino acids of one allotype with those of another allotype and referred to herein as isoallotype mutations, as described in more detail in Stickler et al. ⁇ Genes Immun. 2011 Apr; 12(3): 213-221), which is herein incorporated by reference for all that it teaches.
  • specific amino acids of the Glml allotype are replaced.
  • isoallotype mutations D356E and L358M are made in the CH3 sequence.
  • domain B has a human IgGl CH3 amino acid sequence with the following mutational changes: P343V; Y349C; and a tripeptide insertion, 445P, 446G, 447K.
  • domain B has a human IgGl CH3 sequence with the following mutational changes: T366K; and a tripeptide insertion, 445K, 446S, 447C.
  • domain B has a human IgGl CH3 sequence with the following mutational changes: Y349C and a tripeptide insertion, 445P, 446G, 447K.
  • domain B has a human IgGl CH3 sequence with a 447C mutation incorporated into an otherwise endogenous CH3 sequence.
  • domain B In the antigen-binding CH1- substituted proteins described herein, the N- terminus of domain B is connected to the C-terminus of domain A. In certain embodiments, domain B has a CH3 amino acid sequence that is mutated at its N-terminus at the junction between domain A and domain B, as described in greater detail below in Section 6.4.20.1 and Example 6.
  • domain B In some embodiments of the antigen-binding CH1- substituted proteins, the C- terminus of domain B is connected to the N-terminus of domain D. In certain embodiments, domain B has a CH3 amino acid sequence that is extended at the C-terminus at the junction between domain B and domain D, as described in greater detail below in Section 6.4.20.3.
  • CH2 amino acid sequences are sequences of the third domain of an antibody heavy chain, with reference from the N-terminus to C-terminus.
  • an antigen-binding CH1 -substituted protein has more than one paired set of CH2 domains that have CH2 sequences, wherein a first set has CH2 amino acid sequences from a first isotype and one or more orthologous sets of CH2 amino acid sequences from another isotype.
  • the orthologous CH2 amino acid sequences, as described herein, are able to interact with CH2 amino acid sequences from a shared isotype, but not significantly interact with the CH2 amino acid sequences from another isotype present in the antigen-binding CH1- substituted protein.
  • all sets of CH2 amino acid sequences are from the same species. In preferred embodiments, all sets of CH2 amino acid sequences are human CH2 amino acid sequences. In other embodiments, the sets of CH2 amino acid sequences are from different species.
  • the first set of CH2 amino acid sequences is from the same isotype as the other non-CH2 domains in the antigen-binding CH1 -substituted protein. In a specific embodiment, the first set has CH2 amino acid sequences from an IgG isotype and the one or more orthologous sets have CH2 amino acid sequences from an IgM or IgE isotype. In certain embodiments, one or more of the sets of CH2 amino acid sequences are endogenous CH2 sequences.
  • one or more of the sets of CH2 amino acid sequences are endogenous CH2 sequences that have one or more mutations.
  • the one or more mutations are orthogonal knob-hole mutations, orthogonal charge-pair mutations, or orthogonal hydrophobic mutations.
  • Orthologous CH2 amino acid sequences useful for the antigen-binding CH1- substituted proteins are described in more detail in international PCT applications W02017/011342 and WO2017/106462, herein incorporated by reference in their entirety
  • domain D has a constant region amino acid sequence. Constant region amino acid sequences are described in more detail, e.g., in Section 6.4.3.
  • domain D has a CH2 amino acid sequence.
  • CH2 amino acid sequences as described herein, are CH2 amino acid sequences of the third domain of a native antibody heavy chain, with reference from the N-terminus to C-terminus.
  • the CH2 sequences are mammalian sequences, including but not limited to mouse, rat, hamster, rabbit, camel, donkey, goat, and human sequences.
  • the CH2 sequences are human sequences.
  • the CH2 sequences are from a IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM isotype.
  • the CH2 sequences are from an IgGl isotype.
  • the CH2 sequences are endogenous sequences.
  • the sequence is UniProt accession number P01857 amino acids 111-223.
  • the CH2 sequences have a N-terminal hinge region peptide that connects the N-terminal variable domain-constant domain segment to the CH2 domain, as discussed in more detail below in Section 6.4.20.3.
  • the N- terminus of domain D is connected to the C-terminus of domain B.
  • domain B has a CH3 amino acid sequence that is extended at the C-terminus at the junction between domain D and domain B, as described in greater detail below in Section 6.4.20.3.
  • domain E has a constant region domain amino acid sequence. Constant region amino acid sequences are described in more detail, e.g., in Section 6.4.3.
  • the constant region sequence is a CH3 sequence.
  • the constant region sequence has been mutated to include one or more orthogonal mutations.
  • domain E has a constant region sequence that is a CH3 sequence comprising knob-hole (synonymously,“knob-in-hole,”“KIH”) orthogonal mutations, as described in greater detail below in Section 6.4.15.2, and either a S354C or a Y349C mutation that forms an engineered disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in in greater detail below in Section 6.4.15.1.
  • the knob-hole orthogonal mutation is a T366W mutation.
  • the constant region domain sequence is a CH1 sequence.
  • the CH1 amino acid sequence of domain E is the only CH1 amino acid sequence in the antigen-binding CH1 -substituted protein.
  • the N-terminus of the CH1 domain is connected to the C-terminus of a CH2 domain, as described in greater detail below in 6.4.20.5.
  • the constant region sequence is a CL sequence.
  • the N-terminus of the CL domain is connected to the C-terminus of a CH2 domain, as described in greater detail below in 6.4.20.5. CH1 and CL sequences are described in further detail in Section 6.4.1.
  • domain F has a variable region domain amino acid sequence.
  • Variable region domain amino acid sequences as discussed in greater detail in Section 6.4.2, are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences. VL and VH sequences are described in greater detail above in Sections 6.4.2.1 and 6.4.2.4, respectively.
  • domain F has a VH antibody domain sequence.
  • domain G has a constant region domain sequence. In some embodiments, domain G has a constant region domain sequence that is not a CH1 sequence.
  • domain G has a CH3 amino acid sequence. CH3 sequences are described in greater detail above in Section 6.4.3.1.
  • domain G has a human IgGl CH3 sequence with the following mutational changes: S354C; and a tripeptide insertion, 445P, 446G, 447K.
  • domain G has a human IgGl CH3 sequence with the following mutational changes: S354C; and 445P, 446G, 447K tripeptide insertion.
  • domain G has a human IgGl CH3 sequence with the following changes: L351D, and a tripeptide insertion of 445G, 446E, 447C.
  • domain G comprises an orthologous CH2 amino acid sequence described herein.
  • the constant region sequence is a CH1 or Cl sequence.
  • domain B is a Cl sequence
  • domain G is a CH1 sequence.
  • CH1 and Cl sequences are described herein.
  • the CH1 or Cl sequence comprises one or more CH1 or Cl orthogonal modifications described herein.
  • domain H has a variable region domain amino acid sequence.
  • Variable region domain amino acid sequences as discussed in greater detail in Section 6.4.2, are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences. VL and VH sequences are described in greater detail above in Sections 6.4.2.1. and 6.4.2.4, respectively.
  • domain H has a VL antibody domain sequence. 6.4.9. Domain I (Constant Region)
  • domain I has a constant region domain amino acid sequence. Constant region domain amino acid sequences are described in greater detail above, e.g., in Section 6.4.3. In a series of preferred embodiments of the antigen-binding CH1 -substituted proteins, domain I has a CL amino acid sequence. In another series of embodiments, domain I has a CH1 amino acid sequence. CH1 and CL amino acid sequences are described in further detail in Section 6.4.1.
  • domain J has a CH2 amino acid sequence.
  • CH2 amino acid sequences are described in greater detail above in Section 6.4.4.
  • the CH2 amino acid sequence has a N-terminal hinge region that connects domain J to domain I, as described in more detail below in Section 6.4.20.4.
  • the C- terminus of domain J is connected to the N-terminus of domain K.
  • domain J is connected to the N-terminus of domain K that has a CH1 amino acid sequence or CL amino acid sequence, as described in further detail below in Section 6.4.20.5.
  • domain K has a constant region domain amino acid sequence. Constant region domain amino acid sequences are described in greater detail above in Section 6.4.3.
  • domain K has a constant region sequence that is a CH3 sequence comprising knob-hole orthogonal mutations, as described in greater detail below in Section 6.4.15.2; isoallotype mutations, as described in more detail above in 6.4.3.1.; and either a S354C or a Y349C mutation that forms an engineered disulfide bridge with a CH3 domain containing an orthogonal mutation, as described in in greater detail below in Section 6.4.15.1.
  • the knob-hole orthogonal mutations combined with isoallotype mutations are the following mutational changes: D356E, L358M, T366S, L368A, and Y407V.
  • the constant region domain sequence is a CH1 sequence.
  • the CH1 amino acid sequence of domain K is the only CH1 amino acid sequence in the antigen-binding CH1- substituted protein.
  • the N-terminus of the CH1 domain is connected to the C-terminus of a CH2 domain, as described in greater detail below in 6.4.20.5.
  • the constant region sequence is a CL sequence.
  • the N-terminus of the CL domain is connected to the C-terminus of a CH2 domain, as described in greater detail below in 6.4.20.5. CH1 and CL sequences are described in further detail in Section 6.4.1.
  • domain L has a variable region domain amino acid sequence.
  • Variable region domain amino acid sequences as discussed in greater detail in Section 6.4.2, are variable region domain amino acid sequences of an antibody including VL and VH antibody domain sequences. VL and VH sequences are described in greater detail above in Sections
  • domain L has a VH antibody domain sequence.
  • domain M has a constant region domain amino acid sequence. Constant region domain amino acid sequences are described in greater detail above, e.g., in Section 6.4.3. In a series of preferred embodiments wherein domain I has a CL amino acid sequence, domain M has a CH1 amino acid sequence. In another series of preferred embodiments of the antigen binding CH1- substituted proteins wherein domain I has a CH1 amino acid sequence, domain M has a Cl domain sequence. CH1 and CL amino acid sequences are described in further detail in Section 6.4.1.
  • a domain A VL or VH amino acid sequence and a cognate domain F VL or VH amino acid sequence are associated and form an antigen binding site (ABS).
  • the A:F antigen binding site (ABS) is capable of specifically binding an epitope of an antigen. Antigen binding by an ABS is described in greater detail below in Section 6.4.14.1.
  • the ABS formed by domains A and F is identical in sequence to one or more other ABSs within the antigen-binding CH1- substituted protein and therefore has the same recognition specificity as the one or more other sequence-identical ABSs within the antigen-binding CH1 -substituted protein.
  • the A:F ABS is non-identical in sequence to one or more other ABSs within the antigen-binding CH1- substituted protein.
  • the A:F ABS has a recognition specificity different from that of one or more other sequence-non-identical ABSs in the antigen-binding CH1 -substituted protein.
  • the A:F ABS recognizes a different antigen from that recognized by at least one other sequence-non-identical ABS in the antigen-binding CH1- substituted protein.
  • the A:F ABS recognizes a different epitope of an antigen that is also recognized by at least one other sequence-non-identical ABS in the antigen-binding CH1- substituted protein.
  • the ABS formed by domains A and F recognizes an epitope of antigen, wherein one or more other ABSs within the antigen-binding CH1- substituted protein recognizes the same antigen but not the same epitope.
  • ABS and the antigen-binding CH1 -substituted protein comprising such ABS, is said to“recognize” the epitope (or more generally, the antigen) to which the ABS specifically binds, and the epitope (or more generally, the antigen) is said to be the
  • affinity refers to the strength of interaction of non-covalent interm olecular forces between one molecule and another.
  • the affinity i.e. the strength of the interaction, can be expressed as a dissociation equilibrium constant (KD), wherein a lower KD value refers to a stronger interaction between molecules.
  • KD values of antibody constructs are measured by methods well known in the art including, but not limited to, bio layer interferometry (e.g. Octet/FORTEBIO ® ), surface plasmon resonance (SPR) technology (e.g. Biacore ® ), and cell binding assays.
  • affinities are dissociation equilibrium constants measured by bio-layer interferometry using
  • Specific binding refers to an affinity between an ABS and its cognate antigen or epitope in which the KD value is below 10 6 M, 10 7 M, 10 8 M, 10 9 M, or 10 10 M.
  • the number of ABSs in an antigen-binding CH1 -substituted protein as described herein defines the“valency” of the antigen-binding CH1- substituted protein. As schematized in FIG. 2, an antigen-binding CH1- substituted protein having a single ABS is “monovalent”. An antigen-binding CH1 -substituted protein having a plurality of ABSs is said to be“multivalent”.
  • a multivalent antigen-binding CH1 -substituted protein having two ABSs is“bivalent.”
  • a multivalent antigen-binding CH1 -substituted protein having three ABSs is“trivalent”
  • a multivalent antigen-binding CH1 -substituted protein having four ABSs is“tetravalent.”
  • all of the plurality of ABSs have the same recognition specificity.
  • such an antigen-binding CH1- substituted protein is a“monospecific”“multivalent” binding construct.
  • at least two of the plurality of ABSs have different recognition specificities.
  • Such antigen-binding CH1- substituted proteins are multivalent and
  • the antigen-binding CH1 -substituted protein is“bispecific.” In multivalent embodiments in which the ABSs collectively have three recognition
  • the antigen-binding CH1 -substituted protein is“trispecific.”
  • the antigen binding CH1 -substituted protein is“multiparatopic”
  • Multivalent embodiments in which the ABSs collectively recognize two epitopes on the same antigen are“biparatopic”
  • multivalency of the antigen-binding CH1- substituted protein improves the avidity of the antigen-binding CH1- substituted protein for a specific target.
  • avidity refers to the overall strength of interaction between two or more molecules, e.g. a multivalent antigen-binding CH1- substituted protein for a specific target, wherein the avidity is the cumulative strength of interaction provided by the affinities of multiple ABSs. Avidity can be measured by the same methods as those used to determine affinity, as described above.
  • the avidity of an antigen-binding CH1- substituted protein for a specific target is such that the interaction is a specific binding interaction, wherein the avidity between two molecules has a KD value below 10 6 M, 10 7 M, 10 8 M, 10 9 M, or 10 10 M.
  • the avidity of an antigen-binding CH1- substituted protein for a specific target has a KD value such that the interaction is a specific binding interaction, wherein the one or more affinities of individual ABSs do not have has a KD value that qualifies as specifically binding their respective antigens or epitopes on their own.
  • the avidity is the cumulative strength of interaction provided by the affinities of multiple ABSs for separate antigens on a shared specific target or complex, such as separate antigens found on an individual cell. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABSs for separate epitopes on a shared individual antigen.
  • a domain B constant region amino acid sequence and a domain G constant region amino acid sequence are associated. Constant region domain amino acid sequences are described in greater detail above in Section 6.4.3. Other constant region domain amino acid sequences, including CH1 and Cl amino acid sequences, are described herein in Section 6.4.1.
  • domain B and domain G have CH3 amino acid sequences.
  • CH3 sequences are described in greater detail above in Section 6.4.3.1.
  • the amino acid sequences of the B and the G domains are identical.
  • the sequence is an endogenous CH3 sequence.
  • the amino acid sequences of the B and the G domains are different, and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the B domain interacts with the G domain, and wherein neither the B domain nor the G domain significantly interacts with a CH3 domain lacking the orthogonal modification.
  • orthogonal modifications or synonymously“orthogonal mutations” as described herein are one or more engineered mutations in an amino acid sequence of an antibody domain that increase the affinity of binding of a first domain having orthogonal modification for a second domain having a complementary orthogonal modification.
  • the orthogonal modifications decrease the affinity of a domain having the orthogonal modifications for a domain lacking the complementary orthogonal modifications.
  • orthogonal modifications are mutations in an endogenous antibody domain sequence.
  • orthogonal modifications are modifications of the N-terminus or C-terminus of an endogenous antibody domain sequence including, but not limited to, amino acid additions or deletions.
  • orthogonal modifications include, but are not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations, as described in greater detail below in Sections 6.4.15.1-6.4.15.3.
  • orthogonal modifications include a combination of orthogonal modifications selected from, but not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations.
  • the orthogonal modifications can be combined with amino acid substitutions that reduce immunogenicity, such as isoallotype mutations, as described in greater detail above in Section 6.4.3.1.
  • the orthogonal modifications comprise mutations that generate engineered disulfide bridges between a first and a second domain.
  • “engineered disulfide bridges” are mutations that provide non- endogenous cysteine amino acids in two or more domains such that a non-native disulfide bond forms when the two or more domains associate.
  • Engineered disulfide bridges are described in greater detail in Merchant et al. (. Nature Biotech (1998) 16:677-681), the entirety of which is hereby incorporated by reference for all it teaches.
  • engineered disulfide bridges improve orthogonal association between specific domains.
  • the mutations that generate engineered disulfide bridges are a K392C mutation in one of a first or second CH3 domains, and a D399C in the other CH3 domain.
  • the mutations that generate engineered disulfide bridges are a S354C mutation in one of a first or second CH3 domains, and a Y349C in the other CH3 domain.
  • the mutations that generate engineered disulfide bridges are a 447C mutation in both the first and second CH3 domains that are provided by extension of the C-terminus of a CH3 domain incorporating a KSC tripeptide sequence.
  • knob-hole mutations are mutations that change the steric features of a first domain’s surface such that the first domain will preferentially associate with a second domain having complementary steric mutations relative to association with domains without the complementary steric mutations.
  • Knob-hole mutations are described in greater detail in U.S. Pat. No. 5,821,333 and U.S. Pat. No. 8,216,805, each of which is incorporated herein in its entirety.
  • knob-hole mutations are combined with engineered disulfide bridges, as described in greater detail in Merchant et al. (. Nature Biotech (1998) 16:677-681)), incorporated herein by reference in its entirety.
  • knob-hole mutations, isoallotype mutations, and engineered disulfide mutations are combined.
  • the knob-in-hole mutations are a T366Y mutation in a first domain, and a Y407T mutation in a second domain.
  • the knob- in-hole mutations are a F405A in a first domain, and a T394W in a second domain.
  • the knob-in-hole mutations are a T366Y mutation and a F405A in a first domain, and a T394W and a Y407T in a second domain.
  • the knob-in-hole mutations are a T366W mutation in a first domain, and a Y407A in a second domain.
  • the combined knob-in-hole mutations and engineered disulfide mutations are a S354C and T366W mutations in a first domain, and a Y349C, T366S, L368A, and aY407V mutation in a second domain.
  • the combined knob-in-hole mutations, isoallotype mutations, and engineered disulfide mutations are a S354C and T366W mutations in a first domain, and a Y349C, D356E, L358M, T366S, L368A, and aY407V mutation in a second domain.
  • orthogonal modifications are charge-pair mutations.
  • charge-pair mutations are mutations that affect the charge of an amino acid in a domain’s surface such that the domain will preferentially associate with a second domain having complementary charge-pair mutations relative to association with domains without the complementary charge-pair mutations.
  • charge-pair mutations improve orthogonal association between specific domains. Charge- pair mutations are described in greater detail in U.S. Pat. No. 8,592,562, U.S. Pat. No. 9,248,182, and U.S. Pat. No. 9,358,286, each of which is incorporated by reference herein for all they teach.
  • charge-pair mutations improve stability between specific domains.
  • the charge-pair mutations are a T366K mutation in a first domain, and a L351D mutation in the other domain.
  • the E domain has a CH3 amino acid sequence.
  • the K domain has a CH3 amino acid sequence.
  • amino acid sequences of the E and K domains are identical, wherein the sequence is an endogenous CH3 sequence.
  • the sequences of the E and K domains are different.
  • the different sequences separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, wherein the E domain interacts with the K domain, and wherein neither the E domain nor the K domain significantly interacts with a CH3 domain lacking the orthogonal modification.
  • the orthogonal modifications include, but are not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations, as described in greater detail above in sections 6.4.15.1-6.4.15.3.
  • orthogonal modifications include a combination of orthogonal modifications selected from, but not limited to, engineered disulfide bridges, knob-in-hole mutations, and charge-pair mutations.
  • the orthogonal modifications can be combined with amino acid substitutions that reduce immunogenicity, such as isoallotype mutations.
  • domain I has a CL sequence and domain M has a CH1 sequence.
  • domain H has a VL sequence and domain L has a VH sequence.
  • domain H has a VL amino acid sequence
  • domain I has a CL amino acid sequence
  • domain L has a VH amino acid sequence
  • domain M has a CH1 amino acid sequence.
  • domain H has a VL amino acid sequence
  • domain I has a CL amino acid sequence
  • domain L has a VH amino acid sequence
  • domain M has a CH1 amino acid sequence
  • domain K has a CH3 amino acid sequence.
  • the amino acid sequences of the I domain and the M domain separately comprise respectively orthogonal modifications in an endogenous sequence, wherein the I domain interacts with the M domain, and wherein neither the I domain nor the M domain significantly interacts with a domain lacking the orthogonal modification.
  • the orthogonal mutations in the I domain are in a CL sequence and the orthogonal mutations in the M domain are in CH1 sequence.
  • Orthogonal mutations are in CH1 and CL sequences are described in more detail above in Section 6.4.1.1.
  • the amino acid sequences of the H domain and the L domain separately comprise respectively orthogonal modifications in an endogenous sequence, wherein the H domain interacts with the L domain, and wherein neither the H domain nor the L domain significantly interacts with a domain lacking the orthogonal modification.
  • the orthogonal mutations in the H domain are in a VL sequence and the orthogonal mutations in the L domain are in VH sequence.
  • the orthogonal mutations are charge-pair mutations at the VH/VL interface.
  • the charge-pair mutations at the VH/VL interface are a Q39E in VH with a corresponding Q38K in VL, or a Q39K in VH with a corresponding Q38E in VL, as described in greater detail in Igawa et al. ⁇ Protein Eng. Des. Sel ., 2010, vol. 23, 667-677), herein incorporated by reference for all it teaches.
  • the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen
  • the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen
  • the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen
  • the interaction between the H domain and the L domain form a second antigen binding site specific for the first antigen.
  • the antigen-binding CH1 -substituted proteins have three antigen binding sites and are therefore termed“trivalent.”
  • the first polypeptide chain or the third polypeptide chain further comprises a domain N and a domain O, wherein domain N has a variable region domain amino acid sequence, wherein domain O has a constant region amino acid sequence, wherein domains N and O are arranged, from N-terminus to C-terminus, in a N-0 orientation, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain or to the N-terminus of domain H of the third polypeptide chain; the binding molecule further comprises a fifth polypeptide chain, comprising: a domain P and a domain Q, wherein the domains are arranged, from N-terminus to C- terminus, in a P-Q orientation, and domain P has a variable region domain amino acid sequence and domain Q has a constant region amino acid sequence; and either the first or third polypeptide chain is associated with the
  • the first polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain A of the first polypeptide chain.
  • the third polypeptide chain further comprises domain N and domain O, and wherein the C-terminus of domain O is attached, directly or indirectly, to the N-terminus of domain H of the first polypeptide chain.
  • the amino acid sequences of domain N and domain A are identical, the amino acid sequences of domain H is different from domains N and A, the amino acid sequences of domain O and domain B are identical, the amino acid sequences of domain I is different from domains O and B, the amino acid sequences of domain P and domain F are identical, the amino acid sequences of domain L is different from domains P and F, the amino acid sequences of domain Q and domain G are identical, the amino acid sequences of domain M is different from domains Q and G; and (b) wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the first antigen.
  • the amino acid sequences of domain N, domain A, and domain H are different, the amino acid sequences of domain O, domain B, and domain I are different, the amino acid sequences of domain P, domain F, and domain L are different, and the amino acid sequences of domain Q, domain G, and domain M are different; and (b) the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for a third antigen.
  • domain N , domain A, and domain H each comprise a VL amino acid sequence
  • domain P, domain F, and domain L each comprise a VH amino acid sequence
  • domain O and domain Q each comprise a CH3 amino acid sequence
  • domain B and domain I each comprise a CL amino acid sequence
  • domain G and domain M each comprise a CH1 amino acid sequence.
  • domain N, domain A, and domain H are VL domains
  • domain P, domain F, and domain L are VH domains
  • domain O and domain Q are CH3 domains
  • domain B and domain I are CL domains
  • domain G and domain M are CH1 domains.
  • the amino acid sequences of the O and the Q domains are identical, and the sequences of the O and the Q domains are endogenous CH3 sequences. [00207] In some embodiments, the amino acid sequences of the O and the Q domains are different and separately comprise respectively orthogonal modifications in an endogenous CH3 sequence, the O domain interacts with the Q domain, and neither the O domain nor the Q domain significantly interacts with a CH3 domain lacking the orthogonal modification.
  • the orthogonal modifications of the O and the Q domains comprise mutations that generate engineered disulfide bridges between domain O and G.
  • the mutations of the O and the Q domains that generate engineered disulfide bridges are a S354C mutation in one of the O domain and Q domains, and a 349C in the other domain.
  • the orthogonal modifications of the O and the Q domains comprise knob-in-hole mutations.
  • the knob-in hole mutations of the O and the Q domains are a T366W mutation in one of the O domain and Q domain, and a T366S, L368A, and aY407V mutation in the other domain.
  • the orthogonal modifications of the O and the Q domains comprise charge-pair mutations.
  • the charge-pair mutations of the O and the Q domains are a T366K mutation in one of the O domain and Q domain, and a L351D mutation in the other domain.
  • the first polypeptide chain further comprises domain N and domain O, wherein the domains are arranged, from N-terminus to C-terminus, in a N-O-A-B-D-E orientation, and wherein domain N has a VL amino acid sequence, domain O has a CH3 amino acid sequence;
  • the antigen-binding CH1- substituted protein further comprises a fifth polypeptide chain, comprising: a domain P and a domain Q, wherein the domains are arranged, from N- terminus to C-terminus, in a P-Q orientation, and wherein domain P has a VH amino acid sequence and domain Q has a CH3 amino acid sequence; and
  • the first and the fifth polypeptides are associated through an interaction between the N and the P domains and an interaction between the O and the Q domains to form the antigen-binding CH1 -substituted protein.
  • these trivalent embodiments are associated through an interaction between the N and the P domains and an interaction between the O and the Q domains to form the
  • the antigen-binding CH1- substituted proteins further comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R-S-H-I-J-K orientation, and wherein domain R has a VL amino acid sequence and domain S has a constant domain amino acid sequence; (b) the antigen-binding CH1- substituted protein further comprises a sixth polypeptide chain, comprising: a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation, and wherein domain T has a VH amino acid sequence and domain U has a constant domain amino acid sequence; and (c) the third and the sixth polypeptides are associated through an interaction between the R and the T domains and an interaction between the S and the U domains
  • the domain O is connected to domain A through a peptide linker.
  • the domain S is connected to domain H through a peptide linker.
  • the peptide linker connecting either domain O to domain A or connecting domain S to domain H is a 6 amino acid GSGSGS peptide sequence, as described in more detail in Section 6 4 20 6
  • the amino acid sequences of domain N and domain A are identical, the amino acid sequences of domain H is different from domains N and A, the amino acid sequences of domain O and domain B are identical, the amino acid sequences of domain I is different from domains O and B, the amino acid sequences of domain P and domain F are identical, the amino acid sequences of domain L is different from domains P and F, the amino acid sequences of domain Q and domain G are identical, the amino acid sequences of domain M is different from domains Q and G; and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the first antigen.
  • the amino acid sequences of domain N and domain H are identical, the amino acid sequences of domain A is different from domains N and H, the amino acid sequences of domain O and domain I are identical, the amino acid sequences of domain B is different from domains O and I, the amino acid sequences of domain P and domain L are identical, the amino acid sequences of domain F is different from domains P and L, the amino acid sequences of domain Q and domain M are identical, the amino acid sequences of domain G is different from domains Q and M; and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for the second antigen.
  • the amino acid sequences of domain N, domain A, and domain H are different, the amino acid sequences of domain O, domain B, and domain I are different, the amino acid sequences of domain P, domain F, and domain L are different, and the amino acid sequences of domain Q, domain G, and domain M are different; and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain N and domain P form a third antigen binding site specific for a third antigen.
  • domain O has a constant region sequence that is a CL from a kappa light chain and domain Q has a constant region sequence that is a CH1 from an IgGl isotype, as discussed in more detail in Section 6.4.1.
  • domain O and domain Q have CH3 sequences such that they specifically associate with each other, as discussed in more detail above in Section 6.4.15.
  • the amino acid sequences of domain R and domain A are identical, the amino acid sequences of domain H is different from domain R and A, the amino acid sequences of domain S and domain B are identical, the amino acid sequences of domain I is different from domain S and B, the amino acid sequences of domain T and domain F are identical, the amino acid sequences of domain L is different from domain T and F, the amino acid sequences of domain U and domain G are identical, the amino acid sequences of domain M is different from domain U and G and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the first antigen. 6.4.18.5. Trivalent 1x2 Bispecific Constructs [l(A)x2(B-B)]
  • the antigen-binding CH1- substituted protein further comprises a second CH1 domain, or portion thereof.
  • the amino acid sequences of domain R and domain H are identical, the amino acid sequences of domain A is different from domain R and H, the amino acid sequences of domain S and domain I are identical, the amino acid sequences of domain B is different from domain S and I, the amino acid sequences of domain T and domain L are identical, the amino acid sequences of domain F is different from domain T and L, the amino acid sequences of domain U and domain M are identical, the amino acid sequences of domain G is different from domain U and M and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for the second antigen.
  • amino acid sequences of domain S and domain I are identical to [0100] in particular embodiments.
  • the amino acid sequences of domain U and domain M are CH1 sequences.
  • the amino acid sequences of domain R, domain A, and domain H are different, the amino acid sequences of domain S, domain B, and domain I are different, the amino acid sequences of domain T, domain F, and domain L are different, and the amino acid sequences of domain U, domain G, and domain M are different; and the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen, and the domain R and domain T form a third antigen binding site specific for a third antigen.
  • domain S has a constant region sequence that is a CL from a kappa light chain and domain U has a constant region sequence that is a CH1 from an IgGl isotype, as discussed in more detail in Section 6.4.1.
  • domain S and domain U have CH3 sequences such that they specifically associate with each other, as discussed in more detail above in Section 6.4.15.
  • the antigen-binding CH1 -substituted protein further comprises a second CH1 domain, or portion thereof.
  • the amino acid sequences of domain S and domain I are CH1 sequences.
  • the amino acid sequences of domain U and domain M are CH1 sequences.
  • the antigen-binding CH1- substituted proteins have 4 antigen binding sites and are therefore termed“tetravalent.”
  • the antigen binding CH1 -substituted proteins further comprise a fifth and a sixth polypeptide chain, wherein (a) the first polypeptide chain further comprises a domain N and a domain O, wherein the domains are arranged, from N-terminus to C-terminus, in a N-O-A-B-D-E orientation; (b) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R-S-H-I-J-K orientation; (c) the antigen-binding CH1 -substituted protein further comprises a fifth and a sixth polypeptide chain, wherein the fifth polypeptide chain comprises a domain P and a domain Q, wherein the domains are arranged, from N-terminus to C-terminus, in a P-Q orientation, and the sixth polypeptide chain comprises a domain T
  • the domain O is connected to domain A through a peptide linker and the domain S is connected to domain H through a peptide linker.
  • the peptide linker connecting domain O to domain A and connecting domain S to domain H is a 6 amino acid GSGSGS peptide sequence, as described in more detail in Section 6.4.20.6.
  • the amino acid sequences of domain N and domain A are identical, the amino acid sequences of domain H and domain R are identical, the amino acid sequences of domain O and domain B are identical, the amino acid sequences of domain I and domain S are identical, the amino acid sequences of domain P and domain F are identical, the amino acid sequences of domain L and domain T are identical, the amino acid sequences of domain Q and domain G are identical, the amino acid sequences of domain M and domain U are identical; and wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the domain N and domain P form a second antigen binding site specific for the first antigen, the interaction between the H domain and the L domain form a third antigen binding site specific for a second antigen, and the interaction between the R domain and the T domain form a fourth antigen binding site specific for the second antigen.
  • the amino acid sequences of domain H and domain A are identical, the amino acid sequences of domain N and domain R are identical, the amino acid sequences of domain I and domain B are identical, the amino acid sequences of domain O and domain S are identical, the amino acid sequences of domain L and domain F are identical, the amino acid sequences of domain P and domain T are identical, the amino acid sequences of domain M and domain G are identical, the amino acid sequences of domain Q and domain U are identical; and wherein the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen, the domain N and domain P form a second antigen binding site specific for a second antigen, the interaction between the H domain and the L domain form a third antigen binding site specific for the first antigen, and the interaction between the R domain and the T domain form a fourth antigen binding site specific for the second antigen.
  • the amino acid sequence that forms a junction between the C-terminus of a VL domain and the N-terminus of a CH3 domain is an engineered sequence.
  • one or more amino acids are deleted or added in the C-terminus of the VL domain.
  • the junction connecting the C-terminus of a VL domain and the N-terminus of a CH3 domain is one of the sequences described in Table 3 below in Section 6.13.6.
  • Al 11 is deleted in the C-terminus of the VL domain.
  • one or more amino acids are deleted or added in the N-terminus of the CH3 domain.
  • P343 is deleted in the N-terminus of the CH3 domain.
  • P343 and R344 are deleted in the N-terminus of the CH3 domain.
  • one or more amino acids are deleted or added to both the C-terminus of the VL domain and the N-terminus of the CH3 domain.
  • Al 11 is deleted in the C-terminus of the VL domain and P343 is deleted in the N-terminus of the CH3 domain.
  • Al 11 and VI 10 are deleted in the C-terminus of the VL domain.
  • Al 11 and VI 10 are deleted in the C- terminus of the VL domain and the N-terminus of the CH3 domain has a P343 V mutation.
  • the amino acid sequence that forms a junction between the C-terminus of a VH domain and the N-terminus of a CH3 domain is an engineered sequence.
  • one or more amino acids are deleted or added in the C-terminus of the VH domain.
  • the junction connecting the C-terminus of a VH domain and the N-terminus of the CH3 domain is one of the sequences described in Table 4 below in Section 6.13.6.
  • K177 and Gl 18 are deleted in the C-terminus of the VH domain.
  • one or more amino acids are deleted or added in the N-terminus of the CH3 domain.
  • P343 is deleted in the N-terminus of the CH3 domain.
  • P343 and R344 are deleted in the N-terminus of the CH3 domain.
  • P343, R344, and E345 are deleted in the N-terminus of the CH3 domain.
  • one or more amino acids are deleted or added to both the C-terminus of the VH domain and the N-terminus of the CH3 domain.
  • T166, K177, and Gl 18 are deleted in the C-terminus of the VH domain.
  • the N- terminus of the CH2 domain has a“hinge” region amino acid sequence.
  • hinge regions are sequences of an antibody heavy chain that link the N-terminal variable domain-constant domain segment of an antibody and a CH2 domain of an antibody.
  • the hinge region typically provides both flexibility between the N-terminal variable domain-constant domain segment and CH2 domain, as well as amino acid sequence motifs that form disulfide bridges between heavy chains (e.g. the first and the third polypeptide chains).
  • the hinge region amino acid sequence is SEQ ID NO: 56.
  • a CH3 amino acid sequence is extended at the C- terminus at the junction between the C-terminus of the CH3 domain and the N-terminus of a CH2 domain.
  • a CH3 amino acid sequence is extended at the C- terminus at the junction between the C-terminus of the CH3 domain and a hinge region, which in turn is connected to the N-terminus of a CH2 domain.
  • the CH3 amino acid sequence is extended by inserting a PGK tripeptide sequence followed by the DKTHT motif of an IgGl hinge region.
  • the extension at the C-terminus of the CH3 domain incorporates amino acid sequences that can form a disulfide bond with orthogonal C-terminal extension of another CH3 domain.
  • the extension at the C-terminus of the CH3 domain incorporates a KSC tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region that forms a disulfide bond with orthogonal C-terminal extension of another CH3 domain that incorporates a GEC motif of a kappa light chain.
  • a CL amino acid sequence is connected through its C-terminus to a hinge region, which in turn is connected to the N-terminus of a CH2 domain.
  • Hinge region sequences are described in more detail above in Section 6.4.20.3.
  • the hinge region amino acid sequence is SEQ ID NO:56.
  • a CH2 amino acid sequence is connected through its C-terminus to the N-terminus of a constant region domain. Constant regions are described in more detail above in Section 6.4.5.
  • the CH2 sequence is connected to a CH3 sequence via its endogenous sequence.
  • the CH2 sequence is connected to a CH1 or CL sequence. Examples discussing connecting a CH2 sequence to a CH1 or CL sequence are described in more detail in LT.S. Pat. No. 8,242,247, which is hereby incorporated in its entirety. 6.4.20.6. Junctions Connecting Domain O to Domain A or
  • heavy chains of antibodies are extended at their N-terminus to include additional domains that provide additional ABSs.
  • the C-terminus of the constant region domain amino acid sequence of a domain O and/or a domain S is connected to the N-terminus of the variable region domain amino acid sequence of a domain A and/or a domain H, respectively.
  • the constant region domain is a CH3 amino acid sequence and the variable region domain is a VL amino acid sequence.
  • the constant region domain is a CL amino acid sequence and the variable region domain is a VL amino acid sequence.
  • the constant region domain is connected to the variable region domain through a peptide linker.
  • the peptide linker is a 6 amino acid GSGSGS peptide sequence.
  • light chains of antibodies are extended at their N-terminus to include additional variable domain-constant domain segments of an antibody.
  • the constant region domain is a CH1 amino acid sequence and the variable region domain is a VH amino acid sequence.
  • bivalent antigen-binding CH1 -substituted proteins are provided.
  • the antigen-binding CH1- substituted proteins comprise a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B- D-E orientation, and domain A has a VL amino acid sequence, domain B has a CH3 amino acid sequence, domain D has a CH2 amino acid sequence, and domain E has a constant region domain amino acid sequence; (b) the second polypeptide chain comprises a domain F and a domain G, wherein the domains are arranged, from N-terminus to C-terminus, in a F-G orientation, and wherein domain F has a VH amino acid sequence and domain G has a CH3 amino acid sequence; (c) the third polypeptide chain comprises a domain H, a domain I, a domain
  • domain E has a CH3 amino acid sequence
  • domain H has a VL amino acid sequence
  • domain I has a CL amino acid sequence
  • domain K has a CH3 amino acid sequence
  • domain L has a VH amino acid sequence
  • domain M has a CH1 amino acid sequence.
  • the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen
  • the interaction between the H domain and the L domain form a second antigen binding site specific for a second antigen
  • the antigen-binding CH1 -substituted protein is a bispecific bivalent antigen-binding CH1- substituted protein.
  • the interaction between the A domain and the F domain form a first antigen binding site specific for a first antigen
  • the interaction between the H domain and the L domain form a second antigen binding site specific for the first antigen
  • the antigen-binding CH1- substituted protein is a monospecific bivalent antigen-binding CH1 -substituted protein.
  • the antigen binding CH1 -substituted protein has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B- D-E orientation, and domain A has a first VL amino acid sequence, domain B has a human IgGl CH3 amino acid sequence with a T366K mutation and a C-terminal extension incorporating a KSC tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region, domain D has a human IgGl CH2 amino acid sequence, and domain E has human IgGl CH3 amino acid with a S354C and T366W mutation; (b) the second polypeptide chain has a domain F and a domain G, wherein
  • the first polypeptide chain has the sequence SEQ ID NO: 8
  • the second polypeptide chain has the sequence SEQ ID NO: 9
  • the third polypeptide chain has the sequence SEQ ID NO: 10
  • the fourth polypeptide chain has the sequence SEQ ID NO: 11.
  • the antigen-binding CH1- substituted protein has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, and domain A has a first VL amino acid sequence, domain B has a human IgGl CH3 amino acid sequence with a C-terminal extension incorporating a KSC tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region, domain D has a human IgGl CH2 amino acid sequence, and domain E has human IgGl CH3 amino acid with a S354C and a T366W mutation; (b) the second polypeptide chain has a domain F and a domain G, wherein the domains are arranged, from N
  • the antigen-binding CH1- substituted protein has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, and domain A has a first VL amino acid sequence, domain B has a human IgGl CH3 amino acid sequence with a Y349C mutation and a C-terminal extension incorporating a PGK tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region, domain D has a human IgGl CH2 amino acid sequence, and domain E has a human IgGl CH3 amino acid with a S354C and a T366W mutation; (b) the second polypeptide chain has a domain F and a domain
  • the first polypeptide chain has the sequence SEQ ID NO:24
  • the second polypeptide chain has the sequence SEQ ID NO:25
  • the third polypeptide chain has the sequence SEQ ID NO: 10
  • the fourth polypeptide chain has the sequence SEQ ID NO: 11.
  • the antigen-binding CH1- substituted protein has a first, second, third, and fourth polypeptide chain, wherein (a) the first polypeptide chain comprises a domain A, a domain B, a domain D, and a domain E, wherein the domains are arranged, from N-terminus to C-terminus, in a A-B-D-E orientation, and domain A has a first VL amino acid sequence, domain B has a human IgGl CH3 amino acid sequence with a Y349C mutation, a P343 V mutation, and a C-terminal extension incorporating a PGK tripeptide sequence that is followed by the DKTHT motif of an IgGl hinge region, domain D has a human IgGl CH2 amino acid sequence, and domain E has human IgGl CH3 amino acid with a S354C mutation and a T366W mutation; (b) the second polypeptide chain has
  • the first polypeptide chain has the sequence SEQ ID NO:32
  • the second polypeptide chain has the sequence SEQ ID NO:25
  • the third polypeptide chain has the sequence SEQ ID NO: 10
  • the fourth polypeptide chain has the sequence SEQ ID NO: 11. 6.6.
  • the antigen-binding CH1- substituted proteins further comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R-S-H-I-J-K orientation, and wherein domain R has the first VL amino acid sequence and domain S has a human IgGl CH3 amino acid sequence with a Y349C mutation and a C-terminal extension incorporating a PGK tripeptide sequence that is followed by GSGSGS linker peptide connecting domain S to domain H; (b) the antigen-binding CH1- substituted protein further comprises a sixth polypeptide chain, comprising: a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation, and wherein domain T has the
  • the first polypeptide chain has the sequence SEQ ID NO:24
  • the second polypeptide chain has the sequence SEQ ID NO:25
  • the third polypeptide chain has the sequence SEQ ID NO: 37
  • the fourth polypeptide chain has the sequence SEQ ID NO:l 1
  • the sixth polypeptide chain has the sequence SEQ ID NO:25.
  • the antigen-binding CH1 -substituted proteins further comprise a sixth polypeptide chain, wherein (a) the third polypeptide chain further comprises a domain R and a domain S, wherein the domains are arranged, from N-terminus to C-terminus, in a R- S-H-I-J-K orientation, and wherein domain R has a third VL amino acid sequence and domain S has a human IgGl CH3 amino acid sequence with a T366K mutation and a C- terminal extension incorporating a KSC tripeptide sequence that is followed by GSGSGS linker peptide connecting domain S to domain H; (b) the antigen-binding CH1- substituted protein further comprises a sixth polypeptide chain, comprising: a domain T and a domain U, wherein the domains are arranged, from N-terminus to C-terminus, in a T-U orientation, and where
  • the first polypeptide chain has the sequence SEQ ID NO:24
  • the second polypeptide chain has the sequence SEQ ID NO:25
  • the third polypeptide chain has the sequence SEQ ID NO:45
  • the fourth polypeptide chain has the sequence SEQ ID NO:l 1
  • the sixth polypeptide chain has the sequence SEQ ID NO:
  • the antigen-binding CH1 -substituted protein is a lxl MH2 bivalent bispecific platform, described in, e.g., W02017011342, which is hereby
  • the antigen-binding CH1 -substituted protein is a CH3 Domain Substitution multispecific platform, described in WO2016087650, which is hereby incorporated by reference in its entirety.
  • antigen binding sites of the antigen-binding CH1 -substituted proteins described herein may be chosen to specifically bind a wide variety of molecular targets.
  • an antigen binding site or sites may specifically bind E-Cad, CLDN7, FGFR2b, N-Cad, Cad-l l, FGFR2c, ERBB2, ERBB3, FGFR1, FOLR1, IGF-Ira, GLP1R, PDGFRa, PDGFRb, EPHB6, ABCG2, CXCR4, CXCR7, Integrin-avb3, SPARC, VCAM, ICAM, Annexin, ROR1, ROR2, TNFa, CD 137, angiopoietin 2, angiopoietin 3, BAFF, beta amyloid, C5, CA-125, CD147, CD125, CD147, CD152, CD19, CD20, CD22, CD23,
  • An antigen binding site or sites may be chosen that specifically binds the TNF family of receptors including, but not limited to, TNFR1 (also known as CD 120a and TNFRSF1A), TNFR2 (also known as CDl20b and TNFRSF1B), TNFRSF3 (also known as I ⁇ R), TNFRSF4 (also known as 0X40 and CD 134), TNFRSF5 (also known as CD40), TNFRSF6 (also known as FAS and CD95), TNFRSF6B (also known as DCR3), TNFRSF7 (also known as CD27), TNFRSF8 (also known as CD30), TNFRSF9 (also known as 4- 1BB), TNFRSF10A (also known as TRAILR1, DR4, and CD26), TNFRSF10B (also known as TRAILR2, DR5, and CD262), TNFRSF10C (also known as TRAILR3, DCR1, CD263), TNFRSF10D (
  • TNFRSF17 also known as BCMA and CD269
  • TNFRSF18 also known as GITR and CD357
  • TNFRSF19 also known as TROY, TAJ, and TRADE
  • TNFRSF21 also known as CD358
  • TNFRSF25 also known as Apo-3, TRAMP, LARD, or WS-l
  • EDA2R also known as XEDAR
  • An antigen binding site or sites may be chosen that specifically binds immune- oncology targets including, but not limited to, checkpoint inhibitor targets such as PD1, PDL1, CTLA-4, PDL2, B7-H3, B7-H4, BTLA, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, BY55, and CGEN-15049.
  • checkpoint inhibitor targets such as PD1, PDL1, CTLA-4, PDL2, B7-H3, B7-H4, BTLA, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, BY55, and CGEN-15049.
  • an antigen binding site or sites may be chosen that specifically target tumor-associated cells.
  • the antigen binding site or sites specifically target tumor associated immune cells.
  • the antigen binding site or sites specifically target tumor associated regulatory T cells (Tregs).
  • tumor associated regulatory T cells Tugs
  • an antigen-binding CH1 -substituted protein has antigen binding sites specific for antigens selected from one or more of CD25, 0X40, CTLA-4, and NRP1 such that the antigen-binding CH1 -substituted protein specifically targets tumor associated regulatory T cells.
  • an antigen-binding CH1- substituted protein has antigen binding sites that specifically bind CD25 and 0X40, CD25 and CTLA-4, CD25 and NRP1, 0X40 and CTLA-4, 0X40 and NRP1, or CTLA-4 and NRP1 such that the antigen-binding CH1 -substituted protein specifically targets tumor associated regulatory T cells.
  • a bispecific bivalent antigen-binding CH1 -substituted protein has antigen binding sites that specifically bind CD25 and 0X40, CD25 and CTLA- 4, CD25 and NRP1, 0X40 and CTLA-4, 0X40 and NRP1, or CTLA-4 and NRP1 such that the antigen-binding CH1- substituted protein specifically targets tumor associated regulatory T cells.
  • the specific targeting of the tumor associated regulatory T cells results in depletion (e.g. killing) of the regulatory T cells.
  • the depletion of the regulatory T cells is mediated by an antibody-drug conjugate (ADC) modification, such as an antibody conjugated to a toxin, as discussed in more detail below in Section 6.9.1.
  • ADC antibody-drug conjugate
  • an antigen-binding CH1 -substituted protein has antigen binding sites selected from one or more of CD3, ROR1, and ROR2.
  • a bispecific bivalent has antigen binding sites that specifically bind CD3 and ROR1.
  • a bispecific bivalent has antigen binding sites that specifically bind CD3 and ROR2.
  • a trispecific trivalent has antigen binding sites that specifically bind CD3, ROR1, and ROR2.
  • the antigen-binding CH1- substituted protein has additional modifications.
  • the antigen-binding CH1- substituted protein is conjugated to a therapeutic agent (i.e. drug) to form an antigen-binding CH1- substituted protein-drug conjugate.
  • therapeutic agents include, but are not limited to,
  • chemotherapeutic agents include imaging agents (e.g. radioisotopes), immune modulators (e.g. cytokines, chemokines, or checkpoint inhibitors), and toxins (e.g. cytotoxic agents).
  • imaging agents e.g. radioisotopes
  • immune modulators e.g. cytokines, chemokines, or checkpoint inhibitors
  • toxins e.g. cytotoxic agents.
  • the therapeutic agents are attached to the antigen-binding CH1- substituted protein through a linker peptide, as discussed in more detail below in Section 6.9.3.
  • ADCs antibody-drug conjugates
  • the antigen-binding CH1- substituted protein has modifications that comprise one or more additional binding moieties.
  • the binding moieties are antibody fragments or antibody formats including, but not limited to, full-length antibodies, Fab fragments, Fvs, scFvs, tandem scFvs, Diabodies, scDiabodies, DARTs, tandAbs, minibodies, camelid VHH, and other antibody fragments or formats known to those skilled in the art.
  • Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), herein incorporated by reference for all that it teaches.
  • the one or more additional binding moieties are attached to the C-terminus of the first or third polypeptide chain.
  • the one or more additional binding moieties are attached to the C-terminus of both the first and third polypeptide chain.
  • the one or more additional binding moieties are attached to the C-terminus of both the first and third polypeptide chains.
  • individual portions of the one or more additional binding moieties are separately attached to the C-terminus of the first and third polypeptide chains such that the portions form the functional binding moiety.
  • the one or more additional binding moieties are attached to the N-terminus of any of the polypeptide chains (e.g. the first, second, third, fourth, fifth, or sixth polypeptide chains).
  • individual portions of the additional binding moieties are separately attached to the N-terminus of different polypeptide chains such that the portions form the functional binding moiety.
  • the one or more additional binding moieties are specific for a different antigen or epitope of the ABSs within the antigen-binding CH1- substituted protein. In certain embodiments, the one or more additional binding moieties are specific for the same antigen or epitope of the ABSs within the antigen-binding CH1- substituted protein. In certain embodiments, wherein the modification is two or more additional binding moieties, the additional binding moieties are specific for the same antigen or epitope. In certain embodiments, wherein the modification is two or more additional binding moieties, the additional binding moieties are specific for different antigens or epitopes.
  • the one or more additional binding moieties are attached to the antigen-binding CH1 -substituted protein using in vitro methods including, but not limited to, reactive chemistry and affinity tagging systems, as discussed in more detail below in Section 6.9.3.
  • the one or more additional binding moieties are attached to the antigen-binding CH1 -substituted protein through Fc-mediated binding (e.g. Protein A/G).
  • the one or more additional binding moieties are attached to the antigen-binding CH1 -substituted protein using recombinant DNA techniques, such as encoding the nucleotide sequence of the fusion product between the antigen-binding CH1- substituted protein and the additional binding moieties on the same expression vector (e.g. plasmid). 6.9.3. Functional/Reactive Groups
  • the antigen-binding CH1- substituted protein has modifications that comprise functional groups or chemically reactive groups that can be used in downstream processes, such as linking to additional moieties (e.g. drug conjugates and additional binding moieties, as discussed in more detail above in Sections 6.9.1. and 6.9.2.) and downstream purification processes.
  • additional moieties e.g. drug conjugates and additional binding moieties, as discussed in more detail above in Sections 6.9.1. and 6.9.2.
  • the modifications are chemically reactive groups including, but not limited to, reactive thiols (e.g. maleimide based reactive groups), reactive amines (e.g. A-hydroxy sued ni mi de based reactive groups),“click chemistry” groups (e.g. reactive alkyne groups), and aldehydes bearing formylglycine (FGly).
  • reactive thiols e.g. maleimide based reactive groups
  • reactive amines e.g. A-hydroxy sued ni mi de based reactive groups
  • “click chemistry” groups e.g. reactive alkyne groups
  • aldehydes bearing formylglycine FGly
  • the modifications are functional groups including, but not limited to, affinity peptide sequences (e.g. HA, HIS, FLAG, GST, MBP, and Strep systems etc.).
  • the functional groups or chemically reactive groups have a cleavable peptide sequence.
  • the cleavable peptide is cleaved by means including, but not limited to, photocleavage, chemical cleavage, protease cleavage, reducing conditions, and pH conditions.
  • protease cleavage is carried out by intracellular proteases.
  • protease cleavage is carried out by extracellular or membrane associated proteases.
  • ADC therapies adopting protease cleavage are described in more detail in Choi et al. ( Theranostics , 2012; 2(2): 156-178.), the entirety of which is hereby incorporated by reference for all it teaches.
  • compositions that comprise an antigen-binding CH1- substituted protein as described herein and a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition is sterile.
  • the pharmaceutical composition comprises the antigen-binding CH1 -substituted protein at a concentration of 0.1 mg/ml - 100 mg/ml.
  • the pharmaceutical composition comprises the antigen-binding CH1- substituted protein at a concentration of 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 5 mg/ml, 7.5 mg/ml, or 10 mg/ml.
  • the pharmaceutical composition comprises the antigen-binding CH1 -substituted protein at a concentration of more than 10 mg/ml.
  • the antigen-binding CH1- substituted protein is present at a concentration of 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, or even 50 mg/ml or higher. In particular embodiments, the antigen-binding CH1- substituted protein is present at a concentration of more than 50 mg/ml.
  • compositions are described in more detail in U.S. Pat No. 8,961,964, U.S. Pat No. 8,945,865, U.S. Pat No. 8,420,081,
  • antigen-binding CH1 -substituted proteins described herein can readily be manufactured by expression using standard cell free translation, transient transfection, and stable transfection approaches currently used for antibody manufacture.
  • Expi293 cells can be used for production of the antigen-binding CH1 -substituted proteins using protocols and reagents from ThermoFisher, such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethylenimine as described in detail in Fang et al. ⁇ Biological Procedures Online , 2017, 19: 11), herein incorporated by reference for all it teaches.
  • ExpiCHO ThermoFisher
  • ExpiCHO can be used for production of the antigen-binding CH1 -substituted proteins using protocols and reagents from
  • ThermoFisher such as ExpiFectamine, or other reagents known to those skilled in the art, such as polyethylenimine as described in detail in Fang et al. ⁇ Biological Procedures Online , 2017, 19: 11).
  • the expressed proteins can be readily separated from undesired proteins and protein complexes using a CH1 affinity resin, such as the CaptureSelect CH1 resin and provided protocol from ThermoFisher. Further purification can be affected using ion exchange chromatography as is routinely used in the art.
  • a CH1 affinity resin such as the CaptureSelect CH1 resin and provided protocol from ThermoFisher. Further purification can be affected using ion exchange chromatography as is routinely used in the art.
  • methods of treatment comprising administering an antigen-binding CH1 -substituted protein as described herein to a patient in an amount effective to treat the patient.
  • an antibody of the present disclosure may be used to treat a cancer.
  • the cancer may be a cancer from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may be a neoplasm, malignant; carcinoma; carcinoma,
  • nonencapsulating sclerosing carcinoma adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous
  • adenocarcinoma adenocarcinoma
  • ceruminous adenocarcinoma adenocarcinoma
  • mucoepidermoid carcinoma adenocarcinoma
  • cystadenocarcinoma papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant;
  • paraganglioma malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma;
  • alveolar rhabdomyosarcoma stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;
  • hemangioendothelioma malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma;
  • ameloblastoma malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma;
  • An antibody of the present disclosure may be administered to a subject per se or in the form of a pharmaceutical composition for the treatment of, e.g., cancer, autoimmunity, transplantation rejection, post-traumatic immune responses, graft-versus- host disease, ischemia, stroke, and infectious diseases, for example by targeting viral antigens, such as gpl20 of HIV.
  • a pharmaceutical composition for the treatment of, e.g., cancer, autoimmunity, transplantation rejection, post-traumatic immune responses, graft-versus- host disease, ischemia, stroke, and infectious diseases, for example by targeting viral antigens, such as gpl20 of HIV.
  • the various antigen-binding proteins tested were expressed using the Expi293 transient transfection system according to manufacturer’s instructions. Briefly, four plasmids coding for four individual chains were mixed at 1 : 1 : 1 : 1 mass ratio, unless otherwise stated, and transfected with ExpiFectamine 293 transfection kit into Expi293 cells. Cells were cultured at 37°C with 8% C02, 100% humidity and shaking at 125 rpm. Transfected cells were fed once after 16-18 hours of transfections. The cells were harvested at day 5 by centrifugation at 2000 g for 10 minutes. The supernatant was collected for affinity chromatography purification.
  • ExpiCHO transient transfection system according to manufacturer’s instructions. Briefly, four plasmids coding for four individual chains were mixed at 1 : 1 : 1 : 1 mass ratio, unless otherwise stated, and transfected with ExpiFectamine CHO transfection kit into ExpiCHO. Cells were cultured at 37°C with 8% C02, 100% humidity and shaking at 125 rpm.
  • Transfected cells were fed once after 16-18 hours of transfections.
  • the cells were harvested at day 5 by centrifugation at 2000 g for 10 munities.
  • the supernatant was collected for affinity chromatography purification.
  • the elution was monitored by absorbance at 280 nm and the elution peaks were pooled for analysis.
  • a 1 mL CaptureSelectTM XL column (ThermoFisher) was equilibrated with PBS. The sample was loaded onto the column at 5 ml/min. The sample was eluted using 0.1 M acetic acid pH 4.0. The elution was monitored by absorbance at 280 nm and the elution peaks were pooled for analysis.
  • Samples containing the various separated antigen-binding proteins were analyzed by reducing and non-reducing SDS-PAGE for the presence of complete product, incomplete product, and overall purity. 2 ug of each sample was added to 15 uL SDS loading buffer. Reducing samples were incubated in the presence of 10 mM reducing agent at 75°C for 10 minutes. Non-reducing samples were incubated at 95°C for 5 minutes without reducing agent. The reducing and non-reducing samples were loaded into a 4-15% gradient TGX gel (BioRad) with running buffer and run for 30 minutes at 250 volts. Upon completion of the run, the gel was washed with DI water and stained using GelCode Blue Safe Protein Stain (ThermoFisher). The gels were destained with DI water prior to analysis. Densitometry analysis of scanned images of the destained gels was performed using standard image analysis software to calculate the relative abundance of bands in each sample.
  • the elution was monitored by absorbance at 280 nm and the purity of the samples were calculated by peak integration to identify the abundance of the monomer peak and contaminants peaks.
  • the monomer peak and contaminant peaks were separately pooled for analysis by SDS-PAGE as described above.
  • Samples containing the various separated antigen-binding proteins were analyzed by analytical size exclusion chromatography for the ratio of monomer to high molecular weight product and impurities. Cleared supernatants were analyzed with an industry standard TSK G3000SWxl column (Tosoh Bioscience) on an Agilent 1100 HPLC. The TSK column was equilibrated with PBS. 25 uL of each sample at 1 mg/mL was loaded onto the column at 1 ml/min. The sample was eluted using an isocratic flow of PBS for 1.5 CV. The elution was monitored by absorbance at 280 nm and the elution peaks were analyzed by peak integration.
  • Samples containing the various separated antigen-binding proteins were analyzed by mass spectrometry to confirm the correct species by molecular weight. All analysis was performed by a third-party research organization. Briefly, samples were treated with a cocktail of enzymes to remove glycosylation. Samples were both tested in the reduced format to specifically identify each chain by molecular weight. Samples were all tested under non-reducing conditions to identify the molecular weights of all complexes in the samples. Mass spec analysis was used to identify the number of unique products based on molecular weight.
  • each bispecific antibody was expressed using the Expi293 system, purified from undesired protein products on an anti- CHl column, and run on an SDS-PAGE gel. As shown in Fig.
  • the bivalent bispecific construct has a single CH1 domain at Domain M with the sequence of a human IgGl CH1 region [SEQ ID NO: 23] With reference to a typical native antibody architecture, the CH1 domain typically found at domain B has been substituted with a CH3 amino acid sequence having the noted orthogonal mutations.
  • the A domain (SEQ ID NO: 12) and F domain (SEQ ID NO: 16) form an antigen binding site (A:F) specific for“Antigen A”.
  • the H domain has the VH sequence from nivolumab and the L domain has the VL sequence from nivolumab; H and L associate to form an antigen binding site (H:L) specific for human PD1.
  • the B domain (SEQ ID NO: 13) has the sequence of human IgGl CH3 with several mutations: T366K, 445K, 446S, and 447C insertion.
  • T366K mutation is a charge pair cognate of the L351D residue in Domain G.
  • The“447C” residue on domain B comes from the C-terminal KSC tripeptide insertion.
  • Domain D (SEQ ID NO: 14) has the sequence of human IgGl CH2
  • Domain E (SEQ ID NO: 15) has the sequence of human IgGl CH3 with the mutations T366W and S354C.
  • the 366W is the“knob” mutation.
  • the 354C introduces a cysteine that is able to form a disulfide bond with the cognate 349C mutation in Domain K.
  • Domain G (SEQ ID NO: 17) has the sequence of human IgGl CH3 with the following mutations: L351D, and 445G, 446E, 447C tripeptide insertion.
  • L351D mutation introduces a charge pair cognate to the Domain B T366K mutation.
  • The“447C” residue on domain G comes from the C-terminal GEC tripeptide insertion.
  • Domain I (SEQ ID NO: 19) has the sequence of human C kappa light chain (CK)
  • Domain J (SEQ ID NO: 20) has the sequence of human IgGl CH2 domain, and is identical to the sequence of domain D.
  • Domain K (SEQ ID NO: 21) has the sequence of human IgGl CH3 with the following changes: Y349C, D356E, L358M, T366S, L368A, Y407V.
  • the 349C mutation introduces a cysteine that is able to form a disulfide bond with the cognate 354C mutation in Domain E.
  • the 356E and L358M introduce isoallotype amino acids that reduce immunogenicity.
  • the 366S, 368A, and 407V are“hole” mutations.
  • BC1 could readily be expressed at high levels using mammalian expression at concentrations greater than 100 pg/ml.
  • the“BC1” protein - an antigen-binding CH1- substituted protein that is bivalent (and bispecific) and has a single CH1 domain - could easily be purified in a single step using a CH1 -specific CaptureSelectTM affinity resin from
  • FIG. 5A SEC analysis demonstrates that a single-step CH1 affinity purification step yields a single, monodisperse peak via gel filtration in which >98% is monomer.
  • FIG. 5B shows comparative literature data of SEC analysis of a CrossMab bivalent antibody construct having 2 CH1 domains.
  • FIG. 6A is a cation exchange chromatography (IEX) elution profile of“BC1” following one-step purification using the CaptureSelectTM CH1 affinity resin, showing a single tight peak.
  • FIG. 6B is a cation exchange chromatography elution profile of“BC1” following purification using standard Protein A purification, showing additional elution peaks consistent with the co-purification of incomplete assembly products.
  • IEX ion exchange chromatography
  • lane 4 shows minimal additional purification of the anti- CHleluate with a subsequent cation exchange polishing step, while lanes 8-10
  • FIG. 8 compares SDS-PAGE gels of “BC1” after single-step CHl-affmity purification, under both non-reducing and reducing conditions (Panel A) with SDS-PAGE gels of a CrossMab bispecific antibody under non-reducing and reducing conditions as published in the referenced literature (Panel B).
  • FIG. 9 shows mass spec analysis of“BC1”, demonstrating two distinct heavy chains (FIG. 9A) and two distinct light chains (FIG. 9B) under reducing conditions.
  • the mass spectrometry data in FIG. 10 confirms the absence of incomplete pairing after purification.
  • FIG. 12A shows SEC analysis of“BC6” following one- step purification using the CaptureSelectTM CH1 affinity resin.
  • the data demonstrate that the single step CH1 affinity purification yields a single monodisperse peak, similar to what we observed with“BC1”, demonstrating that the disulfide bonds between polypeptide chains 1 and 2 and between polypeptide chains 3 and 4 are intact.
  • the chromatogram also shows the absence of non-covalent aggregates.
  • FIG. 12B shows a SDS-PAGE gel under non-reducing conditions, with lane 1 loaded with a first lot of“BC6” after a single-step CH1 affinity purification, lane 2 loaded with a second lot of“BC6” after a single-step CH1 affinity purification. Lanes 3 and 4 demonstrate further purification can be achieved with ion exchange chromatography subsequent to CH1 affinity purification. 6.13.5.
  • Polypeptide chain 1 “BC28” chain 1 (SEQ ID NO:24)
  • Polypeptide chain 2 “BC28” chain 2 (SEQ ID NO:25)
  • Polypeptide chain 3:“BC1” chain 3 (SEQ ID NO: 10)
  • Polypeptide chain 4:“BC1” chain 4 (SEQ ID NO: 11)
  • The“BC28” A:F antigen binding site is specific for“Antigen A”.
  • the “BC28” H:L antigen binding site is specific for PD1 (nivolumab sequences).
  • “BC28” domain B has the following changes as compared to wild type CH3: Y349C; 445P, 446G, 447K insertion.
  • “BC28” domain E has the following changes as compared to wild type CH3: S354C, T366W.
  • “BC28” domain G has the following changes as compared to wild type: S354C; 445P, 446G, 447K insertion.
  • BC28 thus has an engineered cysteine at residue 349C of Domain B and engineered cysteine at residue 354C of domain G (“349C-354C”).
  • BC30 has an engineered cysteine at residue 354C of Domain B and 349C of Domain G (“354C-349C”).
  • FIG. 14 shows SEC analysis of“BC28” and“BC30” following one-step purification using the CaptureSelectTM CH1 affinity resin following expression using the Expi293 system.
  • FIG. 17 shows non-reducing SDS-PAGE of protein expressed using the ThermoFisher Expi293 transient transfection system.
  • Lane 1 shows the eluate of the trivalent 2x1“BC1-2X1” protein following one-step purification using the CaptureSelectTM CH1 affinity resin.
  • Lane 2 shows the lower molecular weight, faster migrating, bivalent“BC1” protein following one-step purification using the CaptureSelectTM CH1 affinity resin.
  • Lanes 3-5 demonstrate purification of“BC1- 2x1” using protein A.
  • Lanes 6 and 7 show purification of“BCl-2xl” using CH1 affinity resin. The abundance of lower bands representing incomplete complexes is decreased when purified with the CH1 affinity resin.
  • the A:F antigen binding site is specific for“Antigen A”, as is the H:L binding antigen binding site.
  • the R:T antigen binding site is specific for PD.
  • the specificity of this construct is thus Antigen“A” x (PD 1 -Antigen“A”).
  • Antigen binding site A:F was specific for’’Antigen A”
  • Antigen binding site H:L was specific for PD1 (nivolumab sequence)
  • Antigen binding site R:T was specific for CTLA4.
  • FIG. 20 shows size exclusion chromatography with“BC28-lxlxla” following expression using the Expi293 system and one-step purification using the
  • CaptureSelectTM CH1 affinity resin demonstrating a single well-defined peak.
  • FIG. 22 shows the overall architecture of a 2x2 tetravalent bispecific construct “BC22 -2x2”.
  • the 2x2 tetravalent bispecific was constructed with“BC1” scaffold by duplicating each variable domain-constant domain segment. Domain nomenclature is schematized in FIG. 21.
  • FIG. 23 is a SDS-PAGE gel.
  • Lanes 7-9 show the“BC22-2x2” tetravalent construct respectively following expression using the Expi293 system and one-step purification using the CaptureSelectTM CH1 affinity resin (“CH1 eluate”), and after an additional ion exchange chromatography purification (lane 8,“pk 1 after IEX”; lane 9,“pk 2 after IEX”).
  • Lanes 1-3 are the trivalent 2x1 construct“BC2l-2xl” after CH1 affinity purification (lane 1) and, in lanes 2 and 3, subsequent ion exchange chromatography.
  • Lanes 4-6 are the 1x2 trivalent construct“BCl2-lx2”.
  • FIG. 24 shows a SDS-PAGE gel with various constructs, each following expression using the Expi293 system and one-step purification using the CaptureSelectTM CH1 affinity resin, under non-reducing and reducing conditions.
  • Lanes 1 (nonreducing conditions) and 2 (reducing conditions, + DTT) are the bivalent lxl bispecific construct“BC1”.
  • Lanes 3 (nonreducing) and 4 (reducing) are the bivalent lxl bispecific construct“BC28” (see Example 4).
  • Lanes 5 (nonreducing) and 6 (reducing) are the bivalent lxl bispecific construct“BC44” (see Example 5).
  • Lanes 7 (nonreducing) and 8 (reducing) are the trivalent 1x2 bispecific“BC28-lx2” construct (see Example 9).
  • Lanes 9 (nonreducing) and 10 (reducing) are the trivalent 1x2 trispecific “BC28-lxlxla” construct described in Example 11.
  • the SDS-PAGE gel demonstrates the complete assembly of each construct, with the predominant band in the non-reducing gel appearing at the expected molecular weight for each construct.
  • CH1 expression is considered the rate limiting step in antibody folding and secretion. Therefore, we tested controlling the expression ratio of the four chains, particularly the ratio of the chain having the CH1 domain.
  • the Expi293 Expression system was used to test the assembly efficiency by varying the ratio of transfected expression vectors for each of the four polypeptide chains. In brief, lpg of total plasmid for all chains combined was transfected into 1 mL Expi293 cells. The expression of the CH1 domain was controlled by varying the relative ratio of the expression vector for the polypeptide chain containing the CH1 domain. The construct tested used the BC28 architecture.
  • the ratio of the 4 th polypeptide chain containing the single CH1 was varied in the transfection mixture, as well as the ratio of the other chains in separate samples.
  • the various ratios tested are shown in Table 5.
  • Supernatant from the Expi293 Expression system was run directly on an SDS-PAGE gel.
  • controlling the expression of the CH1 domain containing polypeptide chain was demonstrated to improve the efficiency of expressing and forming the desired complete antigen-binding CH1- substituted proteins.
  • Chain 1 VL (0X40:24) - CH3 (BC1) - GS linker - VL (0X40: 11) - CL -CH2-CH3 (Knob, 354C)
  • VH and VL antigen binding sites (ABSs) from 0X40 ABS clone numbers 24 and 11 (0X40:24 and 0X40: 11) are described in W02019/040791A1, which is hereby incorporated by reference in its entirety.
  • the constructs each contain at least one fewer CH1 than the valency of the construct. Each was expressed using the Expi293 system and subjected to one-step purification using the CaptureSelectTM CH1 affinity resin.
  • FIG. 26B shows an SDS-PAGE gel of the purification products of the constructs described in FIG. 26A (lanes A and B).
  • the SDS-PAGE gel demonstrates the complete assembly of each construct, with the predominant band in the non-reducing gel appearing at the expected molecular weight for each construct.
  • Chain 1 VL (I st antigen binding site (ABS)) - CH3 (T366K, 477C) -CH2-CH3 (Hole, 349C)
  • Chain 2 VH (I st ABS) - CH3 (L351D, 447C)
  • Chain 3 VL (3 rd ABS) - CH3(T366K, 447C) -VL (2 nd ABS) -CH2 -CH3 (Knob,4C)
  • FIG. 27B shows an SDS- PAGE gel of the resulting purification products, demonstrating the complete assembly of each construct, with the predominant band in the non-reducing gel appearing at the expected molecular weight for each construct.
  • FIG. 27C shows size exclusion chromatography results following ExpiCHO expression and one-step purification described above, showing a single peak denoting purity of the sample comprising the correctly assembled product.
  • Antibody architectures are compared using different purification strategies.
  • Antibody architectures that are tested include the various B-body formats described above, other antibody platforms that have substituted CH1 for another domain leaving only a single CH1, and other similar antibody platforms but that still retain a number of CH1 domains equivalent to the valency of the antibody.
  • bivalent bispecific B-Body formats e.g ., those described above including“BC1,”“BC28,” and“BC44,” 2) Abbvie lxl MH2 bivalent bispecific platforms, described in, e.g., W02017011342, which is hereby incorporated by reference in its entirety; 3) Merck Kga CH3 Domain Substitution bivalent bispecific platforms, described in WO2016087650, which is hereby incorporated by reference in its entirety 4) and trivalent bispecific/trispecific B-Body formats described herein.
  • the different platforms are expressed and purified using the anti-CHl binding reagent, as described above.
  • the different platforms are also purified using other standard techniques, such as Protein A purification.
  • the purified antibodies are analyzed using various analytical tools and methods to assess purity and abundance of desired products, as described above.
  • the different platforms are then compared against one another, and from the analysis it is determined that the anti-CHl purification strategy improves purifying antibody platforms having a single CH1 domain and/or antibody platforms having fewer CH1 domains than antibody valencies.
  • VL -VEIKRTPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPGKDKTHTCPP CPAPELLGGPSVFLFPPKPKDTL ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSV HEALHNHYTQKSLSPGK
  • bivalent monospecific construct CHAIN 2 [SEQ ID NO : 2 ]
  • VL -VEIKRTPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV HEALHNHYTQKSLSLSPGKDRrjjrCPP

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Abstract

L'invention concerne des procédés de purification de constructions d'anticorps multivalents, des constructions d'anticorps purifiées à l'aide des procédés, et des compositions pharmaceutiques comprenant les constructions purifiées, ainsi que des méthodes de traitement des constructions purifiées.
PCT/US2019/023447 2018-03-21 2019-03-21 Purification d'anticorps multispécifiques à l'aide d'une résine ch1 WO2019183406A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009080253A1 (fr) * 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2012105833A1 (fr) * 2011-02-01 2012-08-09 Bac Ip B.V. Protéine de liaison d'antigène dirigée contre un épitope dans le domaine ch1 d'anticorps de l'igg humaine
WO2016087650A1 (fr) * 2014-12-05 2016-06-09 Merck Patent Gmbh Anticorps à domaine échangé
US20170129962A1 (en) * 2015-10-02 2017-05-11 Hoffmann-La Roche Inc. Multispecific antibodies
US20180118811A1 (en) * 2016-10-19 2018-05-03 Invenra Inc. Antibody constructs

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009080253A1 (fr) * 2007-12-21 2009-07-02 F. Hoffmann-La Roche Ag Anticorps bivalents bispécifiques
WO2012105833A1 (fr) * 2011-02-01 2012-08-09 Bac Ip B.V. Protéine de liaison d'antigène dirigée contre un épitope dans le domaine ch1 d'anticorps de l'igg humaine
WO2016087650A1 (fr) * 2014-12-05 2016-06-09 Merck Patent Gmbh Anticorps à domaine échangé
US20170129962A1 (en) * 2015-10-02 2017-05-11 Hoffmann-La Roche Inc. Multispecific antibodies
US20180118811A1 (en) * 2016-10-19 2018-05-03 Invenra Inc. Antibody constructs

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