WO2023240067A2 - Multimérisation de molécules de liaison ayant un variant de région constante d'anticorps et des mutations qui réduisent les fonctions effectrices - Google Patents

Multimérisation de molécules de liaison ayant un variant de région constante d'anticorps et des mutations qui réduisent les fonctions effectrices Download PDF

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WO2023240067A2
WO2023240067A2 PCT/US2023/067976 US2023067976W WO2023240067A2 WO 2023240067 A2 WO2023240067 A2 WO 2023240067A2 US 2023067976 W US2023067976 W US 2023067976W WO 2023240067 A2 WO2023240067 A2 WO 2023240067A2
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molecule
igg
region
antigen
human
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PCT/US2023/067976
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WO2023240067A3 (fr
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Bin Fan
Melissa PINARD
Avantika KEKATPURE
Jonathan AGUAYO
Kalyani MONDAL
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Ngb Biopharmaceuticals, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • molecules comprising an engineered IgG C H 2 region and oligomer complexes comprising the same. Also provided herein are mutations that reduce the effector functions of the molecules. Further provided herein are a pharmaceutical composition comprising the molecules or oligomers described herein, a host cell, nucleic acids, a vector that are related to the molecules described herein, and a method of producing and using such molecules and oligomers.
  • a molecule comprising an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine [0006] In an aspect, provided herein is a molecule comprising an IgG C H 2 region, wherein (i) position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine; or position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine; and (ii) the IgG C H 2 region further comprises one or more substitutions by EU numbering selected from the group consisting of: position 228 in the IgG C H 2 region by EU numbering is substituted to be a proline (“228P”), position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine (“234A”), position 235 in the IgG C H 2 region by EU numbering is substituted to be a proline (“228P
  • the molecule further comprises an IgG hinge region. In other embodiments, the molecule further comprises an IgG C H 3 region. In yet other embodiments, the molecule further comprises a human ⁇ tailpiece. In still yet other embodiments, the molecule further comprises an IgG C H 1 region. [0008] In some specific embodiments, the human ⁇ tailpiece comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75%, 80%, 85%, or 90% identity to SEQ ID NO: 1. In other specific embodiments, the human ⁇ tailpiece is conjugated to the C-terminus of the IgG C H 2 region.
  • the human ⁇ tailpiece is conjugated to the C-terminus of the IgG C H 3 region.
  • the IgG is a human IgG.
  • the human IgG is a human IgG1.
  • the human IgG is a human IgG2.
  • the human IgG is a human IgG3.
  • the human IgG is a human IgG4.
  • the molecule further comprises a binding domain that specifically binds to a target.
  • the binding domain is an antibody fragment.
  • the molecule is an antibody or antigen binding fragment thereof.
  • an oligomer comprising two or more molecules (e.g., two, three, four, five, six, seven, eight, or more) as described herein.
  • an isolated nucleic acid encoding the molecule as described herein.
  • a vector comprising the nucleic acid as described herein.
  • an oligomer which comprises two or more molecules (e.g., two, three, four, five, six, seven, eight, or more), and each molecule comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine.
  • an oligomer which comprises two or more molecules (e.g., two, three, four, five, six, seven, eight, or more), and each molecule comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine.
  • the molecule further comprises an IgG hinge region.
  • the molecule further comprises an IgG C H 3 region.
  • the molecule further comprises a human ⁇ tailpiece.
  • the molecule further comprises an IgG C H 1 region.
  • the human ⁇ tailpiece comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO: 1.
  • the human ⁇ tailpiece is conjugated to the C-terminus of the IgG C H 2 region.
  • the human ⁇ tailpiece is conjugated to the C-terminus of the IgG C H 3 region.
  • position 297 in the IgG C H 2 region by EU numbering is substituted to be a glycine (“297G”).
  • position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine (“234A”) and position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine (“235A”).
  • position 228 in the IgG C H 2 region by EU numbering is substituted to be a proline (“228P”).
  • position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine (“234A”) and position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine (“235A”) and one or more of: (a) position 269 in the IgG C H 2 region by EU numbering substituted to be an alanine (“269A”); (b) position 270 in the IgG C H 2 region by EU numbering substituted to be an alanine (“270A”), serine (“270S”), glutamine (“270Q”), glycine (“270G”), threonine (“270T”), valine (“270V”), or lys
  • the molecule comprising the 234A and 235A substitutions further comprises (f) only the 270A substitution, (g) the 270A and 322A substitutions, (h) the 270A and 329A substitutions, (i) the 270A and 331G substitutions, (j) the 270A, 322A and 329A substitutions, (k) the 270A, 322A, and 331G substitutions, (l) the 270A, 329A, and 331G substitutions, or (m) the 270A, 322A, 329A, and 331G substitutions.
  • any one of the foregoing molecules further comprises the 269A substitution and/or the 253C substitution and/or the 309C substitution.
  • a molecule comprising an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine and the IgG C H 2 region further comprises the 234A, 235A, 270A, 329A, and 331G substitutions by EU numbering.
  • the molecule comprises at least one further amino acid substitution.
  • the IgG is a human IgG. In some specific embodiments, the human IgG is a human IgG1. In other specific embodiments, the human IgG is a human IgG2.
  • the human IgG is a human IgG3. In still yet other specific embodiments, the human IgG is a human IgG4. [0023] In some embodiments, the molecule further comprises a binding domain that specifically binds to a target. In some specific embodiments, the binding domain is an antibody fragment. In other embodiments, the molecule is an antibody or antigen binding fragment thereof. [0024] In one embodiment of the molecule provided herein, the molecule exhibits a reduced binding affinity to one or more of human Fc ⁇ RIIIA and/or Fc ⁇ RIIB and/or Fc ⁇ RI compared to a molecule comprising the wildtype IgG Fc region.
  • the molecule exhibits reduced binding to human C1q compared to a molecule comprising the wildtype IgG Fc region.
  • the oligomer is a pentamer. In other embodiments, the oligomer is a hexamer.
  • the oligomer is homomeric and the two or more molecules bind to the same target. In other embodiments, the oligomer is heteromeric. In some specific embodiments, the two or more molecules bind to two or more different targets.
  • a pharmaceutical composition which comprises the molecule as described herein, the oligomer as described herein, the isolated nucleic acid as described herein, or the vector as described herein, and a pharmaceutically acceptable excipient.
  • a method for treating a disease or disorder in a subject comprising administering to the subject the pharmaceutical composition as described herein.
  • a method of making an oligomer comprising two or more molecules each comprising an IgG C H 2 region, and the method comprises introducing into each molecule cysteine amino acid substitution at position 253 by EU numbering in the IgG C H 2 region.
  • a method of making an oligomer comprising two or more molecules each comprising an IgG C H 2 region, and the method comprises introducing into each molecule cysteine amino acid substitution at position 309 by EU numbering in the IgG C H 2 region.
  • a method of producing oligomerized molecules which comprises (i) introducing the vector of claim 40 to a host cell; (ii) cultivating the host cell under suitable conditions for the production of the oligomerized molecules; and (iii) purifying the oligomerized molecules. 4.
  • FIGS.1A-1B depict analysis of transient expression of human IgG1 and variants thereof.
  • FIG.1A shows analysis of transient expression of different human IgG1 (huIgG1) Fc- ⁇ tp and huIgG1 Fc- ⁇ tp variants and controls by SDS-PAGE.
  • Lane 1 depicts protein ladder; lane 2 depicts IgM positive control (250 mg/L); lane 3 depicts Expi293 expression system without transfection; lane 6 depicts huIgG1 Fc with I253C mutation followed by an immunoglobulin ⁇ chain tail piece ( ⁇ tp) (SEQ ID NO: 3); lane 7 depicts huIgG1 Fc with I253C mutation followed by an immunoglobulin ⁇ chain tail piece ( ⁇ tp); lane 8 depicts huIgG1 Fc with Q438C mutation followed by a ⁇ tp (SEQ ID NO: 5); lane 9 depicts huIgG1 Fc with Q438C mutation followed by an ⁇ tp; lane 10 depicts huIgG1 Fc with Y436C mutation followed by a ⁇ tp (SEQ ID NO: 4); lane 11 depicts huIgG1 Fc with Y436C mutation followed by an ⁇ tp; lane 12
  • FIG.1B shows analysis of PAGE.
  • Lane 1 depicts protein ladder;
  • lane 2 depicts purified IgG1 positive control (100 mg/L);
  • lane 3 depicts purified IgG1 positive control (200 mg/L);
  • lane 4 depicts conditioned medium from Expi293 expression system without transfection;
  • lane 5 depicts huIgG1 Fc with H310C mutation followed by an immunoglobulin ⁇ chain tail piece ( ⁇ tp) [huIgG1 Fc (H310C)- ⁇ tp];
  • lane 6 depicts huIgG1 Fc with L251C, I253G and S254C mutations followed by an immunoglobulin ⁇ chain tail piece ( ⁇ tp) [huIgG1 Fc (L251C, I253G, and S254C)- ⁇ tp];
  • lane 7 depicts huIgG1 Fc with S254C and N434C mutations followed by an
  • FIG.1C shows analysis of transient expression of different human IgG1 (huIgG1) Fc- ⁇ tp variants and controls by SDS-PAGE.
  • Lane 1 depicts protein ladder;
  • lane 2 depicts purified IgG1 positive control (100 mg/L);
  • lane 3 depicts purified IgG1 positive control (200 mg/L);
  • lane 4 depicts conditioned medium from Expi293 expression system without transfection;
  • lane 5 depicts huIgG1 Fc with N286C mutation followed by an immunoglobulin ⁇ chain tail piece ( ⁇ tp) [huIgG1 Fc (N286C)- ⁇ tp].
  • FIGS.2A-2C depict purification of huIgG1 Fc with I253C followed by a ⁇ tp (huIgG1 Fc (I253C)- ⁇ tp).
  • FIG.2A shows purification with MabSelect TM protein A resin.
  • FIG. 2B shows purification with CaptureSelectTM FcXL Affinity Matrix.
  • FIG.2C shows purification with anion exchange chromatography.
  • FIGS.3A-3D depict characterization of huIgG1 Fc (I253C)- ⁇ tp.
  • FIG.3A shows intact mass analysis of purified huIgG1 Fc (I253C)- ⁇ tp
  • FIG.3B shows HPLC-SEC analysis of huIgG1 Fc (I253C)- ⁇ tp; BioRad gel filtration protein standard
  • FIG.3C shows HPLC-SEC analysis of huIgG1 Fc (I253C)- ⁇ tp; quantification of purity by integration.
  • FIG.3D shows thermal stability analysis of huIgG1 Fc (I253C)- ⁇ tp by nano-format of Differential Scanning Fluorimetry (nano DSF).
  • FIG.4 depicts expression of IgG hexamers by SDS-PAGE.
  • Lane 1 depicts purified human IgG1 positive control (100 mg/L); lane 2 depicts purified human IgG1 hexamer positive control (100 mg/L); lane 3 depicts conditioned medium from Expi293 expression system without transfection; lane 4 depicts anti-beta blotho huIgG1 hexamer; lane 5 depicts anti-GDNF Family Receptor Alpha Like (GFRAL) huIgG1 hexamer; lane 6 depicts anti-vascular endothelial growth factor (VEGF) huIgG1 hexamer.
  • GFRAL anti-GDNF Family Receptor Alpha Like
  • FIGS.5A-5B depict analysis of transient expression of different human IgG1 (huIgG1) Fc- ⁇ tp effectorless variants and controls by SDS-PAGE.
  • FIG.5A depicts the SDS- PAGE analysis.
  • FIG.5B provides description of various samples as seen in FIG.5A.
  • FIG.6 depicts binding of different human IgG1 (huIgG1) Fc- ⁇ tp effectorless variants to human C1q. Readings were made at 650nm on a spectrometer after incubation of the purified variants with serial dilutions of human C1q.
  • FIG.7 depicts binding of different human IgG1 (huIgG1) Fc- ⁇ tp effectorless variants to human Fc ⁇ R1 dpecited using Meso Scale Discovery (MSD) assay.
  • FIG.8 depicts binding of two huIgG1 Fc (L309C)- ⁇ tp effectorless variants to human C1q.
  • FIGS.9A-9B depict binding of two huIgG1 Fc (L309C)- ⁇ tp effectorless variants to human Fc ⁇ R1 as measured by Biacore analysis.
  • FIG.9A depicts PSC-0821 binding to human Fc ⁇ R1.
  • FIG.9B depicts PSC-1538 binding to human Fc ⁇ R1.
  • the present disclosure is based in part on the surprising finding that certain amino acid substitution at an antibody constant region (i.e., in IgG C H 2 region), alone and in various combinations, reduce various immune effector functions. These mutations are particularly useful when used in monomeric and oligomeric (e.g., pentameric and hexameric) arrangements.
  • amino acid substitutions at an antibody constant region i.e., substitution to cysteine at position 253 by EU numbering in IgG C H 2 region or substitution to cysteine at position 309 by EU numbering in IgG C H 2 region
  • substitution to cysteine at position 253 by EU numbering in IgG C H 2 region or substitution to cysteine at position 309 by EU numbering in IgG C H 2 region that can increase the formation of oligomers such as hexamers as demonstrated in Section 6 below.
  • the number of inter-molecule bonds among congregate of molecules determines the level of the complexity of the corresponding oligomers that can possibly be formed.
  • oligomers with more complex structures may be formed among congregate of such molecules.
  • the nature and location of the amino acid residues within a molecule that can form inter-molecule bonds significantly impact the efficiency for formation of oligomers.
  • an oligomer formed by the disclosed molecules is also provided herein.
  • a pharmaceutical composition comprising the molecules and the oligomers provided herein.
  • a kit comprising the compositions provided herein.
  • nucleic acid molecules, vectors and host cells that express the disclosed molecules.
  • methods of generating the molecules and oligomers provided herein are also provided herein.
  • the bond is formed upon oxidation of the two thiols, thus linking the two residues and their respective main body of the molecules by the covalent disulfide bond.
  • disulfide bonds are typically in a configuration of six atoms, C ⁇ – C ⁇ – S ⁇ – S’ ⁇ – C’ ⁇ – C’ ⁇ , linking two amino acid residues.
  • EU numbering refers to a widely used numbering scheme for the constant domain (including portions of the C H 1, hinge, and the Fc). See Kabat, E.A.
  • oligomer As used herein, the term “oligomer,” “oligomerized molecule,” or “multimerized molecule” used herein refer to a structure of a complex that consists of several similar or identical repeating units.
  • the oligomer can be dimer, triplet, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer, twelve-mer, thirteen-mer, fourteen-mer, fifteen-mer, sixteen-mer, seventeen-mer, eighteen-mer, nineteen-mer, twenty-mer, etc.
  • antibody immunoglobulin
  • Ig immunoglobulin
  • monoclonal antibodies including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies
  • antibody compositions with polyepitopic or monoepitopic specificity polyclonal or monovalent antibodies
  • multivalent antibodies multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments thereof (e.g., domain antibodies) as described below.
  • the term also includes all antibody variants including those carrying various mutations (e.g., in constant regions or Fc region) and other modifications (e.g., with additional peptide sequence to the C-terminus or N-terminus).
  • An antibody can be human, humanized, chimeric and/or affinity matured, as well as an antibody from other species, for example, mouse, rabbit, llama, etc.
  • antibody is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed.1995); and Kuby, Immunology (3d ed.1997).
  • Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies including from Camelidae species (e.g., llama or alpaca) or their humanized variants, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived.
  • synthetic antibodies recombinantly produced antibodies
  • single domain antibodies including from Camelidae species (e.g., llama or alpaca) or their humanized variants
  • intrabodies e.g., anti-idiotypic (anti-Id) antibodies
  • functional fragments e.g., antigen-binding fragments
  • Non-limiting examples of functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab’) fragments, F(ab) 2 fragments, F(ab’) 2 fragments, disulfide- linked Fvs (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody.
  • scFv single-chain Fvs
  • Fab fragments F(ab’) fragments, F(ab) 2 fragments, F(ab’) 2 fragments
  • dsFv disulfide- linked Fvs
  • antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody).
  • an antigen e.g., one or more CDRs of an antibody.
  • antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol.178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990).
  • the antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.
  • Antibodies may be agonistic antibodies or antagonistic antibodies.
  • Antibodies may be neither agonistic nor antagonistic.
  • An “antigen” is a structure to which an antibody can selectively bind.
  • a target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide.
  • an antigen is associated with a cell, for example, is present on or in a cell.
  • the terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions.
  • a complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of dissociation rate (k off ) to association rate (k on ) of a binding molecule (e.g., an antibody) to a monovalent antigen (koff/kon) is the dissociation constant K D , which is inversely related to affinity.
  • K D the dissociation constant
  • the value of K D varies for different complexes of antibody and antigen and depends on both k on and k off .
  • the dissociation constant K D for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.
  • the affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen.
  • a binding molecule or antigen binding domain that binds to or specifically binds to an antigen can be identified, for example, by immunoassays, Octet ® , Biacore ® , or other techniques known to those of skill in the art.
  • a binding molecule or antigen binding domain binds to or specifically binds to an antigen when it binds to an antigen with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme linked immunosorbent assay
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background.
  • the extent of binding of a binding molecule or antigen binding domain to a “non-target” protein is less than about 10% of the binding of the binding molecule or antigen binding domain to its particular target antigen, for example, as determined by fluorescence activated cell sorting (FACS) analysis or RIA.
  • a binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding the antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and/or diagnostic agent in targeting the antigen.
  • a binding molecule or antigen binding domain that binds to an antigen has a dissociation constant (K D ) of less than or equal to 1 ⁇ M, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM.
  • K D dissociation constant
  • a binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among the antigen from different species.
  • affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or an Fc receptor).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody/Fc receptor or antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant ( K D ).
  • the antibodies or antigen binding fragments of the molecules described herein can comprise “chimeric” sequences in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No.
  • the antibodies or antigen binding fragments of the molecules described herein can comprise portions of “humanized” forms of nonhuman (e.g., murine) antibodies that are chimeric antibodies that include human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • a nonhuman species e.g., donor antibody
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibodies or antigen binding fragments of the molecules described herein can comprise portions of a “fully human antibody” or “human antibody,” wherein the terms are used interchangeably herein and refer to an antibody that comprises a human variable region and, for example, a human constant region. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin. “Fully human” antibodies, in certain embodiments, can also encompass antibodies which bind polypeptides and are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al.
  • a “human antibody” is one that possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol.227:381; Marks et al., 1991, J. Mol.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol.6(5):561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. Pat. Nos.6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., 2006, Proc. Natl. Acad. Sci.
  • a typical 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the ⁇ and ⁇ chains and four CH domains for ⁇ and ⁇ isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • the VL is aligned with the VH
  • the CL is aligned with the first constant domain of the heavy chain (CH1).
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • the pairing of a VH and VL together forms a single antigen-binding site.
  • Fab refers to an antibody region that binds to antigens.
  • a conventional IgG usually comprises two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and the light chain.
  • variable region and the constant region of the heavy chain in a Fab region are VH and CH1 regions
  • variable region and the constant region of the light chain in a Fab region are VL and CL regions.
  • the VH, CH1, VL, and CL in a Fab region can be arranged in various ways to confer an antigen binding capability according to the present disclosure.
  • VH and CH1 regions can be on one polypeptide
  • VL and CL regions can be on a separate polypeptide, similarly to a Fab region of a conventional IgG.
  • VH, CH1, VL and CL regions can all be on the same polypeptide and oriented in different orders.
  • variable region refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable region of the heavy chain may be referred to as “VH.”
  • the variable region of the light chain may be referred to as “VL.”
  • variable refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen.
  • variable regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions that are each about 9-12 amino acids long.
  • the variable regions of heavy and light chains each comprise four FRs, largely adopting a ⁇ sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases form part of, the ⁇ sheet structure.
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed.1991)).
  • the constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • the variable regions differ extensively in sequence between different antibodies.
  • the variable region is a human variable region.
  • the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ), and mu ( ⁇ ), based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: ⁇ , ⁇ , and ⁇ contain approximately 450 amino acids, while ⁇ and ⁇ contain approximately 550 amino acids.
  • light chain when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • CDR hypervariable region
  • HVR immunoglobulin
  • L1 variable region sequence
  • CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems.
  • the term “constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the term refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site.
  • the constant region may contain the C H 1, C H 2, and C H 3 regions of the heavy chain and the C L region of the light chain.
  • IgG C H 1 As used herein, the terms “IgG C H 1,” “IgG C H 2,” and “IgG C H 3” refer to the commonly defined first, second and third domains, respectively, in the constant region of a heavy chain of IgG.
  • a heavy chain from an IgG usually contains three constant domains IgG C H 1, C H 2, C H 3, and a spacer hinge region between the C H 1 and C H 2 domains.
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl- terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • a “functional Fc region” possesses an “effector function” of a native sequence Fc region.
  • effector functions include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor), etc.
  • Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those skilled in the art.
  • a “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification (e.g., substituting, addition, or deletion).
  • the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, or from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of a parent polypeptide.
  • the variant Fc region herein can possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, or at least about 90% homology therewith, for example, at least about 95% homology therewith.
  • the term “framework” or “FR” refers to those variable region residues flanking the CDRs.
  • FR residues are present, for example, in chimeric, humanized, human, domain antibodies (e.g., single domain antibodies), diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.
  • an “epitope” is a term in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single domain antibody sequence) can specifically bind.
  • An epitope can be a linear epitope or a conformational, non- linear, or discontinuous epitope.
  • an epitope can be contiguous amino acids of the polypeptide (a “linear” epitope) or an epitope can comprise amino acids from two or more non-contiguous regions of the polypeptide (a “conformational,” “non-linear” or “discontinuous” epitope).
  • a linear epitope may or may not be dependent on secondary, tertiary, or quaternary structure.
  • a binding molecule binds to a group of amino acids regardless of whether they are folded in a natural three dimensional protein structure.
  • a binding molecule requires amino acid residues making up the epitope to exhibit a particular conformation (e.g., bend, twist, turn or fold) in order to recognize and bind the epitope.
  • the term “targeting region” or “binding domain” is used herein in broadest sense and refers to a region of a molecule that can specifically bind to a target (or an antigen).
  • the targeting region can be the antigen-binding fragment without a C H 1 region, or any reformatted antigen-binding fragment such as single-chain variable fragment (scFv), any polypeptide that is non-immunoglobulin but forms specific binding to a target via enzyme-substrate, receptor-ligand or other protein-protein interactions.
  • the targeting region can also be a sequence of nucleic acids that forms specific binding to the target via reverse complementary binding.
  • the targeting region can also be an entity that can form any covalent or non-covalent connection with a target.
  • the targeting region or binding domain is derived from an antibody such as antigen binding domain or fragment of antibody.
  • the terms “antigen-binding fragment,” “antigen-binding domain,” “antigen-binding region,” and similar terms when used in the context of an antibody refer to that portion of the antibody, which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the CDRs).
  • Antigen binding fragment as used herein include “antibody fragment,” which comprise a portion of an intact antibody, such as the antigen binding or variable region of the intact antibody. Examples of antibody fragments are described above, and include, without limitation, Fab, Fab’, F(ab’)2, and Fv fragments; diabodies and di-diabodies; single-chain antibody molecules; dual variable domain antibodies; single variable domain antibodies (sdAbs); and multispecific antibodies formed from antibody fragments.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non- amino acids.
  • polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a “polypeptide” can occur as a single chain or as two or more associated chains.
  • amino acid substitution refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different “replacement” amino acid residue.
  • the replacement residue or residues may be “naturally occurring amino acid residues” (i.e.
  • alanine (Ala): arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln): glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val).
  • non-naturally occurring amino acid residue refers to a residue, other than those naturally occurring amino acid residues listed above, which is able to covalently bind adjacent amino acid residues(s) in a polypeptide chain.
  • non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman, et al., Meth Enzym.202 (1991) 301-336.
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” used herein refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express FcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
  • FcRs e.g. Natural Killer (NK) cells, neutrophils, and macrophages
  • the primary cells for mediating ADCC NK cells, express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • Antibody-dependent cellular phagocytosis and “ADCP” refer to a process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils and dendritic cells) that bind to an immunoglobulin Fc region.
  • phagocytic immune cells e.g., macrophages, neutrophils and dendritic cells
  • C1q is a polypeptide that includes a binding site for the Fc region of an immunoglobulin. C1q together with two serine proteases, C1r and C1s, forms the complex C1, the first component of the complement dependent cytotoxicity (CDC) pathway. Human C1q can be purchased commercially from, e.g. Quidel, San Diego, California.
  • complement-dependent cytotoxicity refers to a mechanism for inducing cell death in which an Fc effector domain(s) of a target-bound antibody activates a series of enzymatic reactions culminating in the formation of holes in the target cell membrane.
  • antigen-antibody complexes such as those on antibody-coated target cells bind and activate complement component C1q which in turn activates the complement cascade leading to target cell death.
  • Activation of complement may also result in deposition of complement components on the target cell surface that facilitate ADCC by binding complement receptors (e.g., CR3) on leukocytes.
  • complement receptors e.g., CR3
  • Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP phagocytosis
  • B cell receptor e.g. B cell receptor
  • a “reduced effector function” as used herein refers to a reduction of a specific effector function, like for example ADCC or CDC, in comparison to a control (for example a polypeptide with a wildtype Fc region), by at least 20% and a “strongly reduced effector function” as used herein refers to a reduction of a specific effector function, like for example ADCC or CDC, in comparison to a control, by at least 50%.
  • “Polynucleotide” or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • Oligonucleotide refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length.
  • oligonucleotide and “polynucleotide” are not mutually exclusive.
  • a cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced.
  • the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction.
  • RNA transcripts The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”
  • An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an “isolated” nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • one or more nucleic acid molecules encoding a single domain antibody or an antibody as described herein are isolated or purified.
  • nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule may include isolated forms of the molecule.
  • a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • vector refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding an antibody or antigen binding fragment as described herein, in order to introduce a nucleic acid sequence into a host cell.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selection control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art.
  • both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.
  • nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA
  • immunoblotting for expression of gene products or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • the term “host cell” refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences.
  • Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the term “specificity” refers to selective recognition of an antigen binding molecule (such as an antibody) for a particular epitope of an antigen. Natural antibodies, for example, are monospecific.
  • the term “multispecific” as used herein denotes that an antigen binding protein has two or more antigen-binding sites of which at least two bind different antigens.
  • Bispecific as used herein denotes that an antigen binding molecule has two different antigen-binding specificities.
  • the term “monospecific” used herein denotes an antigen binding molecule that has one or more binding sites each of which bind the same antigen.
  • the term “valent” as used herein denotes the presence of a specified number of binding sites in an antigen binding molecule.
  • a natural antibody for example or a full length antibody has two binding sites and is bivalent.
  • the terms “trivalent”, “tetravalent”, “pentavalent” and “hexavalent” denote the presence of two binding site, three binding sites, four binding sites, five binding sites, and six binding sites, respectively, in an antigen binding molecule.
  • the term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • excipient means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material.
  • Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof.
  • encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing
  • excipient can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.
  • excipients are pharmaceutically acceptable excipients.
  • Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • a pharmaceutically acceptable excipient is an aqueous pH buffered solution.
  • excipients are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Water is an exemplary excipient when a composition (e.g., a pharmaceutical composition) is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, particularly for injectable solutions.
  • An excipient can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • Oral compositions can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.
  • the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.
  • the term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • a molecule capable of forming connections with two or more other molecules of the same or similar structure, thus forming oligomers comprising two or more units. More specifically, in some embodiments, the molecule provided herein comprises an IgG C H 2 region in which the amino acid residue at position 253 (according to EU numbering) is substituted to be a cysteine residue, thereby generating a site capable of forming a disulfide bond with another molecule having the same amino acid substitution.
  • the molecule provided herein comprises an IgG C H 2 region in which the amino acid residue at position 309 (according to EU numbering) is substituted to be a cysteine residue, thereby generating a site capable of forming a disulfide bond with another molecule having the same amino acid substitution.
  • the molecule provided herein in addition to the cysteine residue at position 253 or 309 of C H 2 region, the molecule provided herein comprises at least one other site capable of forming connection with other molecules of the same or similar structure
  • the type of connection provided herein can be covalent or non-covalent, and the connection provided herein can be direct or indirect.
  • the connection provided herein is a disulfide bond via cysteine residues.
  • the cysteine residues can be introduced into the molecule, for example, by point mutations or by adding a tailpiece. Alternatively, the cysteine residues may be present naturally in antibody region, such as a hinge region. In some embodiments, the above properties are presented in the molecule in combination. [0093] In some embodiments, the type of connections between the molecules provided herein is via covalent bonds. In some specific embodiments, the type of connections between the molecules provided herein is via disulfide bonds. In some other specific embodiments, the type of connections between the molecules provided herein is via electron sharing. In other embodiments, the type of connections between the molecules provided herein is via non-covalent bonds. In some specific embodiments, the type of connections between the molecules provided herein is via hydrogen bonds.
  • the type of connections between the molecules provided herein is via ionic interactions. In some other specific embodiments, the type of connections between the molecules provided herein is via Van der Waals forces. In some other specific embodiments, the type of connections between the molecules provided herein is via hydrophobic bonds. [0094] In some embodiments, the molecule provided herein forms direct connections with each other. In other embodiments, the molecule provided herein forms indirect connections with each other. In some specific embodiments, the molecule provided herein connects with each other via directly formed disulfide bonds. In other embodiments, the molecule provided herein connects with each other via indirectly formed disulfide bonds.
  • the molecule provided herein connects with each other via one or more connecting sites, wherein the connecting molecule forms disulfide bonds with the molecules provided herein.
  • the connecting molecule is the J chain that is found in polymeric IgA and IgM.
  • provided herein is a molecule that forms connections with each other via disulfide bonds, which can be formed between two natural amino acids or unusual amino acids.
  • the connections formed between the molecules provided herein are disulfide bonds between two natural amino acids.
  • the connections formed between the molecules provided herein are disulfide bonds between two cysteine residues.
  • the connections formed between the molecules provided herein are disulfide bonds between two unusual amino acids.
  • the unusual amino acids each process a reactive sulfhydryl group.
  • the unusual amino acids are tyrosine derivatives with para-substituted aliphatic thiols of various lengths (see Liu et al., PNAS, 113 (21) 5910-5915 (2016)).
  • Point Mutations for Forming Disulfide Bonds [0096]
  • provided herein is a molecule that forms connections with each other via disulfide bonds between introduced point mutations.
  • an alanine is substituted with a cysteine.
  • an arginine is substituted with a cysteine.
  • an asparagine is substituted with a cysteine.
  • an aspartic is substituted with a cysteine.
  • a glutamine is substituted with a cysteine.
  • a glutamic acid is substituted with a cysteine.
  • a glycine is substituted with a cysteine.
  • a histidine is substituted with a cysteine.
  • an isoleucine is substituted with a cysteine.
  • a leucine is substituted with a cysteine.
  • a lysine is substituted with a cysteine.
  • a methionine is substituted with a cysteine.
  • a phenylalanine is substituted with a cysteine.
  • a proline is substituted with a cysteine.
  • a serine is substituted with a cysteine.
  • a threonine is substituted with a cysteine.
  • a tryptophan is substituted with a cysteine.
  • a tyrosine is substituted with a cysteine.
  • a valine is substituted with a cysteine.
  • the location of the introduced point mutation satisfies geometric constraints and parameters for stable disulfide bonds.
  • the location of the introduced point mutation ensures a close distance between C ⁇ - C ⁇ ’ in the formed disulfide bond.
  • the location of the introduced point mutation ensures a close distance between C ⁇ - C ⁇ ’ in the formed disulfide bond.
  • the location of the introduced point mutation ensures a proper bond angel between C ⁇ - C ⁇ - S ⁇ in the formed disulfide bond.
  • the location of the introduced point mutation ensures a proper bond angel between C ⁇ - S ⁇ – S’ ⁇ in the formed disulfide bond. In yet other specific embodiments, the location of the introduced point mutation ensures a proper rotation angel of the C ⁇ atoms about the S–S bond in the formed disulfide bond.
  • the molecule provided herein comprises in an IgG C H 1 region with a point mutation to cysteine. [0098] In some specific embodiments, the molecule provided herein comprises in an IgG C H 2 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgG C H 3 region with a point mutation to cysteine.
  • the molecule provided herein comprises in a hinge region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgE C ⁇ 1 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgE C ⁇ 2 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgE C ⁇ 3 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgE C ⁇ 4 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgD C H 1 region with a point mutation to cysteine.
  • the molecule provided herein comprises in an IgD C H 2 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgD C H 3 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgM C ⁇ 1 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgM C ⁇ 2 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgM C ⁇ 3 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgM C ⁇ 4 region with a point mutation to cysteine.
  • the molecule provided herein comprises in an IgA C ⁇ 1 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgA C ⁇ 2 region with a point mutation to cysteine. In some specific embodiments, the molecule provided herein comprises in an IgA C ⁇ 3 region with a point mutation to cysteine. In a specific embodiment, the molecule provided herein comprises in an IgG C H 2 region with a point mutation at location Ile 253 by EU numbering to cysteine. In a specific embodiment, the molecule provided herein comprises in an IgG C H 2 region with a point mutation at location Leu 309 by EU numbering to cysteine.
  • the molecule provided herein comprises two or more the above described point mutations. In some embodiments, the molecule provided herein comprises a point mutation at location Ile 253 by EU numbering to cysteine in an IgG C H 2 region and at least one other point mutation described above. [00100] In some embodiments, the molecule provided herein comprises two or more the above described point mutations. In some embodiments, the molecule provided herein comprises a point mutation at location Leu 309 by EU numbering to cysteine in an IgG C H 2 region and at least one other point mutation described above.
  • the molecule provided herein further comprises an added tailpiece which facilitates the forming of stable disulfide bonds with each other.
  • the added tailpiece comprises the eighteen amino acid long ⁇ tailpiece (SEQ ID NO: 1).
  • the added tailpiece comprises 99% identity to SEQ ID NO: 1 and retains the cysteine residue in the SEQ ID NO: 1.
  • the added tailpiece comprises 95% identity to SEQ ID NO: 1 and retains the cysteine residue in the SEQ ID NO: 1.
  • the added tailpiece comprises 90% identity to SEQ ID NO: 1 and retains the cysteine residue in the SEQ ID NO: 1.
  • the added tailpiece comprises 85% identity to SEQ ID NO: 1 and retains the cysteine residue in the SEQ ID NO: 1. In other embodiments, the added tailpiece comprises 70% identity to SEQ ID NO: 1 and retains the cysteine residue in the SEQ ID NO: 1. In other embodiments, the added tailpiece comprises 65% identity to SEQ ID NO: 1 and retains the cysteine residue in the SEQ ID NO: 1. In other embodiments, the added tailpiece comprises 50% identity to SEQ ID NO: 1 and retains the cysteine residue in the SEQ ID NO: 1. [00102] In some embodiments, the added tailpiece comprises the tailpiece in IgA ⁇ heavy chain ( ⁇ tailpiece).
  • the added tailpiece comprises 99% identity to ⁇ tailpiece and retains the cysteine residue in the ⁇ tailpiece. In other embodiments, the added tailpiece comprises 95% identity to ⁇ tailpiece and retains the cysteine residue in the ⁇ tailpiece. In other embodiments, the added tailpiece comprises 90% identity to ⁇ tailpiece and retains the cysteine residue in the ⁇ tailpiece. In other embodiments, the added tailpiece comprises 85% identity to ⁇ tailpiece and retains the cysteine residue in the ⁇ tailpiece. In other embodiments, the added tailpiece comprises 80% identity to ⁇ tailpiece and retains the cysteine residue in the ⁇ tailpiece.
  • the added tailpiece comprises 75% identity to ⁇ tailpiece and retains the cysteine residue in the ⁇ tailpiece. In other embodiments, the added tailpiece comprises 70% identity to ⁇ tailpiece and retains the cysteine residue in the ⁇ tailpiece.
  • certain polypeptide described herein comprises amino acid sequences with certain percent identity relative to a reference polypeptide. The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci.
  • Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res.25:33893402 (1997).
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Altschul BLAST
  • Gapped BLAST Altschul BLAST
  • PSI Blast programs the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).
  • NCBI National Center for Biotechnology Information
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAM120 weight residue table When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. [00104] In other embodiments, provided herein is a molecule with an added short polypeptide containing one or more cysteine. In some specific embodiments, the added short polypeptide is less than five amino acids long. In some specific embodiments, the added short polypeptide is six, seven, eight, nine, or ten amino acids long.
  • the added short polypeptide is eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty amino acids long. In some specific embodiments, the added short polypeptide is twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty amino acids long. In some specific embodiments, the added short polypeptide is more than thirty amino acids long. [00105] Provided herein is a molecule with an added tailpiece in a location which facilitates the forming of stable disulfide bonds with each other. In some embodiments, the tailpiece is added to the C-terminus of the molecule.
  • the tailpiece is added to the C-terminus of the C H 1 region. In some specific embodiments, the tailpiece is added to the C- terminus of the hinge region. In some specific embodiments, the tailpiece is added to the C- terminus of the C H 2 region. In some specific embodiments, the tailpiece is added to the C- terminus of the C H 3 region. In some embodiments, the tailpiece is added to the N-terminus of the molecule. In some specific embodiments, the tailpiece is added to the N-terminus of the C H 1 region. In some specific embodiments, the tailpiece is added to the N-terminus of the hinge region. In some specific embodiments, the tailpiece is added to the N-terminus of the C H 2 region.
  • the tailpiece is added to the N-terminus of the C H 3 region.
  • Configurations of the Engineered Molecule [00106]
  • the molecule provided herein has both two or more point mutations described above and an added tailpiece described above.
  • the molecule provided herein has both a point mutation described above and two or more added tailpieces described above.
  • the molecule provided herein has both two or more point mutations described above and two or more added tailpieces described above.
  • a molecule comprising different antibody constant regions that are engineered for multimerization.
  • the molecules described herein comprise one or more point mutation(s) in C H 2 region and thus engineered disulfide bond(s) can form between such molecules.
  • the molecules described herein comprise one or more added tailpiece(s) and thus engineered disulfide bond(s) can form between such molecules.
  • the molecules described herein comprise one or more point mutation(s) in C H 2 region and one or more added tailpiece(s) and thus multiple engineered disulfide bonds can form between such molecules.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine. In some specific embodiments, the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, and an IgG hinge region. In some specific embodiments, the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, and an IgG C H 3 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, and an IgG hinge region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, and an IgG C H 3 region. In some specific embodiments, the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, and an IgG C H 1 region. [00110] In some specific embodiments, the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, an IgG hinge, and C H 3 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, an IgG hinge, and an IgG C H 1 region. In some specific embodiments, the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, an IgG C H 3 region, and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, an IgG hinge, an IgG C H 3 region, and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, an IgG hinge, and C H 3 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, an IgG hinge, and an IgG C H 1 region. In some specific embodiments, the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, an IgG C H 3 region, and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, an IgG hinge, an IgG C H 3 region, and an IgG C H 1 region.
  • the order of different regions can follow the order in a wild-type IgG, or can be different from the order in a wild-type IgG.
  • the molecules described herein further comprise one or more added tailpiece(s) as described above.
  • the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1) or a variant thereof.
  • the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG hinge region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG C H 2 region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG C H 3 region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG C H 1 region.
  • the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge, and an IgG C H 2 region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, and an IgG C H 3 region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, and an IgG C H 1 region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG C H 2 region, and an IgG C H 3 region.
  • the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG C H 2 region, and an IgG C H 1 region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG C H 3 region, and an IgG C H 1 region. [00115] In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 2 region, and an IgG C H 3 region.
  • the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 2 region, and an IgG C H 1 region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 3 region, and an IgG C H 1 region. In some specific embodiments, the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG C H 2 region, an IgG C H 3 region, and an IgG C H 1 region.
  • the molecule described herein comprises a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 2 region, an IgG C H 3 region, and an IgG C H 1 region.
  • the order of different regions can follow the order in a wild-type IgG, or can be different from the order in a wild-type IgG, and the human ⁇ tailpiece can be conjugated to the C-terminus or N-terminus of the present molecules.
  • the molecules described herein comprise one or more point mutation(s) and one or more added tailpiece(s) and thus multiple engineered disulfide bonds can form between such molecules.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, and a human ⁇ tailpiece (SEQ ID NO: 1). In some specific embodiments, the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG hinge region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG C H 3 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, and an IgG C H 3 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), an IgG C H 3 region, and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 253 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 3 region, and an IgG C H 1 region.
  • the order of different regions can follow the order in a wild-type IgG, or can be different from the order in a wild-type IgG, and the human ⁇ tailpiece can be conjugated to the C-terminus or N-terminus of the molecules.
  • the molecules described herein comprise one or more point mutation(s) and one or more added tailpiece(s) and thus multiple engineered disulfide bonds can form between such molecules.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, and a human ⁇ tailpiece (SEQ ID NO: 1).
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG hinge region. In some specific embodiments, the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG C H 3 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, and an IgG C H 3 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), an IgG C H 3 region, and an IgG C H 1 region.
  • the molecule described herein comprises an IgG C H 2 region, wherein position 309 in the IgG C H 2 region by EU numbering is substituted to be a cysteine, a human ⁇ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 3 region, and an IgG C H 1 region.
  • the order of different regions can follow the order in a wild-type IgG, or can be different from the order in a wild-type IgG, and the human ⁇ tailpiece can be conjugated to the C-terminus or N-terminus of the molecules.
  • variations may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule.
  • the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues.
  • the variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • the present molecules may further comprises other IgG regions.
  • the molecule provided herein comprises IgG C H 1 region.
  • the molecule provided herein comprises IgG hinge region.
  • the molecule provided herein comprises IgG C H 3 region.
  • the molecule provided herein comprises IgG C H 1 and C H 2 regions.
  • the molecule provided herein comprises IgG C H 1 and C H 3 regions. In some embodiments, the molecule provided herein comprises IgG hinge and C H 2 regions. In some embodiments, the molecule provided herein comprises IgG hinge and C H 3 regions. In some embodiments, the molecule provided herein comprises IgG C H 2 and C H 3 regions. In some embodiments, the molecule provided herein comprises IgG C H 1, hinge, and C H 2 regions. In some embodiments, the molecule provided herein comprises IgG C H 1, hinge, and C H 3 regions. In some embodiments, the molecule provided herein comprises IgG C H 1, C H 2, and C H 3 regions.
  • the molecule provided herein comprises IgG C H 1, hinge, C H 2, and C H 3 regions.
  • the IgG regions disclosed in the preceding paragraph are from human IgG. In some specific embodiments, the IgG regions disclosed in the preceding paragraph are from human IgG1. In some specific embodiments, the IgG regions disclosed in the preceding paragraph are from human IgG2. In some specific embodiments, the IgG regions disclosed in the preceding paragraph are from human IgG3. In some specific embodiments, the IgG regions disclosed in the preceding paragraph are from human IgG4. In some specific embodiments, the IgG regions in one molecule disclosed in the preceding paragraph are mixed with different human IgG isotypes.
  • the IgG regions disclosed in the preceding paragraph are from mouse IgG. In other embodiments, the IgG regions disclosed in the preceding paragraph are from rat IgG. In other embodiments, the IgG regions disclosed in the preceding paragraph are from monkey IgG, donkey IgG, sheep IgG, goat IgG, guinea pig IgG, camel IgG, horse IgG, or chicken IgG. 5.2.2 Molecules Comprising Reduced Effector Functions [00123] In some embodiments, provided herein is a molecule comprising at least one addition, substitution, or deletion of an amino acid.
  • position 297 in the IgG C H 2 region by EU numbering is substituted to be a glycine (“297G”).
  • This aspect may be present alone or in combination with any of the other aspects or embodiments described herein including, for example, monomeric or multimeric molecules comprising the 253C substitution, and/or the 309C substitution, and/or the human ⁇ tailpiece.
  • position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine (“234A”) and position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine (“235A”).
  • position 228 in the IgG C H 2 region by EU numbering is substituted to be a proline (“228P”).
  • This aspect may be present alone or in combination with any of the other aspects or embodiments described herein including, for example, monomeric or multimeric molecules comprising the 253C substitution and/or the 309C substitution, the human ⁇ tailpiece, and/or the 297G substitution.
  • position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine and one or more of: (a) position 269 in the IgG C H 2 region by EU numbering substituted to be an alanine (“269A”); (b) position 270 in the IgG C H 2 region by EU numbering substituted to be an alanine (“270A”), serine, glutamine, glycine, threonine, valine, or lysine; (c) position 322 in the IgG C H 2 region by EU numbering is substituted to be an alanine (“322A”); (d) position 329 in the IgG C H 2 region by EU numbering substituted to be an alanine (“329A”); and/or (e) position 331 in the IgG C H 2 region by
  • the molecule compring the 234A and 235A substitutions further comprises (f) only the 270A substation, (g) the 270A and 322A substitutions, (h) the 270A and 329A substitutions, (i) the 270A and 331G substitutions, (j) the 270A, 322A and 329A substitutions, (k) the 270A, 322A, and 331G substitutions, (l) the 270A, 329A, and 331G substitutions, and (m) the 270A, 322A, 329A, and 331G substitutions.
  • any one of the foregoing molecules further comprises the 269A substitution.
  • a molecule wherein position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • a molecule wherein position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • a molecule wherein position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 322 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • a molecule wherein position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 269 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 322 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be a serine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be a glutamine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be a threonine. In another embodiment, position 270 in the IgG C H 2 region by EU numbering is substituted to be a valine. In one embodiment, position 270 in the IgG C H 2 region by EU numbering is substituted to be a lysine. [00134] In another embodiment, position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine
  • position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine
  • position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • a molecule wherein position 331 in the IgG C H 2 region by EU numbering is substituted to be a serine. In a further embodiment, provided here is a molecule wherein position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine. [00137] In an embodiment, provided here is a molecule position 322 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • a molecule wherein position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 331 in the IgG C H 2 region by EU numbering is substituted to be a serine.
  • position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be a glycine, a serine, a threonine, a glutamine or an alanine.
  • a molecule wherein position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 270 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 329 in the IgG C H 2 region by EU numbering is substituted to be a serine.
  • position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • a molecule wherein position 234 in the IgG C H 2 region by EU numbering is substituted to be an alanine, position 235 in the IgG C H 2 region by EU numbering is substituted to be an alanine and position 329 in the IgG C H 2 region by EU numbering is substituted to be an alanine.
  • position 270 in the IgG C H 2 region by EU numbering is substituted to be a glycine, a serine, a threonine, a glutamine or an alanine.
  • position 331 in the IgG C H 2 region by EU numbering is substituted to be a glycine.
  • the variants of the invention have reduced or ablated affinity for an Fc receptor responsible for an effector function compared to a molecule having the same amino acid sequence as the molecule comprising a Fc variant of the invention but not comprising the addition, substitution, or deletion of at least one amino acid residue to the Fc region (also referred to herein as an “wildtype molecule”).
  • molecules comprising a Fc variant of the invention comprise at least one or more of the following properties: reduced or ablated effector (ADCC and/or CDC and/or ADCP) function, reduced or ablated binding to Fc receptors, reduced or ablated binding to C1q or reduced or ablated toxicities.
  • provided herein is a molecule that exhibits a reduced binding affinity to one or more of Fc receptors. In one embodiment, provided herein is a molecule that exhibits a reduced binding affinity to human Fc ⁇ RIIIA. In one embodiment, provided herein is a molecule that exhibits a reduced binding affinity to human Fc ⁇ RIIB. In one embodiment, provided herein is a molecule that exhibits a reduced binding affinity to human Fc ⁇ RI. In one embodiment, provided herein is a molecule that exhibits a reduced binding affinity to human Fc ⁇ RIIIA and human Fc ⁇ RIIB and human Fc ⁇ RI compared to a molecule comprising the wildtype IgG Fc region.
  • molecules comprising an Fc variant of the invention exhibit affinities for human Fc ⁇ RI that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of a wildtype molecule.
  • molecules comprising an Fc variant of the invention exhibit affinities for human Fc ⁇ RIIB that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of a wildtype molecule.
  • molecules comprising an Fc variant of the invention exhibit affinities for human Fc ⁇ RIIIA that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of a wildtype molecule.
  • molecules provided herein exhibit affinities for Fc ⁇ RI that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than that of a wildtype molecule.
  • molecules provided herein exhibit affinities for Fc ⁇ RIIB that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than that of a wildtype molecule.
  • molecules provided herein exhibit affinities for Fc ⁇ RIIIA that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than that of a wildtype molecule.
  • affinities for Fc ⁇ RIIIA that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than that of a wildtype molecule.
  • molecules comprising an Fc variant of the invention exhibit affinities for C1q receptor that are at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold less than that of a wildtype molecule.
  • molecules provided herein exhibit affinities for C1q that are at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% less than that of a wildtype molecule.
  • the ADCC induced by a molecule provided herein is reduced and in a preferred embodiment the ADCC is reduced to at least 20% of the ADCC induced by the molecule comprising the wildtype Fc polypeptide.
  • the ADCC and CDC induced by the molecule comprising the wildtype Fc polypeptide is decreased or ablated and in a still further aspect the molecule comprising a Fc variant described above exhibit a decreased ADCC, CDC and ADCP compared to the molecule comprising the wildtype Fc region.
  • the ADCC induced by said molecule provided herein is reduced to at least 20% of the ADCC induced by a molecule comprising a wild-type IgG Fc region.
  • the ADCC induced by said molecule provided herein is reduced to at least 30% of the ADCC induced by a molecule comprising a wild-type IgG Fc region.
  • the ADCC induced by said molecule provided herein is reduced to at least 40% of the ADCC induced by a molecule comprising a wild-type IgG Fc region. In one embodiment, the ADCC induced by said molecule provided herein is reduced to at least 50% of the ADCC induced by a molecule comprising a wild-type IgG Fc region. 5.2.3 Binding Molecules [00147] Besides the properties and structures for forming oligomers as disclosed above, the molecules provided herein comprises other properties and functions. In certain embodiments, the present molecules are binding molecules comprising a binding domain. In some embodiments, the present molecules are antibodies or fragments thereof.
  • the binding domain of the molecule provided herein comprises a non-immunoglobulin binding agent. In other embodiments, the binding domain of the molecule provided herein comprises a sequence of nucleic acids that forms specific binding to the target via reverse complementary binding. [00148] In some embodiments, the binding domain of the molecule provided herein comprises an antibody fragment.
  • Exemplary fragments include Fab fragments (e.g., an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab’ (e.g., an antibody fragment containing a single antigen- binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab’)2 (e.g., two Fab’ molecules joined by inter-chain disulfide bonds in the hinge regions of the heavy chains; the Fab’ molecules may be directed toward the same or different epitopes); a bispecific Fab (e.g., a Fab molecule having two antigen binding domains, each of which may be directed to a different epitope); a single chain comprising a variable region, also known as, scFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); a disulfide-linked
  • an antibody is a single chain Fv fragment (scFv) (see, e.g., WO 93/16185; U.S. Pat.
  • Fv and scFv have intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use.
  • scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv (See, e.g., Borrebaeck ed., supra).
  • the antibody fragment may also be a “linear antibody,” for example, as described in the references cited above. Such linear antibodies may be monospecific or multi-specific, such as bispecific.
  • Smaller antibody-derived binding structures are the separate variable domains (V domains) also termed single variable domain antibodies (sdAbs).
  • V-like domains mounted on an Fc equivalent domain structure as part of their immune system.
  • the V-like domains typically display long surface loops, which allow penetration of cavities of target antigens. They also stabilize isolated VH domains by masking hydrophobic surface patches.
  • the antibody fragment in the molecule provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.
  • the antibodies described herein can, for example, include humanized antibodies, e.g., deimmunized or composite human antibodies.
  • a humanized antibody can comprise human framework region and human constant region sequences.
  • a humanized antibody can comprise human constant region sequences.
  • a humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4 (e.g., variants of IgG4 and IgG4 nullbody).
  • a humanized antibody can comprise kappa or lambda light chain constant sequences.
  • Humanized antibodies can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International publication No.
  • a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization may be performed, for example, following the method of Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework.
  • CDR grafting in which the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework.
  • the amino acid sequences of the six CDRs of the parent non-human antibody e.g., rodent
  • SDRs the amino acid sequences of the six CDRs of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity.
  • the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence that is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al., 1993, J. Immunol.151:2296-308; and Chothia et al., 1987, J. Mol. Biol.196:901-17).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
  • the same framework may be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; and Presta et al., 1993, J. Immunol.151:2623-32).
  • the framework is derived from the consensus sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) and VH subgroup III (VHIII).
  • VL6I VL6 subgroup I
  • VHIII VH subgroup III
  • human germline genes are used as the source of the framework regions.
  • FR homology is irrelevant. The method consists of comparison of the non-human sequence with the functional human germline gene repertoire.
  • Those genes encoding the same or closely related canonical structures to the murine sequences are then selected.
  • those with highest homology within the CDRs are chosen as FR donors.
  • the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., 2002, J. Immunol.169:1119-25).
  • antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng.13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol.234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
  • Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes, and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants (Lazar et al., 2007, Mol. Immunol.44:1986-98).
  • Antibody variants may be isolated from phage, ribosome, and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, 2005, Nat. Biotechnol.23:1105-16; Dufner et al., 2006, Trends Biotechnol.24:523-29; Feldhaus et al., 2003, Nat. Biotechnol.21:163-70; and Schlapschy et al., 2004, Protein Eng. Des.
  • residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, 1992, J. Mol. Biol.224:487-99), or from the more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem.272:10678-84).
  • FR shuffling whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall’Acqua et al., 2005, Methods 36:43-60).
  • the libraries may be screened for binding in a two-step process, first humanizing VL, followed by VH.
  • a one-step FR shuffling process may be used.
  • Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physicochemical properties including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., 2007, Mol. Immunol.44:3049-60).
  • the “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non- human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple subclasses with distinct human V-segment CDRs.
  • the “human engineering” method involves altering a non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies.
  • the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk,” “moderate risk,” or “high risk” residues.
  • the classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody’s folding.
  • the particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody’s variable regions with the corresponding region of a specific or consensus human antibody sequence.
  • the amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment.
  • a composite human antibody can be generated using, for example, Composite Human AntibodyTM technology (Antitope Ltd., Cambridge, United Kingdom).
  • variable region sequences are designed from fragments of multiple human antibody variable region sequences in a manner that avoids T cell epitopes, thereby minimizing the immunogenicity of the resulting antibody.
  • Such antibodies can comprise human constant region sequences, e.g., human light chain and/or heavy chain constant regions.
  • the molecule provided herein contains a deimmunized antibody fragment whose T-cell epitopes have been removed.
  • Methods for making deimmunized antibodies have been described. See, e.g., Jones et al., Methods Mol Biol. 2009;525:405-23, xiv, and De Groot et al., Cell. Immunol.244:148-153(2006)).
  • Deimmunized antibodies comprise T-cell epitope-depleted variable regions and human constant regions.
  • VH and VL of an antibody are cloned and T-cell epitopes are subsequently identified by testing overlapping peptides derived from the VH and VL of the antibody in a T cell proliferation assay.
  • T cell epitopes are identified via in silico methods to identify peptide binding to human MHC class II. Mutations are introduced in the VH and VL to abrogate binding to human MHC class II. Mutated VH and VL are then utilized to generate the deimmunized antibody [00169]
  • the molecule described herein is derived from a fully human anti-human antibody. Fully human antibodies may be produced by any method known in the art.
  • Human antibody fragment provided herein can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s).
  • human monoclonal antibodies of the present disclosure can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor, 1984, J. Immunol.133:3001-05; Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987); and Boerner et al., 1991, J. Immunol.147:86-95.
  • transgenic animals e.g., mice
  • transgenic mice that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • Transgenic mice that express human antibody repertoires have been used to generate high-affinity human sequence monoclonal antibodies against a wide variety of potential drug targets (see, e.g., Jakobovits, A., 1995, Curr. Opin. Biotechnol. 6(5):561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol.8(4):455-58; U.S. Pat.
  • the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (e.g., such B lymphocytes may be recovered from an individual or may have been immunized in vitro) (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and U.S. Pat. No.5,750,373).
  • Gene shuffling can also be used to derive human antibodies from non-human, for example, rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody.
  • this method which is also called “epitope imprinting” or “guided selection,” either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras.
  • Examples of guided selection to humanize mouse antibodies towards cell surface antigens include the folate- binding protein present on ovarian cancer cells (see, e.g., Figini et al., 1998, Cancer Res.58:991- 96) and CD147, which is highly expressed on hepatocellular carcinoma (see, e.g., Bao et al., 2005, Cancer Biol. Ther.4:1374-80).
  • a potential disadvantage of the guided selection approach is that shuffling of one antibody chain while keeping the other constant could result in epitope drift. In order to maintain the epitope recognized by the non-human antibody, CDR retention can be applied (see, e.g., Klimka et al., 2000, Br. J.
  • Multispecific antibodies such as bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens.
  • the multispecific antibodies provided herein are bispecific antibodies.
  • bispecific antibodies are mouse, chimeric, human or humanized antibodies.
  • one of the binding specificities is for one target/antigen and the other is for another target/antigen.
  • bispecific antibodies may bind to two different epitopes of the same target/antigen.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibodies).
  • Methods for making multispecific antibodies are known in the art, such as, by co- expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40).
  • bispecific Antibodies see, for example, Bispecific Antibodies (Kontermann ed., 2011).
  • Fc engineering it may be desirable to modify the molecule described herein by Fc engineering, if the molecule described herein contains C H 1, hinge, C H 2, and/or C H 3 region(s).
  • the modification to the above-mentioned regions in the molecule described herein results in the decrease or elimination of an effector function of the antibody.
  • the effector function is antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • the effector function is ADCC.
  • the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP and CDC. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the molecule described herein. [00177] To increase the serum half-life of the molecule described herein, one may incorporate a salvage receptor binding epitope into the molecule, for example, as described in U.S. Pat. No. 5,739,277.
  • Term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • amino acid sequence modification(s) of the molecule provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity, or solubility.
  • antibody variants can be prepared.
  • antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide.
  • amino acid changes may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.
  • the molecule provided herein is chemically modified, for example, by the covalent attachment of any type of other molecule(s) to the molecule.
  • the antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody may contain one or more non-classical amino acids.
  • Variations may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide.
  • Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids.
  • the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule.
  • the substitution is a conservative amino acid substitution made at one or more predicted non- essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined. [00183] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed.1975)): (1) non- polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His(H).
  • Naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • Any cysteine residue not involved in maintaining the proper conformation of the antibody provided herein also may be substituted, for example, with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and to prevent aberrant crosslinking.
  • the molecule with antibody variants having an improved property such as affinity, stability, or expression level as compared to a parent molecule may be prepared by in vitro affinity maturation.
  • in vitro affinity maturation is based on the principles of mutation and selection.
  • Libraries of antibodies are displayed on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cell) or in association (e.g., covalently or non-covalently) with their encoding mRNA or DNA. Affinity selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies.
  • Phage display is a widespread method for display and selection of antibodies.
  • the antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions to the bacteriophage coat protein.
  • Selection involves exposure to antigen to allow phage-displayed antibodies to bind their targets, a process referred to as “panning.” Phage bound to antigen are recovered and used to infect bacteria to produce phage for further rounds of selection.
  • Hoogenboom 2002, Methods. Mol.
  • the antibody may be fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall through disulfide bonds to Aga1p. Display of a protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall.
  • Magnetic separation and flow cytometry are used to screen the library to select for antibodies with improved affinity or stability. Binding to a soluble antigen of interest is determined by labeling of yeast with biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Variations in surface expression of the antibody can be measured through immunofluorescence labeling of either the hemagglutinin or c-Myc epitope tag flanking the scFv. Expression has been shown to correlate with the stability of the displayed protein, and thus antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., 1999, J. Mol. Biol.292:949-56).
  • yeast display An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of the eukaryotic yeast cells, taking advantage of endoplasmic reticulum chaperones and quality-control machinery. Once maturation is complete, antibody affinity can be conveniently “titrated” while displayed on the surface of the yeast, eliminating the need for expression and purification of each clone.
  • a theoretical limitation of yeast surface display is the potentially smaller functional library size than that of other display methods; however, a recent approach uses the yeast cells’ mating system to create combinatorial diversity estimated to be 1014 in size (see, e.g., U.S. Pat. Publication 2003/0186374; and Blaise et al., 2004, Gene 342:211–18).
  • antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system.
  • the DNA library coding for a particular library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold.
  • the resulting complex of mRNA, ribosome, and protein can bind to surface-bound ligand, allowing simultaneous isolation of the antibody and its encoding mRNA through affinity capture with the ligand.
  • ribosome- bound mRNA is then reverse transcribed back into cDNA, which can then undergo mutagenesis and be used in the next round of selection (see, e.g., Fukuda et al., 2006, Nucleic Acids Res. 34:e127).
  • mRNA display a covalent bond between antibody and mRNA is established using puromycin as an adaptor molecule (Wilson et al., 2001, Proc. Natl. Acad. Sci. USA 98:3750-55).
  • the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube.
  • random mutations can be introduced easily after each selection round, for example, by non-proofreading polymerases, as no library must be transformed after any diversification step.
  • mammalian display systems may be used.
  • Diversity may also be introduced into the CDRs of the antibody libraries in a targeted manner or via random introduction. The former approach includes sequentially targeting all the CDRs of an antibody via a high or low level of mutagenesis or targeting isolated hot spots of somatic hypermutations (see, e.g., Ho et al., 2005, J. Biol.
  • Chem.280:607-17 residues suspected of affecting affinity on experimental basis or structural reasons.
  • Diversity may also be introduced by replacement of regions that are naturally diverse via DNA shuffling or similar techniques (see, e.g., Lu et al., 2003, J. Biol. Chem.278:43496-507; U.S. Pat. Nos.5,565,332 and 6,989,250).
  • Alternative techniques target hypervariable loops extending into framework- region residues (see, e.g., Bond et al., 2005, J. Mol. Biol.348:699-709) employ loop deletions and insertions in CDRs or use hybridization-based diversification (see, e.g., U.S. Pat.
  • the antibodies can be immobilized onto solid supports, columns, pins, or cellulose/poly(vinylidene fluoride) membranes/other filters, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin- coated beads or used in any other method for panning display libraries.
  • cellulose/poly(vinylidene fluoride) membranes/other filters expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin- coated beads or used in any other method for panning display libraries.
  • Covalent modifications include reacting targeted amino acid residues of an antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the antibody fragment(s) of the molecule.
  • Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the ⁇ - amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
  • An antibody fragment(s) of the molecule of the present disclosure may also be modified to form chimeric molecules comprising the antibody fragment(s) of the molecule fused to another, heterologous polypeptide or amino acid sequence, for example, an epitope tag (see, e.g., Terpe, 2003, Appl. Microbiol. Biotechnol.60:523-33) or another Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi eds., 1999)).
  • the present disclosure also provides conjugates comprising any one of the antibodies of the present disclosure covalently bound by a synthetic linker to one or more non-antibody agents.
  • antibody fragment(s) of the molecule provided herein are conjugated or recombinantly fused, e.g., to a therapeutic agent (e.g., a cytotoxic agent) or a diagnostic or detectable molecule.
  • a therapeutic agent e.g., a cytotoxic agent
  • the conjugated or recombinantly fused antibody fragment(s) of the molecule can be useful, for example, for treating or preventing a disease or disorder.
  • the conjugated or recombinantly fused antibodies can be useful, for example, for monitoring or prognosing the onset, development, progression, and/or severity of a disease.
  • diagnosis and detection can be accomplished, for example, by coupling the antibody fragment(s) of the molecule to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin or avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent
  • antibody fragment(s) of the molecule that are recombinantly fused or chemically conjugated (covalent or non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, for example, to a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acids) to generate fusion proteins, as well as uses thereof.
  • fusion proteins comprising a targeting region and a heterologous protein, polypeptide, or peptide.
  • the heterologous protein, polypeptide, or peptide that the antibody is fused to is useful for targeting the antibody to a particular cell type.
  • antibodies provided herein can be fused to marker or “tag” sequences, such as a peptide, to facilitate purification.
  • the marker or tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (see, e.g., QIAGEN, Inc.), among others, many of which are commercially available.
  • a pQE vector see, e.g., QIAGEN, Inc.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767-78), and the “FLAG” tag.
  • HA hemagglutinin
  • FLAG FLAG
  • Fusion proteins may be generated, for example, through the techniques of gene- shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”).
  • DNA shuffling may be employed to alter the activities of the antibodies as provided herein, including, for example, antibodies with higher affinities and lower dissociation rates (see, e.g., U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol.8:724-33; Harayama, 1998, Trends Biotechnol.16(2):76- 82; Hansson et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-13).
  • Antibodies, or the encoded antibodies may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination.
  • a polynucleotide encoding an antibody provided herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the antibody fragment(s) of the molecule provided herein can also be conjugated to a second antibody to form an antibody heteroconjugate as described, for example, in U.S. Pat. No. 4,676,980.
  • the antibody fragment(s) of the molecule as provided herein may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • the linker may be a “cleavable linker” facilitating release of the conjugated agent in the cell, but non-cleavable linkers are also contemplated herein.
  • Linkers for use in the conjugates of the present disclosure include, without limitation, acid labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids, for example, valine and/or citrulline such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., Chari et al., 1992, Cancer Res.52:127-31; and U.S. Pat.
  • acid labile linkers e.g., hydrazone linkers
  • disulfide-containing linkers e.g., disulfide-containing linkers
  • peptidase-sensitive linkers e.g., peptide linkers comprising amino acids, for example, valine and/or citrulline such as citrulline-valine or phenylalanine-lysine
  • photolabile linkers
  • Conjugates of the antibody and an agent may be made using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo- MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4- vinylsulfone)benzoate).
  • bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulf
  • conjugates of antibodies and agents may be prepared using any suitable methods as disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed.2008)).
  • Conventional conjugation strategies for antibodies and agents have been based on random conjugation chemistries involving the ⁇ -amino group of Lys residues or the thiol group of Cys residues, which results in heterogeneous conjugates.
  • Recently developed techniques allow site-specific conjugation to antibodies, resulting in homogeneous loading and avoiding conjugate subpopulations with altered antigen-binding or pharmacokinetics.
  • thiomabs comprising cysteine substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., 2008, J. Immunol. Meth.332: 41-52; and Junutula et al., 2008, Nature Biotechnol.26:925-32).
  • selenocysteine is cotranslationally inserted into an antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al., 2008, Proc. Natl. Acad. Sci. USA 105:12451-56; and Hofer et al., 2009, Biochemistry 48(50):12047-57).
  • the targeting region of the molecule provided herein comprises a non-immunoglobulin binding agent.
  • the non-immunoglobulin binding agent forms specific binding to the target via enzyme-substrate, receptor-ligand or other protein-protein interactions.
  • a non-immunoglobulin binding agent is identified as an agent that displaces or is displaced by the molecule of the present disclosure in a competitive binding assay.
  • These alternative binding agents may include, for example, any of the engineered protein scaffolds known in the art.
  • Such scaffolds include, for example, anticalins, which are based upon the lipocalin scaffold, a protein structure characterized by a rigid beta-barrel that supports four hypervariable loops which form the ligand binding site.
  • Novel binding specificities may be engineered by targeted random mutagenesis in the loop regions, in combination with functional display and guided selection (see, e.g., Skerra, 2008, FEBS J.275:2677-83).
  • Other suitable scaffolds may include, for example, adnectins, or monobodies, based on the tenth extracellular domain of human fibronectin III (see, e.g., Koide and Koide, 2007, Methods Mol.
  • Biol.352 95- 109); affibodies, based on the Z domain of staphylococcal protein A (see, e.g., Nygren et al., 2008, FEBS J.275:2668-76); DARPins, based on ankyrin repeat proteins (see, e.g., Stumpp et al., 2008, Drug. Discov. Today 13:695-701); fynomers, based on the SH3 domain of the human Fyn protein kinase (see, e.g., Grabulovski et al., 2007, J. Biol.
  • Chem.282:3196-204 affitins, based on Sac7d from Sulfolobus acidolarius (see, e.g., Krehenbrink et al., 2008, J. Mol. Biol. 383:1058-68); affilins, based on human y-B-crystallin (see, e.g., Ebersbach et al., 2007, J. Mol.
  • Biol.372:172-85 avimers, based on the A domain of membrane receptor proteins (see, e.g., Silverman et al., 2005, Biotechnol.23:1556-61); cysteine-rich knottin peptides (see, e.g., Kolmar, 2008, FEBS J.275:2684-90); and engineered Kunitz-type inhibitors (see, e.g., Nixon and Wood, 2006, Curr. Opin. Drug. Discov. Dev.9:261-68).
  • Kunitz-type inhibitors see, e.g., Nixon and Wood, 2006, Curr. Opin. Drug. Discov. Dev.9:261-68.
  • Gebauer and Skerra 2009, Curr. Opin. Chem. Biol.13:245-55.
  • the molecule disclosed herein comprises a signal peptide (SEQ ID NO.2). In other embodiments, the molecule disclosed herein comprises at least 95% identity to SEQ ID NO.2. In other embodiments, the molecule disclosed herein comprises at least 90% identity to SEQ ID NO.2. In other embodiments, the molecule disclosed herein comprises at least 85% identity to SEQ ID NO.2. In other embodiments, the molecule disclosed herein comprises at least 80% identity to SEQ ID NO.2. In other embodiments, the molecule disclosed herein comprises at least 70% identity to SEQ ID NO.2.
  • the molecule disclosed herein comprises at least 60% identity to SEQ ID NO.2. In other embodiments, the molecule disclosed herein comprises at least 50% identity to SEQ ID NO.2.
  • the binding domain of the present molecules is capable of binding to one or more antigen(s).
  • the antigen can act as a cell surface marker on target cells associated with a special disease state.
  • the antigen is a tumor antigen.
  • the antigen of a target cell is an antigen on the surface of the cancer cell.
  • the antigen is a tumor-specific antigen, a tumor-associated antigen, or a neoantigen.
  • the target cell is a cancer cell, e.g., a cell of an adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic, head and neck cancer, Hodgkin lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumor, non-Hodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma spinal cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer endometrial cancer, vaginal cancer, or vulvar cancer.
  • a cancer cell e.g., a cell of an adrenal cancer, anal cancer, appendix cancer, bile
  • the cancer is an adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic, head and neck cancer, Hodgkin lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumor, non-Hodgkin lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma spinal cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer endometrial cancer, vaginal cancer, or vulvar cancer.
  • the cancer is a adrenal cancer. In some embodiments, the cancer is a anal cancer. In some embodiments, the cancer is an appendix cancer. In some embodiments, the cancer is a bile duct cancer. In some embodiments, the cancer is a bladder cancer. In some embodiments, the cancer is a bone cancer. In some embodiments, the cancer is a brain cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a cervical cancer. In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is a esophageal cancer. In some embodiments, the cancer is a gallbladder cancer. In some embodiments, the cancer is a gestational trophoblastic.
  • the cancer is a head and neck cancer. In some embodiments, the cancer is a Hodgkin lymphoma. In some embodiments, the cancer is an intestinal cancer. In some embodiments, the cancer is a kidney cancer. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is a liver cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is a mesothelioma. In some embodiments, the cancer is a multiple myeloma (MM). In some embodiments, the cancer is a neuroendocrine tumor. In some embodiments, the cancer is a non- Hodgkin lymphoma.
  • the cancer is an intestinal cancer. In some embodiments, the cancer is a kidney cancer. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is a liver cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the
  • the cancer is an oral cancer. In some embodiments, the cancer is an ovarian cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is a prostate cancer. In some embodiments, the cancer is a sinus cancer. In some embodiments, the cancer is a skin cancer. In some embodiments, the cancer is a soft tissue sarcoma spinal cancer. In some embodiments, the cancer is a stomach cancer. In some embodiments, the cancer is a testicular cancer. In some embodiments, the cancer is a throat cancer. In some embodiments, the cancer is a thyroid cancer. In some embodiments, the cancer is a uterine cancer endometrial cancer. In some embodiments, the cancer is a vaginal cancer.
  • the cancer is a vulvar cancer.
  • the adrenal cancer is an adrenocortical carcinoma (ACC), adrenal cortex cancer, pheochromocytoma, or neuroblastoma.
  • the anal cancer is a squamous cell carcinoma, cloacogenic carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma.
  • the appendix cancer is a neuroendocrine tumor (NET), mucinous adenocarcinoma, goblet cell carcinoid, intestinal-type adenocarcinoma, or signet-ring cell adenocarcinoma.
  • NET neuroendocrine tumor
  • the bile duct cancer is an extrahepatic bile duct cancer, adenocarcinomas, hilar bile duct cancer, perihilar bile duct cancer, distal bile duct cancer, or intrahepatic bile duct cancer.
  • the bladder cancer is transitional cell carcinoma (TCC), papillary carcinoma, flat carcinoma, squamous cell carcinoma, adenocarcinoma, small-cell carcinoma, or sarcoma.
  • the bone cancer is a primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer.
  • the brain cancer is an astrocytoma, brain stem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary carcinoma, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal region tumor, medulloblastoma, or primary CNS lymphoma.
  • the breast cancer is a breast adenocarcinoma, invasive breast cancer, noninvasive breast cancer, breast sarcoma, metaplastic carcinoma, adenocystic carcinoma, phyllodes tumor, angiosarcoma, HER2-positive breast cancer, triple-negative breast cancer, or inflammatory breast cancer.
  • the cervical cancer is a squamous cell carcinoma, or adenocarcinoma.
  • the colorectal cancer is a colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma, signet ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma.
  • the esophageal cancer is an adenocarcinoma or squamous cell carcinoma.
  • the gall bladder cancer is an adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma.
  • the gestational trophoblastic disease is a hydatidiform mole, gestational trophoblastic neoplasia (GTN), choriocarcinoma, placental-site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT).
  • the head and neck cancer is a laryngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, oral cancer, oropharyngeal cancer, or tonsil cancer.
  • the Hodgkin lymphoma is a classical Hodgkin lymphoma, nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte-depleted, or nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL).
  • the intestinal cancer is a small intestine cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumors, carcinoid tumors, or lymphoma.
  • the kidney cancer is a renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urothelial cancer, renal pelvis carcinoma, or renal sarcoma.
  • the leukemia is an acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or a myelodysplastic syndrome (MDS).
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myeloid leukemia
  • HCL hairy cell leukemia
  • MDS myelodysplastic syndrome
  • the leukemia is AML.
  • the liver cancer is a hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis.
  • the lung cancer is a small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland- type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor.
  • the melanoma is a superficial spreading melanoma, nodular melanoma, acral-lentiginous melanoma, lentigo maligna melanoma, amelanotic melanoma, desmoplastic melanoma, ocular melanoma, or metastatic melanoma.
  • the mesothelioma is a pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma.
  • the multiple myeloma is an active myeloma or smoldering myeloma.
  • the neuroendocrine tumor is a gastrointestinal neuroendocrine tumor, pancreatic neuroendocrine tumor, or lung neuroendocrine tumor.
  • the non-Hodgkin’s lymphoma is an anaplastic large-cell lymphoma, lymphoblastic lymphoma, peripheral T cell lymphoma, follicular lymphoma, cutaneous T cell lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, MALT lymphoma, small-cell lymphocytic lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), precursor T- lymphoblastic leukemia/lymphoma, acute lymphocytic leukemia (ALL), adult T cell lymphoma/leukemia (ATLL), hairy cell leukemia, B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, primary central nervous system (CNS) lymphoma, mantle cell lymphoma (MCL), marginal zone lymphomas
  • the oral cancer is a squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinomas, lymphoma, benign oral cavity tumor, eosinophilic granuloma, fibroma, granular cell tumor, karatoacanthoma, leiomyoma, osteochondroma, lipoma, schwannoma, neurofibroma, papilloma, condyloma acuminatum, verruciform xanthoma, pyogenic granuloma, rhabdomyoma, odontogenic tumors, leukoplakia, erythroplakia, squamous cell lip cancer, basal cell lip cancer, mouth cancer, gum cancer, or tongue cancer.
  • the ovarian cancer is a ovarian epithelial cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, clear cell epithelial ovarian cancer, undifferentiated epithelial ovarian cancer, ovarian low malignant potential tumors, primary peritoneal carcinoma, fallopian tube cancer, germ cell tumors, teratoma, dysgerminoma ovarian germ cell cancer, endodermal sinus tumor, sex cord- stromal tumors, sex cord-gonadal stromal tumor, ovarian stromal tumor, granulosa cell tumor, granulosa-theca tumor, Sertoli-Leydig tumor, ovarian sarcoma, ovarian carcinosarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, Krukenberg tumor, or ovarian cyst.
  • the pancreatic cancer is a pancreatic exocrine gland cancer, pancreatic endocrine gland cancer, or pancreatic adenocarcinoma, islet cell tumor, or neuroendocrine tumor.
  • the prostate cancer is a prostate adenocarcinoma, prostate sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor.
  • the sinus cancer is a squamous cell carcinoma, mucosa cell carcinoma, adenoid cystic cell carcinoma, acinic cell carcinoma, sinonasal undifferentiated carcinoma, nasal cavity cancer, paranasal sinus cancer, maxillary sinus cancer, ethmoid sinus cancer, or nasopharynx cancer.
  • the skin cancer is a basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi sarcoma (KS), actinic keratosis, skin lymphoma, or keratoacanthoma.
  • basal cell carcinoma a basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi sarcoma (KS), actinic keratosis, skin lymphoma, or keratoacanthoma.
  • KS Kaposi sarcoma
  • the soft tissue cancer is an angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumors (GISTs), Kaposi sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid/round cell liposarcoma (MRCL), well-differentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma.
  • GISTs gastrointestinal stromal tumors
  • Kaposi sarcoma leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid/round cell liposarcoma (MRCL), well-differentiated liposarcom
  • the spinal cancer is a spinal metastatic tumor.
  • the stomach cancer is a stomach adenocarcinoma, stomach lymphoma, gastrointestinal stromal tumors, carcinoid tumor, gastric carcinoid tumors, Type I ECL-cell carcinoid, Type II ECL-cell carcinoid, or Type III ECL-cell carcinoid.
  • the testicular cancer is a seminoma, non-seminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratoma, gonadal stromal tumor, leydig cell tumor, or sertoli cell tumor.
  • the throat cancer is a squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal cancer, pharyngeal cancer, nasopharynx cancer, oropharynx cancer, hypopharynx cancer, laryngeal cancer, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, lymphoepithelioma, spindle cell carcinoma, verrucous cancer, undifferentiated carcinoma, or lymph node cancer.
  • the thyroid cancer is a papillary carcinoma, follicular carcinoma, Hürthle cell carcinoma, medullary thyroid carcinoma, or anaplastic carcinoma.
  • the uterine cancer is an endometrial cancer, endometrial adenocarcinoma, endometroid carcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinosarcoma, uterine sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, or undifferentiated sarcoma.
  • the vaginal cancer is a squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma.
  • the vulvar cancer is a squamous cell carcinoma or adenocarcinoma.
  • Tumor antigens are proteins that are produced by tumor cells that can elicit an immune response, particularly T-cell mediated immune responses.
  • exemplary tumor antigens include, but not limited to, a glioma-associated antigen, carcinoembryonic antigen (CEA), ⁇ - human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY- ESO-1, LAGE-la, p53, prostein, PSMA, HER2/neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2,
  • the cancer antigen is CEA, immature laminin receptor, TAG- 72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EpCAM, EphA3, Her2/neu, telomerase, mesothelin, SAP-1, surviving, a BAGE family antigen, CAGE family antigen, GAGE family antigen, MAGE family antigen, SAGE family antigen, XAGE family antigen, NY-ESO-1/LAGE-1, PRAME, SSX-2, Melan-A, MART-1, Gp100, pmel17, tyrosinase, TRP-1, TRP-2, P.
  • the cancer antigen is CEA. In some embodiments, the cancer antigen is immature laminin receptor. In some embodiments, the cancer antigen is TAG-72. In some embodiments, the cancer antigen is HPV E6. In some embodiments, the cancer antigen is HPV E7. In some embodiments, the cancer antigen is BING-4. In some embodiments, the cancer antigen is calcium-activated chloride channel 2. In some embodiments, the cancer antigen is cyclin-B1.
  • the cancer antigen is 9D7. In some embodiments, the cancer antigen is EpCAM. In some embodiments, the cancer antigen is EphA3. In some embodiments, the cancer antigen is Her2/neu. In some embodiments, the cancer antigen is telomerase. In some embodiments, the cancer antigen is mesothelin. In some embodiments, the cancer antigen is SAP-1. In some embodiments, the cancer antigen is surviving. In some embodiments, the cancer antigen is a BAGE family antigen. In some embodiments, the cancer antigen is CAGE family antigen. In some embodiments, the cancer antigen is GAGE family antigen. In some embodiments, the cancer antigen is MAGE family antigen.
  • the cancer antigen is SAGE family antigen. In some embodiments, the cancer antigen is XAGE family antigen. In some embodiments, the cancer antigen is NY-ESO-1/LAGE-1. In some embodiments, the cancer antigen is PRAME. In some embodiments, the cancer antigen is SSX-2. In some embodiments, the cancer antigen is Melan-A. In some embodiments, the cancer antigen is MART-1. In some embodiments, the cancer antigen is Gp100. In some embodiments, the cancer antigen is pmel17. In some embodiments, the cancer antigen is tyrosinase. In some embodiments, the cancer antigen is TRP-1. In some embodiments, the cancer antigen is TRP-2.
  • the cancer antigen is P. polypeptide. In some embodiments, the cancer antigen is MC1R. In some embodiments, the cancer antigen is prostate-specific antigen. In some embodiments, the cancer antigen is ⁇ -catenin. In some embodiments, the cancer antigen is BRCA1. In some embodiments, the cancer antigen is BRCA2. In some embodiments, the cancer antigen is CDK4. In some embodiments, the cancer antigen is CML66. In some embodiments, the cancer antigen is fibronectin. In some embodiments, the cancer antigen is MART-2. In some embodiments, the cancer antigen is p53. In some embodiments, the cancer antigen is Ras. In some embodiments, the cancer antigen is TGF- ⁇ RII.
  • the cancer antigen is MUC1.
  • the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor.
  • Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and gp100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • Other target molecules belong to the group of transformation-related molecules such as the oncogene HER2/Neu/ErbB-2.
  • the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA).
  • TSA tumor-specific antigen
  • TAA tumor-associated antigen
  • a TSA is unique to tumor cells and does not occur on other cells in the body.
  • a TAA associated antigen is not unique to a tumor cell, and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • TAAs may be antigens that are expressed on normal cells during fetal development, when the immune system is immature, and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.
  • TSA or TAA antigens include: differentiation antigens such as MART-1/MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor- specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor- suppressor genes such as p53, Ras, HER2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • differentiation antigens such as MART-1/MelanA (MART-I), gp 100 (Pmel 17
  • Additional non-limiting exemplary targets of the binding molecules provided herein include GPC2, CD276, Delta-like protein ligand 3 (DLL3), NY-ESO-1, melanoma associated antigen 4; survivin protein, synovial sarcoma X breakpoint protein 2, CD3, epidermal growth factor receptor (EGFR), erbb2 tyrosine kinase receptor, HER2, CEA, CD66, CD66e, ROR1, ntrkr1 tyrosine kinase receptor, GPC3, mesothelin, glutamate carboxypeptidase II, PMSA, PD- L1, folate receptor alpha, PSCA, Mucin 1, HLA antigen (such as HLA class I antigen A-2 alpha, HLA class I antigen A-11 alpha, and HLA class II antigen), c-Met, hepatocyte growth factor receptor, K-Ras GTPase (KRAS), IL-15 receptor, Kit tyros
  • the binding molecule provided herein binds to a B cell antigen.
  • the B cell antigen is a CD1a, CD1b, CD1c, CD1d, CD2, CD5, CD6, CD9, CD11a, CD11b, CD11c, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD29, CD30, CD31, CD32a, CD32b, CD35, CD37, CD38, CD39, CD40, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49b, CD49c, CD49d, CD50, CD52, CD53, CD54, CD55, CD58, CD60a, CD62L, CD63, CD68, CD69, CD70, CD72, CD73, CD74, CD75, CD75S, CD77, CD79a, CD79b, CD80, CD81, CD
  • the B cell antigen is a CD1a antigen. In some embodiments, the B cell antigen is a CD1b antigen. In some embodiments, the B cell antigen is a CD1c antigen. In some embodiments, the B cell antigen is a CD1d antigen. In some embodiments, the B cell antigen is a CD2 antigen. In some embodiments, the B cell antigen is a CD5 antigen. In some embodiments, the B cell antigen is a CD6 antigen. In some embodiments, the B cell antigen is a CD9 antigen. In some embodiments, the B cell antigen is a CD11a antigen. In some embodiments, the B cell antigen is a CD11b antigen.
  • the B cell antigen is a CD11c antigen. In some embodiments, the B cell antigen is a CD17 antigen. In some embodiments, the B cell antigen is a CD18 antigen. In some embodiments, the B cell antigen is a CD19 antigen. In some embodiments, the B cell antigen is a CD20 antigen. In some embodiments, the B cell antigen is a CD21 antigen. In some embodiments, the B cell antigen is a CD22 antigen. In some embodiments, the B cell antigen is a CD23 antigen. In some embodiments, the B cell antigen is a CD24 antigen. In some embodiments, the B cell antigen is a CD25 antigen.
  • the B cell antigen is a CD26 antigen. In some embodiments, the B cell antigen is a CD27 antigen. In some embodiments, the B cell antigen is a CD29 antigen. In some embodiments, the B cell antigen is a CD30 antigen. In some embodiments, the B cell antigen is a CD31 antigen. In some embodiments, the B cell antigen is a CD32a antigen. In some embodiments, the B cell antigen is a CD32b antigen. In some embodiments, the B cell antigen is a CD35 antigen. In some embodiments, the B cell antigen is a CD37 antigen. In some embodiments, the B cell antigen is a CD38 antigen.
  • the B cell antigen is a CD39 antigen. In some embodiments, the B cell antigen is a CD40 antigen. In some embodiments, the B cell antigen is a CD45 antigen. In some embodiments, the B cell antigen is a CD45RA antigen. In some embodiments, the B cell antigen is a CD45RB antigen. In some embodiments, the B cell antigen is a CD45RC antigen. In some embodiments, the B cell antigen is a CD45RO antigen. In some embodiments, the B cell antigen is a CD46 antigen. In some embodiments, the B cell antigen is a CD47 antigen. In some embodiments, the B cell antigen is a CD48 antigen.
  • the B cell antigen is a CD49b antigen. In some embodiments, the B cell antigen is a CD49c antigen. In some embodiments, the B cell antigen is a CD49d antigen. In some embodiments, the B cell antigen is a CD50 antigen. In some embodiments, the B cell antigen is a CD52 antigen. In some embodiments, the B cell antigen is a CD53 antigen. In some embodiments, the B cell antigen is a CD54 antigen. In some embodiments, the B cell antigen is a CD55 antigen. In some embodiments, the B cell antigen is a CD58 antigen. In some embodiments, the B cell antigen is a CD60a antigen.
  • the B cell antigen is a CD62L antigen. In some embodiments, the B cell antigen is a CD63 antigen. In some embodiments, the B cell antigen is a CD68 antigen. In some embodiments, the B cell antigen is a CD69 antigen. In some embodiments, the B cell antigen is a CD70 antigen. In some embodiments, the B cell antigen is a CD72 antigen. In some embodiments, the B cell antigen is a CD73 antigen. In some embodiments, the B cell antigen is a CD74 antigen. In some embodiments, the B cell antigen is a CD75 antigen. In some embodiments, the B cell antigen is a CD75S antigen.
  • the B cell antigen is a CD77 antigen. In some embodiments, the B cell antigen is a CD79a antigen. In some embodiments, the B cell antigen is a CD79b antigen. In some embodiments, the B cell antigen is a CD80 antigen. In some embodiments, the B cell antigen is a CD81 antigen. In some embodiments, the B cell antigen is a CD82 antigen. In some embodiments, the B cell antigen is a CD83 antigen. In some embodiments, the B cell antigen is a CD84 antigen. In some embodiments, the B cell antigen is a CD85E antigen. In some embodiments, the B cell antigen is a CD85I antigen.
  • the B cell antigen is a CD85J antigen. In some embodiments, the B cell antigen is a CD86 antigen. In some embodiments, the B cell antigen is a CD92 antigen. In some embodiments, the B cell antigen is a CD95 antigen. In some embodiments, the B cell antigen is a CD97 antigen. In some embodiments, the B cell antigen is a CD98 antigen. In some embodiments, the B cell antigen is a CD99 antigen. In some embodiments, the B cell antigen is a CD100 antigen. In some embodiments, the B cell antigen is a CD102 antigen. In some embodiments, the B cell antigen is a CD108 antigen.
  • the B cell antigen is a CD119 antigen. In some embodiments, the B cell antigen is a CD120a antigen. In some embodiments, the B cell antigen is a CD120b antigen. In some embodiments, the B cell antigen is a CD121b antigen. In some embodiments, the B cell antigen is a CD122 antigen. In some embodiments, the B cell antigen is a CD124 antigen. In some embodiments, the B cell antigen is a CD125 antigen. In some embodiments, the B cell antigen is a CD126 antigen. In some embodiments, the B cell antigen is a CD130 antigen. In some embodiments, the B cell antigen is a CD132 antigen.
  • the B cell antigen is a CD137 antigen. In some embodiments, the B cell antigen is a CD138 antigen. In some embodiments, the B cell antigen is a CD139 antigen. In some embodiments, the B cell antigen is a CD147 antigen. In some embodiments, the B cell antigen is a CD148 antigen. In some embodiments, the B cell antigen is a CD150 antigen. In some embodiments, the B cell antigen is a CD152 antigen. In some embodiments, the B cell antigen is a CD162 antigen. In some embodiments, the B cell antigen is a CD164 antigen. In some embodiments, the B cell antigen is a CD166 antigen.
  • the B cell antigen is a CD167a antigen. In some embodiments, the B cell antigen is a CD170 antigen. In some embodiments, the B cell antigen is a CD171 antigen. In some embodiments, the B cell antigen is a CD175 antigen. In some embodiments, the B cell antigen is a CD175s antigen. In some embodiments, the B cell antigen is a CD180 antigen. In some embodiments, the B cell antigen is a CD184 antigen. In some embodiments, the B cell antigen is a CD185 antigen. In some embodiments, the B cell antigen is a CD192 antigen. In some embodiments, the B cell antigen is a CD196 antigen.
  • the B cell antigen is a CD197 antigen. In some embodiments, the B cell antigen is a CD200 antigen. In some embodiments, the B cell antigen is a CD205 antigen. In some embodiments, the B cell antigen is a CD201a antigen. In some embodiments, the B cell antigen is a CDw210b antigen. In some embodiments, the B cell antigen is a CD212 antigen. In some embodiments, the B cell antigen is a CD213a1 antigen. In some embodiments, the B cell antigen is a CD213a2 antigen. In some embodiments, the B cell antigen is a CD 215 antigen. In some embodiments, the B cell antigen is a CD217 antigen.
  • the B cell antigen is a CD218a antigen. In some embodiments, the B cell antigen is a CD218b antigen. In some embodiments, the B cell antigen is a CD220 antigen. In some embodiments, the B cell antigen is a CD221 antigen. In some embodiments, the B cell antigen is a CD222 antigen. In some embodiments, the B cell antigen is a CD224 antigen. In some embodiments, the B cell antigen is a CD225 antigen. In some embodiments, the B cell antigen is a CD226 antigen. In some embodiments, the B cell antigen is a CD227 antigen. In some embodiments, the B cell antigen is a CD229 antigen.
  • the B cell antigen is a CD230 antigen. In some embodiments, the B cell antigen is a CD232 antigen. In some embodiments, the B cell antigen is a CD252 antigen. In some embodiments, the B cell antigen is a CD252 antigen. In some embodiments, the B cell antigen is a CD254 antigen. In some embodiments, the B cell antigen is a CD255 antigen. In some embodiments, the B cell antigen is a CD256 antigen. In some embodiments, the B cell antigen is a CD257 CD258 antigen. In some embodiments, the B cell antigen is a CD259 antigen. In some embodiments, the B cell antigen is a CD260 antigen.
  • the B cell antigen is a CD261 antigen. In some embodiments, the B cell antigen is a CD262 antigen. In some embodiments, the B cell antigen is a CD263 antigen. In some embodiments, the B cell antigen is a CD264 antigen. In some embodiments, the B cell antigen is a CD267 antigen. In some embodiments, the B cell antigen is a CD268 antigen. In some embodiments, the B cell antigen is a CD269 antigen. In some embodiments, the B cell antigen is a CD270 antigen. In some embodiments, the B cell antigen is a CD272 antigen. In some embodiments, the B cell antigen is a CD274 antigen.
  • the B cell antigen is a CD275 antigen. In some embodiments, the B cell antigen is a CD277 antigen. In some embodiments, the B cell antigen is a CD279 antigen. In some embodiments, the B cell antigen is a CD283 antigen. In some embodiments, the B cell antigen is a CD289 antigen. In some embodiments, the B cell antigen is a CD290 antigen. In some embodiments, the B cell antigen is a CD295 antigen. In some embodiments, the B cell antigen is a CD298 antigen. In some embodiments, the B cell antigen is a CD300 antigen. In some embodiments, the B cell antigen is a CD300c antigen.
  • the B cell antigen is a CD305 antigen. In some embodiments, the B cell antigen is a CD306 antigen. In some embodiments, the B cell antigen is a CD307a antigen. In some embodiments, the B cell antigen is a CD307b antigen. In some embodiments, the B cell antigen is a CD307c antigen. In some embodiments, the B cell antigen is a CD307d antigen. In some embodiments, the B cell antigen is a CD307e antigen. In some embodiments, the B cell antigen is a CD314 antigen. In some embodiments, the B cell antigen is a CD215 antigen.
  • the B cell antigen is a CD316 antigen. In some embodiments, the B cell antigen is a CD317 antigen. In some embodiments, the B cell antigen is a CD319 antigen. In some embodiments, the B cell antigen is a CD321 antigen. In some embodiments, the B cell antigen is a CD327 antigen. In some embodiments, the B cell antigen is a CD328 antigen. In some embodiments, the B cell antigen is a CD329 antigen. In some embodiments, the B cell antigen is a CD338 antigen. In some embodiments, the B cell antigen is a CD351 antigen. In some embodiments, the B cell antigen is a CD352 antigen.
  • the B cell antigen is a CD353 antigen. In some embodiments, the B cell antigen is a CD354 antigen. In some embodiments, the B cell antigen is a CD355 antigen. In some embodiments, the B cell antigen is a CD356 antigen. In some embodiments, the B cell antigen is a CD357 antigen. In some embodiments, the B cell antigen is a CD358 antigen. In some embodiments, the B cell antigen is a CD360 antigen. In some embodiments, the B cell antigen is a CD361 antigen. In some embodiments, the B cell antigen is a CD362 antigen. In some embodiments, the B cell antigen is a CD363 antigen.
  • target of the present binding molecule is a pathogen.
  • the target cell is a cell comprising a pathogen.
  • the pathogen causes an infectious disease selected from the group consisting of an Acute Flaccid Myelitis (AFM), Anaplasmosis, Anthrax, Babesiosis, Botulism, Brucellosis, Campylobacteriosis, Carbapenem-resistant Infection, Chancroid, Chikungunya Virus Infection, Chlamydia, Ciguatera, Difficile Infection, Perfringens, Coccidioidomycosis fungal infection, coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jacob Disease/transmissible spongiform encephalopathy, Cryptosporidiosis (Crypto), Cyclosporiasis, Dengue 1,2,3 or 4, Diphtheria, E.
  • AMF Acute Flaccid Myelitis
  • Anaplasmosis Anaplasmos
  • coli infection/Shiga toxin-producing (STEC), Eastern Equine Encephalitis, Hemorrhagic Fever (Ebola), Ehrlichiosis, Encephalitis, Arboviral or parainfectious, Non-Polio Enterovirus, D68 Enteroviru(EV-D68), Giardiasis, Glanders, Gonococcal Infection, Granuloma inguinale, Haemophilus Influenza disease Type B (Hib or H- flu), Hantavirus Pulmonary Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes, Herpes Zoster (Shingles), Histoplasmosis infection, Human Immunodeficiency Virus/AIDS (HIV/AIDS), Human Papillomavirus (HPV), Influenza (F
  • the infectious disease is Acute Flaccid Myelitis (AFM).
  • the infectious disease is Anaplasmosis.
  • the infectious disease is Anthrax.
  • the infectious disease is Babesiosis.
  • the infectious disease is Botulism.
  • the infectious disease is Brucellosis.
  • the infectious disease is Campylobacteriosis.
  • the infectious disease is Carbapenem-resistant Infection.
  • the infectious disease is Chancroid.
  • the infectious disease is Chikungunya Virus Infection.
  • the infectious disease is Chlamydia.
  • the infectious disease is Ciguatera. In some embodiments, the infectious disease is Difficile Infection. In some embodiments, the infectious disease is Perfringens. In some embodiments, the infectious disease is Coccidioidomycosis fungal infection. In some embodiments, the infectious disease is coronavirus. In some embodiments, the infectious disease is Covid-19 (SARS-CoV-2). In some embodiments, the infectious disease is Creutzfeldt-Jacob Disease/transmissible spongiform encephalopathy. In some embodiments, the infectious disease is Cryptosporidiosis (Crypto). In some embodiments, the infectious disease is Cyclosporiasis. In some embodiments, the infectious disease is Dengue 1,2,3 or 4.
  • the infectious disease is Diphtheria. In some embodiments, the infectious disease is E. coli infection/Shiga toxin-producing (STEC). In some embodiments, the infectious disease is Eastern Equine Encephalitis. In some embodiments, the infectious disease is Hemorrhagic Fever (Ebola). In some embodiments, the infectious disease is Ehrlichiosis. In some embodiments, the infectious disease is Encephalitis. In some embodiments, the infectious disease is Arboviral or parainfectious. In some embodiments, the infectious disease is Non-Polio Enterovirus. In some embodiments, the infectious disease is D68 Enteroviru(EV-D68). In some embodiments, the infectious disease is Giardiasis.
  • the infectious disease is Glanders. In some embodiments, the infectious disease is Gonococcal Infection. In some embodiments, the infectious disease is Granuloma inguinale. In some embodiments, the infectious disease is Haemophilus Influenza disease Type B (Hib or H-flu). In some embodiments, the infectious disease is Hantavirus Pulmonary Syndrome (HPS). In some embodiments, the infectious disease is Hemolytic Uremic Syndrome (HUS). In some embodiments, the infectious disease is Hepatitis A (Hep A). In some embodiments, the infectious disease is Hepatitis B (Hep B). In some embodiments, the infectious disease is Hepatitis C (Hep C).
  • the infectious disease is Hepatitis D (Hep D). In some embodiments, the infectious disease is Hepatitis E (Hep E). In some embodiments, the infectious disease is Herpes. In some embodiments, the infectious disease is Herpes Zoster (Shingles). In some embodiments, the infectious disease is Histoplasmosis infection. In some embodiments, the infectious disease is Human Immunodeficiency Virus/AIDS (HIV/AIDS). In some embodiments, the infectious disease is Human Papillomavirus (HPV). In some embodiments, the infectious disease is Influenza (Flu). In some embodiments, the infectious disease is Legionellosis (Legionnaires Disease). In some embodiments, the infectious disease is Leprosy (Hansens Disease).
  • the infectious disease is Leptospirosis. In some embodiments, the infectious disease is Listeriosis (Listeria). In some embodiments, the infectious disease is Lyme Disease. In some embodiments, the infectious disease is Lymphogranuloma venereum infection (LGV). In some embodiments, the infectious disease is Malaria. In some embodiments, the infectious disease is Measles. In some embodiments, the infectious disease is Melioidosis. In some embodiments, the infectious disease is Meningitis (Viral). In some embodiments, the infectious disease is Meningococcal Disease (Meningitis (Bacterial)). In some embodiments, the infectious disease is Middle East Respiratory Syndrome Coronavirus (MERS-CoV).
  • the infectious disease is Mumps. In some embodiments, the infectious disease is Norovirus. In some embodiments, the infectious disease is Pediculosis. In some embodiments, the infectious disease is Pelvic Inflammatory Disease (PID). In some embodiments, the infectious disease is Pertussis (Whooping Cough). In some embodiments, the infectious disease is Plague (Bubonic. In some embodiments, the infectious disease is Septicemic. In some embodiments, the infectious disease is Pneumonic). In some embodiments, the infectious disease is Pneumococcal Disease (Pneumonia). In some embodiments, the infectious disease is Poliomyelitis (Polio). In some embodiments, the infectious disease is Powassan.
  • the infectious disease is Psittacosis. In some embodiments, the infectious disease is Pthiriasis. In some embodiments, the infectious disease is Pustular Rash diseases (Small pox). In some embodiments, the infectious disease is monkeypox. In some embodiments, the infectious disease is cowpox). In some embodiments, the infectious disease is Q-Fever. In some embodiments, the infectious disease is Rabies. In some embodiments, the infectious disease is Rickettsiosis (Rocky Mountain Spotted Fever). In some embodiments, the infectious disease is Rubella (German Measles). In some embodiments, the infectious disease is Salmonellosis gastroenteritis (Salmonella).
  • the infectious disease is Scabies. In some embodiments, the infectious disease is Scombroid. In some embodiments, the infectious disease is Sepsis. In some embodiments, the infectious disease is Severe Acute Respiratory Syndrome (SARS). In some embodiments, the infectious disease is Shigellosis gastroenteritis (Shigella). In some embodiments, the infectious disease is Smallpox. In some embodiments, the infectious disease is Staphyloccal Infection Methicillin-resistant (MRSA). In some embodiments, the infectious disease is Staphylococcal Food Poisoning Enterotoxin B Poisoning (Staph Food Poisoning). In some embodiments, the infectious disease is Saphylococcal Infection Vancomycin Intermediate (VISA).
  • SARS Severe Acute Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • the infectious disease is Shigellosis gastroenteritis (Shigella). In some embodiments, the infectious disease is Smallpox. In some embodiments, the infectious disease is Staphy
  • the infectious disease is Staphylococcal Infection Vancomycin Resistant (VRSA). In some embodiments, the infectious disease is Streptococcal Disease Group A (invasive) (Strep A (invasive). In some embodiments, the infectious disease is Streptococcal Disease. In some embodiments, the infectious disease is Group B (Strep-B). In some embodiments, the infectious disease is Streptococcal Toxic-Shock Syndrome STSS Toxic Shock. In some embodiments, the infectious disease is Syphilis (primary. In some embodiments, the infectious disease is secondary. In some embodiments, the infectious disease is early latent. In some embodiments, the infectious disease is late latent. In some embodiments, the infectious disease is congenital).
  • VRSA Staphylococcal Infection Vancomycin Resistant
  • the infectious disease is Streptococcal Disease Group A (invasive) (Strep A (invasive). In some embodiments, the infectious disease is Streptococcal Disease. In some embodiments, the infectious disease is Group B
  • the infectious disease is Tetanus Infection. In some embodiments, the infectious disease is Trichomoniasis. In some embodiments, the infectious disease is Trichonosis Infection. In some embodiments, the infectious disease is Tuberculosis (TB). In some embodiments, the infectious disease is Tuberculosis Latent (LTBI). In some embodiments, the infectious disease is Tularemia. In some embodiments, the infectious disease is Typhoid Fever Group D. In some embodiments, the infectious disease is Vaginosis. In some embodiments, the infectious disease is Varicella (Chickenpox),Vibrio cholerae (Cholera). In some embodiments, the infectious disease is Vibriosis (Vibrio).
  • the infectious disease is Ebola Virus Hemorrhagic Fever. In some embodiments, the infectious disease is Lasa Virus Hemorrhagic Fever. In some embodiments, the infectious disease is Marburg Virus Hemorrhagic Fever. In some embodiments, the infectious disease is West Nile Virus. In some embodiments, the infectious disease is Yellow Fever. In some embodiments, the infectious disease is Yersenia. In some embodiments, the infectious disease is and Zika Virus Infection. [00224] In some embodiments, the pathogen is a bacteria.
  • the bacteria is a bacteria of a bacillus, bartonella, bordetella, borrelia, brucella, campylobacter, chlamydia, chlamydophila, clostridium, corynebacterium, enterococcus, escherichia, francisella, haemophilus, helicobacter, legionella, leptospira, listeria, mycobacterium, mycoplasma, neisseria, pseudomonas, rickettsia, salmonella, shigella, staphylococcus, streptococcus, treponema, ureaplasma, vibrio or yersinia genus.
  • the bacteria is a bacteria of the bacillus genus. In some embodiments, the bacteria is a bacteria of the bartonella genus. In some embodiments, the bacteria is a bacteria of the bordetella genus. In some embodiments, the bacteria is a bacteria of the borrelia genus. In some embodiments, the bacteria is a bacteria of the brucella genus. In some embodiments, the bacteria is a bacteria of the campylobacter genus. In some embodiments, the bacteria is a bacteria of the chlamydia genus. In some embodiments, the bacteria is a bacteria of the chlamydophila genus.
  • the bacteria is a bacteria of the clostridium genus. In some embodiments, the bacteria is a bacteria of the corynebacterium genus. In some embodiments, the bacteria is a bacteria of the enterococcus genus. In some embodiments, the bacteria is a bacteria of the escherichia genus. In some embodiments, the bacteria is a bacteria of the francisella genus. In some embodiments, the bacteria is a bacteria of the haemophilus genus. In some embodiments, the bacteria is a bacteria of the helicobacter genus. In some embodiments, the bacteria is a bacteria of the legionella genus.
  • the bacteria is a bacteria of the leptospira genus. In some embodiments, the bacteria is a bacteria of the listeria genus. In some embodiments, the bacteria is a bacteria of the mycobacterium genus. In some embodiments, the bacteria is a bacteria of the mycoplasma genus. In some embodiments, the bacteria is a bacteria of the neisseria genus. In some embodiments, the bacteria is a bacteria of the pseudomonas genus. In some embodiments, the bacteria is a bacteria of the rickettsia genus. In some embodiments, the bacteria is a bacteria of the salmonella genus. In some embodiments, the bacteria is a bacteria of the shigella genus.
  • the bacteria is a bacteria of the staphylococcus genus. In some embodiments, the bacteria is a bacteria of the streptococcus genus. In some embodiments, the bacteria is a bacteria of the treponema genus. In some embodiments, the bacteria is a bacteria of the ureaplasma genus. In some embodiments, the bacteria is a bacteria of the vibrio genus. In some embodiments, the bacteria is a bacteria of the yersinia genus. [00225] In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is a protozoa, helminth, or ectoparasite.
  • the protozoa is an entamoeba, giardia, leishmania, balantidium, plasmodium, or cryptosporidium.
  • the helminth is a trematode, cestode, acanthocephalan, or round worm.
  • the ectoparasite is a arthropod.
  • the pathogen is a virus.
  • the virus is a virus of the adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, coronaviridae, filoviridae, flaviviridae, hepadnaviridae, hepeviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, rhabdoviridae, or togaviridae family.
  • the virus is a virus of the virus is a virus of the adenoviridae family. In some embodiments, the virus is a virus of the arenaviridae family. In some embodiments, the virus is a virus of the astroviridae family. In some embodiments, the virus is a virus of the bunyaviridae family. In some embodiments, the virus is a virus of the caliciviridae family. In some embodiments, the virus is a virus of the coronaviridae family. In some embodiments, the virus is a virus of the filoviridae family. In some embodiments, the virus is a virus of the flaviviridae family.
  • the virus is a virus of the hepadnaviridae family. In some embodiments, the virus is a virus of the hepeviridae family. In some embodiments, the virus is a virus of the orthomyxoviridae family. In some embodiments, the virus is a virus of the papillomaviridae family. In some embodiments, the virus is a virus of the paramyxoviridae family. In some embodiments, the virus is a virus of the parvoviridae family. In some embodiments, the virus is a virus of the picornaviridae family. In some embodiments, the virus is a virus of the polyomaviridae family.
  • the virus is a virus of the poxviridae family. In some embodiments, the virus is a virus of the reoviridae family. In some embodiments, the virus is a virus of the retroviridae family. In some embodiments, the virus is a virus of the rhabdoviridae family. In some embodiments, the virus is a virus of the togaviridae family.
  • the virus is an adenovirus, coronavirus, coxsackievirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 2, cytomegalovirus, human herpes virus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, or varicella-zoster virus.
  • the virus is an adenovirus.
  • the virus is a coronavirus.
  • the coronavirus virus is Covid-19 (SARS-CoV-2). In some embodiments, the virus is a coxsackievirus. In some embodiments, the virus is a Epstein-Barr virus. In some embodiments, the virus is a hepatitis A virus. In some embodiments, the virus is a hepatitis B virus. In some embodiments, the virus is a hepatitis C virus. In some embodiments, the virus is a herpes simplex virus type 2. In some embodiments, the virus is a cytomegalovirus. In some embodiments, the virus is a human herpes virus type 8. In some embodiments, the virus is a human immunodeficiency virus.
  • the virus is an influenza virus. In some embodiments, the virus is a measles virus. In some embodiments, the virus is a mumps virus. In some embodiments, the virus is a human papillomavirus. In some embodiments, the virus is a parainfluenza virus. In some embodiments, the virus is a poliovirus. In some embodiments, the virus is a rabies virus. In some embodiments, the virus is a respiratory syncytial virus. In some embodiments, the virus is a rubella virus. In some embodiments, the virus is a varicella-zoster virus.
  • antibodies of the invention may have altered (relative to an unmodified antibody) Fc ⁇ R and/or C1q binding properties (examples of binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K D ), dissociation and association rates (k off and k on , respectively) binding affinity and/or avidity) and that certain alterations are more or less desirable.
  • binding properties include but are not limited to, binding specificity, equilibrium dissociation constant (K D ), dissociation and association rates (k off and k on , respectively) binding affinity and/or avidity
  • K D equilibrium dissociation constant
  • One skilled in the art can determine which kinetic parameter is most important for a given antibody application.
  • a modification that reduces binding to one or more positive regulator (e.g., Fc ⁇ RIIIA) and/or enhanced binding to an inhibitory Fc receptor (e.g., Fc ⁇ RIIB) would be suitable for reducing ADCC activity.
  • the ratio of binding affinities e.g., equilibrium dissociation constants (K D ) can indicate if the ADCC activity of an antibody of the invention is enhanced or decreased.
  • a modification that reduces binding to C1q would be suitable for reducing or eliminating CDC activity.
  • the affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-Fc ⁇ R interactions, i.e., specific binding of an Fc region to an Fc ⁇ R including but not limited to, equilibrium methods (e.g., enzyme-linked immuno absorbent assay (ELISA) or radioimmunoassay (RTA)), or kinetics (e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • in vitro assay methods biochemical or immunological based assays
  • ELISA enzyme-linked immuno absorbent assay
  • RTA radioimmunoassay
  • kinetics e.g., BIACORE® analysis
  • indirect binding assays e.g.
  • oligomers may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • a detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999).
  • Oligomers [00229]
  • the oligomer disclosed herein can be formed when the molecules are connected to two other molecules or more than two other molecules.
  • the oligomer disclosed herein can be made up of homomeric or heteromeric molecules.
  • the oligomer disclosed herein can be made up of different numbers of molecules.
  • the present disclosure provides an effective platform for forming multispecific and/or multivalent binding oligomers.
  • the oligomer provided herein is formed by the molecules each of which has one connection site, thus the oligomer is a dimer.
  • the oligomer provided herein is formed by the molecules each of which has two connection sites.
  • the two connection sites of one molecule are directed to two connection sites of one other molecule, thus the oligomer is a dimer.
  • the two connection sites of one molecule are directed to connection sites of two other molecules, thus the oligomer is larger than a dimer.
  • the oligomer provided herein is formed by the molecules each of which has three connection sites. In some specific embodiments, the three connection sites of one molecule are directed to three connection sites of one other molecule, thus the oligomer is a dimer. In some specific embodiments, the three connection sites of one molecule are directed to connection sites of two other molecules thus the oligomer is larger than a dimer. In some specific embodiments, the three connection sites of one molecule are directed to connection sites of three other molecules, thus the oligomer is larger than a dimer. In some embodiments, the oligomer provided herein comprises 3 units. In some embodiments, the oligomer provided herein comprises 4 units. In some embodiments, the oligomer provided herein comprises 5 units.
  • the oligomer provided herein comprises 6 units. In some embodiments, the oligomer provided herein comprises more than 6 units. In some embodiments, the oligomer is a trimer. In some embodiments, the oligomer is a tetramer. In some embodiments, the oligomer is a pentamer. In other embodiments, the oligomer is a hexamer. In other embodiments, the oligomer provided herein is a heptamer. In other embodiments, the oligomer provided herein is an octamer. In other embodiments, the oligomer provided herein is a nonamer. In other embodiments, the oligomer provided herein is a decamer.
  • the oligomer provided herein is an undecamer. In other embodiments, the oligomer provided herein is a twelve-mer. In other embodiments, the oligomer provided herein is a thirteen-mer. In other embodiments, the oligomer provided herein is a fourteen-mer. In other embodiments, the oligomer provided herein is a fifteen-mer. In other embodiments, the oligomer provided herein is a sixteen-mer. In other embodiments, the oligomer provided herein is a seventeen-mer. In other embodiments, the oligomer provided herein is an eighteen-mer. In other embodiments, the oligomer provided herein is a nineteen-mer.
  • the oligomer provided herein is a twenty-mer.
  • the oligomer provided herein can be formed by the same or different molecules. In some embodiments, the oligomer is homomeric. In other embodiments, the oligomer is heteromeric. [00232] In some embodiments, the individual molecules in the oligomer provided herein all bind to the same antigen (or target), therefore the oligomer is monospecific; however at least two molecules are different from each other, e.g., at the binding domain. For example, the molecules are different in terms of format or structure of the binding domains or the molecules are different in the sequences of the binding domains.
  • all the molecules in the oligomer provided herein bind to the same antigen, and thus the oligomer is monospecific. In other embodiments, not all the molecules in the oligomer provided herein bind to the same antigen, and thus the oligomer is multispecific. In some embodiments, the oligomer is bispecific. In some embodiments, the oligomer is trispecific. In other embodiments, the oligomer is tetraspecific. In other embodiments, the oligomer is pentaspecific. In other embodiments, the oligomer is a hexaspeicfic.
  • the oligomer provided herein binds to the same antigen but on different epitopes.
  • the oligomer provided herein is multivalent. In some embodiments, the oligomer is bivalent. In some embodiments, the oligomer is trisvalent. In other embodiments, the oligomer is tetravalent. In other embodiments, the oligomer is pentavalent. In other embodiments, the oligomer is a hexavalent. In other embodiments, the oligomer provided herein is a heptavalent. In other embodiments, the oligomer provided herein is an octavalent.
  • the oligomer provided herein is a nonavalent. In other embodiments, the oligomer provided herein is a decavalent.
  • the present disclosure further provides pharmaceutical compositions comprising at least a molecule of the present disclosure.
  • a pharmaceutical composition comprises therapeutically effective amount of the molecules provided herein and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprises therapeutically effective amount of oligomers provided herein and a pharmaceutically acceptable excipient.
  • the term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete), carrier or vehicle.
  • Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders sustained release formulations and the like Examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.
  • Such compositions will contain a prophylactically or therapeutically effective amount of the active ingredient provided herein, such as in purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration. [00237]
  • the choice of excipient is determined in part by the particular cell, and/or by the method of administration.
  • acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDTA and/or non-ionic surfactants.
  • Buffers may be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent.
  • Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof.
  • buffers may comprise histidine and trimethylamine salts such as Tris.
  • Preservatives may be added to retard microbial growth.
  • Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.
  • Tonicity agents sometimes known as “stabilizers” can be present to adjust or maintain the tonicity of liquid in a composition.
  • tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Additional exemplary excipients include: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) agents preventing denaturation or adherence to the container wall.
  • excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, ⁇ - monothiog
  • Non-ionic surfactants or detergents may be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody.
  • Suitable non- ionic surfactants include, e.g., polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose.
  • Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents include benzalkonium chloride or benzethonium chloride.
  • a pharmaceutical composition can be provided as a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng.14:201-40 (1987); Buchwald et al., Surgery 88:507-16 (1980); and Saudek et al., N. Engl. J. Med.321:569- 74 (1989)).
  • polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., a fusion protein as described herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol.25:351-56 (1989); Howard et al., J.
  • a prophylactic or therapeutic agent e.g., a fusion protein as described herein
  • a composition provided herein see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability,
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol.2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, Science 249:1527-33 (1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT publication Nos.
  • compositions described herein may also contain more than one active compound or agent as necessary for the particular indication being treated.
  • the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent.
  • cytotoxic agent chemotherapeutic agent
  • cytokine cytokine
  • immunosuppressive agent or growth inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or therapeutic molecule provided herein, construction of a nucleic acid as part of a retroviral or other vector, etc.
  • the pharmaceutical composition provided herein contains the binding molecules in amounts effective to treat or prevent the disease or disorder, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.
  • excipient can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete), carrier or vehicle.
  • adjuvant e.g., Freunds’ adjuvant (complete or incomplete)
  • Pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA.
  • compositions will contain a prophylactically or therapeutically effective amount of the active ingredient provided herein, such as in purified form, together with a suitable amount of excipient so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the choice of excipient is determined in part by the particular cell, and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, Vitamin E, sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDTA and/or non-ionic surfactants.
  • Buffers may be used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent.
  • Suitable buffering agents for use with the present disclosure include both organic and inorganic acids and salts thereof.
  • buffers may comprise histidine and trimethylamine salts such as Tris.
  • Preservatives may be added to retard microbial growth.
  • Suitable preservatives for use with the present disclosure include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.
  • Tonicity agents sometimes known as “stabilizers” can be present to adjust or maintain the tonicity of liquid in a composition.
  • tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Additional exemplary excipients include: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) agents preventing denaturation or adherence to the container wall.
  • excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, ⁇ - monothiog
  • Non-ionic surfactants or detergents may be present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody.
  • Suitable non- ionic surfactants include, e.g., polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose.
  • Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents include benzalkonium chloride or benzethonium chloride.
  • a pharmaceutical composition can be provided as a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release (see, e.g., Sefton, Crit. Ref. Biomed. Eng.14:201-40 (1987); Buchwald et al., Surgery 88:507-16 (1980); and Saudek et al., N. Engl. J. Med.321:569- 74 (1989)).
  • polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., a fusion protein as described herein) or a composition provided herein (see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol.25:351-56 (1989); Howard et al., J.
  • a prophylactic or therapeutic agent e.g., a fusion protein as described herein
  • a composition provided herein see, e.g., Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability,
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of a particular target tissue, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release Vol.2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, Science 249:1527-33 (1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents as described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT publication Nos.
  • compositions described herein may also contain more than one active compound or agent as necessary for the particular indication being treated.
  • the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent.
  • cytotoxic agent chemotherapeutic agent
  • cytokine cytokine
  • immunosuppressive agent or growth inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • compositions and delivery systems are known and can be used with the therapeutic agents provided herein, including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or therapeutic molecule provided herein, construction of a nucleic acid as part of a retroviral or other vector, etc.
  • the pharmaceutical composition provided herein contains the binding molecules in amounts effective to treat or prevent the disease or disorder, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.
  • nucleic acid molecules that encode the molecules described herein encompasses a nucleic acid molecule that includes coding sequences for the polypeptide as well as a nucleic acid molecule which includes additional coding and/or non coding sequences.
  • the nucleic acid molecules of the disclosure can be in the form of RNA or in the form of DNA.
  • a nucleic acid molecule comprises the coding sequence for a polypeptide fused in the same reading frame to a nucleic acid molecule which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide).
  • the nucleic acid molecule can have the leader sequence cleaved by the host cell to form a “mature” form of the polypeptide.
  • a nucleic acid molecule comprises the coding sequence for a polypeptide fused in the same reading frame to a marker or tag sequence.
  • a marker sequence is a hexa-histidine tag supplied by a vector that allows efficient purification of the polypeptide fused to the marker in the case of a bacterial host.
  • a marker is used in conjunction with other affinity tags.
  • the vector for the production of the molecule may be produced by recombinant DNA technology using techniques well-known in the art.
  • methods for preparing the molecule provided herein by expressing a polynucleotide containing nucleotide sequence encoding different portions of the molecule as described in Section 5.2 are described herein.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • replicable vectors comprising a nucleotide sequence encoding the molecule provided herein, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos. WO 86/05807 and WO 89/01036; and U.S. Patent No.5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce the molecule provided herein.
  • host cells containing a polynucleotide encoding the molecule provided herein, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • a variety of host-expression vector systems may be utilized to express the molecules provided herein (see, e.g., U.S. Patent No.5,807,715).
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the molecule provided herein in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • Bacterial cells such as Escherichia coli, or, eukaryotic cells, especially for the expression of whole recombinant molecule, can be used for the expression of a recombinant molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2).
  • the molecule provided herein is produced in CHO cells.
  • the expression of nucleotide sequences encoding the molecule provided herein is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the molecule being expressed. For example, when a large quantity of such a molecule is to be produced, for the generation of pharmaceutical compositions of the molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E.
  • coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.24:5503-5509); and the like.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • an AcNPV promoter for example the polyhedrin promoter.
  • the coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing the molecule in infected hosts (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359).
  • Specific initiation signals may also be required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.
  • the molecule provided herein as a fully human antibody or a fragment thereof is produced in mammalian cells, such as CHO cells.
  • stable expression For long-term, high-yield production of recombinant proteins, stable expression can be utilized.
  • cell lines which stably express the molecule described herein may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines that express the molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the molecule.
  • a number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O’Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
  • the expression levels of the molecule described herein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3 (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3 (Academic Press, New York, 1987)).
  • a marker in the vector system When a marker in the vector system is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the gene encoding the molecule described herein, production of the molecule will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:
  • the host cell may be co-transfected with two expression vectors provided herein, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci.
  • the method provided herein comprises a first step for performing the fuction of introducing an amino acid substitution to one or more molecule(s) comprising an IgG C H 2 region, e.g., substitution of the amino acid residue the position 253 or 309 by EU numbering with a cysteine residue; and a second step for performing the fuction of expressing such molecules.
  • the methods provided herein further comprises a step for performing the function of introducing a human ⁇ tailpiece to the molecules.
  • the methods provided herein comprise further purifying or isolating oliogmers comprising the molecules.
  • the methods provided herein comprise constructing a vector encoding a molecule provided herein. [00279]
  • a method of producing oligomerized molecules comprising: (a) introducing the vectors disclosed in Section 5.5 into a host cell; (b) cultivating the host cell under suitable conditions for the production of the oligomerized molecules; and (c) purifying the oligomerized molecules in the conditioned medium of the host cell.
  • the methods provided herein may comprise detecting the existence of oligomerized molecules in the conditioned medium of the host cell eg by SDS polyacrylamide gel electrophoresis (PAGE)
  • the methods provided herein may further comprise characterizing the purified oligomerized molecules with intact mass spectrometry or high-performance liquid chromatography (HPLC)- size exclusion chromatography (SEC).
  • HPLC high-performance liquid chromatography
  • SEC size exclusion chromatography
  • the added tailpiece described in Section 5.2.1 and other genetic modifications described in Section 5.2.3 can be made using recombinant DNA techniques known to one skilled in the art (see, e.g., Zyskind and Berstein, 1989, Recombinant DNA laboratory Manual; Rajagopal, 2012, Recombinant DNA Technology and Genetic Engineering; Sambrook and Russel, Molecular Cloning, A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Kostelny et al., Int. J. Cancer 93:556-565, 2001; Cole et al., J.
  • cell transfection or transduction can be performed in host cells.
  • Introducing polynucleotides into a host cell can be done by any known method, including, for example, packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to CHO cells, HeLa cells, and human hepatocellular carcinoma cells.
  • host cells secret mixture, which contains the monomeric form of the molecule and different levels of oligomers of the molecules, into culture media.
  • culture media is collected 12 hours after introducing vectors to host cells. In some embodiments, culture media is collected 24 hours after introducing vectors to host cells. In some embodiments, culture media is collected 36 hours after introducing vectors to host cells. In some embodiments, culture media is collected 48 hours after introducing vectors to host cells. In some embodiments, culture media is collected 60 hours after introducing vectors to host cells. In some embodiments, culture media is collected 72 hours after introducing vectors to host cells. In some embodiments, culture media is collected 84 hours after introducing vectors to host cells. In some embodiments, culture media is collected 96 hours after introducing vectors to host cells.
  • the molecule provided herein may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the molecule provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • the purified oligomerized molecules need to be characterized and confirmed its identity. Different methods are known in the art.
  • the purified oligomerized molecules can be characterized by mass spectrometry. In specific embodiments, the purified oligomerized molecules can be characterized by intact mass spectrometry. In other embodiments, the purified oligomerized molecules can be characterized by HPLC. In specific embodiments, the purified oligomerized molecules can be characterized by HPLC-SEC. In other embodiments, the purified oligomerized molecules can be characterized by x-ray crystallography. In other embodiments, the purified oligomerized molecules can be characterized by NMR spectroscopy. In other embodiments, the purified oligomerized molecules can be characterized by cryoelectron microscopy.
  • Polynucleic acid sequences encoding the molecules (such as antibodies or fragments thereof) of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleic acid sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present disclosure.
  • Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector.
  • Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides.
  • the vector components generally include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Pat. No. 5,648,237.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • bacteriophage such as GEMTM-11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • the expression vector of the present application may comprise two or more promoter- cistron pairs, encoding each of the polypeptide components.
  • a promoter is an untranslated regulatory sequence located upstream (5′) to a cistron that modulates its expression.
  • Prokaryotic promoters typically fall into two classes, inducible and constitutive.
  • Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g. the presence or absence of a nutrient or a change in temperature.
  • promoters recognized by a variety of potential host cells are well known.
  • the selected promoter can be operably linked to cistron DNA encoding the present antibody by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application.
  • Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes.
  • heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.
  • Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the - galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or the trc promoter.
  • trp tryptophan
  • other promoters that are functional in bacteria such as other known bacterial or phage promoters
  • Their nucleic acid sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target peptide (Siebenlist et al. Cell 20: 269 (1980)) using linkers or adaptors to supply any required restriction sites.
  • each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane.
  • the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector.
  • the signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell.
  • the signal sequence can be substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP.
  • STII heat-stable enterotoxin II
  • the production of the antibodies according to the present disclosure can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron.
  • Certain host strains e.g., the E.
  • Prokaryotic host cells suitable for expressing the molecules (such as antibodies or fragments thereof) of the present disclosure include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive organisms.
  • useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P.
  • E. coli cells are used as hosts. Examples of E.
  • coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol.2 (Washington, D.C.: American Society for Microbiology, 1987), pp.1190-1219; ATCC Deposit No.27,325) and derivatives thereof, including strain 33D3 having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A(nmpc-fepE) degP41 kan R (U.S. Pat. No.5,639,635).
  • Other strains and derivatives thereof such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537) and E.
  • coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
  • plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
  • Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Transformation means introducing DNA into the prokaryotic host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride is generally used for bacterial cells that contain substantial cell-wall barriers.
  • Prokaryotic cells used to produce the antibodies of the present application are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.
  • any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.
  • the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.
  • the prokaryotic host cells are cultured at suitable temperatures and pHs.
  • an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for the activation of the promoter.
  • PhoA promoters are used for controlling transcription of the polypeptides.
  • the transformed host cells are cultured in a phosphate-limiting medium for induction.
  • the phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons et al., J. Immunol. Methods 263:133-147 (2002)).
  • a variety of other inducers may be used, according to the vector construct employed, as is known in the art.
  • the expressed antibodies of the present disclosure are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration.
  • the proteins may be further purified, for example, by affinity resin chromatography.
  • proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced.
  • the expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.
  • PAGE polyacrylamide gel electrophoresis
  • Western blot assay Alternatively, protein production is conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. To improve the production yield and quality of the antibodies of the present disclosure, various fermentation conditions can be modified.
  • the chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. J Bio Chem 274:19601-19605 (1999); U.S. Pat. No.6,083,715; U.S. Pat. No.6,027,888; Bothmann and Pluckthun, J. Biol. Chem. 275:17100-17105 (2000); Ramm and Pluckthun, J. Biol. Chem.275:17106-17113 (2000); Arie et al., Mol. Microbiol.39:199-210 (2001).
  • Standard protein purification methods known in the art can be employed.
  • the following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.
  • Protein A immobilized on a solid phase for example can be used in some embodiments for immunoaffinity purification of binding molecules of the present disclosure.
  • the solid phase to which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column.
  • the column has been coated with a reagent, such as glycerol, in an attempt to prevent nonspecific adherence of contaminants.
  • a reagent such as glycerol
  • the solid phase is then washed to remove contaminants non-specifically bound to the solid phase.
  • the antibodies of interest is recovered from the solid phase by elution.
  • the vector components generally include, but are not limited to, one or more of the following, a signal sequence, an origin of replication, one or more marker genes, and enhancer element, a promoter, and a transcription termination sequence.
  • a vector for use in a eukaryotic host may also an insert that encodes a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, are available.
  • the DNA for such precursor region can be ligated in reading frame to DNA encoding the antibodies of the present application.
  • the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
  • Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Selection genes may encode proteins that confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline; complement auxotrophic deficiencies; or supply critical nutrients not available from complex media.
  • Selection schemes utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up nucleic acid encoding the antibodies of the present application.
  • cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
  • Mtx methotrexate
  • An exemplary appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.
  • host cells transformed or co-transformed with the polypeptide encoding-DNA sequences, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3′-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic.
  • APH aminoglycoside 3′-phosphotransferase
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the desired polypeptide sequences. Eukaryotic genes have an AT-rich region located approximately 25 to 30 based upstream from the site where transcription is initiated.
  • Another sequence found 70 to 80 bases upstream from the start of the transcription of many genes may be included.
  • the 3′ end of most eukaryotic may be the signal for addition of the poly A tail to the 3′ end of the coding sequence. All of these sequences may be inserted into eukaryotic expression vectors.
  • Polypeptide transcription from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterolog
  • Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein, and insulin). Examples include the SV40 enhancer on the late side of the replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters.
  • the enhancer may be spliced into the vector at a position 5′ or 3′ to the polypeptide encoding sequence, but is preferably located at a site 5′ from the promoter.
  • Expression vectors used in eukaryotic host cells also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the polypeptide-encoding mRNA.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.
  • Host cells can be transformed with the above-described expression or cloning vectors for antibodies production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce the antibodies of the present application may be cultured in a variety of media.
  • Re.30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • growth factors such as insulin, transferrin, or epidermal growth factor
  • salts such as sodium chloride, calcium, magnesium, and phosphate
  • buffers such as HEPES
  • nucleotides such as adenosine and thymidine
  • antibiotics such as GENTAMYCINTM drug
  • trace elements defined as
  • the antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a commercially available protein concentration filter for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the protein composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly (styrene-divinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • compositions are administered in an effective amount to effect treatment of the disease or disorder.
  • Uses include uses of the compositions in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods.
  • the methods are carried out by administering the compositions to the subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or disorder in the subject.
  • the treatment provided herein cause complete or partial amelioration or reduction of a disease or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the terms include, but do not imply, complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
  • the treatment provided herein delay development of a disease or disorder, e.g., defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer).
  • This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease or disorder
  • a late stage cancer such as development of metastasis
  • the method or the use provided herein prevents a disease or disorder.
  • the present molecules or oligomers are used for treating solid tumor cancer.
  • the present molecules or oligomers are used for treating blood cancer.
  • the disease or disorder is an autoimmune and inflammatory disease.
  • the disease or disorder is an infectious disease.
  • the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis.
  • diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct
  • the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.
  • the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma.
  • the cancer is selected from a group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lympho
  • NHL non-Hodgkin's lymph
  • the disease or disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from a group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, a liver cancer and a lung cancer.
  • the cancer is an adrenal cancer.
  • the cancer is an anal cancer.
  • the cancer is an appendix cancer. In some embodiments, the cancer is a bile duct cancer. In some embodiments, the cancer is a bladder cancer. In some embodiments, the cancer is a bone cancer. In some embodiments, the cancer is a brain cancer. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a cervical cancer. In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is an esophageal cancer. In some embodiments, the cancer is a gallbladder cancer. In some embodiments, the cancer is a gestational trophoblastic. In some embodiments, the cancer is a head and neck cancer.
  • the cancer is a Hodgkin lymphoma. In some embodiments, the cancer is an intestinal cancer. In some embodiments, the cancer is a kidney cancer. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is a liver cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is a mesothelioma. In some embodiments, the cancer is a multiple myeloma (MM). In some embodiments, the cancer is a neuroendocrine tumor. In some embodiments, the cancer is a non- Hodgkin lymphoma. In some embodiments, the cancer is an oral cancer.
  • the cancer is an ovarian cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is a prostate cancer. In some embodiments, the cancer is a sinus cancer. In some embodiments, the cancer is a skin cancer. In some embodiments, the cancer is a soft tissue sarcoma spinal cancer. In some embodiments, the cancer is a stomach cancer. In some embodiments, the cancer is a testicular cancer. In some embodiments, the cancer is a throat cancer. In some embodiments, the cancer is a thyroid cancer. In some embodiments, the cancer is a uterine cancer endometrial cancer. In some embodiments, the cancer is a vaginal cancer. In some embodiments, the cancer is a vulvar cancer.
  • the adrenal cancer is an adrenocortical carcinoma (ACC), adrenal cortex cancer, pheochromocytoma, or neuroblastoma.
  • the anal cancer is a squamous cell carcinoma, cloacogenic carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma.
  • the appendix cancer is a neuroendocrine tumor (NET), mucinous adenocarcinoma, goblet cell carcinoid, intestinal-type adenocarcinoma, or signet-ring cell adenocarcinoma.
  • NET neuroendocrine tumor
  • the bile duct cancer is an extrahepatic bile duct cancer, adenocarcinomas, hilar bile duct cancer, perihilar bile duct cancer, distal bile duct cancer, or intrahepatic bile duct cancer.
  • the bladder cancer is transitional cell carcinoma (TCC), papillary carcinoma, flat carcinoma, squamous cell carcinoma, adenocarcinoma, small-cell carcinoma, or sarcoma.
  • the bone cancer is a primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer.
  • the brain cancer is an astrocytoma, brain stem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary carcinoma, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal region tumor, medulloblastoma, or primary CNS lymphoma.
  • the breast cancer is a breast adenocarcinoma, invasive breast cancer, noninvasive breast cancer, breast sarcoma, metaplastic carcinoma, adenocystic carcinoma, phyllodes tumor, angiosarcoma, HER2-positive breast cancer, triple-negative breast cancer, or inflammatory breast cancer.
  • the cervical cancer is a squamous cell carcinoma, or adenocarcinoma.
  • the colorectal cancer is a colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma, signet ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma.
  • the esophageal cancer is an adenocarcinoma or squamous cell carcinoma.
  • the gall bladder cancer is an adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma.
  • the gestational trophoblastic disease is a hydatidiform mole, gestational trophoblastic neoplasia (GTN), choriocarcinoma, placental-site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT).
  • the head and neck cancer is a laryngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, oral cancer, oropharyngeal cancer, or tonsil cancer.
  • the Hodgkin lymphoma is a classical Hodgkin lymphoma, nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte-depleted, or nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL).
  • the intestinal cancer is a small intestine cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumors, carcinoid tumors, or lymphoma.
  • the kidney cancer is a renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urothelial cancer, renal pelvis carcinoma, or renal sarcoma.
  • the leukemia is an acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or a myelodysplastic syndrome (MDS).
  • ALL acute lymphocytic leukemia
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myeloid leukemia
  • HCL hairy cell leukemia
  • MDS myelodysplastic syndrome
  • the leukemia is AML.
  • the liver cancer is a hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis.
  • the lung cancer is a small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large-cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland- type lung carcinoma, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor.
  • the melanoma is a superficial spreading melanoma, nodular melanoma, acral-lentiginous melanoma, lentigo maligna melanoma, amelanotic melanoma, desmoplastic melanoma, ocular melanoma, or metastatic melanoma.
  • the mesothelioma is a pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma.
  • the multiple myeloma is an active myeloma or smoldering myeloma.
  • the neuroendocrine tumor is a gastrointestinal neuroendocrine tumor, pancreatic neuroendocrine tumor, or lung neuroendocrine tumor.
  • the non-Hodgkin’s lymphoma is an anaplastic large-cell lymphoma, lymphoblastic lymphoma, peripheral T cell lymphoma, follicular lymphoma, cutaneous T cell lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, MALT lymphoma, small-cell lymphocytic lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), precursor T- lymphoblastic leukemia/lymphoma, acute lymphocytic leukemia (ALL), adult T cell lymphoma/leukemia (ATLL), hairy cell leukemia, B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, primary central nervous system (CNS) lymphoma, mantle cell lymphoma (MCL), marginal zone lymphomas
  • the oral cancer is a squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinomas, lymphoma, benign oral cavity tumor, eosinophilic granuloma, fibroma, granular cell tumor, karatoacanthoma, leiomyoma, osteochondroma, lipoma, schwannoma, neurofibroma, papilloma, condyloma acuminatum, verruciform xanthoma, pyogenic granuloma, rhabdomyoma, odontogenic tumors, leukoplakia, erythroplakia, squamous cell lip cancer, basal cell lip cancer, mouth cancer, gum cancer, or tongue cancer.
  • the ovarian cancer is a ovarian epithelial cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, clear cell epithelial ovarian cancer, undifferentiated epithelial ovarian cancer, ovarian low malignant potential tumors, primary peritoneal carcinoma, fallopian tube cancer, germ cell tumors, teratoma, dysgerminoma ovarian germ cell cancer, endodermal sinus tumor, sex cord- stromal tumors, sex cord-gonadal stromal tumor, ovarian stromal tumor, granulosa cell tumor, granulosa-theca tumor, Sertoli-Leydig tumor, ovarian sarcoma, ovarian carcinosarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, Krukenberg tumor, or ovarian cyst.
  • the pancreatic cancer is a pancreatic exocrine gland cancer, pancreatic endocrine gland cancer, or pancreatic adenocarcinoma, islet cell tumor, or neuroendocrine tumor.
  • the prostate cancer is a prostate adenocarcinoma, prostate sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor.
  • the sinus cancer is a squamous cell carcinoma, mucosa cell carcinoma, adenoid cystic cell carcinoma, acinic cell carcinoma, sinonasal undifferentiated carcinoma, nasal cavity cancer, paranasal sinus cancer, maxillary sinus cancer, ethmoid sinus cancer, or nasopharynx cancer.
  • the skin cancer is a basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi sarcoma (KS), actinic keratosis, skin lymphoma, or keratoacanthoma.
  • basal cell carcinoma a basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi sarcoma (KS), actinic keratosis, skin lymphoma, or keratoacanthoma.
  • KS Kaposi sarcoma
  • the soft tissue cancer is an angiosarcoma , dermatofibrosarcoma, epithelioid sarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumors (GISTs), Kaposi sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid/round cell liposarcoma (MRCL), well-differentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma.
  • GISTs gastrointestinal stromal tumors
  • Kaposi sarcoma leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid/round cell liposarcoma (MRCL), well-differentiated liposar
  • the spinal cancer is a spinal metastatic tumor.
  • the stomach cancer is a stomach adenocarcinoma, stomach lymphoma, gastrointestinal stromal tumors, carcinoid tumor, gastric carcinoid tumors, Type I ECL-cell carcinoid, Type II ECL-cell carcinoid, or Type III ECL-cell carcinoid.
  • the testicular cancer is a seminoma, non-seminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratoma, gonadal stromal tumor, leydig cell tumor, or sertoli cell tumor.
  • the throat cancer is a squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal cancer, pharyngeal cancer, nasopharynx cancer, oropharynx cancer, hypopharynx cancer, laryngeal cancer, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, lymphoepithelioma, spindle cell carcinoma, verrucous cancer, undifferentiated carcinoma, or lymph node cancer.
  • the thyroid cancer is a papillary carcinoma, follicular carcinoma, Hürthle cell carcinoma, medullary thyroid carcinoma, or anaplastic carcinoma.
  • the uterine cancer is an endometrial cancer, endometrial adenocarcinoma, endometroid carcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinosarcoma, uterine sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, or undifferentiated sarcoma.
  • the vaginal cancer is a squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma.
  • the vulvar cancer is a squamous cell carcinoma or adenocarcinoma.
  • the disease or disorder is caused by a pathogen.
  • the pathogen causes an infectious disease selected from the group consisting of an Acute Flaccid Myelitis (AFM), Anaplasmosis, Anthrax, Babesiosis, Botulism, Brucellosis, Campylobacteriosis, Carbapenem-resistant Infection, Chancroid, Chikungunya Virus Infection, Chlamydia, Ciguatera, Difficile Infection, Perfringens, Coccidioidomycosis fungal infection, coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jacob Disease/transmissible spongiform encephalopathy, Cryptosporidiosis (Crypto), Cyclosporiasis, Dengue 1,2,3 or 4, Diphtheria, E.
  • AMF Acute Flaccid Myelitis
  • Anaplasmosis Anaplasmosis
  • Anthrax Anthrax
  • coli infection/Shiga toxin-producing (STEC), Eastern Equine Encephalitis, Hemorrhagic Fever (Ebola), Ehrlichiosis, Encephalitis, Arboviral or parainfectious, Non-Polio Enterovirus, D68 Enteroviru(EV-D68), Giardiasis, Glanders, Gonococcal Infection, Granuloma inguinale, Haemophilus Influenza disease Type B (Hib or H-flu), Hantavirus Pulmonary Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes, Herpes Zoster (Shingles), Histoplasmosis infection, Human Immunodeficiency Virus/AIDS (HIV/AIDS), Human Papillomavirus (HPV), Influenza (F
  • the pathogen is a bacteria.
  • the bacteria is a bacteria of a bacillus, bartonella, bordetella, borrelia, brucella, campylobacter, chlamydia, chlamydophila, clostridium, corynebacterium, enterococcus, escherichia, francisella, haemophilus, helicobacter, legionella, leptospira, listeria, mycobacterium, mycoplasma, neisseria, pseudomonas, rickettsia, salmonella, shigella, staphylococcus, streptococcus, treponema, ureaplasma, vibrio or yersinia genus.
  • the pathogen is a parasite.
  • the parasite is a protozoa, helminth, or ectoparasite.
  • the protozoa is an entamoeba, giardia, leishmania, balantidium, plasmodium, or cryptosporidium.
  • the helminth is a trematode, cestode, acanthocephalan, or round worm.
  • the ectoparasite is an arthropod.
  • the pathogen is a virus.
  • the virus is a virus of the adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, coronaviridae, filoviridae, flaviviridae, hepadnaviridae, hepeviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, rhabdoviridae, or togaviridae family.
  • the virus is an adenovirus, coronavirus, coxsackievirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 2, cytomegalovirus, human herpes virus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, or varicella-zoster virus.
  • the disease or disorder is an immune or autoimmune disorder.
  • Such disorders include autoimmune bullous disease, abetalipoprotemia, acquired immunodeficiency-related diseases, acute immune disease associated with organ transplantation, acquired acrocyanosis, acute and chronic parasitic or infectious processes, acute pancreatitis, acute renal failure, acute rheumatic fever, acute transverse myelitis, adenocarcinomas, aerial ectopic beats, adult (acute) respiratory distress syndrome, AIDS dementia complex, alcoholic cirrhosis, alcohol- induced liver injury, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allergy and asthma, allograft rejection, alpha-l-antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis, anemia, angina pectoris, ankylosing spondylitis-associated lung disease, anterior horn cell degeneration, antibody mediated cytotoxicity, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aortic and peripheral
  • the disease or disorder is an inflammatory disease.
  • Inflammation plays a fundamental role in host defenses and the progression of immune-mediated diseases.
  • the inflammatory response is initiated in response to injury (e.g., trauma, ischemia, and foreign particles) and infection (e.g., bacterial or viral infection) by a complex cascade of events, including chemical mediators (e.g., cytokines and prostaglandins) and inflammatory cells (e.g., leukocytes).
  • the inflammatory response is characterized by increased blood flow, increased capillary permeability, and the influx of phagocytic cells. These events result in swelling, redness, warmth (altered heat patterns), and pus formation at the site of injury or infection.
  • Cytokines and prostaglandins control the inflammatory response, and are released in an ordered and self-limiting cascade into the blood or affected tissues. This release of cytokines and prostaglandins increases the blood flow to the area of injury or infection, and may result in redness and warmth. Some of these chemicals cause a leak of fluid into the tissues, resulting in swelling. This protective process may stimulate nerves and cause pain. These changes, when occurring for a limited period in the relevant area, work to the benefit of the body. A delicate well-balanced interplay between the humoral and cellular immune elements in the inflammatory response enables the elimination of harmful agents and the initiation of the repair of damaged tissue.
  • the inflammatory response may result in considerable damage to normal tissue and may be more harmful than the original insult that initiated the reaction.
  • clinical intervention is needed to prevent tissue damage and organ dysfunction.
  • Diseases such as psoriasis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, Crohn’s disease, asthma, allergies or inflammatory bowel disease, are characterized by chronic inflammation.
  • Inflammatory diseases such as arthritis, related arthritic conditions (e.g., osteoarthritis, rheumatoid arthritis, and psoriatic arthritis), inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), sepsis, psoriasis, atopic dermatitis, contact dermatitis, and chronic obstructive pulmonary disease, chronic inflammatory pulmonary diseases are also prevalent and problematic ailments. 5.7.2.
  • a sample such as a cell, tissue sample, lysate, composition, or other sample derived therefrom is contacted with a binding molecule provided herein and the binding is determined or detected.
  • the sample When binding in the test sample is demonstrated or detected as compared to a reference cell of the same tissue type, it may indicate the presence of an associated disease or disorder, and/or that a therapeutic containing the antibody will specifically bind to a tissue or cell that is the same as or is of the same type as the tissue or cell or other biological material from which the sample is derived.
  • the sample is from human tissues and may be from diseased and/or normal tissue, e.g., from a subject having the disease or disorder to be treated and/or from a subject of the same species as such subject but that does not have the disease or disorder to be treated.
  • the normal tissue or cell is from a subject having the disease or disorder to be treated but is not itself a diseased cell or tissue, such as a normal tissue from the same or a different organ than a cancer that is present in a given subject.
  • exemplary immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA).
  • An indicator moiety, or label group can be used so as to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures.
  • exemplary labels include radionuclides (e.g. 125 I, 131 I, 35 S, 3 H, or 32 P and/or chromium ( 51 Cr), cobalt ( 57 Co), fluorine ( 18 F), gadolinium ( 153 Gd, 159 Gd), germanium ( 68 Ge), holmium ( 166 Ho), indium ( 115 In, 113 In, 112 In, 111 In), iodine ( 125 I, 123 I, 121 I), lanthanium ( 140 La), lutetium ( 177 Lu), manganese ( 54 Mn), molybdenum ( 99 Mo), palladium ( 103 Pd), phosphorous ( 32 P), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium (186Re, 188Re),
  • labeled antibodies provided.
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • antibodies are not labeled, and the presence thereof can be detected using a labeled antibody which binds to any of the antibodies.
  • kits, unit dosages, and articles of manufacture comprising any of the molecules and oligomers described herein.
  • a kit is provided which contains any one of the pharmaceutical compositions described herein and preferably provides instructions for its use.
  • the kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information.
  • the present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating a disease or disorder (such as cancer) described herein and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the composition is used for treating the particular condition in an individual.
  • the label or package insert will further comprise instructions for administering the composition to the individual.
  • the label may indicate directions for reconstitution and/or use.
  • the container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation.
  • Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • kits or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • pharmacies for example, hospital pharmacies and compounding pharmacies.
  • certain abbreviations are used herein.
  • One example is the single letter abbreviation to represent amino acid residues.
  • amino acids and their corresponding three letter and single letter abbreviations are as follows: alanine Ala (A) arginine Arg (R) asparagine Asn (N) aspartic acid Asp (D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycine Gly (G) histidine His (H) isoleucine Ile (I) leucine Leu (L) lysine Lys (K) methionine Met (M) phenylalanine Phe (F) proline Pro (P) serine Ser (S) threonine Thr (T) tryptophan Trp (W) tyrosine Tyr (Y) valine Val (V) [00346] The disclosure is generally disclosed herein using affirmative language to describe the numerous embodiments.
  • Example 1 Plasmid Construction for huIgG1 Fc- ⁇ tp Variants.
  • This example provides the design of plasmid constructs for candidates with different point mutations in the constant region to test for formation of hexamers.
  • Constructs were made where each of the following groups of mutations were introduced into the human IgG1 Fc region based on the EU numbering. Ile 253 was substituted with cysteine (“I253C”). Tyr 436 was substituted with cysteine (“Y436C”). Gln 438 was substituted with cysteine (“Q438C”). His 310 was substituted with cysteine (“H310C”).
  • Leu 251 was substituted with cysteine (“L251C”), Ile 253 was substituted with glycine (“I253G”), and Ser 254 was substituted with cysteine (“S254C”). Ser 254 was substituted with cysteine (“S254C”) and Asn 434 was substituted with cysteine (“N434C”). Leu 251 was substituted with cysteine (“L251C”) and Ser 254 was substituted with cysteine (“S254C”). Asn 286 was substituted with cysteine (“N286C”). Leu 309 was substituted with cysteine (“L309C”).
  • a ⁇ tp (SEQ ID NO: 1) was added to the C-terminus of the C H 3 region.
  • a signal peptide (SEQ ID NO: 2) was added to the N-terminus of the C H 2 region.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with I253C mutation is SEQ ID NO: 3.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with Y436C mutation is SEQ ID NO: 4.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with Q438C mutation is SEQ ID NO: 5.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with H310C mutation is SEQ ID NO: 6.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with L251C, I253G, and S254C mutations is SEQ ID NO: 7.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with S254C and N434C mutations is SEQ ID NO: 8.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with L251C and S254C mutations is SEQ ID NO: 9.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with N286C mutations is SEQ ID NO: 10.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with L309C mutation is SEQ ID NO: 11.
  • the amino acid sequence of huIgG1 Fc- ⁇ tp with L234A, L235A, D270A, L309C, P329A, and P331G mutations is SEQ ID NO: 12.
  • the various sequences described above, each including the signal peptide sequence of SEQ ID NO: 2, are shown in Table 1 below. Table 1.
  • the amino acid sequences of the huIgG1 Fc- ⁇ tp variants were codon optimized for mammalian expression.
  • the codon optimized DNA was synthesized and sub-cloned into mammalian expression vector pTT5.
  • 6.2 Example 2 Production, Purification, and Characterization of Hexamers of huIgG1 Fc- ⁇ tp Variants.
  • the various huIgG1 Fc- ⁇ tp contructs were transiently expressed using the Expi293 expression system (Thermo Fisher Scientific). Transfection was done following manufacturer’s protocol.
  • the transfected cell culture was incubated at 37 o C, 5% CO2 for 4 days.10 ⁇ L of conditioned medium from each of huIgG1 Fc ⁇ tp contructs and controls was mixed with 33 ⁇ L 4X Laemmli buffer and was heated at 95 ⁇ C for 5 min. NuPAGETM 3-8%, Tris-Acetate protein gel was prepared, and used to visualize the transient expression of target proteins in the conditioned medium (see FIG.1). [00354] As shown in FIG.1A, wild type huIgG1- ⁇ tp appeared mainly as monomeric form (54kDa, lane 12).
  • huIgG1 Fc (H310C)- ⁇ tp huIgG1 Fc (L251C, I253G, and S254C)- ⁇ tp
  • huIgG1 Fc (S254C and N434C)- ⁇ tp huIgG1 Fc (L251C and S254C)- ⁇ tp
  • huIgG1 Fc (N286C)- ⁇ tp was expressed mainly as monomer (lanes 5).
  • huIgG1 Fc (I253C)- ⁇ tp was used for further purification and analysis.
  • Purification of huIgG1 Fc (I253C)- ⁇ tp was initiated manually by affinity pulldown using MabSelect TM protein A resin and CaptureSelectTM FcXL Affinity Matrix (Thermo Fisher Scientific) side by side. 200 ⁇ l of protein A and FcXL resin were packed in small spin columns. Culture media with the expressed proteins were applied to the column by pipetting. After binding, the flow-through, wash and elution processes were performed by centrifugation at 700 g for 0.5 min per spin.
  • Buffer 1 50 mM sodium acetate, pH 3.5
  • Buffer 2 20 mM acetic acid, 100 mM glycine, pH 3.5
  • PBS buffer pH 7.5
  • Each elution fraction was at a volume of 200 ⁇ l and neutralized immediately after elution.
  • 20 ⁇ L of sample was mixed with 7 ⁇ L 4X Laemmli buffer (non-reducing), and heat denatured at 95 ⁇ C for 5 min.
  • huIgG1 Fc (I253C)- ⁇ tp can bind to FcXL resin via C H 3 interaction.
  • huIgG1 Fc (I253C)- ⁇ tp was captured and purified using diafiltration followed by anion exchange chromatography (AIEX).
  • AIEX anion exchange chromatography
  • 5 ml culture media with the expressed protein was filtered and buffer exchanged to Buffer A (50 mM Tris, 50 mM NaCl, pH 8.0) via diafiltration using a 30 kDa cutoff concentrator (MilliporeSigma TM Amicon TM Ultra).
  • the protein was then loaded to a 1 ml HiTrap TM Q XL column (Cytiva) on a ⁇ KTA Avant FPLC system at a flow rate of 1 ml/min.
  • the column with bound protein was washed by 10 column volume (CV) of Buffer A and eluted using a liner gradient from 100% Buffer A towards 100% Buffer B with high salt (50 mM Tris, 500 mM NaCl, pH 8.0).
  • the elution fractions were collected at 250 ⁇ l volume.
  • Hexameric huIgG1 Fc (I253C)- ⁇ tp exhibited strong binding to Q XL resin and eluted at a gradient of 75% Buffer B or higher (note that 25% Buffer A/75% Buffer B contains 370 mM NaCl, and the conductivity was equivalent to 18.45 mS/cm).
  • Elution fractions were analyzed by electrophoresis using a 4-20% CriterionTM TGX Stain-FreeTM Protein Gel as shown in FIG.2C.
  • the pooled fractions (right to the marker lane in FIG.2C) were buffer exchanged to standard HBS buffer for further characterization.
  • a single huIgG1 Fc (I253C)- ⁇ tp chain has a detected molecular weight of 26905.70 Da, as shown in FIG.3A, which matches the calculated molar mass of a single chain without post translational modification (26904.50 Da).
  • the purified huIgG1 Fc (I253C)- ⁇ tp hexamer was also analyzed under non-denaturing conditions by HPLC- SEC (Agilent).15 ⁇ l of huIgG1 Fc (I253C)- ⁇ tp (1 mg/ml) was injected to a TSKgel G3000SW column (TOSOH) for a 30-minute run at a flow rate of 0.5 ml/min using 2X PBS as the mobile phase. BioRad gel filtration standard was used before and after the sample runs.
  • the retention time of bovine thyroglobulin (670 kDa) was 13 minutes
  • the retention time of bovine ⁇ -globulin (158 kDa) was 18 minutes
  • the retention time of chicken ovalbumin 44 kDa was 20 minutes.
  • huIgG1 Fc (I253C)- ⁇ tp monomer comprised 2 Fc chains and had an estimated size of 54 kDa
  • the hexamer comprised 12 Fc chains and had a size of 324 kDa.
  • Exemplary hexameric IgG antibodies comprising the huIgG1 Fc (I253C)- ⁇ tp were expressed and purified.
  • the antigen binding portion of the exemplary hexameric IgG antibodies comprised a VH and VL from anti-beta klotho, anti-GFRAL or anti-VEGF as shown in Table 2.
  • Table 2. Proteins were transiently expressed using the Expi293 expression system and analyzed by electrophoresis as described above.
  • IgG hexamers comprising an huIgG1 Fc (I253C)- ⁇ tp and a VH and VL of anti-beta klotho are shown in lane 4; IgG hexamers comprising an huIgG1 Fc (I253C)- ⁇ tp and a VH and VL of anti-GFRAL are shown in lane 5; and IgG hexamers comprising an huIgG1 Fc (I253C)- ⁇ tp and a VH and VL of anti-VEGF are shown in lane 6 of FIG.4. [00360] These hexamers were purified by protein L affinity chromatography followed by mixed mode chromatography.
  • the protein sample was filtered through a 0.2 ⁇ m sterile filter unit and purified by affinity chromatography. Under the right buffer condition (Buffer A: 1xPBS) the sample was loaded to a 5 ml HiTrap ProL column using AKTA AVANT FPLC system at a flow rate of 5 ml/min. The bound protein was washed by 5 CV of Buffer A and eluted from the resin using 15 CV of 100 % Buffer B1 (0.1 M Glycine, 20 mM acetate, pH 3.5) and then 15 CV of Buffer B2 (0.1 M Glycine, 20 mM acetate, pH 3.0). The eluted fractions were pooled and neutralized separately.
  • Buffer A 1xPBS
  • the neutralized pooled fractions were then filtered through a 0.2 ⁇ m sterile filter unit and purified by mixed mode chromatography. Under the right buffer condition (Buffer C: 50 mM HEPES, 30 mM NaCl, 5 mM PO4) the sample was loaded to a EconoFit CHT Type II, 40 ⁇ m prepacked column using AKTA AVANT FPLC system at a flow rate of 5 ml/min. The bound protein was washed by 5 CV of Buffer C and eluted with a 20 CV linear gradient from 0% to 100% Buffer D1 (50 mM HEPES, 2 M NaCl, 5 mM PO4), then with a 10 CV linear gradient back to 0% Buffer D1.
  • Buffer D1 50 mM HEPES, 2 M NaCl, 5 mM PO4
  • huIgG1 LALA, D270A, P329A, K322A (I253C)- ⁇ tp CPPCPAPEAAGGPSVFLFPPKPKDTLMCSRTPEVTCVVVDVSHEAPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALAAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPTLYNVSLVMSD TAGTCY (SEQ ID NO:19) AA: L234A,L235A (LALA) A: K322A A: P329A C: I253C PTLYNVSLVMSDTAGTCY: ⁇ TP (SEQ ID NO:1) [00365] huI
  • huIgG1 Fc (LALA D270A P329A P331G) (I253C)- ⁇ tp (lane 12), huIgG1 Fc (LALA D270A P329A) (I253C)- ⁇ tp (lane 13) and huIgG1 Fc (LALA D270A K322A P329A) (I253C)- ⁇ tp (lane 9) all showed good expression. 6.6 Example 6 – huIgG1 Fc (I253C)- ⁇ tp Effectorless Variants binding to Human C1q [00377] The variants were expressed and purified as described in Example 3.
  • Example 8 huIgG1 Fc (L309C)- ⁇ tp In Combination With Effectorless Variants binding to Human C1q
  • Exemplary hexameric IgG antibodies comprising the huIgG1 Fc (L309C)- ⁇ tp were expressed and purified and point mutations were introduced in the constant region to test for reduction in effector functions.
  • the huIgG1 Fc (L309C)- ⁇ TP had a strong binding to C1q, but C1q binding was eliminated by the addition of the other LALA, D270A, P329A, and P331G mutation combination.
  • Biacore analysis was performed against Fc ⁇ R1.
  • a Biacore Series S CM5 Sensor Chip had its surface coupled with an anti-His capture antibody (Cytiva 28995056) via EDC-NHS Chemistry.
  • Flow cell 1 of the sensor chip served as a reference while the remaining flow cells captured Fc ⁇ RI-His (R&D Systems 1257-FC).
  • Human Fc ⁇ RI was captured by running 1 ⁇ g/mL of the molecule at a flow rate of 10 ⁇ L/min over the Anti-His modified CM5 flow cell surface for 30 seconds. Subsequently, PSC- 0821 and PSC-1538 were each injected at a high flow rate of 30 ⁇ L/min for 120 seconds over all flow cells at five different concentrations (6.25nM-100nM, 2-fold dilutions). An accurate KD measurement could not be evaluated with the 1:1 Langmuir Binding model. Instead, the sensorgrams were qualitatively assessed to determine a binary yes/no binding interaction between Fc ⁇ RI and each of the two hexamers.
  • ABX1963/ABX2149 (anti-VEGF) VH QVQLVQSGAEVKKPGASVKVSCKASGYDFTHYGMNWVRQAPGQRLEWMGWINTYTG EPTYAADFKRRVTITRDTSASTAYMELSSLRSEDTAVYYCAKYPYYYGTSHWYFDVWG QGTLVTVSS (SEQ ID NO:33)
  • the Fc region of ABX1963 contained only the L234A/L235A effect
  • the Fc region of ABX2149 comprised the identical amino acid sequence as ABX1963 except further introduced the D270A, P329A, and P331G substitutions, resulting in a final construct of: huIgG1 (LALA, D270A, P329A, P331G) (I253C)- ⁇ tp (SEQ ID NO: 20).
  • huIgG1 LALA, D270A, P329A, P331G
  • I253C I253C- ⁇ tp
  • VEGF binding was assessed using a bioluminescent cell-based reporter assay (Promega Inc., Cat.#GA2001) that measured VEGF stimulation of KDR (VEGFR2) using the NFAT-RE as a readout.
  • the assay was performed following manufacturer’s protocol. Briefly, serial dilutions (10 pM to 75 nM) of each anti-VEGF construct were added to pre-plated KDR/NFAT-RE HEK293 cells followed by addition of recombinant human VEGF molecule at concentration of 3nM. After a 6-hour incubation, Bio-GloTM Reagent was added and luminescence was quantified using luminescence plate reader. The IC50 values for each construct are provided in Table 4.

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Abstract

Selon certains aspects, l'invention concerne des molécules qui sont modifiées pour former une structure Fc oligomère et contiennent des mutations pour réduire les fonctions effectrices.
PCT/US2023/067976 2022-06-07 2023-06-06 Multimérisation de molécules de liaison ayant un variant de région constante d'anticorps et des mutations qui réduisent les fonctions effectrices WO2023240067A2 (fr)

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