Multimerization of binding molecules having antibody constant region variants

Abstract

Molecules engineered to form oligomers, each of which has an IgG C 3 nucleotide at position 253 according to EU numbering substituted to a cysteine. H 2 domain and / or the human μ tailpiece.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 180,969, filed April 28, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application incorporates by reference in its entirety the Sequence Listing submitted with this application as text entitled "13370-120-228_Sequence_Listing_ST25.txt", which was created on April 25, 2022 and is 26,426 bytes in size.

[0003] 1. Engineered IgG C H Provided herein are molecules comprising the two regions and oligomeric complexes comprising the same. Further provided herein are pharmaceutical compositions comprising the molecules or oligomers described herein, host cells, nucleic acids, vectors related to the molecules described herein, and methods of making and using such molecules and oligomers. [Background technology]

[0004] 2. In the field of antibody therapeutics, the design and generation of novel recombinant antibodies or derivatives has attracted considerable attention and is making continuous progress. Specifically, novel designs are desired to overcome the limitations of native antibodies and to provide advantages such as improvements in antibody half-life, pharmacokinetics, stability, avidity, blood clearance, tissue or target cell penetration and retention, etc. The compositions and methods described herein fulfill this need and provide related advantages. Summary of the Invention

[0005] 3. In one embodiment, the present invention provides IgG C H A molecule containing two IgG domains according to EU numbering: H A molecule is provided in which position 253 in the 2 region is substituted to be a cysteine.

[0006] In some embodiments, the molecule further comprises an IgG hinge region. H In yet other embodiments, the molecule further comprises a human μ tailpiece. In yet other embodiments, the molecule further comprises an IgG C H It further includes one region.

[0007] In some specific embodiments, the human μ tailpiece comprises the 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 μ tailpiece comprises the amino acid sequence of SEQ ID NO: 1. H In yet another specific embodiment, the human μ tailpiece is conjugated to the C-terminus of the IgG C2 domain. H It is conjugated to the C-terminus of the 3 domain.

[0008] In some embodiments, the IgG is human IgG. In some specific embodiments, the human IgG is human IgG1. In other specific embodiments, the human IgG is human IgG2. In yet other specific embodiments, the human IgG is human IgG3. In yet still other specific embodiments, the human IgG is human IgG4.

[0009] In some embodiments, the molecule further comprises a binding domain that specifically binds to a target. In some particular embodiments, the binding domain is an antibody fragment. In other embodiments, the molecule is an antibody or an antigen-binding fragment thereof.

[0010] In one aspect, provided herein is an oligomer comprising two or more molecules described herein.In another aspect, provided herein is an isolated nucleic acid encoding a molecule described herein.In another aspect, provided herein is a vector comprising a nucleic acid described herein.

[0011] In another aspect, provided herein is an oligomer comprising two or more molecules, each molecule being an IgG C H IgG C according to EU numbering, which contains 2 regions H Position 253 in the 2 domain is substituted to be a cysteine.

[0012] In some embodiments, the molecule further comprises an IgG hinge region. H In yet other embodiments, the molecule further comprises a human μ tailpiece. In yet other embodiments, the molecule further comprises an IgG C H It further includes one region.

[0013] In some specific embodiments, the human μ tailpiece comprises the 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 μ tailpiece comprises the amino acid sequence of SEQ ID NO: 1. H In yet another specific embodiment, the human μ tailpiece is conjugated to the C-terminus of the IgG C2 domain. H It is conjugated to the C-terminus of the 3 domain.

[0014] In some embodiments, the IgG is human IgG. In some specific embodiments, the human IgG is human IgG1. In other specific embodiments, the human IgG is human IgG2. In yet other specific embodiments, the human IgG is human IgG3. In yet still other specific embodiments, the human IgG is human IgG4.

[0015] In some embodiments, the molecule further comprises a binding domain that specifically binds to a target. In some particular embodiments, the binding domain is an antibody fragment. In other embodiments, the molecule is an antibody or an antigen-binding fragment thereof.

[0016] In some embodiments, the oligomer is a pentamer, hi other embodiments, the oligomer is a hexamer.

[0017] In some embodiments, the oligomer is homomeric, and two or more molecules bind to the same target. In other embodiments, the oligomer is heteromeric. In some specific embodiments, two or more molecules bind to two or more different targets.

[0018] In another aspect, provided herein is a pharmaceutical composition comprising a molecule described herein, an oligomer described herein, an isolated nucleic acid described herein, or a vector described herein, and a pharma- ceutically acceptable excipient.In another aspect, provided herein is a method for treating a disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition described herein.

[0019] In yet another aspect, the present invention provides an IgG H

[0023] A method is provided for making an oligomer comprising two or more molecules each comprising an IgG C region, the method comprising: H This involves the introduction into each molecule of a cysteine ​​amino acid substitution at position 253 according to EU numbering in region 2.

[0020] In yet another aspect, provided herein is a method of producing an oligomerized molecule, comprising: (i) introducing into a host cell the vector of claim 40; (ii) culturing the host cell under suitable conditions for production of the oligomerized molecule; and (iii) purifying the oligomerized molecule. [Brief description of the drawings]

[0021] 4. [Figure 1A]Transient expression analysis of human IgG1 and its variants. Analysis of the transient expression of different human IgG1 (huIgG1) Fc-μtp and huIgG1 Fc-αtp variants and controls by SDS-PAGE. Lane 1 shows a protein ladder; lane 2 shows an IgM positive control (250 mg / L); lane 3 shows the Expi293 expression system without transfection; lane 6 shows huIgG1 Fc with an I253C mutation followed by an immunoglobulin μ chain tailpiece (μtp) (SEQ ID NO: 3); lane 7 shows huIgG1 Fc with an I253C mutation followed by an immunoglobulin α chain tailpiece (αtp); lane 8 shows huIgG1 Fc with a Q438C mutation followed by μtp (SEQ ID NO: 5); lane 9 shows huIgG1 Fc with a Q438C mutation followed by αtp; lane 10 shows huIgG1 Fc with a Y436C mutation followed by μtp (SEQ ID NO: 4); lane 11 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 12 shows huIgG1 Fc with a Y436C mutation followed by μtp (SEQ ID NO: 5); lane 13 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 14 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 15 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 16 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 17 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 18 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 19 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 20 shows huIgG1 Fc with a Y436C mutation followed by αtp; lane 21 shows huIgG1 Fc with a Y436 lane 13 shows huIgG1 Fc followed by μtp; lane 14 shows huIgG1 Fc followed by αtp. [Figure 1B]Transient expression analysis of human IgG1 and its variants. Analysis of transient expression of different human IgG1 (huIgG1) Fc-μtp variants and controls by SDS-PAGE. Lane 1 shows the protein ladder; lane 2 shows the purified IgG1 positive control (100 mg / L); lane 3 shows the purified IgG1 positive control (200 mg / L); lane 4 shows the conditioned medium from the Expi293 expression system without transfection; lane 5 shows huIgG1 Fc with H310C mutation followed by the immunoglobulin μ chain tailpiece (μtp) [huIgG1 Fc(H310C)-μtp]; lane 6 shows huIgG1 Fc with L251C, I253G, and S254C mutations followed by the immunoglobulin μ chain tailpiece (μtp) [huIgG1 Fc(L251C, I253G, and S254C)-μtp]; lane 7 shows huIgG1 Fc with S254C and N434C mutations. lane 7 shows huIgG1 Fc with the L251C and S254C mutations followed by the immunoglobulin μ chain tailpiece (μtp) [huIgG1 Fc(L251C and S254C)-μtp]; lane 8 shows huIgG1 Fc with the L251C and S254C mutations followed by the immunoglobulin μ chain tailpiece (μtp) [huIgG1 Fc(L251C and S254C)-μtp]. [Figure 1C] Analysis of transient expression of different human IgG1 (huIgG1) Fc-μtp variants and controls by SDS-PAGE. Lane 1 shows the protein ladder; lane 2 shows purified IgG1 positive control (100 mg / L); lane 3 shows purified IgG1 positive control (200 mg / L); lane 4 shows conditioned medium from the Expi293 expression system without transfection; lane 5 shows huIgG1 Fc with the N286C mutation followed by the immunoglobulin μ chain tailpiece (μtp) [huIgG1 Fc(N286C)-μtp].

[0022] [Figure 2A]Purification of huIgG1 Fc with I253C followed by μtp (huIgG1 Fc(I253C)-μtp). Purification using MabSelect™ Protein A resin. [Figure 2B] Purification of huIgG1 Fc with I253C followed by μtp (huIgG1 Fc(I253C)-μtp). Purification using CaptureSelect™ FcXL Affinity Matrix. [Figure 2C] Purification of huIgG1 Fc with I253C and subsequent μtp (huIgG1 Fc(I253C)-μtp). Purification using anion exchange chromatography.

[0023] [Figure 3A] Characterization of huIgG1 Fc(I253C)-μtp. Intact mass spectrometry of purified huIgG1 Fc(I253C)-μtp. [Figure 3B-1] Characterization of huIgG1 Fc(I253C)-μtp. HPLC-SEC analysis of huIgG1 Fc(I253C)-μtp overlaid with BioRad gel filtration protein standards. [Figure 3B-2] Characterization of huIgG1 Fc(I253C)-μtp. HPLC-SEC analysis of huIgG1 Fc(I253C)-μtp overlaid with BioRad gel filtration protein standards. [Figure 3B-3] Characterization of huIgG1 Fc(I253C)-μtp. HPLC-SEC analysis of huIgG1 Fc(I253C)-μtp overlaid with BioRad gel filtration protein standards. [Figure 3C] Characterization of huIgG1 Fc(I253C)-μtp. HPLC-SEC analysis of huIgG1 Fc(I253C)-μtp: Quantification of purity by integration. [Figure 3D] Characterization of huIgG1 Fc(I253C)-μtp. Thermal stability analysis of huIgG1 Fc(I253C)-μtp by differential scanning fluorescence nanoformat (nanoDSF).

[0024] [Figure 4] Expression of IgG hexamers by SDS-PAGE. Lane 1 shows purified human IgG1 positive control (100 mg / L); lane 2 shows purified human IgG1 hexamer positive control (100 mg / L); lane 3 shows conditioned medium from the Expi293 expression system without transfection; lane 4 shows anti-beta blotho huIgG1 hexamer; lane 5 shows anti-GDNF family receptor alpha-like (GFRAL) huIgG1 hexamer; lane 6 shows anti-vascular endothelial growth factor (VEGF) huIgG1 hexamer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] 5. The present disclosure provides methods for determining amino acid substitutions in antibody constant regions (i.e., IgG C H This is based in part on the surprising finding that substitution of cysteine ​​at position 253 according to EU numbering in the 2 region of the hexamer can increase the formation of oligomers such as hexamers, as demonstrated in section 6 below. The number of intermolecular bonds in a collection of molecules determines the level of complexity of the corresponding oligomers that can possibly be formed. If a molecule contains only one intermolecular binding site, only dimers can be formed, because each molecule is occupied after forming a bond with the other component of the dimer, and no other binding sites are available to form oligomers with more complex structures. In contrast, if a molecule contains multiple intermolecular binding sites, multiple bonds can be formed with multiple binding partners for this given molecule. Thus, oligomers with more complex structures can be formed in the collection of such molecules. Also, the nature and location of amino acid residues in a molecule that can form intermolecular bonds significantly affect the efficiency for the formation of oligomers.

[0026] In one aspect, provided herein are molecules engineered to form oligomers. Also provided herein are oligomers formed by the disclosed molecules. Also provided herein are pharmaceutical compositions comprising the molecules and oligomers provided herein. Also provided herein are kits comprising the compositions provided herein. Further provided herein are nucleic acid molecules, vectors, and host cells expressing the disclosed molecules. Further provided herein are methods of producing the molecules and oligomers provided herein. In yet a further aspect, provided herein are methods for using the molecules and oligomers.

[0027] 5.1 Definition The techniques and procedures described or referenced herein include those that are generally well understood and / or commonly used by those of skill in the art using conventional methodologies, such as the widely used methodologies described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2d ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those of skill in the art. For the purposes of interpreting this specification, the following explanations of terms apply, and whenever appropriate, terms used in the singular include the plural and vice versa. In the event that any explanation of a term provided conflicts with any document incorporated herein by reference, the explanation of the term provided below shall prevail.

[0028] As used herein, the term "disulfide bond" or "disulfide bond" refers to the covalent linkage of sulfur atoms of thiol groups (-SH) at a given residue of a molecule. The bond is formed by the oxidation of two thiols, thus linking two residues of a molecule and their respective bodies by a covalent disulfide bond. Specifically, when the molecule refers to a polypeptide or a derivative thereof, the disulfide bond is typically of the six-atom configuration Cα-Cβ-Sγ-S'γ-C'β-C'α, linking two amino acid residues.

[0029] As used herein, the term "EU numbering" refers to the constant domain (C H 1, hinge, and Fc portions). See Kabat, EA (1991) Sequences of Proteins of Immunological Interest: Tabulation and Analysis of Amino Acid and Nucleic Acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen T-Cell Surface Antigens, [Beta]2-Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoietin, Integrins, PostGamma Globulin, [Alpha]2-Macroglobulins, and Other Related Proteins, 5th edn, National Institutes of Health.

[0030] As used herein, the term "oligomer", "oligomerized molecule" or "multimerized molecule" refers to a complex structure consisting of several similar or identical repeating units. An oligomer can be a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, eleven-mer, dodecamer, trimer, tetramer, fifteen-mer, sixteen-mer, seventeen-mer, eighteen-mer, nineteen-mer, decamer, etc., which corresponds to a complex consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. similar or identical repeating units.

[0031] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and are used in the broadest sense, specifically including, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), single chain antibodies, and fragments thereof (e.g., domain antibodies) as described below. The term also includes all antibody variants, including those bearing various mutations (e.g., in the constant or Fc region) and other modifications (e.g., those with additional peptide sequences to the C-terminus or N-terminus). Antibodies can be human, humanized, chimeric and / or affinity matured, as well as antibodies from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides, capable of binding to a specific molecular antigen, and composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and the carboxy-terminal portion of each chain containing 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 or humanized variants thereof, including those from Camelidae species (e.g., llama or alpaca), intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above (e.g., antigen-binding fragments), which refer 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 is derived.Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules that contain an antigen-binding domain or antigen-binding site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in 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; Pluckthun 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 molecules. The antibodies can be agonist or antagonist antibodies. An antibody may be neither an agonist nor an antagonist.

[0032] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0033] The term "bind" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the association 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, e.g., an antigen, is the affinity of the antibody or functional fragment for that epitope. The association rate (k) of a binding molecule (e.g., an antibody) to a monovalent antigen is the rate at which the binding molecule binds to the antigen. on ) to dissociation rate (k off ) ratio (k off / k on ) is the dissociation constant K D which correlates inversely with affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different complexes of antibody and antigen. on and k off The dissociation constant K for the antibodies provided herein depends on both D 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 antibody and antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction of the antibody and antigen at one site increases the possibility of reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0034] With respect to the binding molecules described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to refer to binding molecules of an antigen-binding domain that specifically binds to an antigen, e.g., a polypeptide. Binding molecules or antigen-binding domains that bind or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those of skill in the art. In some embodiments, a binding molecule or antigen-binding domain binds or specifically binds to an antigen if it binds to the antigen with a higher affinity than any cross-reactive antigens, as determined using experimental techniques, e.g., radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). Typically, a specific or selective response will be at least twice the background signal or noise, and may exceed 10 times background. For a discussion of binding specificity, see, e.g., Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, 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, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. Binding molecules or antigen-binding domains that bind to an antigen include those that are capable of binding to an antigen with sufficient affinity such that the binding molecule is useful, for example, as a therapeutic and / or diagnostic agent in targeting the antigen. In certain embodiments, a binding molecule or antigen-binding domain that binds to an antigen has a dissociation constant (K D In certain embodiments, the binding molecule or antigen binding domain binds to an epitope of an antigen that is conserved among antigens from different species.

[0035] In certain embodiments, antibodies or antigen-binding fragments of the molecules described herein can include "chimeric" sequences in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to sequences in antibodies 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. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55).

[0036] In certain embodiments, the antibodies or antigen-binding fragments of the molecules described herein may include portions of "humanized" forms of non-human (e.g., murine) antibodies, which are chimeric antibodies comprising a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced by residues from a corresponding CDR of a non-human species (e.g., donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. A humanized antibody heavy or light chain may 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 non-human immunoglobulin and all or substantially all of the FRs are those of human immunoglobulin sequences. In certain embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-29; Presta, 1992, Curr. Op. Struct. Biol. 2:593-96; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; U.S. Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0037] In certain embodiments, an antibody or antigen-binding fragment of a molecule described herein may comprise a portion of a "fully human antibody" or "human antibody," which terms are used interchangeably herein to refer to an antibody that comprises a human variable region and, for example, a human constant region. In certain embodiments, the term refers to an antibody that comprises a variable region and a constant region of human origin. A "fully human" antibody may also encompass, in certain embodiments, an antibody that binds a polypeptide and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies having variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that possesses amino acid sequences that correspond to those of an antibody generated by a human and / or that has been generated using techniques that produce human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1:755-68).Methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5:368-74 can also be used for the preparation of human monoclonal antibodies. Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, whose endogenous loci have been disabled but which have been modified to produce such antibodies in response to antigen challenge (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., 2006, Proc. Natl. Acad. Sci. USA 103:3557-62 regarding human antibodies generated via human B-cell hybridoma technology.

[0038] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. For IgG, the four-chain unit is usually about 150,000 daltons. Each L chain is linked to the 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 interchain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. The pairing of VH and VL together forms a single antigen-binding site. The structure and properties of different classes of antibodies are described, for example, in Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5th ed. 1995). th Please refer to the following:

[0039] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. A conventional IgG usually contains two Fab regions, each in 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 light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL in the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, as in the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders.

[0040] The term "variable region", "variable domain", "V region", or "V domain" refers to a portion of an antibody light or heavy chain, usually located at the amino terminus of the light or heavy chain, having a length of about 120-130 amino acids in the heavy chain and about 100-110 amino acids in the light chain, that is 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". The term "variable" refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable region. Instead, V 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 higher variability (e.g., hypervariability) called "hypervariable regions", each about 9-12 amino acids long. The variable regions of the heavy and light chains each contain four FRs that generally adopt a β-sheet configuration connected by three hypervariable regions that form loop connections, and in some cases, form part of a β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence among different antibodies, hi certain embodiments, the variable regions are human variable regions.

[0041] The term "heavy chain", when used in reference to an antibody, refers to a polypeptide chain of about 50-70 kDa that includes a variable region of about 120-130 or more amino acids at the amino-terminal portion and a constant region at the carboxy-terminal portion. The constant region can be one of five different types (e.g., isotypes) designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. Different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, whereas μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains give rise to the five well-known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

[0042] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of about 25 kDa that contains a variable region of about 100 to about 110 or more amino acids at the amino-terminal portion and a constant region at the carboxy-terminal portion. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain.

[0043] As used herein, the terms "hypervariable region", "HVR", "complementarity determining region" and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of the β-sheet framework of an immunoglobulin (Ig or antibody) VH, or one of the three hypervariable regions (L1, L2 or L3) in the non-framework region of the β-sheet framework of an antibody VL. Thus, CDRs are variable region sequences interspersed within framework region sequences. CDR regions are well known to those skilled in the art and are defined by a well-known numbering system.

[0044] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as interaction with the Fc receptor. The term refers to the portion of the immunoglobulin molecule that has a more conserved amino acid sequence than the other part of the immunoglobulin, the variable region, which contains the antigen-binding site. The constant region is the C of the heavy chain. H 1. C H 2, and C H 3 domain and light chain C L As used herein, the term "IgG C H 1," "IgG C H 2" and "IgG C H "3" refers to the commonly defined first, second and third domains, respectively, in the constant region of the heavy chain of IgG. Heavy chains from IgG typically have three constant domains: IgG C H 1. C H 2. C H 3, and C H 1 and C H The spacer between the two domains contains a hinge region.

[0045] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is often defined as extending from an amino acid residue at position Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include an antibody population with all K447 residues removed, an antibody population without the K447 residue removed, and an antibody population with a mixture of antibodies with and without the K447 residue. A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions typically require an Fc region in combination with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may retain at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0046] The term "framework" or "FR" refers to those variable region residues which flank 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.

[0047] As used herein, "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. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more discontinuous regions of a polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). In general, it is understood by those skilled in the art that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids, regardless of whether they are folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires the amino acid residues that make up the epitope to exhibit a particular conformation (eg, a bend, twist, turn or fold) in order to recognize and bind to the epitope.

[0048] The term "targeting region" or "binding domain" is used herein in the broadest sense to refer to a region of a molecule that can specifically bind to a target (or antigen). A targeting region is a C HThe targeting region may be an antigen-binding fragment that does not have one region, or any reformatted antigen-binding fragment, e.g., a single chain variable fragment (scFv), any polypeptide that is non-immunoglobulin but forms a specific binding to a target via enzyme-substrate, receptor-ligand or other protein-protein interactions. The targeting region may also be a sequence of nucleic acid that forms a specific binding to a target via reverse complementation. The targeting region may also be any entity that can form any covalent or non-covalent connection with the target. In some embodiments, the targeting region or binding domain is derived from an antibody, e.g., an antigen-binding domain or fragment of an 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 a portion of an antibody (e.g., CDRs) that includes amino acid residues that interact with the antigen and confer to the binding agent its specificity and affinity for the antigen. "Antigen-binding fragment," as used herein, includes a portion of an intact antibody, e.g., an "antibody fragment" that includes the antigen-binding or variable region of an intact antibody. Examples of antibody fragments are described above and include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies and didiabodies; single-chain antibody molecules; dual variable domain antibodies; single variable domain antibodies (sdAbs); and multispecific antibodies formed from antibody fragments.

[0049] As used herein, the terms "polypeptide" and "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or through intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are polypeptides that contain 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. The polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, and it is understood that in certain embodiments, a "polypeptide" may occur as a single chain or as two or more associated chains.

[0050] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. 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. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. "Oligonucleotide", as used herein, refers to a short, usually single-stranded, synthetic polynucleotide, usually, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells that produce the binding molecules of the present disclosure can include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding an antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of the nascent RNA transcript is referred to as the transcription direction; the region of sequences on the DNA strand that have the same sequence as the RNA transcript that is 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence"; the region of sequences on the DNA strand that have the same sequence as the RNA transcript that is 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence."

[0051] An "isolated nucleic acid" is a nucleic acid, e.g., RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may be substantially free of other cellular material, or culture medium if produced by recombinant techniques, or may be substantially free of chemical precursors or other chemicals if chemically synthesized. In certain embodiments, one or more nucleic acid molecules encoding a single domain antibody or an antibody described herein are isolated or purified. The term encompasses nucleic acid sequences that have been removed from their naturally occurring environment, including recombinant or cloned DNA isolates and chemically synthesized analogs or biologically synthesized analogs by heterologous systems. A substantially pure molecule may include molecules in isolated form.

[0052] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that encode the same amino acid sequence, with degenerate versions of each other. The phrase nucleotide sequence encoding a protein or RNA can also include introns to the extent that the nucleotide sequence encoding the protein contains an intron(s) in some versions.

[0053] As used herein, the term "vector" refers to a material used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid sequence encoding an antibody or antigen-binding fragment described herein, to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include, for example, a selection sequence or marker operable for stable integration into a host cell chromosome. In addition, the vector may include one or more selection marker genes and appropriate expression control sequences. Selection marker genes that may be included provide, for example, resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients that are absent in the medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are co-expressed (e.g., both antibody heavy and light chains or antibody VH and VL), both nucleic acid molecules may be inserted, for example, into a single expression vector or into separate expression vectors. For single vector expression, the encoding nucleic acids may be operably linked to one common expression control sequence, or may be linked to different expression control sequences, e.g., one inducible promoter and one constitutive promoter. Introduction of the nucleic acid molecules into the host cell may be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, e.g., Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblots for expression of gene products, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. It will be understood by those skilled in the art that the nucleic acid molecule is expressed in sufficient amounts to produce the desired product, and it will be further understood that the expression level may be optimized to obtain sufficient expression using methods well known in the art.

[0054] As used herein, the term "host cell" refers to a particular subject cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell that has not been transfected with the nucleic acid molecule due to mutations or environmental influences that may occur during successive generations or integration of the nucleic acid molecule into the host cell genome.

[0055] As used herein, the term "identity" refers to the 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" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to 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 without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, 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 required to achieve maximum alignment over the entire length of the sequences being compared.

[0056] The term "specificity" refers to the selective recognition of an antigen-binding molecule (e.g., an antibody) to a particular epitope of an antigen. For example, a natural antibody is monospecific. The term "multispecific" as used herein refers to an antigen-binding protein having two or more antigen-binding sites, at least two of which bind to different antigens. "Bispecific" as used herein refers to an antigen-binding molecule having two different antigen-binding specificities. The term "monospecific" as used herein refers to an antigen-binding molecule having one or more binding sites, each of which binds to the same antigen.

[0057] The term "valency" as used herein refers to the presence of a specific number of binding sites in an antigen-binding molecule. For example, a natural antibody, or a full-length antibody, has two binding sites and is bivalent. Thus, the terms "trivalent", "tetravalent", "pentavalent" and "hexavalent" refer to the presence of two, three, four, five and six binding sites in an antigen-binding molecule, respectively.

[0058] As used herein, the term "pharmaceutical acceptable" as used herein means approved by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, and more specifically, in humans.

[0059] As used herein, the term "excipient" refers to a pharma- ceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating substance. Excipients include, for example, encapsulating substances or additives, such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, flavorings, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete) or vehicle.

[0060] In some embodiments, the excipient is a pharma- ceutically acceptable excipient. Examples of pharma-ceutically acceptable excipients include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; 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 dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™. Other examples of pharma- ceutically acceptable excipients are described by Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0061] In one embodiment, each component is "pharmaceutical acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for use in contact with human and animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutical acceptable excipient is non-toxic to cells or mammals exposed to it at the dosages and concentrations employed. In some embodiments, the pharma- ceutically acceptable excipient is an aqueous pH buffered solution.

[0062] In some embodiments, the excipients may be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like). Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid excipients, particularly for injectable solutions. Excipients may 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 compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral composition containing formulations can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

[0063] As used herein, 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.

[0064] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context indicates otherwise.

[0065] As used in this specification, the term "between" when used in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0066] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or," when used in phrases 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).

[0067] 5.2 Molecules engineered to form oligomers 5.2.1 Molecules containing engineered constant regions In one aspect, provided herein are molecules capable of forming connections with two or more other molecules of the same or similar structure, thus forming oligomeric molecules comprising two or more units. More specifically, in some embodiments, the molecules provided herein are IgG C IgG1A, IgG2A, IgG3A, IgG4A, IgG5A, IgG6A, IgG7A, IgG8A, IgG9A, IgG10A, IgG11A, IgG12A, IgG13A, IgG14A, IgG15A, IgG16A, IgG17A, IgG18A, IgG19A, IgG20A, IgG21A, IgG22A, IgG23A, IgG24A, IgG25A, IgG35A, IgG46A, IgG47A, IgG48A, IgG49A, IgG50A, IgG51A, IgG52A, IgG53A, IgG6A, IgG6B, IgG7C, IgG8A, IgG8C, IgG10A, IgG11A, IgG12A, IgG13A, IgG14A, IgG15B, IgG15C, IgG16A, IgG17B, IgG18A, IgG19A, IgG19B, IgG15C, IgG16A, IgG17B, IgG18C, IgG19C, IgG19C, IgG20A, IgG11B, IgG13A, IgG14A, IgG15C, IgG15C, IgG16A, IgG17B, IgG18C, IgG19C, IgG19C, IgG20A, IgG15C, IgG16A, IgG17B, IgG18C, IgG19C, IgG20A, IgG19C, IgG19C, IgG20A, IgG19C, H It contains two regions, thereby creating a site capable of forming a disulfide bond with another molecule having the same amino acid substitutions.

[0068] In some embodiments, C H In addition to the cysteine ​​residue at position 253 of the 2 region, the molecules provided herein contain at least one other site capable of forming a connection with other molecules of the same or similar structure. The types of connections provided herein can be covalent or non-covalent, and the connections provided herein can be direct or indirect. In some embodiments, the connections provided herein are disulfide bonds through cysteine ​​residues. The cysteine ​​residues can be introduced into the molecules, for example, by point mutation or by adding a tailpiece. Alternatively, the cysteine ​​residues can be naturally present in antibody regions, for example, in the hinge region. In some embodiments, the above properties are provided in the molecules in combination.

[0069] In some embodiments, the type of connection between molecules provided herein is via a covalent bond. In some particular embodiments, the type of connection between molecules provided herein is via a disulfide bond. In some other embodiments, the type of connection between molecules provided herein is via electron sharing. In other embodiments, the type of connection between molecules provided herein is via a non-covalent bond. In some particular embodiments, the type of connection between molecules provided herein is via hydrogen bonds. In some other embodiments, the type of connection between molecules provided herein is via ionic interactions. In some other embodiments, the type of connection between molecules provided herein is via van der Waals forces. In some other particular embodiments, the type of connection between molecules provided herein is via hydrophobic bonds.

[0070] In some embodiments, the molecules provided herein form direct connections with each other. In other embodiments, the molecules provided herein form indirect connections with each other. In some particular embodiments, the molecules provided herein connect with each other through directly formed disulfide bonds. In other embodiments, the molecules provided herein connect with each other through indirectly formed disulfide bonds. In some particular embodiments, the molecules provided herein connect with each other through one or more connection sites, where the connecting molecule forms a disulfide bond with the molecule provided herein. In one particular embodiment, the connecting molecule is a J chain found in polymeric IgA and IgM.

[0071] In certain embodiments, provided herein are molecules that form connections with each other through disulfide bonds, which may be formed between two natural or unusual amino acids. In some embodiments, the connections formed between the molecules provided herein are disulfide bonds between two natural amino acids. In some specific embodiments, the connections formed between the molecules provided herein are disulfide bonds between two cysteine ​​residues. In some embodiments, the connections formed between the molecules provided herein are disulfide bonds between two unusual amino acids. In some specific embodiments, each of the unusual amino acids possesses a reactive sulfhydryl group. In some specific embodiments, 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)).

[0072] Point mutations to form disulfide bonds In some embodiments, provided herein are molecules that form a connection between each other via a disulfide bond between the introduced point mutations. In one embodiment, alanine is replaced with cysteine. In another embodiment, arginine is replaced with cysteine. In another embodiment, asparagine is replaced with cysteine. In another embodiment, aspartic acid is replaced with cysteine. In another embodiment, glutamine is replaced with cysteine. In another embodiment, glutamic acid is replaced with cysteine. In another embodiment, glycine is replaced with cysteine. In another embodiment, histidine is replaced with cysteine. In another embodiment, isoleucine is replaced with cysteine. In another embodiment, leucine is replaced with cysteine. In another embodiment, lysine is replaced with cysteine. In another embodiment, methionine is replaced with cysteine. In another embodiment, phenylalanine is replaced with cysteine. In another embodiment, proline is replaced with cysteine. In another embodiment, serine is replaced with cysteine. In another embodiment, threonine is replaced with cysteine. In another embodiment, tryptophan is replaced with cysteine, in another embodiment, tyrosine is replaced with cysteine, in another embodiment, valine is replaced with cysteine.

[0073] In some embodiments, provided herein are molecules with point mutations introduced at positions that facilitate the formation of stable disulfide bonds with each other. In some embodiments, the positions of the introduced point mutations satisfy the geometric constraints and parameters for a stable disulfide bond. In some particular embodiments, the positions of the introduced point mutations ensure a close distance between Cα-Cα' in the formed disulfide bond. In some particular embodiments, the positions of the introduced point mutations ensure a close distance between Cβ-Cβ' in the formed disulfide bond. In some particular embodiments, the positions of the introduced point mutations ensure a proper bond angle between Cα-Cβ-Sγ in the formed disulfide bond. In other embodiments, the positions of the introduced point mutations ensure a proper bond angle between Cβ-Sγ-S′γ in the formed disulfide bond. In yet other particular embodiments, the positions of the introduced point mutations ensure a proper rotation angle of the Cβ atom around the S-S bond in the formed disulfide bond. In some particular embodiments, the molecules provided herein are IgG Cα with point mutations to cysteine. H Includes 1 region.

[0074] In some specific embodiments, the molecules provided herein are IgG C H In some specific embodiments, the molecules provided herein comprise an IgG C region having a point mutation to cysteine. HIn some particular embodiments, the molecules provided herein comprise an IgE Cε3 region. In some particular embodiments, the molecules provided herein comprise a hinge region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgE Cε1 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgE Cε2 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgE Cε3 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgE Cε4 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgE Cε5 region with a point mutation to cysteine. H In some specific embodiments, the molecules provided herein comprise an IgD C region having a point mutation to cysteine. H In some specific embodiments, the molecules provided herein comprise an IgD C region having a point mutation to cysteine. H In some particular embodiments, the molecules provided herein comprise an IgM Cμ1 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgM Cμ2 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgM Cμ3 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgM Cμ4 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgA Cα1 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgA Cα2 region with a point mutation to cysteine. In some particular embodiments, the molecules provided herein comprise an IgA Cα3 region with a point mutation to cysteine. In certain embodiments, the molecules provided herein comprise an IgG Cα1 region with a point mutation to cysteine ​​at position Ile253 according to EU numbering. H Includes 2 areas.

[0075] In some embodiments, the molecules provided herein comprise two or more of the above-mentioned point mutations. H In region 2, it contains a point mutation at position Ile253 according to EU numbering to cysteine ​​and at least one other point mutation as described above.

[0076] Added tailpiece for forming disulfide bonds In certain embodiments, the molecules provided herein further comprise an added tailpiece that facilitates the formation of a stable disulfide bond with one another. In some embodiments, the added tailpiece comprises an 18 amino acid long μ tailpiece (SEQ ID NO:1). In other embodiments, the added tailpiece comprises 90% identity to SEQ ID NO:1 and retains a cysteine ​​residue in SEQ ID NO:1. In other embodiments, the added tailpiece comprises 85% identity to SEQ ID NO:1 and retains a cysteine ​​residue in SEQ ID NO:1. In other embodiments, the added tailpiece comprises 70% identity to SEQ ID NO:1 and retains a cysteine ​​residue in SEQ ID NO:1. In other embodiments, the added tailpiece comprises 65% identity to SEQ ID NO:1 and retains a cysteine ​​residue in SEQ ID NO:1. In other embodiments, the added tailpiece comprises 50% identity to SEQ ID NO:1 and retains a cysteine ​​residue in SEQ ID NO:1.

[0077] In some embodiments, the added tailpiece comprises a tailpiece in an IgA alpha heavy chain (the alpha tailpiece). In other embodiments, the added tailpiece comprises 85% identity with the alpha tailpiece and retains a cysteine ​​residue in the tailpiece. In other embodiments, the added tailpiece comprises 70% identity with the alpha tailpiece and retains a cysteine ​​residue in the tailpiece. In other embodiments, the added tailpiece comprises 65% identity with the alpha tailpiece and retains a cysteine ​​residue in the tailpiece. In other embodiments, the added tailpiece comprises 50% identity with the alpha tailpiece and retains a cysteine ​​residue in the alpha tailpiece. In other embodiments, the added tailpiece comprises 35% identity with the alpha tailpiece and retains a cysteine ​​residue in the alpha tailpiece. In other embodiments, the added tailpiece comprises 20% identity with the alpha tailpiece and retains a cysteine ​​residue in the tailpiece. In other embodiments, the added tailpiece comprises 5% identity with the alpha tailpiece and retains a cysteine ​​residue in the alpha tailpiece.

[0078] In some embodiments, a given polypeptide as described herein comprises an amino acid sequence with a given percent identity compared 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. USA 87:2264 2268 (1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed, for example, using the NBLAST nucleotide program parameters set for score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed, for example, using the XBLAST program parameters set for score=50, word length=3 to obtain amino acid sequences homologous to the molecules proteins described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997). Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (supra). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., those of XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).Another non-limiting example of a mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weighted 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 the percent identity, typically only exact matches are counted.

[0079] In other embodiments, the present disclosure provides molecules having an added short polypeptide containing one or more cysteines. In some particular embodiments, the added short polypeptide is less than 5 amino acids long. In some particular embodiments, the added short polypeptide is 6, 7, 8, 9, or 10 amino acids long. In some particular embodiments, the added short polypeptide is 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some particular embodiments, the added short polypeptide is 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In some particular embodiments, the added short polypeptide is more than 30 amino acids long.

[0080] Provided herein are molecules having tailpieces attached at positions that facilitate the formation of stable disulfide bonds with each other. In some embodiments, the tailpiece is attached to the C-terminus of the molecule. In some particular embodiments, the tailpiece is attached to the C-terminus of the molecule. H In some particular embodiments, the tailpiece is added to the C-terminus of the hinge region. In some particular embodiments, the tailpiece is added to the C-terminus of the hinge region. H In some particular embodiments, the tailpiece is added to the C-terminus of the C2 region.H In some embodiments, the tailpiece is added to the C-terminus of the C3 region. In some embodiments, the tailpiece is added to the N-terminus of the molecule. In some particular embodiments, the tailpiece is added to the C3 region. H In some particular embodiments, the tailpiece is added to the N-terminus of the hinge region. In some particular embodiments, the tailpiece is added to the N-terminus of the hinge region. H In some particular embodiments, the tailpiece is added to the N-terminus of the C2 domain. H It is added to the N-terminus of the 3 domain.

[0081] Engineered molecular configurations In some embodiments, provided herein are molecules that include both the point mutations described above and the additional tailpieces described above. In other embodiments, provided herein are molecules that have both two or more point mutations described above and the additional tailpieces described above. In yet other embodiments, provided herein are molecules that have both the point mutations described above and the two or more additional tailpieces described above. In yet other embodiments, provided herein are molecules that have both two or more point mutations described above and the two or more additional tailpieces described above.

[0082] In some embodiments, provided herein are molecules that comprise different antibody constant regions engineered for multimerization. In some embodiments, the molecules described herein comprise C H In another embodiment, the molecules described herein have one or more additional tailpiece(s) so that engineered disulfide bond(s) can form between such molecules. In another embodiment, the molecules described herein have one or more additional tailpiece(s) so that engineered disulfide bond(s) can form between such molecules. H With one or more point mutation(s) in the two regions and one or more added tailpiece(s), multiple engineered disulfide bonds can form between such molecules.

[0083] In some specific embodiments, the molecules described herein are IgG H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H In some specific embodiments, the molecules described herein comprise an IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H In some specific embodiments, the molecules described herein comprise an IgG C2 region, an IgG hinge region, and an IgG β region. H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domains, and IgG C H In some specific embodiments, the molecules described herein comprise IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domains, and IgG C H Includes 1 region.

[0084] In some specific embodiments, the molecules described herein are IgG H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domain, IgG hinge, and C H In some specific embodiments, the molecules described herein comprise IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domain, IgG hinge, and IgG C HIn some specific embodiments, the molecules described herein comprise an IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 regions, IgG C H 3 domains, and IgG C H In some specific embodiments, the molecules described herein comprise an IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domain, IgG hinge, IgG C H 3 domains, and IgG C H Includes 1 region.

[0085] In the above embodiments, the order of the different regions may follow the order in the wild type IgG or may differ from the order in the wild type IgG.

[0086] In some embodiments, the molecules described herein further comprise one or more additional tailpiece(s) as described above. In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO:1) or a variant thereof. In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO:1) and an IgG hinge region. In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO:1) and an IgG hinge region. H In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1) and an IgG C H In some specific embodiments, the molecules described herein comprise three regions: the human μ tailpiece (SEQ ID NO: 1), the IgG C H Includes 1 region.

[0087] In certain embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG hinge, and an IgG CH In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG hinge region, and an IgG C H In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG hinge region, and an IgG C H In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG C H 2 domains, and IgG C H In some specific embodiments, the molecules described herein comprise three regions: the human μ tailpiece (SEQ ID NO: 1), the IgG C H 2 domains, and IgG C H In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG C H 3 domains, and IgG C H Includes 1 region.

[0088] In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 2 domains, and IgG C H In some specific embodiments, the molecules described herein comprise three regions: a human μ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 2 domains, and IgG C H In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 3 domains, and IgG C H In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG C H 2 regions, IgG C H 3 domains, and IgG C H Includes 1 region.

[0089] In some specific embodiments, the molecules described herein comprise a human μ tailpiece (SEQ ID NO: 1), an IgG hinge region, an IgG C H 2 regions, IgG C H 3 domains, and IgG C H Includes 1 region.

[0090] In the above embodiments, the order of the different regions may follow or differ from the order in wild-type IgG, and the human μ tailpiece may be conjugated to the C-terminus or N-terminus of the molecule.

[0091] In some embodiments, the molecules described herein have one or more point mutation(s) and one or more added tailpiece(s), such that multiple engineered disulfide bonds can form between such molecules. In some specific embodiments, the molecules described herein have one or more point mutation(s) and one or more added tailpiece(s), such that multiple engineered disulfide bonds can form between such molecules. H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H In some specific embodiments, the molecules described herein comprise an IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 region, a human μ tailpiece (SEQ ID NO: 1), and an IgG hinge region. In some specific embodiments, the molecules described herein comprise an IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domains, the human μ tailpiece (SEQ ID NO: 1), and IgG C H In some specific embodiments, the molecules described herein comprise IgG C H 2 regions, IgG C according to EU numbering HPosition 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domains, the human μ tailpiece (SEQ ID NO: 1), and IgG C H In some specific embodiments, the molecules described herein comprise an IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domains, the human μ tailpiece (SEQ ID NO: 1), the IgG hinge domain, and the IgG C H In some specific embodiments, the molecules described herein comprise IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domains, the human μ tailpiece (SEQ ID NO: 1), the IgG hinge domain, and the IgG C H In some specific embodiments, the molecules described herein comprise an IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domains, human μ tailpiece (SEQ ID NO: 1), IgG C H 3 domains, and IgG C H In some specific embodiments, the molecules described herein comprise an IgG C H 2 regions, IgG C according to EU numbering H Position 253 in the IgG C2 domain is substituted to be a cysteine. H 2 domains, human μ tailpiece (SEQ ID NO: 1), IgG hinge domain, IgG C H 3 domains, and IgG C H In the above embodiments, the order of the different regions may follow or differ from the order in wild type IgG, and the human μ tailpiece may be conjugated to the C-terminus or N-terminus of the molecule.

[0092] For all of the IgG, IgE, IgD, IgM, and IgA regions described herein, the variation can be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. The amino acid substitution can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, e.g., replacing a leucine with a serine, e.g., a conservative amino acid substitution. For example, standard techniques known to those of skill in the art, including site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions, can be used to introduce mutations in the nucleotide sequence encoding the molecules provided herein. The insertion or deletion can optionally range from about 1 to 5 amino acids. In certain embodiments, the substitution, insertion, 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 compared to the original molecule. In certain embodiments, the substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. The variations permitted can 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.

[0093] IgG C H In addition to the IgG C2 region, the molecule may further comprise other IgG regions. In some embodiments, the molecules provided herein comprise an IgG C2 region. H In some embodiments, the molecules provided herein comprise an IgG hinge region. In some embodiments, the molecules provided herein comprise an IgG C hinge region. H In some embodiments, the molecules provided herein comprise an IgG C H 1 and C H In some embodiments, the molecules provided herein comprise an IgG CH 1 and C H In some embodiments, the molecules provided herein comprise an IgG hinge and C3 region. H In some embodiments, the molecules provided herein comprise an IgG hinge and C2 domain. H In some embodiments, the molecules provided herein comprise an IgG C H 2 and C H In some embodiments, the molecules provided herein comprise an IgG C H 1, hinge, and C H In some embodiments, the molecules provided herein comprise an IgG C H 1, hinge, and C H In some embodiments, the molecules provided herein comprise an IgG C H 1. C H 2, and C H In some embodiments, the molecules provided herein comprise an IgG C H 1, hinge, C H 2, and C H Includes 3 areas.

[0094] In some embodiments, the IgG regions disclosed in the preceding paragraphs are from human IgG. In some embodiments, the IgG regions disclosed in the preceding paragraphs are from human IgG1. In some embodiments, the IgG regions disclosed in the preceding paragraphs are from human IgG2. In some embodiments, the IgG regions disclosed in the preceding paragraphs are from human IgG3. In some embodiments, the IgG regions disclosed in the preceding paragraphs are from human IgG4. In some specific embodiments, the IgG regions in one molecule disclosed in the preceding paragraphs are mixed with different human IgG isotypes. In other embodiments, the IgG regions disclosed in the preceding paragraphs are from mouse IgG. In other embodiments, the IgG regions disclosed in the preceding paragraphs are from rat IgG. In other embodiments, the IgG regions disclosed in the preceding paragraphs are from monkey IgG, donkey IgG, sheep IgG, goat IgG, guinea pig IgG, camel IgG, horse IgG, or chicken IgG.

[0095] 5.2.2 Binding molecules In addition to the properties and structures for forming oligomers disclosed above, the molecules provided herein include other properties and functions. In certain embodiments, the molecules are binding molecules that include a binding domain. In some embodiments, the molecules are antibodies or fragments thereof. In other embodiments, the binding domain of the molecules provided herein includes a non-immunoglobulin binding agent. In other embodiments, the binding domain of the molecules provided herein includes a nucleic acid sequence that forms a specific binding to a target via reverse complementation.

[0096] In some embodiments, the binding domain of the molecules provided herein comprises an antibody fragment. Exemplary fragments include Fab fragments (e.g., an antibody fragment containing an antigen-binding domain and a portion of a light chain and a heavy chain cross-linked by a disulfide bond); Fab' (e.g., an antibody fragment containing a single antigen-binding domain including a Fab and an additional portion of a heavy chain up to the hinge region); F(ab')2 (e.g., two Fab' molecules connected by an interchain disulfide bond at the hinge region of the heavy chain; the Fab' molecules can be directed to the same or different epitopes); bispecific Fab (e.g., a fragment containing two antigen-binding domains and a portion of a heavy chain cross-linked by a disulfide bond); Fab molecules, each of which can be directed to a different epitope; single chains containing variable regions, also known as scFvs (e.g., the variable antigen-binding determining regions of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); disulfide-linked Fvs, or dsFvs (e.g., the variable antigen-binding determining regions of a single light and heavy chain of an antibody linked together by a disulfide bond); camelidized VHs (e.g., some amino acids at the VH interface are replaced by those of naturally occurring camelid antibodies). variable antigen-binding determining regions of a single heavy chain of an antibody, which are those found in the heavy chain of a single scFv; bispecific scFvs (e.g., scFv or dsFv molecules having two antigen-binding domains, each of which can be directed to a different epitope); diabodies (e.g., dimerized scFvs formed when the VH domain of a first scFv assembles with the VL domain of a second scFv and the VL domain of the first scFv assembles with the VH domain of the second scFv; these two antigen-binding regions of a diabody can be the same or These include triabodies (e.g., trimerized scFvs formed in a similar manner to diabodies, but where three antigen-binding domains are generated in a single complex; the three antigen-binding domains may be directed to the same or different epitopes); and tetrabodies (e.g., tetramerized scFvs formed in a similar manner to diabodies, but where four antigen-binding domains are generated in a single complex; the four antigen-binding domains may be directed to the same or different epitopes).

[0097] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can be expressed in and secreted from E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with extended in vivo half-life containing salvage receptor binding epitope residues are described, for example, in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv) (see, for example, WO93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458). Fvs and scFvs have intact integration sites that lack constant regions; thus, they may be suitable for reduced non-specific binding during in vivo use. scFv fusion proteins can be constructed to generate fusion of effector proteins at either the amino or carboxy terminus of the scFv (see, for example, Borrebaeck ed., supra). An antibody fragment may also be a "linear antibody," e.g., as described in the references cited above. Such linear antibodies may be monospecific or multispecific, e.g., bispecific.

[0098] Smaller antibody-derived binding structures are the separate variable domains (V domains), also called single variable domain antibodies (sdAbs). Certain types of organisms, camelids and cartilaginous fish, possess high affinity single V-like domains mounted on Fc-equivalent domain structures as part of their immune systems (Woolven et al., 1999, Immunogenetics 50:98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49). V-like domains (called VhH in camelids and V-NAR in sharks) typically display long surface loops that allow the penetration of the cavity of the target antigen. They also stabilize the isolated VH domain by masking hydrophobic surface patches.

[0099] These VhH and V-NAR domains have been used to engineer sdAbs. Human V domain variants have been designed using selection from phage libraries and other approaches that have yielded stable, high-binding VL and VH derived domains.

[0100] The antibody fragment in the molecules 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.

[0101] The antibodies described herein can include, for example, humanized antibodies, such as deimmunized or composite human antibodies.

[0102] A humanized antibody may comprise human framework regions and human constant region sequences. For example, a humanized antibody may comprise human constant region sequences. In certain embodiments, the humanized antibody may be selected from any class, including immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4 (e.g., IgG4 variants and IgG4 nullbodies). In certain embodiments, the humanized antibody may comprise kappa or lambda light chain constant sequences.

[0103] Humanized antibodies can be produced by a variety of techniques, including CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Patent No. 5,565,332), and, for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, WO93 / 17105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu JS, Gene 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Publication No. US2005 / 0042664A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0104] Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may 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 can be performed, for example, by replacing hypervariable region sequences with the corresponding sequences of a human antibody according to the methods 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).

[0105] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of parent non-human antibodies (e.g., rodents) are grafted onto human antibody frameworks. For example, Padlan et al. determined that only about one-third of the residues in CDRs actually contact antigens, and called them "specificity determining residues," or SDRs (Padlan et al., 1995, FASEB J.9:133-39). In the technique of SDR grafting, only SDR residues are grafted onto human antibody frameworks (see, for example, Kashmiri et al., 2005, Methods 36:25-34).

[0106] The choice of human variable domains, both light and heavy, used in making humanized antibodies can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, 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 can 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 can 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). In some cases, the framework is derived from the consensus sequence of the most abundant human subclasses, VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII). In other methods, human germline genes are used as the source of the framework regions.

[0107] In an alternative paradigm based on CDR comparison, called superhumanization, FR homology is irrelevant. The method consists of comparing non-human sequences with functional human germline gene repertoire. Then, those genes that code the same or closely related canonical structure as the murine sequence are selected. Next, among the genes that share canonical structure with non-human antibodies, those with the highest homology in CDR are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., 2002, J.Immunol.169:1119-25).

[0108] It is further usually desired that antibodies be humanized with retention of their affinity for the antigen and other desirable biological properties. To achieve this goal, according to one method, 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 that illustrate and display predicted 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, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, 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. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0109] Another method for antibody humanization is based on a metric of antibody humanity called Human String Content (HSC). This method compares mouse sequences with a repertoire of human germline genes, and 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).

[0110] In addition to the methods described above, empirical methods can be used to generate and select humanized antibodies. These methods include those based on generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high-throughput screening techniques. Antibody variants can 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. Sel. 17:847-60).

[0111] In the FR library approach, a collection of residue variants is introduced at a specific position in the FR, and then the library is screened to select the FR that best supports the grafted CDR. The residues to be replaced can include some or all of the "Vernier" residues identified as potentially contributing to CDR structure (see, for example, Foote and Winter, 1992, J. Mol. Biol. 224: 487-99), or from a more limited set of target residues identified by Baca et al. (1997, J. Biol. Chem. 272: 10678-84).

[0112] In FR shuffling, entire FRs are combined with non-human CDRs instead to generate a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., 2005, Methods 36:43-60). The library can be screened in a two-step process, first humanizing the VL, followed by the VH. Alternatively, a one-step FR shuffling process can be used. Such a process has been shown to be more efficient than 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).

[0113] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs) and the sequential replacement and binding evaluation of non-human fragments into a library of human FRs. It starts with the CDR3 regions of the non-human VH and VL chains and gradually replaces other regions of the non-human antibody with human FRs, including 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. Humaneering allows the isolation of antibodies that are 91-96% homologous to human germline antibodies (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0114] "Human engineering" methods involve modifying non-human antibodies or antibody fragments, e.g., murine or chimeric antibodies or antibody fragments, by making specific changes to the amino acid sequence of the antibody to generate modified antibodies with reduced immunogenicity in humans that nevertheless retain the desired binding properties of the original non-human antibody. Typically, the techniques involve classifying amino acid residues of a non-human (e.g., murine) antibody as "low risk," "medium risk," or "high risk" residues. The classification is performed using a global risk / benefit calculation that evaluates the predicted benefit (e.g., for immunogenicity in humans) of making a particular substitution against the risk that the substitution will affect folding of the resulting antibody. A particular human amino acid residue (e.g., low or medium risk) to be substituted at a given position of a non-human (e.g., murine) antibody sequence can be selected by aligning an amino acid sequence from the variable region of the non-human antibody with the corresponding region of a particular or consensus human antibody sequence. The amino acid residue at the low or medium risk position in the non-human sequence can be substituted with the corresponding residue in the human antibody sequence according to the alignment. Techniques for producing engineered proteins are described in more detail in Studnicka et al., 1994, Protein Engineering 7:805-14; U.S. Patent Nos. 5,766,886; 5,770,196; 5,821,123; and 5,869,619; and PCT Publication WO 93 / 11794.

[0115] Composite human antibodies can be generated, for example, using Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To generate composite human antibodies, the variable region sequences are engineered from fragments of multiple human antibody variable region sequences in a manner that avoids T-cell epitopes, thereby minimizing immunogenicity of the resulting antibody. Such antibodies can include human constant region sequences, e.g., human light and / or heavy chain constant regions.

[0116] In some embodiments, the molecules provided herein contain deimmunized antibody fragments from which T cell epitopes have been removed. Methods for making deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 2009;525:405-23, xiv, and De Groot et al., Cell. Immunol. 244:148-153 (2006). Deimmunized antibodies contain variable regions from which T cell epitopes have been deleted and human constant regions. Briefly, the VH and VL of an antibody are cloned, and then T cell epitopes are 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 peptides that bind to human MHC class II. Mutations are introduced into the VH and VL to abolish binding to human MHC class II. The mutated VH and VL are then used to generate deimmunized antibodies.

[0117] In certain embodiments, the molecules described herein are derived from fully human anti-human antibodies. Fully human antibodies can be generated by any method known in the art. Human antibody fragments provided herein can be constructed by combining Fv clone variable domain sequence(s) selected from a human-derived phage display library with known human constant domain sequence(s). Alternatively, human monoclonal antibodies of the present disclosure can be produced by hybridoma technology. Human myeloma and mouse-human heteromyeloma cell lines for the generation of human monoclonal antibodies are 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.

[0118] It is also possible to generate transgenic animals (e.g., mice) that, upon immunization, are capable of generating a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. Transgenic mice expressing human antibody repertoires have been used to generate high affinity human sequence monoclonal antibodies against a variety of potential drug targets (see, e.g., Jakobovits, A., 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; U.S. Patent Nos. 6,075,181 and 6,150,584; and Lonberg et al., 2005, Nature Biotechnol. 23:1117-25).

[0119] Alternatively, human antibodies can be prepared through immortalization of human B lymphocytes that produce antibodies directed against a target antigen (e.g., such B lymphocytes can be harvested from an individual or 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. Patent No. 5,750,373).

[0120] Gene shuffling can also be used to derive human antibodies from non-human, e.g., rodent, antibodies, where the human antibodies have similar affinities and specificities as the starting non-human antibody. According to this method, also called "epitope imprinting" or "guided selection", either the heavy or light chain variable regions of a non-human antibody fragment obtained by phage display technology as described herein are replaced with a repertoire of human V domain genes to generate a population of non-human chain / human chain chimeric scFv or Fab chimeras. Selection on antigen leads to the isolation of non-human chain / human chain chimeric scFv or Fab, where the human chain restores the antigen binding site destroyed with the removal of the corresponding non-human chain in the primary phage display clone (e.g., the epitope guides (imprints) the selection of the human chain partner). If the process is repeated to replace the remaining non-human chains, a human antibody is obtained (see, e.g., PCTWO93 / 06213; and Osbourn et al., 2005, Methods 36:61-68). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies with no FR or CDR residues of non-human origin. Examples of guided selection for humanizing mouse antibodies against cell surface antigens include 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).

[0121] A potential drawback of the guided selection approach is that shuffling one antibody chain while maintaining the other constant regions may result in epitope drift. In order to maintain the epitopes recognized by the non-human antibody, CDR retention may be applied (see, for example, Klimka et al., 2000, Br. J. Cancer. 83:252-60; and Beiboer et al., 2000, J. Mol. Biol. 296:833-49). In this method, the non-human VH CDR3 is commonly retained, since this CDR may be the center of the antigen-binding site and may be the most important region of the antibody for antigen recognition. However, in some examples, the VH CDR3 and VL CDR3 of the non-human antibody, as well as VH CDR2, VL CDR2, and VL CDR1 may be retained.

[0122] A multispecific antibody, e.g., a bispecific antibody, is a monoclonal antibody that has binding specificities for at least two different antigens. In some embodiments, the multispecific antibody provided herein is a bispecific antibody. In some embodiments, the bispecific antibody is a murine, chimeric, human, or humanized antibody. In some embodiments, one of the binding specificities is for one target / antigen and the other is for another target / antigen. In some embodiments, the bispecific antibody can bind to two different epitopes of the same target / antigen. The bispecific antibody can be prepared as a full-length antibody or an antibody fragment (e.g., F(ab')2 bispecific antibody).

[0123] Methods for producing multispecific antibodies are known in the art, such as by co-expression of two immunoglobulin heavy-light chain pairs (wherein the two heavy chains have different specificities) (see, e.g., Milstein and Cuello, 1983, Nature 305:537-40). For further details on generating multispecific antibodies (e.g., bispecific antibodies), see, e.g., Bispecific Antibodies (Kontermann ed., 2011).

[0124] The molecules described herein are H 1, hinge, C H 2, and / or C H It may be desirable to modify the molecules described herein by Fc engineering when they contain three region(s). In certain embodiments, modifications to the above regions in the molecules described herein result in the reduction or elimination of the effector function of the antibody. In certain embodiments, the effector function is antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). In some embodiments, the effector function is ADCC. In other embodiments, 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 the Fc region of the molecules described herein.

[0125] To extend the serum half-life of the molecules described herein, a salvage receptor binding epitope can be incorporated into the molecule, for example, as described in U.S. Patent No. 5,739,277. The term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that contributes to extending the in vivo serum half-life of the IgG molecule.

[0126] In some embodiments, amino acid sequence modification(s) of the molecules 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 levels, effector functions, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the molecules and antibody fragments thereof described herein, it is contemplated that antibody variants may be prepared. For example, antibody variants may be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesis of the desired antibody or polypeptide. One skilled in the art will appreciate that 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 properties.

[0127] In some embodiments, the molecules provided herein are chemically modified, for example, by covalent attachment of any type of other molecule(s) to the molecule. Antibody derivatives can include antibodies that have been chemically modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibodies can contain one or more non-classical amino acids.

[0128] The variation may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. The amino acid substitution may be the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, e.g., replacement of a leucine with a serine, e.g., a conservative amino acid substitution. Standard techniques known to those of skill in the art, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions, may be used to introduce mutations in the nucleotide sequence encoding the molecules provided herein. The insertion or deletion may optionally range from 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 compared to the original molecule. In certain embodiments, the substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. The variations permitted can 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.

[0129] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is 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 extends the serum half-life of the antibody.

[0130] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues with similarly charged side chains 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, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.

[0131] Substantial modification of the biological properties of antibodies can be achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chains. Alternatively, conservative (e.g., within amino acid groups with similar properties and / or side chains) substitutions can be made to maintain or not significantly alter properties. Amino acids can be grouped according to the similarity of the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: 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).

[0132] Alternatively, naturally occurring residues can 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 affect chain orientation: Gly, Pro, and (6) aromatic: Trp, Tyr, Phe.

[0133] Any cysteine ​​residue not involved in maintaining the proper conformation of the antibodies provided herein can also be substituted, for example with another amino acid, e.g., alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0134] In some embodiments, molecules with antibody variants with improved properties, e.g., affinity, stability, or expression level, compared to the parent molecule can be prepared by in vitro affinity maturation. Like natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. Libraries of antibodies are displayed (e.g., covalently or non-covalently) on the surface of an organism (e.g., phage, bacteria, yeast, or mammalian cells) or in association with their encoding mRNA or DNA. Affinity selection of the displayed antibodies allows the isolation of organisms or complexes that carry the genetic information encoding the antibody. Two or three rounds of mutation and selection using display methods such as phage display usually result in antibody fragments with affinities in the low nanomolar range. Molecules with affinity-matured antibodies can have nanomolar or even picomolar affinities for the target antigen.

[0135] Phage display is a widely used method for the display and selection of antibodies. Antibodies are displayed on the surface of Fd or M13 bacteriophage as fusions to bacteriophage coat proteins. Selection involves exposure of antigens to allow phage-displayed antibodies to bind to their targets, a process called "panning." Phage that bind antigens are recovered and used to infect bacteria to generate phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, 2002, Methods. Mol. Biol. 178:1-37; and Bradbury and Marks, 2004, J. Immunol. Methods 290:29-49.

[0136] In the yeast display system (see, e.g., Boder et al., 1997, Nat. Biotech. 15:553-57; and Chao et al., 2006, Nat. Protocols 1:755-68), antibodies can be fused to the adhesive subunit of the yeast agglutinin protein Aga2p, which binds the yeast cell wall to Aga1p via a disulfide bond. Display of the protein via Aga2p projects the protein 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 the soluble antigen of interest is determined by labeling the yeast with biotinylated antigen and a secondary reagent, e.g., streptavidin conjugated to a fluorophore. Variations in surface expression of the antibody can be measured via immunofluorescence labeling of hemagglutinin or c-Myc epitope tags adjacent to the scFv. It has been shown that expression correlates with the stability of the displayed protein, and thus antibodies can be selected for improved stability and affinity (see, e.g., Shusta et al., 1999, J. Mol. Biol. 292:949-56). An additional advantage of yeast display is that the displayed protein is folded in the endoplasmic reticulum of the eukaryotic yeast cell, utilizing endoplasmic reticulum chaperones and quality control mechanisms. Once maturation is complete, antibody affinity can be conveniently "titrated" while displayed on the yeast surface, 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, recent approaches use a yeast cell mating system to generate combinatorial diversity estimated to be 1014 in size (e.g., U.S. Patent Publication 2003 / 0186374; and Blaise et al., 2004, Gene 342:211-18).

[0137] In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system. A DNA library encoding a specific library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, still binds to peptidyl-tRNA and occupies the ribosomal tunnel, thus allowing the protein of interest to pop out of the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to a surface-bound ligand, allowing for the simultaneous isolation of the antibody and its encoding mRNA via affinity capture with the ligand. The ribosome-bound mRNA is then reverse transcribed into cDNA, which can then be subjected to mutagenesis and used in the next round of selection (see, for example, Fukuda et al., 2006, Nucleic Acids Res. 34:e127). In mRNA display, a covalent link between the antibody and the mRNA is established using puromycin as an adapter molecule (Wilson et al., 2001, Proc. Natl. Acad. Sci. USA 98:3750-55).

[0138] Because these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, 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. Second, since no library has to be transformed after any diversification step, random mutations can be easily introduced after each selection round, for example by non-proofreading polymerases. In some embodiments, a mammalian display system can be used.

[0139] Diversity can also be introduced into the CDRs of antibody libraries in a targeted manner or through random introduction. The former approach includes sequentially targeting all of the CDRs of an antibody through high or low level mutagenesis, or targeting isolated hotspots of somatic hypermutation (see, for example, Ho et al., 2005, J. Biol. Chem. 280:607-17) or residues suspected to affect affinity for experimental or structural reasons. Diversity can also be introduced by replacement of naturally occurring regions through DNA shuffling or similar techniques (see, for example, Lu et al., 2003, J. Biol. Chem. 278:43496-507; U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops that extend into framework region residues (see, e.g., Bond et al., 2005, J. Mol. Biol. 348:699-709), use loop deletions and insertions in the CDRs, or use hybridization-based diversification (see, e.g., U.S. Patent Publication No. 2004 / 0005709). Additional methods for generating diversity in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Methods that can be used to generate antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Patent Nos. 8,685,897 and 8,603,930, and U.S. Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated by reference herein.

[0140] Screening of libraries can be accomplished by a variety of techniques known in the art. For example, antibodies can be immobilized on solid supports, columns, pins, or cellulose / poly(vinylidene fluoride) membranes / other filters, expressed on host cells added to adsorption plates or used in cell sorting, or conjugated to biotin for capture on streptavidin-coated beads, or used in any other method for panning display libraries.

[0141] For reviews of in vitro affinity maturation methods, see, e.g., Hoogenboom, 2005, Nature Biotechnology 23:1105-16; Quiroz and Sinclair, 2010, Revista Ingeneria Biomedia 4:39-51; and references therein.

[0142] Covalent modifications of the antibody fragment(s) of the molecules provided herein are included within the scope of this disclosure. Covalent modifications include reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or 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 the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, for example, Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0143] Other types of covalent modifications of the antibody fragment(s) of the molecules provided herein that are within the scope of the disclosure include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482-501; and Walsh, 2010, Drug Discov. Today 15:773-80) and linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in, e.g., U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[0144] The antibody fragment(s) of the molecules of the disclosure can also be modified to form chimeric molecules comprising the antibody fragment(s) of the molecule fused to another heterologous polypeptide or amino acid sequence, such as 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)).

[0145] The present disclosure also provides conjugates comprising any one of the antibodies of the present disclosure covalently attached to one or more non-antibody agents by a synthetic linker.

[0146] In some embodiments, the antibody fragment(s) of the molecules provided herein are conjugated or recombinantly fused to, for example, a therapeutic agent (e.g., a cytotoxic agent) or a diagnostic or detectable molecule. The conjugated or recombinantly fused antibody fragment(s) of the molecules can be useful, for example, for treating or preventing a disease or disorder. The conjugated or recombinantly fused antibody can be useful, for example, for monitoring or predicting the onset, development, progression, and / or severity of a disease.

[0147] Such diagnosis and detection can be accomplished using a variety of enzymes, including but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; artificial attachment groups, including but not limited to, streptavidin / biotin or avidin / biotin; fluorescent substances, including but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent substances, including but not limited to, luminol; bioluminescent substances, including but not limited to, luciferase, luciferin, or aequorin; chemiluminescent substances, including but not limited to, acridinium-based compounds or HALOTAG; radioactive substances, including but not limited to, iodine (I, I, I, and I), carbon dioxide, and the like. (14C), sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga and 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, This can be achieved by attachment of the antibody fragment(s) of the molecule to a detectable substance, including, but not limited to, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, or 117Sn; positron emitting metals using various positron emission tomography techniques; and non-radioactive paramagnetic metal ions.

[0148] Also provided herein are antibody fragment(s) recombinantly fused or chemically conjugated (covalent or non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, e.g., 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 a fusion protein, and uses thereof. In particular, provided herein are fusion proteins comprising a targeting region and a heterologous protein, polypeptide, or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide to which the antibody is fused is useful for targeting the antibody to a particular cell type.

[0149] Moreover, the antibodies provided herein can be fused to a marker or "tag" sequence, e.g., a peptide, to facilitate purification. In certain embodiments, the marker or tag amino acid sequence is, inter alia, a hexa-histidine peptide, e.g., a tag provided in a pQE vector (see, e.g., QIAGEN, Inc.), many of which are commercially available. For example, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexa-histidine provides for convenient purification of the fusion protein. Other 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.

[0150] Methods for fusing or conjugating moieties, including polypeptides, to antibodies are known (see, e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, in Monoclonal Antibodies for Cancer Detection and Therapy 475-506 (Pinchera et al. eds., 1985); 303-16 (Baldwin et al. eds., 1985); Thorpe et al., 1982, Immunol. Rev. 62:119-58; U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,723,125; 5,783,181; 5,908,626; 5,844,095; and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO91 / 06570, WO96 / 04388, WO96 / 22024, WO97 / 34631, and WO 99 / 04813;Ashkenazi et al.,1991,Proc.Natl.Acad.Sci.USA 88:10535-39; Traunecker et al., 1988, Nature 331:84-86; Zheng et al., 1995, J. Immunol. 154:5590-600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-41).

[0151] Fusion proteins can be generated through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activities of the antibodies provided herein, including, for example, antibodies with higher affinities and lower off-rates (see, e.g., U.S. Patent 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, can be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. Polynucleotides encoding the antibodies provided herein can be recombined with one or more components, motifs, sections, portions, domains, fragments, etc., of one or more heterologous molecules.

[0152] The antibody fragment(s) of the molecules provided herein can also be conjugated to a second antibody to form an antibody heteroconjugate, e.g., as described in U.S. Patent No. 4,676,980.

[0153] The antibody fragment(s) of the molecules provided herein can also be attached to a solid support, which is particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0154] The linker may be a "cleavable linker" that facilitates the release of the conjugated drug in cells, although non-cleavable linkers are also contemplated herein.Linkers for use in the conjugates of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers that contain amino acids such as valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., Chari et al., 1992, Cancer Res. 52:127-31; and U.S. Patent No. 5,208,020), thioether linkers, or hydrophilic linkers designed to avoid multidrug transporter-mediated resistance (see, e.g., Kovtun et al., 2010, Cancer Res. 70:2528-37).

[0155] Antibody and drug conjugates can be made using a variety of bifunctional protein-linking 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). The present disclosure further contemplates that antibody and drug conjugates can be prepared using any suitable method as disclosed in the art (see, e.g., Bioconjugate Techniques (Hermanson ed., 2d ed.2008)).

[0156] Conventional conjugation strategies for antibodies and drugs are based on random conjugation chemistry with the ε-amino group of Lys residues or the thiol group of Cys residues, resulting 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. These include engineering "thiomabs" that contain cysteine ​​substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not interfere with 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). In another method, selenocysteine ​​is co-translationally inserted into the antibody sequence by recoding the stop codon UGA from the terminus to a selenocysteine ​​insertion, allowing site-specific covalent conjugation with the nucleophilic selenol group of selenocysteine ​​in the presence of 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).

[0157] In some embodiments, the targeting region of the molecules provided herein comprises a non-immunoglobulin binding agent. The non-immunoglobulin binding agent forms a specific binding to the target through enzyme-substrate, receptor-ligand or other protein-protein interactions. In some embodiments, the non-immunoglobulin binding agent is identified as an agent that replaces or can be replaced by the molecules of the present disclosure in a competitive binding assay. These alternative binding agents can include, for example, any of the engineered protein scaffolds known in the art. Such scaffolds include, for example, anticalins, which are based on the lipocalin scaffold, a protein structure characterized by a rigid beta-barrel that supports four hypervariable loops that form the ligand binding site. Novel binding specificities can be engineered by targeted random mutagenesis in the loop regions in combination with functional display and guided selection. (See, for example, Skerra, 2008, FEBS J. 275:2677-83).Other suitable scaffolds include, for example, adnectins or monobodies based on the 10th 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 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., 2007, J. Biol. Chem. 282:3196-204); al., 2008, J. Mol. Biol. 383:1058-68); affilins based on human yB-crystallin (see, e.g., Ebersbach et al., 2007, J. Mol. Biol. 372:172-85); avimers based on the A domains 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). For review, see, e.g., Gebauer and Skerra, 2009, Curr. Opin. Chem. Biol. 13:245-55.

[0158] To ensure the maximum level of yield of the molecules described herein secreted from the host cell, a fragment of the signal peptide may be added. In some embodiments, the molecules disclosed herein comprise a signal peptide (SEQ ID NO:2). In other embodiments, the molecules disclosed herein comprise at least 95% identity to SEQ ID NO:2. In other embodiments, the molecules disclosed herein comprise at least 90% identity to SEQ ID NO:2. In other embodiments, the molecules disclosed herein comprise at least 85% identity to SEQ ID NO:2. In other embodiments, the molecules disclosed herein comprise at least 80% identity to SEQ ID NO:2. In other embodiments, the molecules disclosed herein comprise at least 70% identity to SEQ ID NO:2. In other embodiments, the molecules disclosed herein comprise at least 60% identity to SEQ ID NO:2. In other embodiments, the molecules disclosed herein comprise at least 50% identity to SEQ ID NO:2.

[0159] The binding domain of the molecule is capable of binding to one or more antigen(s). In some embodiments, the antigen may act as a cell surface marker on target cells associated with a particular disease state. In some embodiments, the antigen is a tumor antigen.

[0160] In some embodiments, the antigen of the target cell is an antigen on the surface of a cancer cell. In some embodiments, the antigen is a tumor-specific antigen, a tumor-associated antigen, or a neoantigen. In some embodiments, the target cell is a cancer cell, such as a cell of adrenal gland 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 cancer, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumor, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, nasal 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. In some embodiments, the cancer is adrenal gland 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's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, nasal 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. In some embodiments, the cancer is adrenal gland cancer. In some embodiments, the cancer is anal cancer. In some embodiments, the cancer is appendix cancer. In some embodiments, the cancer is bile duct cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is bone cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is gallbladder cancer. In some embodiments, the cancer is gestational trophoblastic. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is Hodgkin's lymphoma. In some embodiments, the cancer is intestinal cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is lung cancer.In some embodiments, the cancer is melanoma. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is multiple myeloma (MM). In some embodiments, the cancer is a neuroendocrine tumor. In some embodiments, the cancer is non-Hodgkin's lymphoma. In some embodiments, the cancer is oral cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is nasal cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is soft tissue sarcoma spinal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is throat cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is uterine cancer endometrial cancer. In some embodiments, the cancer is vaginal cancer. In some embodiments, the cancer is vulvar cancer.

[0161] In some embodiments, the adrenal cancer is an adrenocortical carcinoma (ACC), adrenocortical carcinoma, pheochromocytoma, or neuroblastoma. In some embodiments, the anal cancer is a squamous cell carcinoma, a cloacogenic carcinoma, an adenocarcinoma, a basal cell carcinoma, or a melanoma. In some embodiments, the appendix cancer is a neuroendocrine tumor (NET), a mucinous adenocarcinoma, a goblet cell carcinoid, an intestinal type adenocarcinoma, or a signet ring cell adenocarcinoma. In some embodiments, the cholangiocarcinoma is an extrahepatic cholangiocarcinoma, an adenocarcinoma, a perihilar cholangiocarcinoma, a distal cholangiocarcinoma, or an intrahepatic cholangiocarcinoma. In some embodiments, the bladder cancer is a transitional cell carcinoma (TCC), a papillary carcinoma, a flat carcinoma, a squamous cell carcinoma, an adenocarcinoma, a small cell carcinoma, or a sarcoma. In some embodiments, the bone cancer is primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer. In some embodiments, the brain cancer is astrocytoma, brain stem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary carcinoma, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal tumor, medulloblastoma, or primary CNS lymphoma. In some embodiments, the breast cancer is breast adenocarcinoma, invasive breast cancer, non-invasive breast cancer, breast sarcoma, metaplastic carcinoma, adenoid cystic carcinoma, phyllodes tumor, angiosarcoma, HER2 positive breast cancer, triple negative breast cancer, or inflammatory breast cancer. In some embodiments, the cervical cancer is squamous cell carcinoma, or adenocarcinoma. In some embodiments, the colorectal cancer is colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma, signet ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma. In some embodiments, the esophageal cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments, the gallbladder cancer is adenocarcinoma, papillary adenocarcinoma, adenosquamous cell carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma. In some embodiments, the gestational trophoblastic disease (GTD) is hydatidiform mole, gestational trophoblastic neoplasm (GTN), choriocarcinoma, placental site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT). In some embodiments, the head and neck cancer is laryngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, oral cavity cancer, oropharyngeal cancer, or tonsil cancer.In some embodiments, the Hodgkin's lymphoma is classical Hodgkin's lymphoma, nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte-depleted, or nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL). In some embodiments, the intestinal cancer is small intestine cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumor, carcinoid tumor, or lymphoma. In some embodiments, the kidney cancer is renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urothelial carcinoma, renal pelvis carcinoma, or kidney sarcoma. In some embodiments, the leukemia is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or myelodysplastic syndrome (MDS). In certain embodiments, the leukemia is AML. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis. In some embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, mixed small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, large cell undifferentiated carcinoma, lung nodule, metastatic lung carcinoma, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland type lung cancer, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor. In some embodiments, the melanoma is superficial spreading melanoma, nodular melanoma, acral lentigo melanoma, lentigo maligna melanoma, amelanotic melanoma, adhesive melanoma, ocular melanoma, or metastatic melanoma. In some embodiments, the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In some embodiments, the multiple myeloma is active or smoldering myeloma. In some embodiments, the neuroendocrine tumor is a gastrointestinal neuroendocrine tumor, a pancreatic neuroendocrine tumor, or a pulmonary neuroendocrine tumor.In some embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of 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's 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 lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, primary central nervous system (CNS) lymphoma, and / or primary idiopathic leukemia. CNS) lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma, B-cell non-Hodgkin lymphoma, T-cell non-Hodgkin lymphoma, natural killer cell lymphoma, cutaneous T-cell lymphoma, Alibert-Bazin syndrome, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), systemic ALCL, enteropathy T-cell lymphoma (EATL), or hepatosplenic gamma / delta T-cell lymphoma. In some embodiments, the oral cancer is squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinoma, lymphoma, benign oral tumor, eosinophilic granuloma, fibroma, granular cell tumor, keratoacanthoma, leiomyoma, osteochondroma, lipoma, schwannoma, neurofibroma, papilloma, genital warts, verrucous xanthomas, pyogenic granuloma, rhabdomyoma, odontogenic tumor, leukoplakia, erythroplakia, squamous cell carcinoma of the lip, basal cell carcinoma of the lip, oral cavity carcinoma, gingival carcinoma, or tongue carcinoma. In some embodiments, the ovarian cancer is epithelial ovarian cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, clear cell epithelial ovarian cancer, undifferentiated epithelial ovarian cancer, ovarian tumor of low malignant potential, primary peritoneal carcinoma, fallopian tube carcinoma, germ cell tumor, teratoma, dysgerminoma ovarian germ cell carcinoma, endodermal sinus tumor, sex cord stromal tumor, 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.In some embodiments, the pancreatic cancer is exocrine pancreatic cancer, endocrine pancreatic cancer, or adenocarcinoma, islet cell tumor, or neuroendocrine tumor. In some embodiments, the prostate cancer is prostate adenocarcinoma, prostate sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor. In some embodiments, the nasal cancer is squamous cell carcinoma, mucosal cell carcinoma, adenoid cystic cell carcinoma, acinar cell carcinoma, undifferentiated paranasal sinus carcinoma, nasal cavity carcinoma, paranasal sinus carcinoma, maxillary sinus carcinoma, ethmoid sinus carcinoma, or nasopharyngeal carcinoma. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi's sarcoma (KS), actinic keratosis, cutaneous lymphoma, or keratoacanthoma. In some embodiments, the soft tissue cancer is angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid / round cell liposarcoma (MRCL), well-differentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma. In some embodiments, the spinal cancer is a spinal metastatic tumor. In some embodiments, the gastric cancer is gastric adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumor, carcinoid tumor, gastric carcinoid tumor, type I ECL cell carcinoid, type II ECL cell carcinoid, or type III ECL cell carcinoid. In some embodiments, the testicular cancer is a seminoma, a non-seminoma, an embryonal carcinoma, a yolk sac carcinoma, a choriocarcinoma, a teratoma, a gonadal stromal tumor, a Leydig cell tumor, or a Sertoli cell tumor. In some embodiments, the laryngeal cancer is a squamous cell carcinoma, an adenocarcinoma, a sarcoma, a laryngeal carcinoma, a pharyngeal carcinoma, a nasopharyngeal carcinoma, an oropharyngeal carcinoma, a hypopharyngeal carcinoma, a laryngeal carcinoma, a laryngeal squamous cell carcinoma, a laryngeal adenocarcinoma, a lymphoepithelioma, a spindle cell carcinoma, a verrucous carcinoma, an undifferentiated carcinoma, or a lymph node carcinoma. In some embodiments, the thyroid cancer is a papillary carcinoma, a follicular carcinoma, a Hürzl cell carcinoma, a medullary thyroid carcinoma, or an undifferentiated carcinoma. In some embodiments, the uterine cancer is an endometrial carcinoma, an endometrial adenocarcinoma, an endometrioid carcinoma, a serous adenocarcinoma, an adenosquamous carcinoma, a uterine carcinosarcoma, a uterine sarcoma, a uterine leiomyosarcoma, an endometrial stromal sarcoma, or an undifferentiated sarcoma. In some embodiments, the vaginal cancer is squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In some embodiments, the vulvar cancer is squamous cell carcinoma or adenocarcinoma.

[0162] Tumor antigens are proteins produced by tumor cells that can induce immune responses, specifically T cell-mediated immune responses. Exemplary tumor antigens include, but are not limited to, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, 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 cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0163] In some embodiments, 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, survivin, BAGE family antigens, CAGE family antigens, GAGE ​​family antigens, MAGE family antigens, SAGE family antigens, XAGE family antigens, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A, MART-1, Gp100, pmel17, tyrosinase, TRP-1, TRP-2, P. polypeptide, MC1R, prostate specific antigen, β-catenin, BRCA1, BRCA2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, or MUC1. In some embodiments, 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. In some embodiments, 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 survivin. In some embodiments, the cancer antigen is a BAGE family antigen. In some embodiments, the cancer antigen is a CAGE family antigen. In some embodiments, the cancer antigen is a GAGE ​​family antigen. In some embodiments, the cancer antigen is a MAGE family antigen. In some embodiments, the cancer antigen is a SAGE family antigen.In some embodiments, the cancer antigen is a 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. In some embodiments, 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 β-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-βRII. In some embodiments, the cancer antigen is MUC1.

[0164] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for 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 oncogene HER2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA).

[0165] In some embodiments, tumor antigens are tumor-specific antigens (TSA) or tumor-associated antigens (TAA). TSAs are unique to tumor cells and do not occur in other cells in the body. TAA-associated antigens are not unique to tumor cells, but instead are also expressed on normal cells under conditions that cannot induce a state of immunological tolerance to the antigen. Expression of antigens on tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be antigens that are expressed on normal cells during fetal development when the immune system is immature and cannot respond, or they can be antigens that are usually present on normal cells at very low levels, but are expressed on tumor cells at much higher levels.

[0166] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), 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 Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.

[0167] Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0168] 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 antigens (e.g., HLA class I antigen A-2 alpha, HLA class I antigen A-11 alpha, and HLA class II antigens), c-Met, hepatocyte growth factor receptor, K-Ras GTPases (KRAS), IL-15 receptor, Kit tyrosine kinase, PDGF receptor beta, RET tyrosine kinase receptor; Raf1 protein kinase, RafB protein kinase, thymidylate synthase, topoisomerase II, Brachyury protein, Flt3 tyrosine kinase, VEGF, VEGF receptors (VEGF-1 receptor, VEGF-2 receptor, and VEGF-3 receptor), estrogen receptors, neoantigens, human papillomavirus E6, and heat shock proteins.

[0169] In some embodiments, the binding molecules provided herein bind to a B cell antigen. In some embodiments, the B cell antigen is selected from the group consisting of 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 ... D45RC, CD45RO, CD46, CD47, CD48, CD49b, CD49c, CD49d, CD50, CD52, CD53, CD54, CD55, CD58, CD60a, CD62L, CD63, CD68, CD 69, CD70, CD72, CD73, CD74, CD75, CD75S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85E, CD85I, CD85J, CD86, CD 92, CD95, CD97, CD98, CD99, CD100, CD102, CD108, CD119, CD120a, CD120b, CD121b, CD122, CD124, CD125, CD126, CD130, CD 132, CD137, CD138, CD139, CD147, CD148, CD150, CD152, CD162, CD164, CD166, CD167a, CD170, CD171, CD175, CD175s, CD180 , CD184, CD185, CD192, CD196, CD197, CD200, CD205, CD201a, CDw210b, CD212, CD213a1, CD213a2, CD215, CD217, CD218a, C D218b, CD220, CD221, CD222, CD224, CD225, CD226, CD227, CD229, CD230, CD232, CD252, CD252, CD254, CD255, CD256, CD257 CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD267, CD268, CD269, CD270, CD272, CD274, C D275, CD277, CD279, CD283, CD289, CD290, CD295, CD298, CD300, CD300c, CD305, CD306, CD307a,In some embodiments, the B cell antigen is a CD307b, CD307c, CD307d, CD307e, CD314, CD215, CD316, CD317, CD319, CD321, CD327, CD328, CD329, CD338, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD360, CD361, CD362, or CD363 antigen. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 isIn some embodiments, the B cell antigen is a CD37 antigen. In some embodiments, the B cell antigen is a CD38 antigen. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 isIn 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 isThe 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. In some embodiments, 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. In some embodiments, 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 CD215 antigen. In some embodiments, the B cell antigen is a CD217 antigen. In some embodiments, 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 the CD225 antigen. In some embodiments, the B cell antigen is the CD226 antigen. In some embodiments, the B cell antigen isIn some embodiments, the B cell antigen is a CD227 antigen. In some embodiments, the B cell antigen is a CD229 antigen. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0170] In one embodiment, the target of the binding molecule is a pathogen. In certain embodiments, the target cell is a cell that contains a pathogen.

[0171] In some embodiments, the pathogen is acute flaccid myelitis (AFM), anaplasmosis, anthrax, babesiosis, botulism, brucellosis, campylobacter infection, carbapenem resistant infection, chancroid, chikungunya virus infection, chlamydia, ciguatera, difficile infection, perfringens, coccidioides fungal infection, coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jakob disease / transmissible spongiform encephalopathy, cryptosporidiosis (C rypto), Cyclosporiasis, Dengue 1, 2, 3 or 4, Diphtheria, E. coli infection / Shiga toxin production (STEC), Eastern equine encephalitis, Hemorrhagic fever (Ebola), Ehrlichiosis, Encephalitis, Arbovirus or parainfectious, Non-polio enterovirus, D68 enterovirus (EV-D68), Giardiasis, Glanders, Gonococcal infection, Inguinal granuloma, Haemophilus influenzae type B (Hib or H-flu), Hantavirus pulmonary syndrome (HPS), Hemolytic uremic syndrome (HUS), Hepatitis A (Hep Hepatitis A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes, Herpes Zoster (Shingles), Histoplasmosis, Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papilloma Virus (HPV), Influenza (Flu), Legionnaires' Disease, Leprosy (Hansens Disease), Leptospirosis, Listeriosis (Listeria), Lyme Disease, Lymphogranuloma Inguinalis (LGV), Malaria, Measles, Melioidosis, Meningitis (Viral), Meningococcal Disease (Meningitis (Bacterial)), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Mumps, Norovirus, Pediculosis, Pelvic Inflammatory Disease (PID), Whooping Cough (Pertussis) Cough), Bubonic plague (bubo, septicemic, pulmonary), Pneumococcal disease (pneumonia), Poliomyelitis (polio), Powassan, Psittacosis, Pubic lice, Pubic lice disease (smallpox, monkeypox, cowpox), Q fever, Rabies, Rickettsiosis (Rocky Mountain spotted fever), Rubella (German measles), Salmonella gastroenteritis (Salmonella), Scabies, Scombroid, Septicemia, Severe Acute Respiratory Syndrome (SARS),Shigella, Smallpox, Methicillin-resistant Staphylococcus aureus infections (MRSA), Staphylococcus aureus food poisoning Enterotoxin B poisoning (Staph food poisoning), Vancomycin-intermediate-susceptible Staphylococcus aureus infections (VISA), Vancomycin-resistant Staphylococcus aureus infections (VRSA), Group A streptococcal disease (invasive) (Strep A (invasive), Group B streptococcal disease (Strep-B), Streptococcal toxic shock syndrome (STSS), Syphilis (primary, secondary, early latent, late latent, congenital), Tetanus infection, Trichomoniasis, Trichinella infection, Tuberculosis (TB), Latent tuberculosis (LTBI), Tularemia, Typhoid fever group D, Vaginosis, Chickenpox, Vibrio cholerae, vibriosis (vibrio), Ebola virus hemorrhagic fever, Lassa virus hemorrhagic fever, Marburg virus hemorrhagic fever, West Nile virus, yellow fever, Yersinia, and Zika virus infection. In some embodiments, the infectious disease is acute flaccid myelitis (AFM). In some embodiments, the infectious disease is anaplasmosis. In some embodiments, the infectious disease is anthrax. In some embodiments, the infectious disease is babesiosis. In some embodiments, the infectious disease is botulism. In some embodiments, the infectious disease is brucellosis. In some embodiments, the infectious disease is Campylobacter ... In some embodiments, the infectious disease is a carbapenem-resistant infection. In some embodiments, the infectious disease is chancroid. In some embodiments, the infectious disease is chikungunya virus infection. In some embodiments, the infectious disease is chlamydia. In some embodiments, the infectious disease is ciguatera poisoning. In some embodiments, the infectious disease is a C. difficile infection. In some embodiments, the infectious disease is C. perfringens. In some embodiments, the infectious disease is a Coccidioides fungal infection. In some embodiments, the infectious disease is a coronavirus. In some embodiments, the infectious disease is Covid-19 (SARS-CoV-2). In some embodiments, the infectious disease isIn some embodiments, the infectious disease is Creutzfeldt-Jakob disease / transmissible spongiform encephalopathy. In some embodiments, the infectious disease is Cryptosporidiosis (Crypto). In some embodiments, the infectious disease is Cyclosporia. In some embodiments, the infectious disease is Dengue type 1, 2, 3 or 4. In some embodiments, 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 an arbovirus or parainfectious. In some embodiments, the infectious disease is a non-polio enterovirus. In some embodiments, the infectious disease is D68 enterovirus (EV-D68). In some embodiments, the infectious disease is Giardiasis. In some embodiments, the infectious disease is glanders. In some embodiments, the infectious disease is gonorrhea. In some embodiments, the infectious disease is granuloma groin. In some embodiments, the infectious disease is Haemophilus influenzae 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). In some embodiments, 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 a Histoplasmosis infection. In some embodiments, the infectious disease isIn some embodiments, the infectious disease is Human Immunodeficiency Virus / AIDS (HIV / AIDS). In some embodiments, the infectious disease is Human Papilloma Virus (HPV). In some embodiments, the infectious disease is Influenza (Flu). In some embodiments, the infectious disease is Legionnaires' Disease (Legionnaires' Disease). In some embodiments, the infectious disease is Leprosy (Leprosy). In some embodiments, 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). In some embodiments, 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 Bubonic Plague (Bubolic. In some embodiments, the infectious disease is Septicemic. In some embodiments, the infectious disease is Pulmonary). In some embodiments, the infectious disease is Pneumococcal Disease (Pneumonia). In some embodiments, the infectious disease is Polio (Polio). In some embodiments, the infectious disease is Powassan. In some embodiments, the infectious disease is Psittacosis. In some embodiments, the infectious disease is Pubic Louse. In some embodiments, the infectious disease is variola (smallpox). In some embodiments, the infectious disease is monkeypox. In some embodiments, the infectious disease isIn 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 rickettsioses (Rocky Mountain spotted fever). In some embodiments, the infectious disease is rubella (German measles). In some embodiments, the infectious disease is Salmonella enterica (Salmonella). In some embodiments, the infectious disease is scabies. In some embodiments, the infectious disease is scombroids. In some embodiments, the infectious disease is septicemia. In some embodiments, the infectious disease is Severe Acute Respiratory Syndrome (SARS). In some embodiments, the infectious disease is Shigella enterica (Shigella). In some embodiments, the infectious disease is smallpox. In some embodiments, the infectious disease is Methicillin-resistant Staphylococcus aureus infection (MRSA). In some embodiments, the infectious disease is Staphylococcus aureus food poisoning enterotoxin B poisoning (Staph food poisoning). In some embodiments, the infectious disease is a vancomycin-intermediate Staphylococcus aureus infection (VISA). In some embodiments, the infectious disease is a vancomycin-resistant Staphylococcus aureus infection (VRSA). In some embodiments, the infectious disease is Group A Streptococcal Disease (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). 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). In some embodiments, the infectious disease is Tetanus Infection. In some embodiments, the infectious disease is Trichomoniasis. In some embodiments, the infectious disease is Trichinella Infection. In some embodiments, the infectious disease is Tuberculosis (TB). In some embodiments, the infectious disease isIn some embodiments, the infectious disease is latent tuberculosis (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 chickenpox, Vibrio cholerae. In some embodiments, the infectious disease is vibriosis (vibrio), In some embodiments, the infectious disease is Ebola virus hemorrhagic fever. In some embodiments, the infectious disease is Lassa 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 Yersinia. In some embodiments, the infectious disease is Zika virus infection.

[0172] In some embodiments, the pathogen is a bacterium. In some embodiments, the bacterium is 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. In some embodiments, the bacterium is a bacterium of the genus bacillus. In some embodiments, the bacterium is a bacterium of the genus bartonella. In some embodiments, the bacterium is a bacterium of the genus bordetella. In some embodiments, the bacterium is a bacterium of the genus borrelia. In some embodiments, the bacterium is a bacterium of the genus brucella. In some embodiments, the bacterium is a bacterium of the genus campylobacter. In some embodiments, the bacterium is a bacterium of the genus chlamydia. In some embodiments, the bacterium is a bacterium of the genus chlamydophila. In some embodiments, the bacterium is a bacterium of the genus clostridium. In some embodiments, the bacterium is a bacterium of the genus corynebacterium. In some embodiments, the bacterium is a bacterium of the genus enterococcus. In some embodiments, the bacterium is a bacterium of the genus escherichia. In some embodiments, the bacterium is a bacterium of the genus francisella. In some embodiments, the bacterium is a bacterium of the genus haemophilus. In some embodiments, the bacterium is a bacterium of the genus helicobacter. In some embodiments, the bacterium is a bacterium of the genus legionella. In some embodiments, the bacterium is a bacterium of the genus leptospira.In some embodiments, the bacterium is a bacterium of the genus Listeria. In some embodiments, the bacterium is a bacterium of the genus Mycobacterium. In some embodiments, the bacterium is a bacterium of the genus Mycoplasma. In some embodiments, the bacterium is a bacterium of the genus Neisseria. In some embodiments, the bacterium is a bacterium of the genus Pseudomonas. In some embodiments, the bacterium is a bacterium of the genus Rickettsia. In some embodiments, the bacterium is a bacterium of the genus Salmonella. In some embodiments, the bacterium is a bacterium of the genus Shigella. In some embodiments, the bacterium is a bacterium of the genus Staphylococcus. In some embodiments, the bacterium is a bacterium of the genus Streptococcus. In some embodiments, the bacterium is a bacterium of the genus Treponema. In some embodiments, the bacterium is a bacterium of the genus Ureaplasma. In some embodiments, the bacterium is a bacterium of the genus Vibrio. In some embodiments, the bacterium is a bacterium of the genus Yersinia.

[0173] In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is a protozoan, a helminth, or an ectoparasite. In some embodiments, the protozoan is an entamoeba, a giardia, a leishmania, a balantidium, a plasmodium, or a cryptosporidium. In some embodiments, the helminth is a trematode, a tapeworm, an anchocephalan, or a roundworm. In some embodiments, the ectoparasite is an arthropod.

[0174] In some embodiments, the pathogen is a virus. In some embodiments, the virus is a virus of the family adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, coronaviridae, filoviridae, flaviviridae, hepadnaviridae, hepeviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, rhabdoviridae, or togaviridae. In some embodiments, the virus is a virus of the family adenoviridae. In some embodiments, the virus is a virus of the family arenaviridae. In some embodiments, the virus is a virus of the family astroviridae. In some embodiments, the virus is a virus of the family bunyaviridae. In some embodiments, the virus is a virus of the family caliciviridae. 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. In some embodiments, 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.In some embodiments, 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.

[0175] In some embodiments, the virus is an adenovirus, a coronavirus, a coxsackievirus, an Epstein-Barr virus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a herpes simplex virus type 2, a cytomegalovirus, a human herpes virus type 8, a human immunodeficiency virus, an influenza virus, a measles virus, a mumps virus, a human papillomavirus, a parainfluenza virus, a poliovirus, a rabies virus, a respiratory syncytial virus, a rubella virus, or a varicella zoster virus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus virus is Covid-19 (SARS-CoV-2). In some embodiments, the virus is a coxsackievirus. In some embodiments, the virus is an 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 herpes simplex virus type 2. In some embodiments, the virus is cytomegalovirus. In some embodiments, the virus is human herpes virus type 8. In some embodiments, the virus is human immunodeficiency virus. In some embodiments, the virus is influenza virus. In some embodiments, the virus is measles virus. In some embodiments, the virus is mumps virus. In some embodiments, the virus is human papillomavirus. In some embodiments, the virus is parainfluenza virus. In some embodiments, the virus is poliovirus. In some embodiments, the virus is rabies virus. In some embodiments, the virus is respiratory syncytial virus. In some embodiments, the virus is rubella virus. In some embodiments, the virus is varicella zoster virus.

[0176] 5.3 Oligomers In another aspect, provided herein are oligomers formed by the molecules provided herein, such as those disclosed in Section 5.2. In some embodiments, the oligomers disclosed herein can be formed when the molecules are connected to two other molecules or more than two other molecules. The oligomers disclosed herein can be composed of homomeric or heteromeric molecules. The oligomers disclosed herein can be composed of different numbers of molecules. Thus, the present disclosure provides an effective platform for forming multispecific and / or multivalent binding oligomers.

[0177] In some embodiments, the oligomers provided herein are formed by molecules each having one connection site, and thus the oligomer is a dimer. In some embodiments, the oligomers provided herein are formed by molecules each having two connection sites. In some particular embodiments, two connection sites of one molecule are directed to two connection sites of one other molecule, and thus the oligomer is a dimer. In some particular embodiments, two connection sites of one molecule are directed to connection sites of two other molecules, and thus the oligomer is more than a dimer. In some embodiments, the oligomers provided herein are formed by molecules each having three connection sites. In some particular embodiments, three connection sites of one molecule are directed to three connection sites of one other molecule, and thus the oligomer is a dimer. In some particular embodiments, three connection sites of one molecule are directed to connection sites of two other molecules, and thus the oligomer is more than a dimer. In some specific embodiments, three connection sites of one molecule are directed to connection sites of three other molecules, thus making the oligomer more than a dimer. In some embodiments, the oligomers provided herein contain 3 units. In some embodiments, the oligomers provided herein contain 4 units. In some embodiments, the oligomers provided herein contain 5 units. In some embodiments, the oligomers provided herein contain 6 units. In some embodiments, the oligomers provided herein contain more than 6 units. In some embodiments, the oligomers are trimers. In some embodiments, the oligomers are tetramers. In some embodiments, the oligomers are pentamers. In other embodiments, the oligomers are hexamers. In other embodiments, the oligomers provided herein are heptamers. In other embodiments, the oligomers provided herein are octamers. In other embodiments, the oligomers provided herein are nonamers. In other embodiments, the oligomers provided herein are decamers. In other embodiments, the oligomers provided herein are undecamers.In other embodiments, the oligomers provided herein are 12-mers. In other embodiments, the oligomers provided herein are 13-mers. In other embodiments, the oligomers provided herein are 14-mers. In other embodiments, the oligomers provided herein are 15-mers. In other embodiments, the oligomers provided herein are 16-mers. In other embodiments, the oligomers provided herein are 17-mers. In other embodiments, the oligomers provided herein are 18-mers. In other embodiments, the oligomers provided herein are 19-mers. In other embodiments, the oligomers provided herein are decamers.

[0178] The oligomers provided herein can be formed by the same or different molecules. In some embodiments, the oligomers are homomers. In other embodiments, the oligomers are heteromers.

[0179] In some embodiments, all of the individual molecules in an oligomer provided herein bind to the same antigen (or target), such that the oligomer is monospecific; however, at least two of the molecules differ from each other, e.g., at the binding domain. For example, the molecules differ in terms of the format or structure of the binding domain, or the molecules differ in the sequence of the binding domain.

[0180] In some embodiments, all of the molecules in the oligomers provided herein bind to the same antigen, and thus the oligomers are monospecific. In some embodiments, not all of the molecules in the oligomers provided herein bind to the same antigen, and thus the oligomers are multispecific. In some embodiments, the oligomers are bispecific. In some embodiments, the oligomers are trispecific. In other embodiments, the oligomers are tetraspecific. In other embodiments, the oligomers are pentaspecific. In other embodiments, the oligomers are hexaspecific.

[0181] In some embodiments, all of the molecules in the oligomers provided herein bind the same antigen, but on different epitopes. In some embodiments, the oligomers provided herein are multivalent. In some embodiments, the oligomers are bivalent. In some embodiments, the oligomers are trivalent. In other embodiments, the oligomers are tetravalent. In other embodiments, the oligomers are pentavalent. In other embodiments, the oligomers are hexavalent. In other embodiments, the oligomers provided herein are heptavalent. In other embodiments, the oligomers provided herein are octavalent. In other embodiments, the oligomers provided herein are nonavalent. In other embodiments, the oligomers provided herein are decavalent.

[0182] 5.4 Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising at least a molecule of the present disclosure. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a molecule provided herein and a pharma- ceutical acceptable excipient. In some particular embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an oligomer provided herein and a pharma- ceutical acceptable excipient.

[0183] In certain embodiments, the term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's 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 used as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, and the like. Examples of suitable pharmaceutical excipients include malt, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions may also contain small amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, 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 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 to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0184] In some embodiments, the choice of excipient will be determined in part by the particular cells and / or by the method of administration. Accordingly, a wide variety of suitable formulations exist.

[0185] Typically, 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, isotonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents, such as EDTA and / or nonionic surfactants.

[0186] Buffers can be used to control pH within a range that optimizes therapeutic effect, especially when stability is pH dependent. Suitable buffers for use with the present disclosure include both organic and inorganic acids and their salts. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers can include histidine and trimethylamine salts, such as Tris.

[0187] Preservatives may be added to prevent microbial growth.Suitable preservatives for use with the present disclosure include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0188] Tonicity agents, sometimes known as "stabilizers", may be present to adjust or maintain the liquid tension in the composition.When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with charged amino acid side groups, thereby reducing the possibility of inter- and intra-molecular interactions.Exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0189] Additional exemplary excipients include (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (as listed above); amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; 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, glycerol ... monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides (e.g., raffinose; and polysaccharides (e.g., dextrin or dextran).

[0190] Non-ionic surfactants or detergents (also known as "wetting agents") may be present to aid in solubilizing the therapeutic agent and to protect the therapeutic protein against perturbation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Suitable non-ionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (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 esters, methylcellulose and carboxymethylcellulose. Anionic detergents that may be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0191] The route of administration will follow known and accepted methods in a suitable manner, for example, by injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intraarticular routes, topical administration, inhalation, or by single or multiple boluses or infusions over time via sustained or sustained release means.

[0192] In another embodiment, the pharmaceutical composition can be provided as a controlled release or sustained release system. In one embodiment, a pump can 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)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., a fusion protein described herein) or compositions 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. Macromol. Sci. Rev. 20:1-12 (1989)). al., J. Neurosurg. 71:105-12 (1989); U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; see PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253.) Polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.In one embodiment, the polymer used in sustained release formulation is inert, does not contain leachable impurities, is stable during storage, is sterile, and is biodegradable.In yet another embodiment, controlled or sustained release system can be placed near specific target tissue, such as the nostril or lung, and thus requires only a fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release Vol.2,115-38(1984)).Controlled release system is, for example, discussed by Langer, Science 249:1527-33(1990). Any technique known to one of skill in the art may be used to produce sustained release formulations containing one or more of the agents described herein (see, e.g., U.S. Patent No. 4,526,938, PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698, Ning et al., Radiotherapy & Oncology 39:179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50:372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60 (1997)).

[0193] The pharmaceutical compositions described herein may also contain multiple active compounds or agents as necessary for the particular indication being treated. Alternatively, or in addition, the compositions may contain cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, or growth inhibitory agents. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0194] The active ingredient may also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, prepared by droplet formation techniques or interfacial polymerization, colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0195] Various compositions and delivery systems are known and can be used in conjunction with the therapeutic agents provided herein, including, but not limited to, liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibodies or therapeutic molecules provided herein, construction of a nucleic acid as part of a retrovirus or other vector, etc.

[0196] In some embodiments, the pharmaceutical compositions provided herein contain the binding molecule in an amount effective for treating or preventing a disease or disorder, for example, a therapeutically effective amount or a prophylactically effective amount.Therapeutic or prophylactic effectiveness is monitored in some embodiments by periodic evaluation of the subject being treated.For repeated administration over several days or more, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs.However, other dosing regimens may be useful and can be determined.

[0197] In certain embodiments, the term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's 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 used as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, and the like. Examples of suitable pharmaceutical excipients include malt, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions may also contain small amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, 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 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 to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0198] In some embodiments, the choice of excipient will be determined in part by the particular cells and / or by the method of administration. Accordingly, a wide variety of suitable formulations exist.

[0199] Typically, 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, isotonicity agents, stabilizers, metal complexes (e.g., Zn-protein complexes); chelating agents, such as EDTA and / or nonionic surfactants.

[0200] Buffers can be used to control pH within a range that optimizes therapeutic effect, especially when stability is pH dependent. Suitable buffers for use with the present disclosure include both organic and inorganic acids and their salts. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers can include histidine and trimethylamine salts, such as Tris.

[0201] Preservatives may be added to prevent microbial growth.Suitable preservatives for use with the present disclosure include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0202] Tonicity agents, sometimes known as "stabilizers", may be present to adjust or maintain the liquid tension in the composition.When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with charged amino acid side groups, thereby reducing the possibility of inter- and intra-molecular interactions.Exemplary tonicity agents include polyhydric sugar alcohols, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0203] Additional exemplary excipients include (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (as listed above); amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; 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, glycerol ... monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharides (e.g., raffinose; and polysaccharides (e.g., dextrin or dextran).

[0204] Non-ionic surfactants or detergents (also known as "wetting agents") may be present to aid in solubilizing the therapeutic agent and to protect the therapeutic protein against perturbation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Suitable non-ionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (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 esters, methylcellulose and carboxymethylcellulose. Anionic detergents that may be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0205] The route of administration will follow known and accepted methods in a suitable manner, for example, by injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intraarticular routes, topical administration, inhalation, or by single or multiple boluses or infusions over time via sustained or sustained release means.

[0206] In another embodiment, the pharmaceutical composition can be provided as a controlled release or sustained release system. In one embodiment, a pump can 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)). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of prophylactic or therapeutic agents (e.g., a fusion protein described herein) or compositions 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. Macromol. Sci. Rev. 20:1-12 (1989)). al., J. Neurosurg. 71:105-12 (1989); U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; see PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253.) Polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.In one embodiment, the polymer used in sustained release formulation is inert, does not contain leachable impurities, is stable during storage, is sterile, and is biodegradable.In yet another embodiment, controlled or sustained release system can be placed near specific target tissue, such as the nostril or lung, and thus requires only a fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release Vol.2,115-38(1984)).Controlled release system is, for example, discussed by Langer, Science 249:1527-33(1990). Any technique known to one of skill in the art may be used to produce sustained release formulations containing one or more of the agents described herein (see, e.g., U.S. Patent No. 4,526,938, PCT Publication Nos. WO 91 / 05548 and WO 96 / 20698, Ning et al., Radiotherapy & Oncology 39:179-89 (1996); Song et al., PDA J. of Pharma. Sci. & Tech. 50:372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997); and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-60 (1997)).

[0207] The pharmaceutical compositions described herein may also contain multiple active compounds or agents as necessary for the particular indication being treated. Alternatively, or in addition, the compositions may contain cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, or growth inhibitory agents. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0208] The active ingredient may also be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, prepared by droplet formation techniques or interfacial polymerization, colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0209] Various compositions and delivery systems are known and can be used in conjunction with the therapeutic agents provided herein, including, but not limited to, liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibodies or therapeutic molecules provided herein, construction of a nucleic acid as part of a retrovirus or other vector, etc.

[0210] In some embodiments, the pharmaceutical compositions provided herein contain the binding molecule in an amount effective for treating or preventing a disease or disorder, for example, a therapeutically effective amount or a prophylactically effective amount.Therapeutic or prophylactic effectiveness is monitored in some embodiments by periodic evaluation of the subject being treated.For repeated administration over several days or more, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs.However, other dosing regimens may be useful and can be determined.

[0211] 5.5 Nucleic Acid Molecules, Vectors, and Host Cells In certain embodiments, the present disclosure encompasses nucleic acid molecules that code for the molecules described herein. The term "nucleic acid molecule encoding a polypeptide" encompasses nucleic acid molecules that include a coding sequence for a polypeptide as well as nucleic acid molecules that include additional coding and / or non-coding sequences. The nucleic acid molecules of the present disclosure may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; it may be double-stranded or single-stranded, and if single-stranded, it may be the coding strand or the non-coding (antisense) strand.

[0212] In certain embodiments, a nucleic acid molecule comprises a coding sequence for a polypeptide fused in the same reading frame to a nucleic acid molecule that aids in the expression and secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide). The nucleic acid molecule can have a leader sequence that is cleaved by the host cell to form a "mature" form of the polypeptide.

[0213] In certain embodiments, the nucleic acid molecule comprises the coding sequence for the polypeptide fused to the marker or tag sequence in the same reading frame.For example, in some embodiments, the marker sequence is a hexa-histidine tag provided by the vector, which allows efficient purification of the marker-fused polypeptide in bacterial host.In some embodiments, the marker is used in conjunction with other affinity tags.

[0214] Recombinant expression of the molecules provided herein requires the construction of an expression vector containing a polynucleotide encoding the molecule. Once a polynucleotide encoding the molecules provided herein is obtained, a vector for the production of the molecules can be generated by recombinant DNA technology using techniques well known in the art. Thus, methods are described herein for preparing the molecules provided herein by expressing polynucleotides containing nucleotide sequences encoding different parts of the molecules described in section 5.2. Methods well known to those skilled in the art can be used to construct expression vectors containing coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided is a replicable vector that contains a nucleotide sequence encoding the molecules provided herein operably linked to a promoter. Such vectors can include nucleotide sequences encoding the constant region of an 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 can be cloned into such a vector for expression of an entire heavy chain, an entire light chain, or both the entire heavy and light chains.

[0215] The expression vector is transfected into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the molecules provided herein. Thus, also provided herein is a host cell containing a polynucleotide encoding a molecule provided herein operably linked to a heterologous promoter. In certain embodiments for expression of molecules having an antigen-binding fragment as described herein, vectors encoding both heavy and light chains can be co-expressed in a host cell for expression of the entire immunoglobulin molecule, as described in more detail below.

[0216] A variety of host-expression vector systems can be utilized to express the molecules provided herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles in which coding sequences of interest can be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, can express the molecules 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 the coding sequence; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence; 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 mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). Bacterial cells, such as Escherichia coli, or eukaryotic cells, can be used for the expression of recombinant molecules, particularly for the expression of whole recombinant molecules. Mammalian cells, such as Chinese hamster ovary cells (CHO), in conjunction with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990, Bio / Technology 8:2). In some embodiments, the molecules provided herein are produced in CHO cells.In certain embodiments, expression of a nucleotide sequence encoding a molecule provided herein is controlled by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0217] In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use for the molecule to be expressed. For example, if large quantities of such molecules are to be produced, a vector that induces expression of a high level of a fusion protein product that is easily purified for the production of pharmaceutical compositions of the molecule may be desired. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO 12:1791); pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509), in which coding sequences may be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Typically, such 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 contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0218] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. Coding sequences can be cloned individually into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0219] In mammalian host cells, a number of viral-based expression systems can be utilized. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion of non-essential regions of the viral genome (e.g., regions El or E3) results in recombinant viruses that are viable and capable of expressing molecules in infected hosts (e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation signals can also be required for efficient translation of the inserted coding sequence. 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. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0220] In addition, a host cell strain may be selected which modulates the expression of the inserted sequences or modifies and processes the gene product in the specific manner 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 post-translational processing and modification of proteins and gene products. An appropriate cell line or host system may be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such 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. In some embodiments, the molecules provided herein as fully human antibodies or fragments thereof are produced in mammalian cells, e.g., CHO cells.

[0221] For long-term, high-yield production of recombinant proteins, stable expression may be utilized. For example, cell lines may be engineered that stably express the molecules described herein. Rather than using expression vectors containing viral origins of replication, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selection marker. Following introduction of the foreign DNA, engineered cells are grown in rich medium for 1-2 days and then switched to selective medium. The selection marker in the recombinant plasmid confers resistance to the selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. This method may be advantageously used to engineer cell lines that express the molecules. Such engineered cell lines may be particularly useful in screening and evaluating compositions that interact directly or indirectly with the molecules.

[0222] A number of selection systems may be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes, which may be used in tk cells, hgprt cells, or aprt cells, respectively. Antimetabolic resistance can also 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. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; 1993, TIB TECH 11(5):155-215); and confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1 (incorporated herein by reference in their entireties).

[0223] The expression levels of the molecules described herein can be increased by vector amplification (for 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)). If the marker in the vector system is amplifiable, increasing the level of inhibitor present in the host cell culture increases 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 can also be increased (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

[0224] For the molecules described in section 5.2.2, including antigen-binding fragments, host cells can 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 can contain identical selectable markers that allow for equal expression of heavy and light chain polypeptides. Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides can be used. In such a situation, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA.

[0225] 5.6 Methods for producing oligomerized molecules In another aspect, provided herein is a method for producing oligomers. More specifically, provided herein is a method for producing IgG C H The method includes a first step for performing a function of introducing an amino acid substitution, e.g., a substitution with a cysteine ​​residue at amino acid residue position 253 according to EU numbering, into one or more molecule(s) comprising the two regions; and a second step for performing a function of expressing such molecule. In some embodiments, the method provided herein further includes a step for performing a function of introducing a human μ tailpiece into the molecule. In some embodiments, the method provided herein further includes purifying or isolating an oligomer comprising the molecule. In some embodiments, the method provided herein includes constructing a vector encoding the molecule provided herein.

[0226] In some specific embodiments, provided herein are methods for producing oligomerized molecules, comprising: (a) introducing into a host cell a vector as disclosed in Section 5.5; (b) culturing 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. In some embodiments, prior to step (c), the methods provided herein may include detecting the presence of the oligomerized molecules in the conditioned medium of the host cell, for example, by SDS-polyacrylamide gel electrophoresis (PAGE). In some embodiments, the methods provided herein may further include characterizing the purified oligomerized molecules by intact mass spectrometry or high performance liquid chromatography (HPLC)-size exclusion chromatography (SEC).

[0227] Point mutations as described in Section 5.2.1 and other single residue modifications as described in Section 5.2.2 can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7; and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34:315-23), or other known techniques can be performed on cloned DNA to generate targeted point mutations as described in Section 5.2.1. The added tailpieces described in Section 5.2.1 and other genetic modifications described in Section 5.2.2 can be made using recombinant DNA techniques known to those of skill 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, NY; Kostelny et al., Int. J. Cancer 93:556-565, 2001; Cole et al., J. Immunol. 159:3613-3621, 1997, and Tsurushita et al., Methods 36:69-83, 2005).

[0228] After obtaining a vector encoding an engineered molecule as disclosed in Section 5.5, cell transfection or transduction can be performed in a host cell. Introducing a polynucleotide into a host cell can be performed by any known method, including, for example, packaging the polynucleotide into a virus (or viral vector) and transducing the host cell with the virus (or vector) or by transfection procedures known in the art. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into the nucleus. 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.

[0229] After introduction of a vector of interest, the host cells secrete a mixture containing the monomeric form of the molecule and different levels of oligomers of the molecule into the medium. In some embodiments, the medium is collected 12 hours after introducing the vector into the host cells. In some embodiments, the medium is collected 24 hours after introducing the vector into the host cells. In some embodiments, the medium is collected 36 hours after introducing the vector into the host cells. In some embodiments, the medium is collected 48 hours after introducing the vector into the host cells. In some embodiments, the medium is collected 60 hours after introducing the vector into the host cells. In some embodiments, the medium is collected 72 hours after introducing the vector into the host cells. In some embodiments, the medium is collected 84 hours after introducing the vector into the host cells. In some embodiments, the medium is collected 96 hours after introducing the vector into the host cells.

[0230] As an initial step to confirm the presence of oligomerized molecules in the conditioned medium prior to further purification and characterization, detection of the molecular weight profile of the different components of the mixture is performed. Since the monomeric and oligomeric forms of the molecules described herein differ at least in terms of mass, mass-based detection techniques are well known in the art and include, but are not limited to, gel electrophoresis, e.g., SDS-PAGE, centrifugation, and chromatography, e.g., gel filtration columns.

[0231] Once a molecule provided herein is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for a particular antigen following Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Additionally, the molecules provided herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0232] The purified oligomerized molecules need to be characterized and their identity confirmed. Different methods are known in the art. In some embodiments, the purified oligomerized molecules can be characterized by mass spectrometry. In certain 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 certain 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 cryo-electron microscopy.

[0233] Recombinant production in prokaryotic cells Polynucleic acid sequences encoding molecules of the present disclosure (e.g., antibodies or fragments thereof) can be obtained using standard recombinant techniques. The desired polynucleic acid sequences can be isolated and sequenced from antibody-producing cells, e.g., hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present disclosure. The selection of an appropriate vector depends primarily on the size of the nucleic acid 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 polynucleotides, or both) and its compatibility with the particular host cell in which it resides. Vector components typically include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and a transcription termination sequence.

[0234] Generally, plasmid vectors containing replicon and control sequences derived from a species compatible with the host cell are used in connection with these hosts. The vector usually carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. Examples of pBR322 derivatives used for the expression of specific antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

[0235] In addition, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as GEM™-11 can be utilized in generating recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0236] The expression vector of the present application may contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is a non-translated control sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes, inducible promoters and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription of the cistron under their control in response to a change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.

[0237] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter may be operably linked to the cistron DNA encoding the 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. Either the native promoter sequence or many heterologous promoters may be used to induce amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because heterologous promoters generally result in greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0238] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the -lactamase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters, such as the tac or trc promoter. However, other promoters functional in bacteria are also suitable, such as other known bacterial or phage promoters. Their nucleic acid sequences have been published, allowing one skilled in the art to operably ligate them to the cistron encoding the target peptide, using linkers or adapters to provide any required restriction sites (Siebenlist et al. Cell 20:269 (1980)).

[0239] In one embodiment, each cistron in a recombinant vector contains a secretory signal sequence component that directs translocation of an expressed polypeptide across a membrane. In general, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and process the native signal sequence for the heterologous polypeptide, the signal sequence may be substituted by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA, and MBP.

[0240] In some embodiments, production of 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. - The strain (SEQ ID NO: 1) provides favorable cytoplasmic conditions for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits.

[0241] Suitable prokaryotic host cells for expressing the molecules (e.g., antibodies or fragments thereof) of the present disclosure include archaea and eubacteria, e.g., gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In some embodiments, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts. Exemplary E. coli strains include those of the genotype W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompT A(nmpc-fepE)degP41 kan RExamples of suitable strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325) and derivatives thereof, including strain 33D3 having the genotype (U.S. Patent No. 5,639,635). Other strains and derivatives thereof may also be suitable, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608). These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having a defined genotype are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, it is necessary to select an appropriate bacterium taking into consideration the replicability of the replicon in the bacterial cell. For example, when a well-known plasmid such as pBR322, pBR325, pACYC177, or pKN410 is used to provide the replicon, E. coli, Serratia, or Salmonella species can be suitably used as the host.

[0242] Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture.

[0243] Host cells are transformed with the expression vectors described above and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Transformation means introducing DNA into a prokaryotic host so that the DNA is replicable either as an extrachromosomal element or by chromosomal integrant. Depending on the host cells used, transformation is performed using standard techniques appropriate for such cells. Generally, calcium treatment using calcium chloride is used for bacterial cells that contain substantial cell wall barriers. Another method of transformation uses polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0244] The prokaryotic cells used to produce the antibodies of the present application are grown in media known in the art and suitable for culturing the selected host cells. An example of a suitable medium is Luria Broth (LB) with necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively permit the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium to grow cells expressing an ampicillin resistance gene.

[0245] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may also be included at appropriate concentrations introduced alone or in mixtures with other supplements or media, such as complex nitrogen sources. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol. The prokaryotic host cells are cultured at a suitable temperature and pH.

[0246] When 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. In one embodiment of the present application, the PhoA promoter is used to control the transcription of the polypeptide. Thus, the transformed host cell is cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited medium is CRAP medium (e.g., Simmons et al., J.Immunol.Methods 263:133-147(2002)). Various other inducers can be used according to the vector construction used, as known in the art.

[0247] The expressed antibodies of the present disclosure are secreted and recovered from the periplasm of the host cells. Protein recovery typically involves disruption of the microorganisms, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein is further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported to and isolated in the culture medium. The cells can be removed from the culture, and the culture supernatant filtered and concentrated for further purification of the produced protein. The expressed polypeptides can be further isolated and characterized using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0248] Alternatively, protein production is carried out in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation techniques are available for the production of recombinant proteins. Various fermentation conditions can be modified to improve the production efficiency and quality of the antibodies of the present disclosure. For example, chaperone proteins have been demonstrated to facilitate proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. J Bio Chem 274:19601-19605 (1999); US Pat. No. 6,083,715; US Patent 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).

[0249] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains that are deficient in proteolytic enzymes can be used for the present invention, for example, as described in U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996). E. coli strains that are deficient in proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins can be used as host cells in the expression system encoding the antibodies of the present application.

[0250] The antibodies generated herein may be further purified to obtain preparations that are substantially homogeneous for further assays and uses. Standard protein purification methods known in the art may be used. 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 cation exchange resins such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, for example using Sephadex G-75. Protein A immobilized on a solid phase may, for example, be used in some embodiments for immunoaffinity purification of the binding molecules of the present disclosure. The solid phase on 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. In some embodiments, the column may be coated with a reagent such as glycerol, intended to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants that are nonspecifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0251] Recombinant production in eukaryotic cells For eukaryotic expression, the vector components usually 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 an enhancer element, a promoter, and a transcription termination sequence.

[0252] Vectors for use with eukaryotic hosts may also have inserts encoding a signal sequence or other polypeptides with a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences are available, as well as viral secretory leaders, such as the herpes simplex gD signal. DNA for such precursor regions may be ligated in reading frame to DNA encoding the antibody of the present application.

[0253] Generally, 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).

[0254] Expression and cloning vectors may contain a selection gene, also known as a selectable marker, that encodes a protein that confers resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, complements an auxotrophic deficiency, or supplies nutrients not available from complex media.

[0255] One example of a selection scheme utilizes a drug to arrest the growth of the host cell. Cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0256] Another example of a suitable selection marker for mammalian cells is one that allows the identification of cells that are capable of incorporating nucleic acid encoding the antibody of the present application. For example, cells transformed with a DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An exemplary suitable host cell when wild-type DHFR is used is a Chinese Hamster Ovary (CHO) cell line that is deficient in DHFR activity. Alternatively, host cells transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selection marker, such as aminoglycoside 3'-phosphotransferase (APH), particularly wild-type hosts containing endogenous DHFR, can be selected by growing the cells in a medium containing a selection factor for the selection marker, such as an aminoglycoside antibiotic.

[0257] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding a desired polypeptide sequence. Eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins. Additional sequences found 70-80 bases upstream from the start of transcription of many genes may be included. The 3' end of most eukaryotes may be a signal for addition of a polyA tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0258] Transcription of a polypeptide from a vector in a mammalian host cell can be controlled by a promoter derived, for example, from the genome of a virus, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), from a heterologous mammalian promoter, such as the actin promoter or an immunoglobulin promoter, from a heat shock promoter (so long as such a promoter is compatible with the host cell system).

[0259] Transcription of DNA encoding the antibody of the present disclosure by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-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) for enhancing elements for activation of eukaryotic promoters. The enhancer can be spliced ​​into the vector at a 5′ or 3′ position of the polypeptide coding sequence, but is preferably located at a site 5′ from the promoter.

[0260] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and occasionally the 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments within the untranslated portion of the polypeptide-encoding mRNA. One useful transcription termination component is the bovine growth hormone polyadenylation region.

[0261] Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic 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 the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); 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 NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0262] Host cells can be transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0263] The host cells used to produce the antibodies of the present application can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. See also Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., J. Med. Soc. 1999, 14:1311 (1999); and others. Any of the media described in U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. Reissue No. 30,985 may be used as a culture medium for the host cells. Any of these media may optionally contain hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (sodium chloride, calcium chloride, The culture medium may be supplemented with essential oils (such as phosphate, magnesium, and salts), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds normally present at minimum 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 one of skill in the art. Culture conditions, e.g., temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to one of skill in the art.

[0264] When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If antibodies are produced intracellularly, as a first step, particulate debris (either host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. If antibodies are secreted into the medium, the supernatant of such expression systems is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.

[0265] Protein compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The substrate to which the affinity ligand is attached is most often agarose, but other substrates 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. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered. After any pre-purification step(s), the mixture containing the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography.

[0266] 5.7 How to use 5.7.1. Treatment methods and uses In another aspect, provided herein are methods of using the molecules and oligomers provided herein and uses thereof. Such methods and uses include, for example, therapeutic methods and uses involving administration of the molecules or oligomers, or compositions containing same, to a subject having a disease or disorder. In some embodiments, the compositions are administered in an effective amount to achieve treatment of the disease or disorder. Uses include use of the compositions in such methods and treatments, and in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the methods are carried out by administering the compositions to a subject having or suspected of having a disease or condition. In some embodiments, the methods thereby treat the disease or disorder in the subject.

[0267] In some embodiments, the treatment provided herein causes a complete or partial improvement or reduction of a disease or disorder, or symptoms, adverse effects or outcomes, or phenotypes associated therewith. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or remission of the disease, and remission or improvement of prognosis. The term includes, but does not imply, complete cure of the disease or complete elimination of any symptoms or effect(s) on all symptoms or outcomes.

[0268] As used herein, in some embodiments, the treatment provided herein delays the progression of a disease or disorder, e.g., stops, hinders, slows down, prevents, stabilizes, inhibits, and / or postpones a disease (e.g., cancer). This delay may be of different duration depending on the disease history and / or the individual being treated. As will be apparent to one skilled in the art, a sufficient or significant delay may actually encompass prevention, in that the individual does not develop the disease or disorder. For example, late-stage cancer, such as the onset of metastasis, may be delayed. In other embodiments, the method or use provided herein prevents a disease or disorder.

[0269] In some embodiments, the molecules or oligomers are used to treat solid tumor cancers. In other embodiments, the molecules or oligomers are used to treat hematological cancers. In other embodiments, the disease or disorder is an autoimmune or inflammatory disease. In other embodiments, the disease or disorder is an infectious disease.

[0270] In some embodiments, the disease or disorder is a disease of abnormal cell growth and / or dysregulated apoptosis. Examples of such diseases include cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, bone cancer, colon cancer, rectal cancer, anal region cancer, gastric cancer, gastrointestinal (gastric, colorectal and / or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, testicular cancer, hepatocellular (liver and / or bile duct cancer), primary or secondary central nervous system tumors, primary or secondary brain tumors, hodgkin lymphoma, leukemia ... These include, but are not limited to, J.D. Kinesiology, chronic or acute leukemia, chronic myelogenous leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, kidney and / or ureteral cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder carcinoma, splenic carcinoma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination thereof.

[0271] In some embodiments, 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 carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, and splenic cancer.

[0272] In some embodiments, the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma. In some embodiments, the cancer is 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 lymphoma (PTCL), cutaneous T-cell lymphoma, mantle zone lymphoma, cystic ... The present invention is directed to a method for treating or preventing the development of a leukemia, comprising administering to the patient a therapeutically effective amount of a leukemia, a leukemia having a therapeutic effect against a leukemia having a pulmonary fibrosis, a pulmonary fibrosis, or a pulmonary fibrosis, selected from the group consisting of acute myeloid leukemia, low-grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T-cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt's leukemia (Burkitt's lymphoma), acute biphenotypic leukemia, chronic myelogenous leukemia, chronic myelogenous leukemia (CML), and chronic monocytic leukemia.

[0273] In other embodiments, the disease or disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of carcinoma, adenocarcinoma, adrenocortical carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, colorectal carcinoma, ductal cell carcinoma, lung cancer, thyroid cancer, nasopharyngeal carcinoma, melanoma, non-melanoma skin cancer, liver cancer, and lung cancer.

[0274] In some embodiments, the cancer is adrenal cancer. In some embodiments, the cancer is anal cancer. In some embodiments, the cancer is appendix cancer. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is bone cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is gallbladder cancer. In some embodiments, the cancer is gestational trophoblastic. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is Hodgkin's lymphoma. In some embodiments, the cancer is intestinal cancer. In some embodiments, the cancer is kidney cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is multiple myeloma (MM). In some embodiments, the cancer is a neuroendocrine tumor. In some embodiments, the cancer is non-Hodgkin's lymphoma. In some embodiments, the cancer is oral cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is nasal cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is soft tissue sarcoma spinal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is throat cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is uterine cancer endometrial cancer. In some embodiments, the cancer is vaginal cancer. In some embodiments, the cancer is vulvar cancer.

[0275] In some embodiments, the adrenal cancer is an adrenocortical carcinoma (ACC), adrenocortical carcinoma, pheochromocytoma, or neuroblastoma. In some embodiments, the anal cancer is a squamous cell carcinoma, a cloacogenic carcinoma, an adenocarcinoma, a basal cell carcinoma, or a melanoma. In some embodiments, the appendix cancer is a neuroendocrine tumor (NET), a mucinous adenocarcinoma, a goblet cell carcinoid, an intestinal type adenocarcinoma, or a signet ring cell adenocarcinoma. In some embodiments, the cholangiocarcinoma is an extrahepatic cholangiocarcinoma, an adenocarcinoma, a perihilar cholangiocarcinoma, a distal cholangiocarcinoma, or an intrahepatic cholangiocarcinoma. In some embodiments, the bladder cancer is a transitional cell carcinoma (TCC), a papillary carcinoma, a flat carcinoma, a squamous cell carcinoma, an adenocarcinoma, a small cell carcinoma, or a sarcoma. In some embodiments, the bone cancer is primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer. In some embodiments, the brain cancer is astrocytoma, brain stem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary carcinoma, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal tumor, medulloblastoma, or primary CNS lymphoma. In some embodiments, the breast cancer is breast adenocarcinoma, invasive breast cancer, non-invasive breast cancer, breast sarcoma, metaplastic carcinoma, adenoid cystic carcinoma, phyllodes tumor, angiosarcoma, HER2 positive breast cancer, triple negative breast cancer, or inflammatory breast cancer. In some embodiments, the cervical cancer is squamous cell carcinoma, or adenocarcinoma. In some embodiments, the colorectal cancer is colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma, signet ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma. In some embodiments, the esophageal cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments, the gallbladder cancer is adenocarcinoma, papillary adenocarcinoma, adenosquamous cell carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma. In some embodiments, the gestational trophoblastic disease (GTD) is hydatidiform mole, gestational trophoblastic neoplasm (GTN), choriocarcinoma, placental site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT). In some embodiments, the head and neck cancer is laryngeal cancer, nasopharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, oral cavity cancer, oropharyngeal cancer, or tonsil cancer.In some embodiments, the Hodgkin's lymphoma is classical Hodgkin's lymphoma, nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte-depleted, or nodular lymphocyte-predominant Hodgkin's lymphoma (NLPHL). In some embodiments, the intestinal cancer is small intestine cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumor, carcinoid tumor, or lymphoma. In some embodiments, the kidney cancer is renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urothelial carcinoma, renal pelvis carcinoma, or kidney sarcoma. In some embodiments, the leukemia is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or myelodysplastic syndrome (MDS). In certain embodiments, the leukemia is AML. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis. In some embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, mixed small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, large cell undifferentiated carcinoma, lung nodule, metastatic lung carcinoma, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland type lung cancer, lung carcinoid, mesothelioma, sarcomatoid carcinoma of the lung, or malignant granular cell lung tumor. In some embodiments, the melanoma is superficial spreading melanoma, nodular melanoma, acral lentigo melanoma, lentigo maligna melanoma, amelanotic melanoma, adhesive melanoma, ocular melanoma, or metastatic melanoma. In some embodiments, the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In some embodiments, the multiple myeloma is active or smoldering myeloma. In some embodiments, the neuroendocrine tumor is a gastrointestinal neuroendocrine tumor, a pancreatic neuroendocrine tumor, or a pulmonary neuroendocrine tumor.In some embodiments, the non-Hodgkin's lymphoma is selected from the group consisting of 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's 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 lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, primary central nervous system (CNS) lymphoma, and / or primary idiopathic leukemia. CNS) lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, lymphoplasmacytic lymphoma, B-cell non-Hodgkin lymphoma, T-cell non-Hodgkin lymphoma, natural killer cell lymphoma, cutaneous T-cell lymphoma, Alibert-Bazin syndrome, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), systemic ALCL, enteropathy T-cell lymphoma (EATL), or hepatosplenic gamma / delta T-cell lymphoma. In some embodiments, the oral cancer is squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinoma, lymphoma, benign oral tumor, eosinophilic granuloma, fibroma, granular cell tumor, keratoacanthoma, leiomyoma, osteochondroma, lipoma, schwannoma, neurofibroma, papilloma, genital warts, verrucous xanthomas, pyogenic granuloma, rhabdomyoma, odontogenic tumor, leukoplakia, erythroplakia, squamous cell carcinoma of the lip, basal cell carcinoma of the lip, oral cavity carcinoma, gingival carcinoma, or tongue carcinoma. In some embodiments, the ovarian cancer is epithelial ovarian cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, clear cell epithelial ovarian cancer, undifferentiated epithelial ovarian cancer, ovarian tumor of low malignant potential, primary peritoneal carcinoma, fallopian tube carcinoma, germ cell tumor, teratoma, dysgerminoma ovarian germ cell carcinoma, endodermal sinus tumor, sex cord stromal tumor, 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.In some embodiments, the pancreatic cancer is exocrine pancreatic cancer, endocrine pancreatic cancer, or adenocarcinoma, islet cell tumor, or neuroendocrine tumor. In some embodiments, the prostate cancer is prostate adenocarcinoma, prostate sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor. In some embodiments, the nasal cancer is squamous cell carcinoma, mucosal cell carcinoma, adenoid cystic cell carcinoma, acinar cell carcinoma, undifferentiated paranasal sinus carcinoma, nasal cavity carcinoma, paranasal sinus carcinoma, maxillary sinus carcinoma, ethmoid sinus carcinoma, or nasopharyngeal carcinoma. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi's sarcoma (KS), actinic keratosis, cutaneous lymphoma, or keratoacanthoma. In some embodiments, the soft tissue cancer is angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid / round cell liposarcoma (MRCL), well-differentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma. In some embodiments, the spinal cancer is a spinal metastatic tumor. In some embodiments, the gastric cancer is gastric adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumor, carcinoid tumor, gastric carcinoid tumor, type I ECL cell carcinoid, type II ECL cell carcinoid, or type III ECL cell carcinoid. In some embodiments, the testicular cancer is a seminoma, a non-seminoma, an embryonal carcinoma, a yolk sac carcinoma, a choriocarcinoma, a teratoma, a gonadal stromal tumor, a Leydig cell tumor, or a Sertoli cell tumor. In some embodiments, the laryngeal cancer is a squamous cell carcinoma, an adenocarcinoma, a sarcoma, a laryngeal carcinoma, a pharyngeal carcinoma, a nasopharyngeal carcinoma, an oropharyngeal carcinoma, a hypopharyngeal carcinoma, a laryngeal carcinoma, a laryngeal squamous cell carcinoma, a laryngeal adenocarcinoma, a lymphoepithelioma, a spindle cell carcinoma, a verrucous carcinoma, an undifferentiated carcinoma, or a lymph node carcinoma. In some embodiments, the thyroid cancer is a papillary carcinoma, a follicular carcinoma, a Hürzl cell carcinoma, a medullary thyroid carcinoma, or an undifferentiated carcinoma. In some embodiments, the uterine cancer is an endometrial carcinoma, an endometrial adenocarcinoma, an endometrioid carcinoma, a serous adenocarcinoma, an adenosquamous carcinoma, a uterine carcinosarcoma, a uterine sarcoma, a uterine leiomyosarcoma, an endometrial stromal sarcoma, or an undifferentiated sarcoma. In some embodiments, the vaginal cancer is squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In some embodiments, the vulvar cancer is squamous cell carcinoma or adenocarcinoma.

[0276] In some embodiments, the disease or disorder is caused by a pathogen. In some embodiments, the pathogen is acute flaccid myelitis (AFM), anaplasmosis, anthrax, babesiosis, botulism, brucellosis, campylobacter infection, carbapenem-resistant infection, chancroid, chikungunya virus infection, chlamydia, ciguatera, difficile infection, perfringens, coccidioides fungal infection, coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jakob disease / transmissible spongiform encephalopathy, cryptosporidiosis (C rypto), Cyclosporiasis, Dengue 1, 2, 3 or 4, Diphtheria, E. coli infection / Shiga toxin production (STEC), Eastern equine encephalitis, Hemorrhagic fever (Ebola), Ehrlichiosis, Encephalitis, Arbovirus or parainfectious, Non-polio enterovirus, D68 enterovirus (EV-D68), Giardiasis, Glanders, Gonococcal infection, Inguinal granuloma, Haemophilus influenzae type B (Hib or H-flu), Hantavirus pulmonary syndrome (HPS), Hemolytic uremic syndrome (HUS), Hepatitis A (Hep Hepatitis A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes, Herpes Zoster (Shingles), Histoplasmosis, Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papilloma Virus (HPV), Influenza (Flu), Legionnaires' Disease, Leprosy (Hansens Disease), Leptospirosis, Listeriosis (Listeria), Lyme Disease, Lymphogranuloma Inguinalis (LGV), Malaria, Measles, Melioidosis, Meningitis (Viral), Meningococcal Disease (Meningitis (Bacterial)), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Mumps, Norovirus, Pediculosis, Pelvic Inflammatory Disease (PID), Whooping Cough (Pertussis)Cough), Bubonic Plague (Bubo, Septicemic, Pulmonary), Pneumococcal Disease (Pneumonia), Poliomyelitis (Polio), Powassan, Psittacosis, Pubic Louse, Pubic Pussy, Pubic Pussy, Q Fever, Rabies, Rickettsiosis (Rocky Mountain Spotted Fever), Rubella (German Measles), Salmonella Gastroenteritis (Salmonella), Scabies, Scombroid, Septicemia, Severe Acute Respiratory Syndrome (SARS), Shigella Gastroenteritis (Shigella), Smallpox, Methicillin-Resistant Staphylococcus Aureus Infections (MRSA), Staphylococcus Aureus Food Poisoning Enterotoxin B Poisoning (Staph Food Poisoning), Vancomycin-Intermediate-Sensitive Staphylococcus Aureus Infections (VISA), Vancomycin-Resistant Staphylococcus Aureus Infections (VRSA), Group A Streptococcal Disease (Invasive) (Strep The infectious diseases are selected from the group consisting of invasive, group B streptococcal disease (Strep-B), streptococcal toxic shock syndrome (STSS), syphilis (primary, secondary, early latent, latent, congenital), tetanus infection, trichomoniasis, trichinella infection, tuberculosis (TB), latent tuberculosis (LTBI), tularemia, typhoid fever group D, vaginosis, chickenpox, Vibrio cholerae, vibriosis, Ebola virus hemorrhagic fever, Lassa virus hemorrhagic fever, Marburg virus hemorrhagic fever, West Nile virus, yellow fever, Yersinia, and Zika virus infection.

[0277] In some embodiments, the pathogen is a bacterium, hi some embodiments, the bacterium is of the genus 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.

[0278] In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is a protozoan, a helminth, or an ectoparasite. In some embodiments, the protozoan is an entamoeba, a giardia, a leishmania, a balantidium, a plasmodium, or a cryptosporidium. In some embodiments, the helminth is a trematode, a tapeworm, an anchocephalan, or a roundworm. In some embodiments, the ectoparasite is an arthropod.

[0279] In some embodiments, the pathogen is a virus. In some embodiments, the virus is a virus of the family adenoviridae, arenaviridae, astroviridae, bunyaviridae, caliciviridae, coronaviridae, filoviridae, flaviviridae, hepadnaviridae, hepeviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, rhabdoviridae, or togaviridae.

[0280] In some embodiments, the virus is an adenovirus, a coronavirus, a coxsackievirus, an Epstein-Barr virus, a hepatitis A virus, a hepatitis B virus, a hepatitis C virus, a herpes simplex virus type 2, a cytomegalovirus, a human herpes virus type 8, a human immunodeficiency virus, an influenza virus, a measles virus, a mumps virus, a human papillomavirus, a parainfluenza virus, a poliovirus, a rabies virus, a respiratory syncytial virus, a rubella virus, or a varicella zoster virus.

[0281] In another embodiment, the disease or disorder is an immune or autoimmune disorder. Such disorders include autoimmune bullous diseases, abetalipoproteinemia, acquired immune deficiency-related diseases, acute immune diseases associated with organ transplantation, acquired acrocyanosis, acute and chronic parasitic or infectious processes, acute pancreatitis, acute renal failure, acute rheumatic fever, acute transverse myelitis, adenocarcinoma, atrial ectopic beats, adult (acute) 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-1- Antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis, anemia, angina, ankylosing spondylitis-related lung disease, anterior horn cell degeneration, antibody-mediated cytotoxicity, antiphospholipid syndrome, antireceptor hypersensitivity reaction, aortic and peripheral aneurysms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, arthropathy, asthenia, asthma, ataxia, atopic allergy, atrial fibrillation (persistent or paroxysmal), atrial flutter, atrioventricular block, atrophic autoimmune hypothyroidism, autoimmune hemolytic anemia, autoimmune hepatitis, type 1 autoimmune hepatitis (classical or lupoid hepatitis) , autoimmune-mediated hypoglycemia, autoimmune neutropenia, autoimmune thrombocytopenia, autoimmune thyroid disease, B-cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bronchiolitis obliterans, bundle branch block, burns, cachexia, cardiac arrhythmias, cardiac syncope syndrome, cardiac tumors, cardiomyopathy, inflammatory responses to cardiopulmonary bypass, cartilage graft rejection, cerebellar cortical degeneration, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy-related disorders, chlamydia, cholecystitis (choleosatatis), chronic alcoholism, chronic active hepatitis, chronic fatigue syndrome, organ transplant-related Chronic immune diseases, chronic eosinophilic pneumonia, chronic inflammatory lesions, chronic mucocutaneous candidiasis, chronic obstructive pulmonary disease (COPD), chronic salicylate poisoning, colorectal common variable immunodeficiency (common variable hypogammaglobulinemia), conjunctivitis, connective tissue disease-associated interstitial lung disease, contact dermatitis, Coombs positive hemolytic anemia, cor pulmonale, Creutzfeldt-Jakob disease, idiopathic autoimmune hepatitis, idiopathic fibrosing alveolitis, culture negative sepsis, cystic fibrosis, cytokine therapy-associated disorders, Crohn's disease, dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, scleroderma, skin conditions,Dermatomyositis / Polymyositis associated lung disease, diabetes mellitus, diabetic arteriosclerotic disease, diabetes mellitus, diffuse Lewy body disease, dilated cardiomyopathy, dilated congestive cardiomyopathy, discoid lupus erythematosus, basal ganglia disorders, disseminated intravascular coagulation, Down's syndrome in middle age, drug-induced interstitial lung disease, drug-induced hepatitis, drug-induced movement disorders induced by drugs that block CNS dopamine receptors, drug hypersensitivity, eczema, encephalomyelitis, endocarditis, endocrine disorders, enteropathic synovitis, epiglottitis, Epstein-Barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hemophagocytic lymphohistiocytosis, fetal thymic implant rejection , Friedreich's ataxia, functional peripheral arterial disease, female infertility, fibrosis, fibrotic lung disease, fungal sepsis, gas gangrene, gastric ulcer, giant cell arteritis, glomerulonephritides, Goodpasture's syndrome, goiter, autoimmune hypothyroidism (Hashimoto's disease), gouty arthritis, rejection of any organ or tissue transplant, graft-versus-host disease, gram-negative sepsis, gram-positive sepsis, granulomas due to intracellular organisms, group B streptococcus (GBS) infection, Graves' disease, hemosiderosis-associated lung disease, Hairy cell leukemia, Hallervorden-Spatz disease, Hashimoto's thyroiditis, hay fever, heart transplant rejection, hemochromatosis, hematopoietic malignancies (leukemia and lymphoma), hemolytic anemia, hemolytic uraemic syndrome / thrombolytic thrombocytopenic purpura, hemorrhage, Henoch-Schonlein purpura, Hepatitis A, Hepatitis B, Hepatitis C, HIV infection / HIV neuropathy, Hodgkin's disease, hypoparathyroidism, Huntington's chorea, hyperkinetic movement disorder, hypersensitivity reactions, hypersensitivity pneumonitis, hyperthyroidism, hypokinetic movement disorder, hypothalamic-pituitary-adrenal axis evaluation, idiopathic Addison's disease, idiopathic leukopenia, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia, idiosyncratic liver disease, childhood spinal muscular atrophy, infectious diseases, aortic inflammation, inflammatory bowel disease, insulin-dependent diabetes mellitus, interstitial pneumonia, iridocyclitis / uveitis / optic neuritis, ischemia-reperfusion injury, ischemic stroke, juvenile pernicious anemia, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, Kawasaki disease, kidney transplant rejection, Legionella, leishmaniasis, leprosy, corticospinal system lesions, linear IgA disease, lipidemia, liver transplant rejection,Lyme disease, lymphedema, lymphocytic infiltrative lung disease, malaria, idiopathic or NOS male infertility, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, renal microscopic vasculitis, migraine, mitochondrial multisystem disorder, mixed connective tissue disease, mixed connective tissue disease-associated lung disease, monoclonal gammopathy, multiple myeloma, multisystem degeneration (Mencel, Dejerine-Thomas, Shai-Drager and Machado-Joseph), myalgic encephalitis / Royal-Free disease, myasthenia gravis, renal microscopic vasculitis, intracellular mycobacterium, mycobacterium avium tuberculosis, myelodysplastic syndrome, myocardial infarction, myocardial ischemic disorder, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, nephrotic syndrome, neurodegenerative diseases, neurogenic type I muscular atrophy, neutropenic fever, nonalcoholic steatohepatitis, occlusion of abdominal aorta and its branches, occlusive arterial disease, organ transplant rejection, orchitis / epididymitis, orchitis / vasectomy reversal, organomegaly, osteoarthritis, osteoporosis, ovarian failure, pancreatic transplant rejection, parasitic diseases, parathyroid transplant rejection, Parkinson's disease, pelvic inflammatory disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, perennial rhinitis , pericardial disease, peripheral atherosclerosis, peripheral vascular disease, peritonitis, pernicious anemia, phacogenic uveitis, Pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathies, monoclonal gammopathy, and skin changes syndrome), post-perfusion syndrome, post-pump syndrome, post-MI open-heart syndrome, post-infectious interstitial lung disease, premature menopause, primary biliary cirrhosis, primary sclerosing hepatitis, primary myxedema, primary pulmonary hypertension, primary sclerosing cholangitis, primary vasculitis, progressive supranuclear palsy, psoriasis, psoriasis type 1, psoriasis type 2, psoriasis arthropathy, pulmonary hypertension secondary to connective tissue disease, pulmonary manifestations of polyarteritis nodosa, post-inflammatory interstitial lung disease, radiation fibrosis, radiation therapy, Raynaud's phenomenon and disease, Raynaud's disease, Refsum's disease, regular narrow QRS tachycardia, Reiter's disease, renal disease NOS, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, rheumatoid arthritis-associated interstitial lung disease, rheumatoid spondylitis, sarcoidosis, Schmidt's syndrome, scleroderma, senile chorea, senile dementia with Lewy bodies, septic syndrome, septic shock, seronegative arthropathy, shock, sickle cell anemia, T-cell or FAB ALL, Takayasu's disease / arteritis, telangiectasia,Th2 and Thl mediated diseases, thromboangiitis obliterans, thrombocytopenia, thyroiditis, toxicity, toxic shock syndrome, transplantation, trauma / hemorrhage, type 2 autoimmune hepatitis (anti-LKM antibody hepatitis), type B insulin resistance with acanthosis nigricans, type III hypersensitivity reactions, type IV hypersensitivity, ulcerative colitis arthropathy, ulcerative colitis, unstable angina, uremia, uric sepsis, urticaria, uveitis, valvular heart disease, varicose veins, vasculitis, vasculitic diffuse lung disease, venous thrombosis, venous thrombosis , ventricular fibrillation, vitiligo, acute liver disease, viral and fungal infections, viral encephalitis / aseptic meningitis, virus-associated hemophagocytic syndrome, Wegener's granulomatosis, Wernicke-Korsakoff syndrome, Wilson's disease, any organ or tissue xenograft rejection, Yersinia and Salmonella-associated arthropathy, acquired immune deficiency syndrome (AIDS), autoimmune lymphoproliferative syndrome, hemolytic anemia, inflammatory diseases, thrombocytopenia, acute and chronic immune disorders associated with organ transplantation Disease, Addison's disease, Allergic diseases, Alopecia, Alopecia areata, Atherosclerosis / arteriosclerosis, Atherosclerosis, Arthritis (including osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis and reactive arthritis), Sjogren's disease-related lung disease, Sjogren's syndrome, Skin allograft rejection, Skin change syndrome, Small intestine transplant rejection, Sperm autoimmunity, Multiple sclerosis (all subtypes), Spinal ataxia, Spinal small These include cerebral degeneration, spondyloarthropathy, sporadic polyglandular deficiency type I, sporadic polyglandular deficiency type II, Still's disease, streptococcal myositis, stroke, structural lesions of the cerebellum, subacute sclerosing panencephalitis, sympathetic ophthalmia, fainting, cardiovascular syphilis, systemic anaphylaxis, systemic inflammatory response syndrome, systemic-onset juvenile rheumatoid arthritis, systemic lupus erythematosus, systemic lupus erythematosus-associated lung disease, lupus nephritis, systemic sclerosis, and systemic sclerosis-associated interstitial lung disease.

[0282] In some embodiments, the disease or disorder is an inflammatory disease. Inflammation plays a fundamental role in host defense 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 involving 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 an influx of phagocytes. These events result in swelling, redness, warmth (altered heat patterns), and pus formation at the site of injury or infection.

[0283] Cytokines and prostaglandins control the inflammatory response and are released into the blood or affected tissue in an ordered, self-limiting cascade. This release of cytokines and prostaglandins can increase blood flow to the area of ​​injury or infection, resulting in redness and warmth. Some of these chemicals can cause leakage of fluid into the tissue, resulting in swelling. This protective process can irritate nerves and cause pain. These changes are beneficial to the body when they oc...

Claims

1. IgG C Ｈ A molecule comprising two regions, said IgG C according to EU numbering Ｈ The molecule wherein position 253 in the .DELTA..times ...

2. The molecule comprises: (a) an IgG hinge region; (b) an IgG C Ｈ 3 region, (c) IgG C Ｈ 11. The molecule of claim 1, further comprising (a) a region and / or (b) a human μ tailpiece.

3. The molecule of claim 2, wherein the human μ tailpiece comprises an amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 75%, at least 80%, at least 85%, or at least 90% identity to the amino acid sequence of SEQ ID NO:

1.

4. The human μ tailpiece is Ｈ or the human μ tailpiece is conjugated to the C-terminus of the IgG C domain. Ｈ The molecule of claim 2, conjugated to the C-terminus of the three regions.

5. The molecule of claim 1 , wherein the IgG is human IgG, human IgG1, human IgG2, human IgG3, or human IgG4.

6. The molecule of claim 1 , further comprising a binding domain that specifically binds to a target.

7. The molecule of claim 6 , wherein the binding domain is an antibody fragment.

8. The molecule of claim 1 , wherein the molecule is an antibody or an antigen-binding fragment thereof.

9. 10. An oligomer comprising two or more molecules according to claim 1.

10. An oligomer comprising two or more molecules, each of said two or more molecules being an IgG C Ｈ 2 regions, and the IgG C according to EU numbering Ｈ The oligomer, wherein position 253 in the 2 region is substituted to be a cysteine.

11. Each of the two or more molecules comprises: (a) an IgG hinge region; (b) an IgG C Ｈ 3 region, (c) IgG C Ｈ 11. The oligomer of claim 10, further comprising (a) a region and / or (b) a human μ tailpiece.

12. 12. The oligomer of claim 11, wherein the human μ tailpiece comprises an amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% identity to the amino acid sequence of SEQ ID NO:

1.

13. The human μ tailpiece is Ｈ or the human μ tailpiece is conjugated to the C-terminus of the IgG C domain. Ｈ The oligomer of claim 11 , wherein the oligomer is conjugated to the C-terminus of the three regions.

14. The oligomer of claim 10, wherein the IgG is human IgG, human IgG1, human IgG2, human IgG3, or human IgG4.

15. The oligomer of claim 10 , wherein each of the two or more molecules further comprises a binding domain that specifically binds to a target.

16. The oligomer according to claim 15 , wherein the binding domain is an antibody fragment.

17. 11. The oligomer according to any one of claims 10, wherein each of the two or more molecules is an antibody or an antigen-binding fragment thereof.

18. The oligomer of claim 10, wherein the oligomer is a pentamer or a hexamer.

19. The oligomer of claim 10 , wherein the oligomer is a homomer, and the two or more molecules bind to the same target, or the oligomer is a heteromer.

20. 20. The oligomer of claim 19, wherein the oligomer is a heteromer, and the two or more molecules bind to two or more different targets.

21. An isolated nucleic acid encoding the molecule of claim 1.

22. A vector comprising the nucleic acid of claim 21.

23. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 8, an oligomer according to any one of claims 9 to 20, an isolated nucleic acid according to claim 21, or a vector according to claim 22.

24. 24. The pharmaceutical composition of claim 23 for use in treating a disease or disorder.

25. IgG C Ｈ A method for making an oligomer comprising two or more molecules each comprising two regions, said method comprising: Ｈ introducing a cysteine ​​amino acid substitution at position 253 according to EU numbering in region 2 into each of said two or more molecules.

26. 1. A method for producing an oligomerized molecule, comprising: i. introducing the vector of claim 22 into a host cell; ii. culturing the host cell under conditions suitable for the production of the oligomerized molecule; and iii. Purifying the oligomerized molecules. The method comprising:

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