Enhanced targeting using antibody-oligonucleotide conjugates

Abstract

The present disclosure provides antibodies, and fragments thereof, conjugated to oligonucleotides that do not hybridize to DNA dendrimers, compositions comprising same, and their use in detection, cell isolation, depletion, diagnostic and therapeutic methods.

Description

[Technical field]

[0001] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as a text file named 19128800602SEQ, with a size of 1 kilobyte, created on March 2, 2022. The Sequence Listing is incorporated herein by reference.

[0002] The present disclosure is directed to antibodies, and fragments thereof, conjugated to oligonucleotides that do not hybridize to DNA dendrimers, compositions comprising same, and their use in detection, cell isolation, depletion, diagnostic and therapeutic methods. [Background technology]

[0003] Antibodies are widely used as therapeutic compounds for the treatment of numerous diseases and conditions, such as cancer, autoimmune, and inflammatory diseases. In addition, antibodies are also used for drug delivery to target antigens. Therapeutic antibodies recognize and bind to antigen receptors to activate or inhibit a series of biological processes to inhibit the proliferation of cancer cells or trigger the immune system. Therapeutic antibodies have the characteristic properties of high affinity, high specificity, and low immunogenicity. Early monoclonal antibodies (mabs) were generated by fusing myeloma cells and spleen cells to obtain hybridomas that secrete mabs. Early therapeutic antibodies were usually produced in mouse hosts, resulting in severe human anti-mouse antibody expression that may interfere with the desired therapeutic effect. The shortcomings of short half-life, xenogeneicity, and limited activity have been gradually improved with the development of genetic engineering, in vitro cell culture techniques, and a better understanding of antibody function. In the past 40 years, therapeutic antibodies have been improved through antibody engineering. Further improvements in antibody function and / or activity are still desired. Summary of the Invention

[0004] The present disclosure provides an antibody or antigen-binding fragment thereof conjugated to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein the antibody or antigen-binding fragment thereof binds to an antigen to a greater extent or faster than the same antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer, or, upon intravenous administration of a physiologically acceptable amount of the antibody or antigen-binding fragment thereof to a subject, the antibody or antigen-binding fragment thereof is present to a lesser extent in the blood of the subject during a period following intravenous administration than the same antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0005] The present disclosure also provides compositions comprising the antibodies or antigen-binding fragments thereof.

[0006] The disclosure also provides a method for increasing the extent to which an antibody or antigen-binding fragment thereof binds to an antigen, comprising conjugating an antibody or antigen-binding fragment thereof in need thereof to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein the antibody or antigen-binding fragment thereof binds to the antigen to a greater extent or faster than the same antibody or antigen-binding fragment thereof that is not hybridized to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0007] The disclosure also provides a method of reducing non-specific binding of an antibody or antigen-binding fragment thereof, the method comprising conjugating an antibody or antigen-binding fragment thereof in need thereof to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein upon intravenous administration of a physiologically acceptable amount of the antibody or antigen-binding fragment thereof to a subject, the antibody or antigen-binding fragment thereof is present in the blood of the subject to a lesser extent during a period following intravenous administration than an identical antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0008] The disclosure also provides a method of treating or preventing a disease in a subject, the method comprising administering to a subject in need thereof a physiologically tolerable amount of an antibody or antigen-binding fragment thereof conjugated to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein the antibody or antigen-binding fragment thereof binds to an antigen to a greater extent or faster than an identical antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer, or wherein upon intravenous administration of a physiologically tolerable amount of the antibody or antigen-binding fragment thereof to a subject, the antibody or antigen-binding fragment thereof is present to a lesser extent in the blood of the subject during a period following intravenous administration than an identical antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0009] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments as claimed herein.

[0010] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0011] [Figure 1] We show that IgG-oligos enhance removal from the bloodstream. Compared to IgG, faster clearance from the circulation was observed for non-specific IgG-oligos, which may help reduce the potential for systemic side effects associated with the use of antibodies. [Figure 2-1] It shows that IgG-oligo enhances splenic and hepatic clearance. Compared to IgG, an increase in the accumulation of non-specific IgG-oligo in the liver and spleen is observed, which may be due to a faster removal from the blood circulation to these organs responsible for clearance, as shown in Figure 1, which may help to attenuate the possibility of side effects from the use of antibodies against non-clearing organs. There is also a decrease in the levels of IgG-oligo in the bladder, kidney, and stomach. This may be due to the removal occurring in the spleen and liver, where the levels are high, or excretion through the kidney and stomach route may be faster, as IgG-oligo is no longer present in these organs, which is also sufficient to remove the antibody from the body and avoid side effects. [Figure 2-2] Continued from Figure 2-1. [Figure 3-1] It shows that anti-ICAM-oligos are rapidly cleared from the bloodstream. Specific anti-ICAM antibodies show faster clearance from the circulation when conjugated to oligos compared to non-oligo anti-ICAMs, which should also help protect against systemic side effects as described above. Furthermore, the faster clearance of anti-ICAM-oligos compared to IgG-oligos indicates that this protective effect is more pronounced for antibodies that recognize specific antigens. This is relevant since specific antibodies are antibodies that recognize antigens and are therefore antibodies that are intended to be used in research, diagnostic, prophylactic or therapeutic settings. [Figure 3-2] Continued from Figure 3-1. [Figure 4-1] Anti-ICAM-oligos enhance lung targeting. Specific anti-ICAM antibodies target the lung (the primary site for ICAM-1 expression) significantly better than anti-ICAM controls, and demonstrate enhanced absolute targeting and specificity, much greater than non-specific IgG (whether or not conjugated to an oligo). [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 4-4] Continued from Figure 4-3. [Diagram 5] This shows that lung targeting of anti-ICAM-oligo was highly specific compared to other formulations, providing minimal accumulation in lymph nodes. The enhanced specificity for the primary site of ICAM-1 expression (lung) provided by anti-ICAM-oligo is shown compared to all other species injected. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] DNA oligonucleotides may be attached to antibodies for several purposes and applications, which may include analytical in vitro systems, ex vivo tissues, cell cultures, or in vivo organisms, such as: a) oligonucleotides can be used as linkers between antibodies and diagnostic or therapeutic cargos (antibody probes or antibody-drug conjugates); b) oligonucleotides can be used as linkers between antibodies and drug delivery vehicles (e.g., liposomes, polymers, dendrimers, inorganic nanoparticles, etc.) that are loaded with diagnostic or therapeutic cargos; c) oligonucleotides can be annealed (fully or partially) to complementary oligonucleotides, and the resulting double-stranded DNA moieties can be used to bind DNA-intercalating drugs (e.g., For example, doxorubicin), d) the oligonucleotides can be annealed with complementary oligonucleotides, and the resulting double-stranded DNA or DNA / RNA chimeras can express or block the expression of one / a group of gene(s) or messenger RNA(s) (e.g., siRNA, microRNA effect, etc.), e) the oligonucleotides can be designed with sequences that provide themselves with the functions described in d), f) the oligonucleotides can be designed with sequences that fold into aptamers and provide specific binding to a different antigen than the antibody to which the oligonucleotide is bound, or to the same antigen as the antibody, increasing its affinity, or activating / blocking the antigen at the same / different domain. These designs can thus be used as tools in research or applied in the diagnostic and / or therapeutic fields.

[0013] It was observed that the coupling of a monoclonal antibody recognizing ICAM-1, a cell surface protein mainly overexpressed in the lungs but also in other organs in diseases involving inflammation, to DNA oligonucleotides significantly increased the binding of this antibody to ICAM-1 target sites in the body (mainly in the lungs) after intravenous injection in mice. Furthermore, the antibody-oligonucleotide conjugate was cleared from the mouse circulation faster and distributed more abundantly than a normal antibody, which may help to avoid systemic side effects of antibodies applied to the human body. This observation was surprising because coupling to oligonucleotides would be expected to reduce the binding and / or targeting ability of the antibody due to a) steric hindrance, b) possible modification or interference with the variable region that recognizes the antigen, c) changes in the physicochemical properties of other antibody regions, and / or d) the negative charge provided by the oligonucleotide (nucleic acids are negatively charged species) being repelled by the negative charge present on the surface of cells in the body (provided by sugar moieties of glycosylated proteins and lipids in the cell membrane). Although the mechanism behind this unexpected finding is unknown, it is nevertheless potentially transformative.

[0014] The terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Various terms relating to aspects of the present disclosure are used throughout this specification and claims. Such terms should be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein.

[0015] Unless expressly stated otherwise, it is never intended that any method or embodiment described herein be interpreted as requiring that its steps be performed in any particular order. If a method claim does not specifically state in the claim or description that the steps are to be limited to a particular order, no order is ever intended to be inferred in any respect. This applies to any possible non-expressive basis for interpretation, including questions of logic regarding the arrangement of steps or operational flow, the apparent meaning derived from grammatical organization or punctuation, or the number or type of embodiments described herein.

[0016] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0017] As used herein, the term "about" means that the recited numerical values ​​are approximate and that small variations do not significantly affect the practice of the disclosed embodiments. When numerical values ​​are used, unless otherwise indicated by context, "about" means that the numerical values ​​can vary by ±10% and remain within the range of the disclosed embodiments.

[0018] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen, including polyclonal, monoclonal, engineered, and otherwise modified forms thereof, including, but not limited to, nanobodies, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies).

[0019] As used herein, the phrase "antigen-binding fragment thereof" refers to a fragment of an antibody capable of binding to an antigen. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, scFv-Fc, diabodies, bispecific diabodies, trispecific triabodies, minibodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, nanobodies, IgNAR, V-NAR, hcIgG, and VhH fragments.

[0020] As used herein, the phrase "chimeric antibody" refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically selected from a human immunoglobulin template.

[0021] As used herein, the term "Fv" fragment is the minimum antibody fragment that contains a complete target recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in non-covalent association (V H- V L The three CDRs of each variable domain interact to form a V H -V L It is in this configuration that the target binding sites on the surface of the dimer are defined.

[0022] As used herein, the phrase "human antibody" refers to an antibody having the amino acid sequence of a human immunoglobulin and includes antibodies isolated from human immunoglobulin libraries or from animals that are transgenic for one or more human immunoglobulins and do not express endogenous immunoglobulins.

[0023] As used herein, the phrase "humanized antibody" refers to a chimeric antibody or antigen-binding fragment thereof that contains minimal sequence derived from a non-human immunoglobulin. In general, a humanized antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of its CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of its framework (FR) regions of a human immunoglobulin sequence corresponding to those of a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin consensus sequence.

[0024] As used herein, the phrase "in need thereof" means that a "subject" or "patient" has been identified as needing a particular method, prevention, or treatment. In some embodiments, identification can be by any diagnostic means. A "subject" or "patient" may be in need of any of the methods, preventions, and treatments described herein.

[0025] As used herein, a "nucleic acid molecule" is a polymeric form of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multi-stranded. A strand of a nucleic acid also refers to its complementary strand.

[0026] As used herein, the phrase "primatized antibody" refers to an antibody that comprises monkey variable regions and human constant regions.

[0027] As used herein, the phrase "regulatory sequence" is intended to include other expression control elements, such as promoters, enhancers, and polyadenylation signals that control the transcription or translation of antibody chain genes.

[0028] As used herein, the term "single-chain Fv" or "scFv" refers to a single chain Fv that is composed of the heavy and light chain variable domains (V H and VL Fv refers to a single-chain Fv antibody in which the V domain (V domain) is linked to form a single chain. Generally, the Fv polypeptide comprises a V domain that enables the scFv to form the desired structure for target binding. H Domain and V L It further comprises a polypeptide linker between the domains.

[0029] As used herein, the terms "subject" and "patient" are used interchangeably. A subject includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), companion animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, rabbits), and non-human primates (e.g., monkeys). In some embodiments, the subject or patient is a human.

[0030] As used herein, "V H The term "variable region" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab fragment, for example.

[0031] As used herein, "V L " refers to the variable region of an immunoglobulin light chain of an antibody, including the light chain of an Fv, scFv, dsFv, or Fab fragment, for example.

[0032] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complementary strand.

[0033] As used herein, an "internal portion" of an antibody or antigen-binding fragment thereof refers to amino acids other than the N-terminal and C-terminal amino acids of an antibody or antigen-binding fragment thereof, respectively.

[0034] As used herein, the term "therapeutic antibody" refers to an antibody or antigen-binding fragment thereof suitable for administration to a subject to treat and / or prevent a disease or disorder.

[0035] As used herein, a "DNA dendrimer" is a highly ordered, branched polymeric molecule having at least three DNA strands, one of which is at least partially complementary to one of the other strands for at least 10 consecutive nucleotides.

[0036] As used herein, a first oligonucleotide is "hybridized" to a second oligonucleotide when at least 10 contiguous nucleotides in the first oligonucleotide are bound by Crick-Watson base pairs to at least 10 contiguous complementary nucleotides in the second oligonucleotide.

[0037] As used herein, a first antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that has an increased affinity, increased binding constant, K, for an antigen, compared to a second antibody or antigen-binding fragment thereof. A (k on / k off ) or increased avidity, it binds to an antigen "to a greater extent or faster" than a second antibody or antigen-binding fragment thereof.

[0038] As used herein, a first antibody or antigen-binding fragment thereof is "identical" to a second antibody or antigen-binding fragment thereof if the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof (i) have the same amino acid sequence(s), (ii) have the same type of derivatization(s), if any, (iii) have the same type of covalent linkage, if any, to an effector moiety, (iv) are fused, via the same type of covalent linkage, if any, to the same type of amino acid sequence of another protein or portion of the same type, (v) are conjugated to the same type of small molecule toxin, if any, (vi) are conjugated to the same type of liposome, (vii) are linked to the same type of polyethylene glycol (PEG) moiety, if any, (viii) have the same type of modification(s), if any, and / or (ix) are linked to the same type of detectable substance(s). However, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof may be identical except for at least one specified difference. As a non-limiting example, a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof can be identical except for certain differences in amino acid sequence.

[0039] As used herein, a first oligonucleotide is "identical" to a second oligonucleotide if the first oligonucleotide and the second oligonucleotide (i) have the same nucleotide sequence(s), and / or (ii) contain the same type of non-natural nucleotide(s), modified nucleotide(s), nucleotide analog(s), or nucleotide substitute(s) at the same position(s).However, the first oligonucleotide and the second oligonucleotide can be identical except for a specific difference.As a non-limiting example, the first oligonucleotide and the second oligonucleotide can be identical except for a specific difference in nucleotide sequence.

[0040] For example, attaching oligonucleotides to therapeutic antibodies such as checkpoint inhibitors, EGFR, Her2, Herceptin, or antibody probes or antibody drug conjugates can significantly improve the target localization and binding or solubility of the antibody, thus improving the diagnostic or therapeutic index of the drug while reducing toxicity because lower doses can be administered or because of more efficient clearing and avoidance of off-target effects. This same result can be observed when antibody fragments or scFvs are used with diagnostic probes and / or therapeutic agents. In certain embodiments, antibody-like molecules are also contemplated, including but not limited to minibodies, nanobodies, diabodies, triabodies, and tetrabodies. In certain embodiments, antibody-based molecules are also contemplated, including but not limited to bispecific, trispecific, or tetraspecific antibodies.

[0041] The present disclosure provides an antibody or antigen-binding fragment thereof conjugated to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein the antibody or antigen-binding fragment thereof binds to an antigen to a greater extent or faster than the same antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer, or, upon intravenous administration of a physiologically acceptable amount of the antibody or antigen-binding fragment thereof to a subject, the antibody or antigen-binding fragment thereof is present to a lesser extent in the blood of the subject during a period following intravenous administration than the same antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0042] The disclosure also provides a method of increasing the extent or rate at which an antibody or antigen-binding fragment thereof binds to an antigen, comprising conjugating an antibody or antigen-binding fragment thereof in need thereof to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein the antibody or antigen-binding fragment thereof binds to an antigen to a greater extent or faster than the same antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0043] The disclosure also provides a method of reducing non-specific binding of an antibody or antigen-binding fragment thereof, comprising conjugating an antibody or antigen-binding fragment thereof in need thereof to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein upon intravenous administration of a physiologically acceptable amount of the antibody or antigen-binding fragment thereof to a subject, the antibody or antigen-binding fragment thereof is present to a lesser extent in the blood of the subject during a period following intravenous administration than an identical antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0044] The disclosure also provides a method of reducing non-specific binding of an antibody or antigen-binding fragment thereof, comprising conjugating an antibody or antigen-binding fragment thereof in need thereof to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein upon intravenous administration of a physiologically acceptable amount of the antibody or antigen-binding fragment thereof to a subject, the antibody or antigen-binding fragment thereof binds to its target to a greater extent or more quickly than the same antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0045] The disclosure also provides a method of diagnosing, treating, or preventing a disease in a subject, comprising administering to a subject in need thereof a physiologically tolerable amount of an antibody or antigen-binding fragment thereof conjugated to an oligonucleotide that is not hybridized to a DNA dendrimer, wherein the antibody or antigen-binding fragment thereof binds to an antigen to a greater extent or faster than the same antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer, or wherein upon intravenous administration of a physiologically tolerable amount of the antibody or antigen-binding fragment thereof to a subject, the antibody or antigen-binding fragment thereof is present to a lesser extent in the blood of the subject during a period following intravenous administration than the same antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide that is not hybridized to the same DNA dendrimer.

[0046] The antibody or antigen-binding fragment thereof can be of any isotype. In some embodiments, the antibody is an IgM, IgA, IgD, IgE, or IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody. In some embodiments, the IgG1 antibody is an IgG1 G1m1, G1m2, G1m3, G1m17, nG1mI, nG1m2, or nG1m17 allotype antibody. In some embodiments, the antibody is an IgG1 G1m17 allotype antibody. In some embodiments, the IgA antibody is an IgA1 or IgA2 antibody. In some embodiments, the antibody has a variant heavy chain.

[0047] In some embodiments, the mutated IgG1 heavy chain is paired with a kappa light chain of the allotype Km1, Km2, or Km3.In some embodiments, the mutated IgG1 heavy chain is paired with a lambda light chain.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof is humanized and is an IgG. In some embodiments, the antibody or antigen-binding fragment thereof is humanized and is an IgG1.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof.Methods for producing chimeric antibodies are known in the art (see Morrison, Science, 1985, 229, 1202-1207; Oi et al., BioTechniques, 1986, 4, 214-221; Gillies et al., J. Immunol. Methods, 1985, 125, 191-202, and U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397).

[0050] In some embodiments, the chimeric antibody or antigen-binding fragment thereof is a primatized chimeric antibody or antigen-binding fragment thereof. Methods for producing primatized antibodies are known in the art (see U.S. Patent Nos. 5,658,570, 5,681,722, and 5,693,780).

[0051] In some embodiments, the chimeric antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. Methods for producing humanized antibodies are known in the art (Riechmann et al., Nature, 1988, 332, 323-327; Padlan, Mol. Immunol., 1991, 28, 489-498; Studnicka et al., Prot. Eng., 1994, 7, 805-814; Roguska et al., (see, e.g., E. et al., Proc. Natl. Acad. Sci., 1994, 91, 969-973; European Patent Nos. EP 239400, EP 592106, and EP 519596; PCT Publication WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, 5,585,089, 5,693,761, 5,693,762, 6,180,370, and 5,565,332).

[0052] In some embodiments, the antibody or antigen-binding fragment thereof is fully humanized. Fully "human" antibodies may be desirable for therapeutic treatment of human patients. Methods for producing fully human antibodies are known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos. 4,444,887 and 4,716,111, and PCT Publication Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741). Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see PCT Publication Nos. WO 98 / 24893, WO 92 / 01047, WO 96 / 34096, and WO 96 / 33735, as well as U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598). Completely human antibodies that recognize a selected epitope can also be generated using a technique referred to as "directed selection" (see Jespers et al., Biotechnology, 1988, 12, 899-903).

[0053] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody. A bispecific antibody has binding specificities for at least two different antigens (i.e., one of the binding specificities is directed to one antigen and the other binding specificity is for any other antigen, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacteria-derived protein, or a bacterial surface protein).

[0054] In some embodiments, the antibody or antigen-binding fragment thereof is a derivatized antibody. For example, the derivatized antibody may be an antibody modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative can contain one or more unnatural amino acids, such as by using ambrx technology (see Wolfson, Chem. Biol., 2006, 13, 1011-1012).

[0055] In some embodiments, the antibody or antigen-binding fragment thereof is derivatized through glycosylation. A suitable biantennary complex may be composed of a core structure with two N-acetylglucosamine (G1cNAc), three mannose, and two G1cNAc residues β-1,2 linked to α-6 mannose and α-3 mannose to form two antennae. One or more fucose (Fuc), galactose (Gal), high mannose glycans Man-5 or Man-9, biantennary G1cNAc, and sialic acid containing N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) residues may be attached to the core. N-linked glycoforms can include G0 (proteins with a core biantennary glycosylation structure), G0F (fucosylated G0), G0F G1cNAc, G1 (proteins with a core glycosylation structure with one galactose residue), G1F (fucosylated G1), G2 (proteins with a core glycosylation structure with two galactose residues), and / or G2F (fucosylated G2). In some embodiments, the antibody has a G0F glycan.

[0056] Antibodies or antigen-binding fragments thereof can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cell and, optionally, secreted into the medium in which the host cell is cultured, from which the antibody can be recovered. Standard recombinant DNA methodologies can be used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells (Molecular Cloning; A Laboratory Manual, Second Edition, Sambrook, Fritsch and Maniatis (eds.), Cold Spring Harbor, NY, 1989; Current Protocols in Molecular Biology, Ausubel et al., eds., Greene Publishing Associates, 1989, and U.S. Patent No. 4,816,397).

[0057] In some embodiments, to generate a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof described herein, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained, for example, by amplifying and modifying germline DNA or cDNA encoding the light and heavy chain variable sequences using the polymerase chain reaction (PCR). Germline DNA sequences of human heavy and light chain variable region genes are known in the art (see the "VBASE" human germline sequence database; Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., J. Mol. Biol., 1992, 22T, 116-198; and Cox et al., Eur. J. Immunol., 1994, 24, 827-836). A DNA fragment encoding a heavy or light chain variable region can be synthesized and used as a template for mutagenesis to generate the variants described herein using routine mutagenesis techniques, or a DNA fragment encoding the variant can be directly synthesized.

[0058] These DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. H or V L The DNA fragment encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The phrase "operably linked" as used in this context is intended to mean that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0059] V HAn isolated DNA molecule encoding the V H The DNA encoding the heavy chain constant region can be converted to a full-length heavy chain gene by operably linking it to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3, and optionally CH4). The sequences of human heavy chain constant region genes are known in the art (see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In some embodiments, the heavy chain constant region is an IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain CH1 constant region can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0060] V L The isolated DNA encoding the region is L The DNA encoding the light chain constant region, C LThe light chain constant region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking it to another DNA molecule encoding the human light chain constant region. The sequences of human light chain constant region genes are known in the art (see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition (USDepartment of Health and Human Services, NIH Publication No. 91-3242)), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. In some embodiments, the light chain constant region is a kappa constant region. To generate an scFv gene, the V H and V L The DNA fragment encoding the V is operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4Ser)3, resulting in V H and V L The sequence can be expressed as a continuous single chain protein, L and V H The domains are joined by flexible linkers (see Bird et al., Science, 1988, 242, 423-426; Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 5879-5883; and McCafferty et al., Nature, 1990, 348, 552-554).

[0061] To express the antibodies or antigen-binding fragments thereof described herein, DNA molecules encoding partial or full-length light and heavy chains obtained as described herein can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the phrase "operably linked" is intended to mean that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes can be inserted into the same expression vector.

[0062] The antibody genes can be inserted into an expression vector by standard methods, such as, for example, ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present. Prior to insertion of the light or heavy chain sequences, the expression vector can already carry antibody constant region sequences. For example, the V H and V L One approach to converting sequences into full-length antibody genes is to H The segment is C in the vector H operatively coupled to the segment(s), V L The segment is C in the vector L The first step is to insert them into an expression vector already encoding the heavy and light chain constant regions, respectively, so that they are operably linked to the segments. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide, e.g., a signal peptide from a non-immunoglobulin protein.

[0063] In addition to the antibody chain genes, the recombinant expression vector can carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990). It will be appreciated by those of skill in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (such as the adenovirus major late promoter (AdMLP)), and polyoma. Viral regulatory elements and sequences thereof are known in the art (see, for example, US Pat. Nos. 5,168,062, 4,510,245, and 4,968,615).

[0064] The recombinant expression vectors of the present disclosure can also carry additional sequences, such as sequences that regulate replication of the vector in a host cell (such as origins of replication) and selectable marker genes. The selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically the selectable marker genes confer resistance to drugs, such as G418, puromycin, blastacidin, hygromycin, or methotrexate, on the host cells into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (DHFR -The host cell may include the gene for expression of the light and heavy chains, such as the ribozyme (for use with methotrexate selection / amplification) and the NEO gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection, etc.

[0065] The antibodies or antigen-binding fragments thereof can be expressed in either prokaryotic or eukaryotic host cells. In some embodiments, expression of the antibodies or antigen-binding fragments thereof can be in eukaryotic cells, such as mammalian host cells, for secretion of properly folded and immunologically active antibodies. Exemplary mammalian host cells for expressing recombinant antibodies or antigen-binding fragments thereof of the present disclosure include Chinese Hamster Ovary (CHO) cells (DHFR), used with a DHFR selection marker (see Kaufman, Mol. Biol., 1982, 159, 601-621). These include CHO cells (see Urlaub, Proc. Natl. Acad. Sci. USA, 1980, 77, 4216-4220), NS0 myeloma cells, COS cells, 293 cells, and SP2 / 0 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody within the host cell or secretion of the antibody into the medium in which the host cell is grown. The antibody or antigen-binding fragment thereof can be recovered from the medium using standard protein purification methods. The host cell can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations of the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect the host cell with DNA encoding either the light or heavy chain (but not both) of the antibodies or antigen-binding fragments thereof described herein.

[0066] Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigen. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies or antigen-binding fragments thereof described herein.

[0067] Furthermore, bifunctional antibodies or antigen-binding fragments thereof can be produced in which one heavy chain and one light chain are antibodies of the present disclosure capable of binding to a first antigen, and the other heavy and light chains are specific for an antigen other than the first antigen, by crosslinking the antibody of the present disclosure to a second antibody by standard chemical crosslinking methods. Bifunctional antibodies can also be made by expressing a nucleic acid engineered to encode the bifunctional antibody.

[0068] For recombinant expression of the antibodies or antigen-binding fragments thereof described herein, a host cell can be co-transfected with two expression vectors of the present disclosure, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. Typically, each of the two vectors contains a separate selectable marker. Alternatively, a single vector can be used that encodes both heavy and light chain polypeptides.

[0069] Once a nucleic acid encoding one or more portions of an antibody or antigen-binding fragment thereof with desired CDR sequences has been generated, further changes can be introduced into the coding sequence, for example, to generate nucleic acid encoding antibodies with different CDR sequences, antibodies with reduced affinity for Fc receptors, or antibodies of a different subclass.

[0070] The antibodies or antigen-binding fragments thereof described herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Variant antibodies or antigen-binding fragments thereof can also be produced using cell-free platforms (Chu et al., Biochemia, 2001, 2).

[0071] Once the antibodies or antigen-binding fragments thereof described herein have been produced by recombinant expression, they can be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (such as by ion exchange, affinity, particularly affinity by Protein A, Protein G or Protein L selection, as well as sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, the antibodies or antigen-binding fragments thereof described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0072] Once isolated, the antibodies or antigen-binding fragments thereof can be further purified, if desired, by, for example, high performance liquid chromatography (Fisher, Laboratory Techniques In Biochemistry And Molecular Biology (Work and Burdon, eds. (Elsevier, 1980)) or gel filtration chromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0073] The present disclosure provides a vector comprising the nucleic acid molecule described herein. The present disclosure also provides a prokaryotic host cell transfected with the vector. The present disclosure also provides a eukaryotic host cell transfected with the vector. In some embodiments, the eukaryotic host cell is a mammalian host cell.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an effector moiety. In some embodiments, the antibody or antigen-binding fragment thereof is modified by covalent attachment of any type of molecule to the antibody or antigen-binding fragment thereof, such that the covalent attachment does not prevent binding to the antigen. In some embodiments, the effector moiety is a detectable label, a cytotoxic agent, a chemotherapeutic agent, or a nucleic acid molecule. The effector moiety may also be an antitumor agent, a drug, a toxin, a biologically active protein (such as an enzyme), another antibody or antibody fragment, a synthetic or natural polymer, a nucleic acid molecule, a radionuclide (such as radioactive iodine), a radioisotope, a chelated metal, a nanoparticle, or a reporter group (such as a fluorescent compound or a compound that can be detected by NMR or ESR spectroscopy).

[0075] In some embodiments, the antibodies or antigen-binding fragments thereof may be conjugated to cytotoxic agents, radionuclides, or drug moieties to modify a particular biological response. Effector moieties include, for example, toxins (e.g., abrin, ricin A, saporin, Pseudomonas exotoxin, diphtheria toxin, ethidium bromide or PE40, PE38, gelonin, RNAse, peptide nucleic acid (PNA), ribosome-inactivating protein (RIP) type 1 or 2, pokeweed antiviral protein (PAP), bryodin, momordin, chemotherapeutic agents, and buganin), signaling molecules (e.g., α-interferon, β-interferon, nerve growth factor, hematopoietin, and erythrocyte-derived toxins), and / or cytotoxic agents (e.g., erythrocyte-derived toxins). The antibody may be a protein or polypeptide, such as a thrombogenic or anti-angiogenic agent (e.g., angiostatin or endostatin), a cytokine or growth factor (e.g., interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), or nerve growth factor (NGF).

[0076] In some embodiments, the cytotoxic agent is a small molecule, a prodrug, a maytansinoid, or a toxin. In some embodiments, the antibody or antigen-binding fragment thereof comprises 3-5 maytansinoid molecules per antibody or antigen-binding fragment thereof. In some embodiments, the maytansinoid is conjugated to the antibody or antigen-binding fragment thereof by a chemical linker selected from N-succinimidyl-3-(2-pyridyldithio)propionate, N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. In some embodiments, the cytotoxic agent is taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorabicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, or puromycin.

[0077] In some embodiments, the detectable label is a radioactive compound, a fluorescent compound, a chromophore, an enzyme, an imaging agent, a metal ion, or a substrate. In some embodiments, fluorescent moieties include, but are not limited to, fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Useful enzyme labels include, but are not limited to, alkaline phosphatase, horseradish peroxidase, glucose oxidase, and the like.

[0078] In some embodiments, the effector moiety is an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil, decarbazine), an alkylating agent (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclosporine, busulfan, dibromomannitol, streptozotocin, mitomycin C5, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), an anthracycline (e.g., daunorubicin (formerly daunomycin) and doxorubicin), an antibiotic (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicin, or duocarmycin), or an antimitotic agent (e.g., vincistine and vinblastine).

[0079] In some embodiments, the radionuclide is 13 N, 18 F, 32 P, 64 Cu, 66 Ga, 67 Ga, 68 Ga, 67 Cu, 77 Br, 80m Br, 82 Rb, 86 Y, 90 Y, 95 Ru, 97 Ru, 99m Tc, 103 Ru, 105 Ru, 111 In, 113m In, 113 Sn, 121m Te, 122m Te, 125m Te, 123 I, 124 I, 125 I, 126 I, 131 I, 133 I, 165 Tm, 167 Tm, 168 Tm, 177 Lu, 186 Re,188 Re, 195m Hg, 211 At, 212 Bi, 213 Bi, and 225 Ac, but is not limited to these.

[0080] In some embodiments, the chemotherapeutic agent is cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, methotrexate, vincristine, doxorubicin, tunicamycin, oligomycin, bortezomib, MG132, 5-flurouracil, sorafenib, flavopiridol, gemcitabine, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, mitomycin, cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, procarbazine. The drugs are cyclosporine, etoposide, campatecin, bleomycin, idarubicin, daunorubicin, dactinomycin, distamycin A, ethidium, netropsin, auristatin, amsacrine, prodigiosin, bortezomib, pipendimol, tomaymycin, duocarmycin SA, plicamycin, mitoxantrone, asparaginase, vinblastine, vinorelbine, paclitaxel, docetaxel, CPT-11, Gleevec, erlotinib, gefitinib, ibrutinib, crizotinib, ceritinib, lapatinib, navitoclax, or regorafenib.

[0081] In some embodiments, the nucleic acid molecule is a single layer nucleic acid carrier, a 1.5 layer nucleic acid carrier, a two layer nucleic acid carrier, a 2.5 layer nucleic acid carrier, or a three layer nucleic acid carrier (such as those disclosed in PCT Publication Nos. WO17 / 143156 and WO17 / 143171).

[0082] Techniques for conjugating such effector moieties to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., pages 623-53 (Robinson et al., eds., 1987); Thorpe et al., Immunol. Rev., 1982, 62, 119-58, and Dubowchik et al., Pharmacology and Therapeutics, 1999, 83, 67-123).

[0083] In some embodiments, the antibody or antigen-binding fragment thereof may be fused to the amino acid sequence of another protein (or a portion thereof; e.g., at least a 10, 20, or 50 amino acid portion of a protein) via a covalent bond (e.g., a peptide bond), through the N-terminus or C-terminus of the antibody, or internally. The antibody or antigen-binding fragment of the present invention may be linked to the other protein at the N-terminus of the antibody's constant domain. Recombinant DNA procedures may be used to create such fusions, for example, as described in PCT Publication No. WO86 / 01533 and European Patent No. EP0392745. In some embodiments, the effector moiety may increase the half-life in vivo and / or enhance delivery of the antibody across an epithelial barrier to the immune system. Examples of suitable effector moieties of this type include polymers, albumin, albumin-binding proteins, or albumin-binding compounds as described in PCT Publication No. WO2005 / 117984.

[0084] In some embodiments, the antibody or antigen-binding fragment thereof may be conjugated to a small molecule toxin. In some embodiments, the antibody or antigen-binding fragment thereof may be conjugated to a dolostatin or dolostatin peptide analog or derivative, such as an auristatin (see U.S. Pat. Nos. 5,635,483 and 5,780,588). The dolostatin or auristatin drug moiety may be attached to the antibody via its N-terminus, C-terminus, or internally (see PCT Publication No. WO02 / 088172). Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF, as disclosed in U.S. Pat. No. 7,498,298 (which discloses linkers and methods of preparing monomethylvaline compounds such as MMAE and MMAF conjugated to linkers).

[0085] Antibodies or antigen-binding fragments thereof can also be conjugated to liposomes for targeted delivery (see Park et al., Adv. Pharmacol., 1997, 40, 399-435, and Marty et al., Methods Molec. Med., 2004, 109, 389-401).

[0086] In some embodiments, the antibody or antigen-binding fragment thereof can be conjugated to a PEG moiety. In some embodiments, the antibody or antigen-binding fragment thereof and the PEG moiety can be attached via any available amino acid side chain or terminal amino acid functional group located on the antibody or antigen-binding fragment thereof, such as any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids can be naturally present in the antibody or antigen-binding fragment thereof or can be engineered into the fragment using recombinant DNA methods (see U.S. Pat. No. 5,219,996). Multiple sites can be used to attach two or more PEG moieties. The PEG moiety can be covalently attached via a thiol group of at least one cysteine ​​residue located on the antibody or antigen-binding fragment thereof. When a thiol group is used as the attachment point, an appropriately activated effector moiety can be used, such as a thiol-selective derivative, such as maleimide and cysteine ​​derivatives.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof can comprise a modified Fab' fragment that is PEGylated. The PEG moiety can be attached to a cysteine ​​in the hinge region. In some embodiments, the PEG-modified Fab' fragment has a maleimide group covalently attached to a single thiol group in the modified hinge region. A lysine residue is covalently attached to the maleimide group, and each amine group on the lysine residue can be attached to a methoxypoly(ethylene glycol) polymer having a molecular weight of about 20,000 Da. Thus, the total molecular weight of the PEG attached to the Fab' fragment can be about 40,000 Da.

[0088] In some embodiments, the antigen-binding fragment can be a Fab, F(ab')2, Fv, scFv, scFv-Fc, diabody, or minibody fragment. In some embodiments, the antigen-binding fragment can be a Fab fragment. In some embodiments, the antigen-binding fragment can be a F(ab')2 fragment. In some embodiments, the antigen-binding fragment can be an Fv fragment. In some embodiments, the antigen-binding fragment can be an scFv fragment. In some embodiments, the antigen-binding fragment can be an scFv-Fc fragment. In some embodiments, the antigen-binding fragment can be a diabody fragment. In some embodiments, the antigen-binding fragment can be a minibody fragment.

[0089] In some embodiments, the antibody or antigen-binding fragment thereof is a therapeutic antibody. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of 3F8, 8H9, abagovomab, abciximab, avituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afacevicumab, afelimomab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, amivantamab, anatumomab mafenatox, andecaliximab, anetumab ravtansine, anifrolumab, ansuvimab, anrukinzumab, apolizumab, apurutumab Ixadotin, arcitumomab, asclinbacumab, acelizumab, atezolizumab, atidortoxumab, atinumab, atortivimab, maftivimab, odesivimab, atorlimumab, avelumab, azintuxizumab Vedotin, bamlanivimab, bapineuzumab, basiliximab, bavituximab, BCD-100, bectumomab, begelomab, belantamab mafodotin, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bermekimab, belsanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, vilutamimab, bivatuzumab, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, brentuximab Vedotin, briakinumab, brodalumab, brolucizumab, broticutuzumab, burosumab, cabilalizumab, camidanlumab tesirin, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, casirivimab, capromab, carumab, carotuximab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, cemiplimab, sergituzumab amnaleukin, certolizumab pegol, cetrelimab, cetuximab, civisatamab, sirmutuzumab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab Tetraxetane, codrituzumab, cofetuzumab peridotin, coltuximabRavtansine, conatumumab, concizumab, cosfrobiximab, crenezumab, crizanlizumab, clotezumab, CR6261, cusatuzumab, dacetuzumab, daclizumab, darotuzumab, dapirorizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, delrotuximab biotin, detumomab, desamizumab, dinutuximab, dinutuximab beta, dilidumab, domagurozumab, dorlimomab Aritox, dostallimab, drozitumab, DS-8201, durigotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, ersilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab vedotin, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enoticumab, ensituximab, epcolitamab, epitumomab Sitaxetan, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etesevimab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faricimab, faretuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficratuzumab figitumumab, filibumab, framvotumab, fretikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, furovocimab, fulnevetomab, fullanumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavirimomab, gedivumab, gemtuzumab ozogamicin, gevokizumab, gilvetomab, gimsilumab, girentuximab, glenbatumumab vedotin, golimumab, gomilikimab, goslanemab, guselkumab, inalumab, ibalizumab, IBI308, ibritumomabTiuxetan, Icrucumab, Idarucizumab, Ifavotuzumab, Igovomab, Iradatuzumab vedotin, IMAB362, Imalumab, Imaprelimab, Imusiromab, Imdevimab, Imgatuzumab, Incracumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab Ozogamicin, ipilimumab, iomab-B, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacinotuzumab, ladiratuzumab vedotin, lampalizumab, lanadelumab, landgrozumab, laprituximab emtansine, ralcaviximab, lebrikizumab, remaresomab, lendalizumab, lenbervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, ribivirumab, rifatuzumab vedotin, ligelizumab, roncatuximab tesirin, rosatuximab vedotin, rilotomab Satetraxetan, Lintuzumab, Lirilumab, Roderucizumab, Lokivetumab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumuletuzumab, Rupartumab, Rupartumab amadotin, Rutikizumab, Maftivimab, Mapatumumab, Margetuximab, Marstacimab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minletumomab, Mirikizumab, Mirvetuximab soravtansine, Mitsumomab, Modotuximab, Mogamulizumab, Monalizumab, Morolimumab, Mosnetuzumab, Motavizumab, Moxetumomab Passudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, naratuximab emtansine, narutumab, natalizumab, navicixizumab, nabivumab, naxitamab, nebacumab, necitumumab, nemolizumab, NEOD001, nerelimomab, nesbacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomabMerpentan, obilutoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odesivimab, odulimomab, ofatumumab, olaratumab, oleculab, orendalizumab, olokizumab, omalizumab, omburtamab, OMS721, onartuzumab, ontuxizumab, ombatilimab, opicinumab, oportuzumab Monatox, oregovomab, olticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placumab, prezalumab, prozalizumab, pogalizumab, polatuzumab Vedotin, ponezumab, polgabiximab, prasinezumab, prezarizumab, priliximab, plitoxaximab, pritumumab, PRO140, kirisumab, racotumomab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranevetomab, ranibizumab, raxibacumab, ravagalimab, ravotolimab, refanezumab, regavirumab, regdanvimab, relatimab, lemtolumab, reslizumab, rilotumumab, rinukumab, risankizumab, rituximab, rivavazumab pegol, lobatumumab, SII RMAb, Loredumab, Romilximab, Romosozumab, Rontalizumab, Rosmantuzumab, Robalpituzumab Tecilin, Rovelizumab, Rozanolixizumab, Ruplizumab, SA237, Sacituzumab govitecan, Samalizumab, Samlotamab vedotin, Sarilumab, Satralizumab, Satumomab pendetide, Secukinumab, Celicrelumab, Seribantumab, Setoxaximab, Setrusumab, Sevirumab, Sibrotuzumab, SGN-CD19A, SHP647, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Siltratumab vedotin, Silkumab, SofituzumabVedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, sputabumab, stimulimab, subizumab, sublatoxumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, tafasitamab, talacotuzumab, talizumab, taliketamab, tamtubetomab, tanezumab, tapritumomab paptox, talextuzumab, taborimab, teclistamab, tefibazumab, terimomab aritox, terisotuzumab, terisotuzumab Vedotin, Tenatumomab, Teneliximab, Teplizumab, Tepositamab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Tiburizumab, Tildrakizumab, Tigatuzumab, Timigtuzumab, Timolumab, Tiragolumab, Tiragotumab, Tislelizumab, Tisotumab Vedotin, TNX-650, Tocilizumab, Tomzotuximab, Toralizumab, Tosatoxumab, Tositumomab, Tobetumab, Tralokinumab, Trastuzumab, Trastuzumab Duocarmazine, Trastuzumab Emtansine, TRBS07, Tregalizumab, Tremelimumab, Trevoglumab, Tucotuzumab Celmoleukin, tubilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab talilline, banalimab, bundletuzumab vedotin, vanticutumab, vanucizumab, bapaliximab, balisakumab, varlilumab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, bovalilizumab, volociximab, bonlerolizumab, bopratelimab, borsetuzumab In some particularly preferred embodiments, the therapeutic antibody comprises ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, spartalizumab, or atezolizumab. In some embodiments, the therapeutic antibody comprises pembrolizumab. In some embodiments, the therapeutic antibody comprises velsanlimab or enlimomab pegol.

[0090] The present disclosure also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises an isotonicity agent, a surfactant, a preservative, and / or a buffer system having a pH of about 4.0 to about 8.0. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents, e.g., a combination therapeutic agent described herein. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition.

[0091] In some embodiments, the pharmaceutical compositions may be presented in a unit dose form containing a predetermined amount of an antibody or antigen-binding fragment thereof described herein per dose. Pharmaceutically acceptable carriers for use in pharmaceutical compositions can take a wide variety of forms, depending on the condition to be treated or the route of administration.

[0092] Pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof described herein can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibodies or antigen-binding fragments thereof having the desired purity with any pharma- ceutically acceptable carriers, excipients or stabilizers typically used in the art, such as, for example, buffers, stabilizers, preservatives, isotonicity agents, non-ionic detergents, antioxidants, and other miscellaneous additives (all of which are referred to herein as "carriers").

[0093] Buffering agents serve to maintain the pH in a range close to physiological conditions. They may be present in concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the pharmaceutical compositions described herein include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, trisodium citrate-citric acid mixtures, monosodium citrate-citric acid mixtures, etc.), succinate buffers (e.g., monosodium succinate-succinic acid mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartrate-sodium tartrate mixtures, potassium tartrate mixtures, tartrate-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumarate-monosodium fumarate mixtures, potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), and the like. buffers (such as sodium mixtures, fumaric acid-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconic acid buffers (such as gluconic acid-sodium glyconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium glyconate mixtures, etc.), oxalic acid buffers (such as oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (such as lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.) and acetate buffers (such as acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

[0094] Preservatives can be added to pharmaceutical compositions to reduce microbial growth and can be added in amounts ranging from about 0.2% to about 1% (w / v). Suitable preservatives for use with the pharmaceutical compositions described herein include, but are not limited to, phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0095] Tonicity agents, sometimes known as "stabilizers," can be added to ensure isotonicity of liquid compositions and include polyhydric sugar alcohols, e.g., trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients, which can range in function from bulking agents to additives that help solubilize the therapeutic agent or prevent denaturation or adhesion to the container walls. Exemplary stabilizers include polyhydric sugar alcohols (listed above), amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, and the like, organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, and the like, including cyclotolyls such as inositol, polyethylene glycols, polymeric amino acids, urea, glutathione, and the like. The stabilizer may be a sulfur-containing reducing agent such as thiocyanate, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate, a protein such as a low molecular weight polypeptide (such as a peptide of 10 residues or less), human serum albumin, bovine serum albumin, gelatin, or immunoglobulin, an aqueous polymer such as polyvinylpyrocidone, a monosaccharide such as xylose, mannose, fructose, glucose, a disaccharide such as lactose, maltose, sucrose, and a trisaccharide such as raffinose, and a polysaccharide such as dextran. The stabilizer may be present in a range of about 0.1 to about 10,000 parts by weight per part by weight active protein.

[0096] Non-ionic surfactants or detergents (also called "wetting agents") can be added to the pharmaceutical compositions to help solubilize the antibodies or antigen-binding fragments thereof, as well as to protect the antibodies or antigen-binding fragments thereof described herein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surfaces without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN™-20, TWEEN™-80, etc.). The non-ionic surfactants can be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, e.g., about 0.07 mg / mL to about 0.2 mg / mL.

[0097] Additional excipients, such as bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, and vitamin E), and cosolvents can also be added to the pharmaceutical composition.

[0098] The present disclosure also provides pharmaceutical kits containing the antibodies or antigen-binding fragments thereof described herein, including antibody conjugates. The pharmaceutical kits can be packages containing the antibodies or antigen-binding fragments thereof described herein (e.g., in either lyophilized or aqueous form) and one or more of the following: a combination therapeutic agent, a device for administering the antibody or antigen-binding fragment thereof (such as a pen, needle, and / or syringe), and, if the antibody is in lyophilized form, pharmaceutical grade water or buffer for resuspending the antibody or antigen-binding fragment thereof.

[0099] In some embodiments, each unit dose of the antibody or antigen-binding fragment thereof is packaged separately and the kit can include one or more unit doses (e.g., 2 unit doses, 3 unit doses, 4 unit doses, 5 unit doses, 8 unit doses, 10 unit doses, or more). In some embodiments, the one or more unit doses are each contained in a syringe or pen.

[0100] Also encompassed herein are diagnostic kits containing the antibodies or antigen-binding fragments thereof (including antibody conjugates) described herein. The diagnostic kits can be packages that contain the antibodies or antigen-binding fragments thereof described herein (such as either lyophilized or in aqueous solution) and one or more reagents useful for performing diagnostic assays. If the antibodies or antigen-binding fragments thereof are labeled with an enzyme, the kits can include substrates and cofactors required by the enzyme (e.g., substrate precursors that provide detectable chromophores or fluorophores). In addition, other additives can be included, such as stabilizers, buffers (such as block buffers or lysis buffers). In some embodiments, the antibodies or antigen-binding fragments thereof included in the diagnostic kits can be immobilized on a solid surface, or the kits can include a solid surface (such as a slide or plate) on which the antibodies or antigen-binding fragments thereof can be immobilized. The relative amounts of the various reagents can be varied to provide concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. In some embodiments, the antibodies or antigen-binding fragments thereof and one or more reagents can be provided (individually or in combination) as dry powders, usually lyophilized, with excipients that provide a reagent solution having the appropriate concentrations upon dissolution.

[0101] The present disclosure also provides a method of detecting a cell expressing an antigen, comprising contacting the cell with an antibody or antigen-binding fragment thereof described herein and detecting the antibody or antigen-binding fragment thereof. In some embodiments, the cell is present in a biological sample obtained from a human and the cell is contacted with the antibody or antigen-binding fragment thereof in vitro. In some embodiments, the cell is present in a human and the cell is contacted with the antibody or antigen-binding fragment thereof in vivo.

[0102] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have high binding affinity to the antigen. In some embodiments, the antibodies or antigen-binding fragments thereof have a specific association rate constant (k on or k Avalue), dissociation rate constant (k off or k D value), affinity constant (K A value), dissociation constant (K D value) and / or IC 50 It has a value.

[0103] In some embodiments, the antibody or antigen-binding fragment thereof has a nucleotide sequence of at least about 10 10 M -1 , at least about 4 × 10 11 M -1 , at least about 10 11 M -1 , at least about 4 × 10 12 M -1 , at least about 10 12 M -1 , at least about 4 × 10 13 M -1 , at least about 10 13 M -1 , at least about 4 × 10 14 M -1 , at least about 10 14 M -1 , at least about 4 × 10 15 M -1 , or at least about 10 15 M -1 K A (k on / k off ), or any pair of the preceding values ​​(approximately 4×10 11 M -1 ~4×10 13 M -1 , or about 4 × 10 12 M -1 ~Approx. 4×10 15 M -1 Any range of K A binds to the antigen.

[0104] In some embodiments, the antibody or antigen-binding fragment thereof is -10 Below, approximately 4×10 -11 M or less, about 10 -11 M or less, approximately 4 x 10 -12 M or less, about 10 -12 M or less, approximately 4 x 1013 M or less, about 10 -13 M or less, approximately 4 x 10 14 M or less, about 10 -14 M or less, approximately 4 x 10 -15 M or less, or about 10 -15 K below M D (k off / k on ), or any pair of the preceding values ​​(e.g., about 4×10 -11 M ~ approx. 4×10 -13 M or approx. 4 x 10 -12 M ~ approx. 4×10 -15 M) any range of K D binds to the antigen.

[0105] In some embodiments, K D (k off / k on ) values ​​are determined by assays well known in the art such as ELISA, isothermal titration calorimetry (ITC), fluorescence polarization assay, or any other biosensor such as BIAcore.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof has a concentration of less than about 0.02 nM, less than about 0.01 nM, less than about 0.005 nM, less than about 0.002 nM, less than about 0.001 nM, less than about 5×10 -4 Less than nM, approximately 2 × 10 -4 Less than nM, approximately 1 × 10 -4 Less than nM, approximately 5 × 10 -5 Less than nM, approximately 2 × 10 -5 Less than nM, approximately 1 × 10 -4 Less than nM, approximately 5 × 10 -6 Less than nM, approximately 2 × 10 -6 Less than nM, approximately 1 × 10 -6 Less than nM, approximately 5 × 10 -7 Less than nM, approximately 2 × 10 -7 Less than nM, or approximately 1 × 10 -7 Less than nM, IC 50 or any range of any pair of the preceding values ​​(e.g., from about 0.02 nM to about 2 x 10 -5 nM, or approximately 5 × 10 -5 nM ~ approx. 1×10 -7 IC50 IC binds to antigens and inhibits the binding of antigen ligands. 50 can be measured according to methods well known in the art, such as ELISA.

[0107] For example, antibodies or antigen-binding fragments thereof, including those modified with biotinylation, horseradish peroxidase, or any other detectable moiety (including those described above), can be used for diagnostic purposes.

[0108] In some embodiments, the antibodies or antigen-binding fragments thereof can be used to purify or detect antigens, including both in vitro and in vivo diagnostic methods. For example, the antibodies or antigen-binding fragments thereof can be used in immunoassays to qualitatively and quantitatively measure the levels of an antigen in a biological sample, or to identify the location, amount, behavior, and / or the like of an antigen in an animal. For example, measuring the level of an antigen using the antibodies or antigen-binding fragments thereof described herein can be used, for example, to 1) diagnose or determine an increased risk of developing cancer in a patient, 2) determine the prognosis of a patient, including the stage and grade of the tumor (particularly whether the cancer is or is likely to be metastatic) and / or its potential sensitivity to antibody therapy, 3) determine the origin of the tumor, and / or 4) determine the effectiveness of a treatment of the patient.

[0109] In some embodiments, the antibodies or antigen-binding fragments thereof can be used in conjunction with compound screening assays, e.g., to evaluate the effect of pharmaceutical agents on antigen expression and / or activity. Additionally, the antibodies or antigen-binding fragments thereof can be used in conjunction with gene therapy techniques, e.g., to evaluate the success of transfection of normal and / or engineered antigen expression.

[0110] The present disclosure also provides a method for diagnosing a neurological disease, comprising detecting the amount or activity of an antigen expressed in nervous tissue or any tissue associated with a non-CNS target organ, such as the lung, liver, kidney, spleen, etc. The diagnostic method can use an antibody or antigen-binding fragment thereof conjugated to a diagnostic agent. The antibody or antigen-binding fragment thereof can be used diagnostically, for example, to detect the expression of an antigen in a particular cell, tissue, or serum, or to monitor the development or progression of an immunological response as part of a clinical trial procedure, for example, to determine the effectiveness of a particular treatment regimen. Detection can be facilitated by coupling the antibody or antigen-binding fragment thereof to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent substances (such as fluorescein and rhodamine and their derivatives), luminescent substances, bioluminescent substances, optical agents (such as porphyrins, anthraquinones, anthrapyrazoles, perylenequinones, xanthenes, cyanines, acridines, phenoxazines and phenothiazines derivatives), radioactive substances, various positron emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions (such as Gd(III), Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III), and V(IV)). The detectable substance can be coupled or conjugated directly to the antibody or antigen-binding fragment thereof using techniques known in the art, or indirectly through an intermediate (such as, for example, a linker known in the art).Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase, see U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, acetylcholinesterase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include, but are not limited to, 125 I, 131 I, 111 In or 99 Examples of such antibodies include, but are not limited to, Tc.

[0111] The present disclosure also provides a method for detecting the expression of an antigen on a cell, comprising contacting a biological sample from a patient with one or more of the antibodies or antigen-binding fragments thereof described herein (optionally conjugated to a detectable moiety) and detecting whether the sample is positive for antigen expression or whether the sample has altered (e.g., decreased or increased) expression compared to a control sample. The biological sample may include biopsies of various tissues, including, but not limited to, skin, muscle, breast, prostate, cervix, ovary, brain, testis, gastrointestinal tract, eye, liver, kidney, and lung. Exemplary cells of the biological sample include tumor cells, skin cells, muscle cells, blood cells, ovarian cells, brain cells, prostate cells, breast cells, testicular cells, gastrointestinal cells, eye cells, liver cells, kidney cells, cervical cells, and lung cells. The biological sample may also be a biological fluid.

[0112] The presence of antigen-expressing cells in a biological sample can indicate the presence of cancer, especially when present in amounts greater than those of normal healthy subjects, and may also indicate metastasis. Disappearance of antigen-expressing cells over time in a patient, especially a patient undergoing treatment, can indicate remission (i.e., successful treatment), while lack of change in antigen-expressing cell levels in a patient undergoing treatment can indicate resistance to treatment and that a different treatment strategy can be used. Similarly, an increase in antigen-expressing cells over time in a patient can indicate recurrence. Additionally, the imaging techniques described herein can be utilized to monitor tumor size to determine the effectiveness of treatment. In some embodiments, other cancer diagnostic assays can be performed to confirm the results obtained with the methods described herein.

[0113] In some embodiments, a biological sample (e.g., a tumor sample) can be obtained from a subject to determine the presence of antigen-expressing cells. The number of antigen-expressing cells can be correlated with tumor grade. In some embodiments, the number of antigen-expressing cells in the biological sample is compared to the number of antigen-expressing cells in a corresponding biological sample from a healthy individual to determine modulation of antigen-expressing cells in the tumor. Subjects containing tumors can be treated with pharmaceutical agents to modulate the activity of antigen-expressing cells to normal, healthy levels.

[0114] Diseases that can be diagnosed using the present methods include, but are not limited to, neuronal cancers such as gliomas (glioblastomas), meningiomas, neurofibromatosis, primary brain tumors including pituitary adenomas, medulloblastomas, craniopharyngiomas, hemangiomas, epidermoid tumors, sarcomas, and intracranial metastases from other tumor sources. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to diagnose glioblastoma multiforme (GBM).

[0115] The disclosure also provides a method of treating an antigen-expressing cancer comprising administering to a human patient in need thereof an antibody or antigen-binding fragment thereof described herein, in some embodiments, the method comprises administering to a human patient having a solid tumor an amount of an antibody or antigen-binding fragment thereof described herein to provide a therapeutic benefit.

[0116] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be administered to patients by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intraarterial, intramuscular, intraocular, topical, local, intrathecal, intracerebroventricular, intrathecal, and intracranial. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. In some embodiments, the antibodies or antigen-binding fragments thereof can be formulated as an aqueous solution. In some embodiments, the antibodies or antigen-binding fragments thereof are administered intravenously or intracranially.

[0117] The antibodies or antigen-binding fragments thereof described herein can be used to treat various antigen-expressing tumors. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat antigen-expressing cancers, such as sarcomas with skeletal muscle characteristics, and skin tumors. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat antigen-expressing neural cancers, such as brain tumors or cerebellar lesions, e.g., micrometastases, in patients. In some embodiments, the antigen-expressing cancer is a neural cancer. In some embodiments, the neural cancer is a primary brain tumor, glioblastoma, glioma, meningioma, neurofibromatosis, pituitary adenoma, medulloblastoma, craniopharyngioma, vascular tumor, epidermoid tumor, sarcoma, or intracranial metastasis from other tumor sources. In some embodiments, the neural cancer is a glioblastoma. In some embodiments, the glioblastoma is glioblastoma multiforme (GBM).

[0118] In some embodiments, the antigen-expressing brain tumor is a GBM tumor that contains GBM tumor-initiating cells. In some embodiments, treatment with an antibody or antigen-binding fragment thereof described herein results in inhibition of proliferation of GBM tumor-initiating cells. In some embodiments, the antibody or antigen-binding fragment thereof further inhibits self-renewal of GBM tumor-initiating cells. Inhibition of cell proliferation and / or self-renewal may result in amelioration of signs or symptoms of the disease. For example, such therapy may result in improved survival (overall survival and / or progression-free survival) and / or may result in an objective clinical response (partial or complete). In some embodiments, the antibody or antigen-binding fragment thereof is internalized by GBM tumor cells, resulting in increased therapeutic efficacy of the antibody or antigen-binding fragment thereof in killing GBM tumor cells to which it binds. In some embodiments, the antibody or antigen-binding fragment thereof acts as an antagonist of antigen biological activity and may further be used as a method for inhibition of aberrant antigen activity.

[0119] In some embodiments, the antibodies or antigen-binding fragments thereof are useful in the treatment of cancers and non-neurological antigen-expressing tumors, including benign tumors. Cancers amenable to treatment with the antibodies or antigen-binding fragments thereof described herein include cancers that overexpress the antigen. In some embodiments, cancers amenable to treatment with the antibodies or antigen-binding fragments thereof described herein include epithelial cell cancers. In some embodiments, cancers amenable to treatment with the antibodies or antigen-binding fragments thereof described herein include, but are not limited to, breast cancer, ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, ovarian cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer, testicular cancer, thymic cancer, head and neck cancer, and colorectal cancer. The cancer may be newly diagnosed, treatment naive, or may be recurrent, refractory, or recurrent and refractory, or may be a metastatic form of a solid tumor.

[0120] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat antigen-expressing hematological malignancies, including, but not limited to, myelomas (such as multiple myeloma), lymphomas (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, and mantle cell lymphoma), leukemias (such as chronic lymphocytic leukemia, acute myeloid leukemia, and acute lymphocytic leukemia), and myelodysplastic syndromes. In some embodiments, the methods of the invention comprise administering to a human patient having a hematological malignancy an antibody or antigen-binding fragment thereof described herein to provide a therapeutic benefit.

[0121] In some embodiments, administration of the antibody or antigen-binding fragment thereof described herein is repeated after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. Repeated administrations can be the same dose or different doses. Administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a particular dosing regimen, a patient can receive antibody therapy for an extended period of time, such as 6 months, 1 year, or more. The amount of the antibody or antigen-binding fragment thereof described herein administered to a patient is a therapeutically effective amount. As used herein, a "therapeutically effective" amount of the antibody or antigen-binding fragment thereof described herein can be administered as a single dose or over the course of a treatment regimen, such as 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, 1 year, or more. Exemplary treatment regimens are further described herein. Treatment of a disease includes treatment of a patient already diagnosed with any type of disease at any clinical stage or manifestation, delaying the onset or progression or worsening of symptoms or signs of the disease, and / or preventing and / or reducing the severity of the disease.

[0122] The use of the antibodies or antigen-binding fragments thereof described herein to treat cancer in patients can result in any demonstrated clinical benefit compared to no treatment (if appropriate) or compared to known standard of care. Clinical benefit can be assessed by any method known to one of skill in the art. In some embodiments, clinical benefit is assessed based on objective response rate (ORR) (determined using RECIST version 1.1), duration of response (DOR), progression-free survival (PFS), and / or overall survival (OS). In some embodiments, a complete response indicates therapeutic benefit. In some embodiments, a partial response indicates therapeutic benefit. In some embodiments, stable disease indicates therapeutic benefit. In some embodiments, an increase in overall survival indicates therapeutic benefit. In some embodiments, therapeutic benefit may constitute an improvement in time to disease progression and / or an improvement in symptoms or quality of life. In some embodiments, therapeutic benefit may not translate to an increase in duration of disease control, but rather a significant reduction in symptom burden resulting in an improvement in quality of life. As will be apparent to one of skill in the art, therapeutic benefit may be observed using the antibodies or antigen-binding fragments thereof described herein alone (monotherapy), or adjunctively to, or in conjunction with, other anti-cancer therapies and / or targeted or non-targeted anti-cancer agents.

[0123] In some embodiments, the therapeutic benefit can be evaluated using standard clinical trials designed to measure the response of cancer to new treatments. To evaluate the therapeutic benefit of the antibodies or antigen-binding fragments thereof described herein, one or a combination of the following tests can be used: 1) Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1, 2) immune-related RECIST (irRECIST), 3) Eastern Cooperative Oncology Group (ECOG) performance status, 4) immune-related response criteria (irRC); 5) disease evaluable by assessment of tumor antigens; 6) validated patient-reported outcome measures; and / or 7) Kaplan-Meier estimates of overall survival and progression-free survival.

[0124] The present disclosure also provides a combination therapy comprising administering to a patient at least two agents, a first agent being an antibody or antigen-binding fragment thereof as described herein, and a second agent being a combination therapeutic agent. The antibody or antigen-binding fragment thereof as described herein and the combination therapeutic agent can be administered simultaneously, sequentially, or separately. The combination therapy can produce additive or more than additive effects.

[0125] In the method, the antibody or antigen-binding fragment thereof described herein and the combination therapeutic agent can be administered at the same time (either simultaneously or sequentially). The antibody or antigen-binding fragment thereof described herein and the combination therapeutic agent are administered sequentially when they are administered to the same patient on the same day, e.g., during a patient visit. Sequential administration can be performed 1, 2, 3, 4, 5, 6, 7, or 8 hours apart. In contrast, the antibody or antigen-binding fragment thereof described herein and the combination therapeutic agent can be administered on different days when they are administered to the patient on different days, e.g., the antibody or antigen-binding fragment thereof and the combination therapeutic agent can be administered one day apart, two days apart, or three days apart, one week apart, two weeks apart, or one month apart. In the method of the present disclosure, administration of the antibody or antigen-binding fragment thereof described herein can be performed before or after administration of the combination therapeutic agent. In some embodiments, the antibody or antigen-binding fragment thereof described herein and the combination therapeutic agent can be administered simultaneously for a period of time, followed by a second period during which administration of the antibody or antigen-binding fragment thereof and the combination therapeutic agent alternate.

[0126] In some embodiments, the co-therapeutic agent is a chemotherapeutic agent, an anti-angiogenic agent, an anti-rheumatic drug, an anti-inflammatory agent, a radiotherapeutic agent, an immunosuppressant, or a cytotoxic drug. The antibodies or antigen-binding fragments thereof described herein can be used in combination with conventional cancer therapies, such as surgery, radiation therapy, chemotherapy, or a combination thereof.

[0127] In some embodiments, the methods of the invention further comprise one or both of surgically removing tumor cells and / or administering radiation therapy. In some embodiments, other therapeutic agents useful for combining tumor therapy with the antibodies or antigen-binding fragments thereof described herein include antagonists, e.g., antibodies, of other factors involved in tumor growth, such as HER2, HER3, HER4, VEGF, or TNF-α. In some embodiments, it may also be beneficial to administer one or more cytokines to the patient for the treatment of cancer. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are co-administered with a growth inhibitory agent.

[0128] For the treatment of cancer, anti-inflammatory agents may be suitably used in combination with the antibodies or antigen-binding fragments thereof described herein, including, but not limited to, acetaminophen, diphenhydramine, meperidine, dexamethasone, pentasa, mesalazine, asacol, codeine phosphate, benorylate, fenbufen, naprosyn, diclofenac, etodolac and indomethacin, aspirin, and ibuprofen.

[0129] For the treatment of cancer, chemotherapeutic agents may be used in combination with the antibodies or antigen-binding fragments thereof described herein. Chemotherapeutic agents include, but are not limited to, radioactive molecules, toxins (such as cytotoxins or cytotoxic agents), including any agent, drug, and liposomes or other vesicles containing chemotherapeutic compounds that are detrimental to cell survival. Examples of suitable chemotherapeutic agents include 1-dehydrotestosterone, 5-fluorouracil, decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin, anti-α5β1 integrin antibodies, alkylating agents, allopurinol sodium, altretamine, amifostine, anastrozole, anthramycin (AMC), antimitotic agents, cis-dichlorodiamineplatinum(II) (DDP) cisplatin, diaminodichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase, live BCG (intravesical), beta-lactamase, cyclosporine ... betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan, calcium leuqualin, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), chlorambucil, cisplatin, cladribine, colchicine, conjugated estrogens, cyclophosphamide, cyclosofamide, cytarabine, cytochalasin B, cytoxan, dacarbazine, dactinomycin, dactinomycin (formerly actinomycin), daunirubicin, daunorcubicin citrate, denileukin diftitox, dexrazoxane, dibromomannitol, dihydroxyanthracin dione, docetaxel, doracetolone mesylate, doxorubicin, dronabinol, E.coli L-asparaginase, eolociximab, emetine, epoetin-α, Erwinia L-asparaginase, esterified estrogens, estradiol, estramustine sodium phosphate, ethidium bromide, ethinyl estradiol, etidronate, etoposide Citroloram factor, etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinic acid, gemcitabine, glucocorticoids, goserelin acetate, gramicidin D, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha-2b, irinotecan, letrozole, leucovorin calcium, leuprolide acetate, levamisole, lidocaine, lomustine, maytansinoids, mechlorethamine, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptiprine, mesna, methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane, mitoxantrone , nilutamide, octreotide acetate, ondansetron, paclitaxel, pamidronate disodium, pentostatin, pilocarpine, primycin, polipheprosan 20 with carmustine implant, porfimer sodium, procaine, procarbazine, propranolol, rituximab, sargramostim, streptozotocin, tamoxifen, taxol, teniposide, tenoposide, testolactone, tetracaine, thioepa, chlorambucil, thioguanine, thiotepa, topotecan, toremifene citrate, trastuzumab, tretinoin, barbicine, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate, or any salt thereof. In some embodiments, the method of the present invention comprises administering to the patient at least one chemotherapeutic agent.

[0130] Any anti-angiogenic agent can be used in combination with the antibody or antigen-binding fragment thereof described herein. In some embodiments, the anti-angiogenic agent is a VEGF antagonist or another VEGF receptor antagonist, such as a VEGF variant, a soluble VEGF receptor fragment, an aptamer that can block VEGF or VEGFR and neutralize anti-VEGFR antibodies, a low molecular weight inhibitor of VEGFR tyrosine kinase, and any combination thereof. Alternatively, or in addition, an anti-VEGF antibody can be co-administered to the patient.

[0131] The treatment regimen administered to a patient can vary depending on the age, weight, and disease state of the patient. The treatment regimen can last from 2 weeks to indefinitely. In some embodiments, the treatment regimen continues for a period of about 2 weeks to about 6 months, about 3 months to about 5 years, about 6 months to about 1 year or about 2 years, about 8 months to about 18 months, etc. The treatment regimen can be a non-variable dose regimen or a multivariate dose regimen.

[0132] The amount of the antibody or antigen-binding fragment thereof described herein to be administered may depend on a variety of factors, including, but not limited to, the specific type of solid tumor being treated, the stage of the solid tumor being treated, the mode of administration, the frequency of administration, the desired therapeutic effect, and other parameters such as the age, weight, and other characteristics of the patient. Determination of an effective dose to provide a therapeutic benefit for a particular mode of administration and frequency of administration is within the capabilities of one of ordinary skill in the art. Effective doses to provide a therapeutic benefit may be initially estimated from in vivo animal models or clinical trials. Suitable animal models for a wide variety of diseases are known in the art. The antibody or antigen-binding fragment thereof described herein may be administered by any route appropriate for the condition being treated.

[0133] In some embodiments, the antibody or antigen-binding fragment thereof described herein is provided as a lyophilized powder in a vial. The vial may contain about 100 mg, about 125 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, or about 400 mg of the antibody or antigen-binding fragment thereof. Prior to administration, the lyophilized powder can be reconstituted with sterile water for injection (SWFI) or other suitable medium to provide a solution containing the antibody or antigen-binding fragment thereof described herein. In some embodiments, the reconstituted solution obtained is further diluted with saline or other suitable medium for injection and administered via IV infusion, for example, twice every 7 days, once every 7 days, once every 14 days, once every 21 days, once every 28 days, once every 35 days, once every 42 days, once every 49 days, or once every 56 days. In some embodiments, for the first cycle, the infusion is administered over a period of 90 minutes. In some embodiments, subsequent infusions are longer than 60 minutes.

[0134] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are administered as an IV infusion once every 7 days at about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 8.0 mg / kg, or about 10.0 mg / kg. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are administered as an IV infusion once every 14 days at about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 8.0 mg / kg, or about 10.0 mg / kg. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are administered as an IV infusion once every 21 days at about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 8.0 mg / kg, or about 10.0 mg / kg. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are administered as an IV infusion once every 28 days at about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.0 mg / kg, about 3.0 mg / kg, about 4.0 mg / kg, about 5.0 mg / kg, about 6.0 mg / kg, about 8.0 mg / kg, or about 10.0 mg / kg.

[0135] When administered adjunctively to or together with other agents, e.g., other chemotherapeutic agents, the antibodies or antigen-binding fragments thereof described herein can be administered on the same schedule as the other agent(s) or on a different schedule. When administered on the same schedule, the antibodies or antigen-binding fragments thereof described herein can be administered before, after, or simultaneously with the other agents. In some embodiments, when the antibodies or antigen-binding fragments thereof described herein are administered adjunctively to or together with a standard of care, the antibodies or antigen-binding fragments thereof described herein can be initiated before the initiation of the standard of care therapy, e.g., one day, several days, one week, several weeks, one month, or even several months before the initiation of the standard of care therapy. In some embodiments, when the antibodies or antigen-binding fragments thereof described herein are administered adjunctively to or together with a standard of care, the antibodies or antigen-binding fragments thereof described herein can be initiated after the initiation of the standard of care therapy, e.g., one day, several days, one week, several weeks, one month, or even several months after the initiation of the standard of care therapy.

[0136] Dosing schedules for subcutaneous administration can vary from once every six months to once daily, depending on numerous clinical factors, including the type of disease, the severity of the disease, and the patient's sensitivity to the antibodies or antigen-binding fragments thereof described herein.

[0137] The present disclosure also provides a method of treating posterior capsule opacification (PCO), comprising administering to a human patient in need thereof an antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof is administered to the eye.

[0138] The present disclosure also provides a method of treating fibrosis, comprising administering to a human patient in need thereof an antibody or antigen-binding fragment thereof described herein. In some embodiments, the antibody or antigen-binding fragment thereof is administered to an organ. In some embodiments, the organ is a kidney or a lung.

[0139] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used in methods to isolate and / or purify cells, such as myofibroblast precursors, by selecting cells that bind to them. Such cells can rapidly migrate to wounds in the skin, lens, retina, and brain, thereby aiding in wound healing. Such cells have the potential to develop into contractile myofibroblasts. The isolated antigen-positive cells can be administered to humans or transplanted into slowly healing or non-healing wounds, such as diabetic and decubitus ulcers and severe surgical resections, to facilitate wound closure.

[0140] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used in methods to isolate neuroprotective cells by selecting cells that bind to them. Cells expressing the antigen can increase in number in response to injury. Such cells can be administered to the retina and brain to reduce neuronal death. Antigen-positive cells and / or the molecule(s) they produce can be injected or otherwise introduced into a subject following injury.

[0141] The oligonucleotides disclosed herein can include nucleotides or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides. The oligonucleotides disclosed herein can include DNA, RNA, or both DNA and RNA.

[0142] The oligonucleotides disclosed herein can also include one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides that contain modifications to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl, etc. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0143] Nucleotide analogs can also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10Exemplary 2' sugar modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n

[0036] Other modifications at the 2' position include, but are not limited to, C 1-10 Alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF 3、 OCF 3、 Modifications include, but are not limited to, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or on 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those that contain modifications at the bridging ring oxygen, e.g., CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0144] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the bond between two nucleotides can be modified to include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, thionoalkyl phosphotriesters, and boranophosphates. These phosphate or modified phosphate bonds between two nucleotides can be via 3'-5' or 2'-5' bonds, and the bonds can include reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide surrogates also include peptide nucleic acids (PNAs).

[0145] Different lengths of oligonucleotides, different base compositions, different sequence(s) of nucleotides, and / or different oligonucleotide densities on an antibody can have different effects allowing for modulation of molecular properties and ultimately the therapeutic efficacy or therapeutic index of the biologic.

[0146] The chemistry of oligonucleotide attachment may also offer unique regulatory properties or flexibility in adapting the design of novel therapeutic moieties with properties related to both biodistribution and clearance rate.

[0147] The disclosed nucleic acid molecules can include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0148] The nucleic acid molecules disclosed herein can also include one or more nucleotide analogs or substitutions. A nucleotide analog is a nucleotide that contains modifications to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications such as A, C, G, and T / U, as well as different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (ploid uracil), uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0149] Nucleotide analogs can also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl, ... 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10 Exemplary 2' sugar modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Also included, but not limited to, are the following: 1-10 Alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF 3、 OCF 3、Modifications include, but are not limited to, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, as well as other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or on 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those that contain modifications at the bridging ring oxygen, e.g., CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0150] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the bond between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriates, thionoalkyl phosphotriesters, and boranophosphates. These phosphate or modified phosphate bonds between two nucleotides can be via 3'-5' or 2'-5' bonds, and these bonds can include reverse polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide surrogates also include peptide nucleic acids (PNAs).

[0151] In some embodiments, the oligonucleotide has the nucleotide sequence set forth in SEQ ID NO:1.

[0152] In order to allow the subject matter disclosed herein to be more efficiently understood, the following examples are provided. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the claimed subject matter in any manner. Throughout these examples, molecular cloning reactions and other standard recombinant DNA techniques were carried out according to the methods described in Maniatis et al., Molecular Cloning-A Laboratory Manual, 2nd ed. Cold Spring Harbor Press (1989), using commercially available reagents, unless otherwise noted. As used herein, immunoglobulin amino acid residue numbering is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.

[0153] To study the circulation and biodistribution of anti-ICAM / 3DNA in vivo, anti-ICAM antibodies were conjugated to the 3DNA outer branches. Since the outer branches of 3DNA are single-stranded DNA, the means of coupling anti-ICAM to 3DNA is to first conjugate anti-ICAM to oligonucleotides whose sequences are complementary to the outer branches of 3DNA, so that the antibody-oligo conjugates can simply be coupled to 3DNA through annealing by mixing them together. Since specific targeting of ICAM-1 had to be demonstrated, a similar construct using a non-specific antibody (IgG control) was also comparatively studied. However, before coupling anti-ICAM-oligos or IgG-oligos to 3DNA for subsequent injection of anti-ICAM / 3DNA or anti-ICAM / 3DNA into mice, it was necessary to demonstrate that the conjugated species (anti-ICAM-oligos or IgG-oligos) were still able to function as the original anti-ICAM and IgG, i.e., they target ICAM-1, which is mainly expressed in the lung, or function as a non-specific control antibody, respectively. To this end, mice were injected with IgG, IgG-oligo, anti-ICAM, or anti-ICAM-oligo, and their circulation and biodistribution were then addressed and compared. Surprisingly, the results demonstrated that the antibody oligo species behaved differently from the original antibody species, and the presence of the oligo increased the targeting specificity of anti-ICAM.

[0154] The disclosed studies show that coupling of antibodies to DNA oligonucleotides modifies the behavior of the antibodies in an unexpected way. For the example of a specific antibody, of which a monoclonal antibody recognizing ICAM-1 is exemplary, conjugation to oligonucleotides significantly increased both the absolute binding and the binding specificity of the antibody to its respective antigen (ICAM-1) demonstrated in vivo. In addition, the antibody oligoconjugates (both specific and non-specific) are cleared from the circulation faster and distributed more abundantly than the original non-conjugated antibody, which may help to avoid systemic side effects of antibodies applied in vivo. This unexpected discovery holds important translational potential to enhance the recognition / targeting ability of an antibody to its antigen, which may be used as a means to enhance the research, therapeutic and / or diagnostic potential of said antibodies, whether used alone or in combination with drugs, tracers, carriers, devices, materials, or cells for several purposes and applications, which may include analytical in vitro systems, ex vivo tissues, cell cultures, or in vivo organisms. EXAMPLES

[0155] Example 1: In vivo biodistribution of non-specific IgG antibody, IgG antibody-oligonucleotide, specific anti-ICAM antibody, and anti-ICAM antibody-oligonucleotide reagent: Rat monoclonal immunoglobulin G Abs (IgG) against mouse ICAM-1 (anti-ICAM) were cloned YN1 produced by the respective hybridomas from the American Type Culture Collection (Manassas, VA). Nonspecific rat IgG Abs (hereafter referred to as IgG) were obtained from Jackson Immunoresearch (Pike West Grove, PA). DNA oligonucleotides (72-mers) modified with thiols at the 5' end were obtained from Oligo Factory (Holliston, MA). Pierce Bond-Breaker TCEP Solution, Pierce LC-SMCC Crosslinker, Pierce Zeba Spin Columns (7k molecular weight cutoff, MWCO), Pierce Thiophilic Adsorption Resin, Amicon spin filters (10k MWCO), Pierce BCA Protein Assay Kit and Heterobifunctional Pierce Crosslinking Kit, bovine serum albumin (BSA), and TCA were obtained from Fisher Scientific (Kerrville, TX). Iodine source iodination tubes were obtained from Pierce (Rockford, IL) and BioSpin Tris columns were obtained from BioRad (Hercules, CA). 125 I was obtained from Perkin-Elmer (Waltham, Mass.). All other reagents were obtained from Sigma Chemical (St. Louis, Mo.).

[0156] Antibody-oligonucleotide conjugates: Antibody-oligonucleotide (Ab-oligo) conjugation for anti-ICAM or non-specific control IgG Ab was performed using N-hydroxysuccinimide (NHS)-maleimide chemistry. Briefly, 72-mer DNA oligonucleotides with 5' thiol modifications were first reduced by adding 50 mM tris(2-carboxyethyl)phosphine (TCEP) and incubating at 25°C for 1 h. Excess TCEP was removed by ethanol precipitation and the oligonucleotides were resuspended in phosphate-buffered saline (PBS) + 5 mM ethylenediaminetetraacetic acid (EDTA), pH 7.2. In parallel, Abs were reacted with excess LC-SMCC crosslinker (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproic acid)) for 1 h at 25°C. Unreacted LC-SMCC crosslinker was removed from the Ab-LC-SMCC reaction using Zeba spin columns equilibrated with PBS+5mM EDTA pH 7.2 according to the vendor's instructions. Reduced oligonucleotide was then added to the Ab-LC-SMCC and the conjugation reaction was incubated at 25°C for 12 hours. Thiophilic adsorption chromatography was utilized to remove excess unreacted thiol oligonucleotide from the Ab-oligonucleotide conjugate, and fractions containing the conjugate were pooled and concentrated using an Amicon 10kDa MWCO spin filter. The final protein concentration of the Ab-oligonucleotide conjugate was determined using a (bicinchoninic acid) BCA protein assay kit with bovine gamma globulin standard.

[0157] Radiolabeling of antibodies and antibody oligoconjugates: The Ab (non-oligonucleotide) or Ab-oligonucleotide conjugate was 125 Using I and iodine source coated iodination tubes 125 I-radiolabeled. 20 μCi of Na 125I was incubated in an iodized tube containing 100 μL of 1 μg / μL Ab in PBS for 5 min at 4° C. The sample was then subjected to size exclusion chromatography in a 6 kDa cutoff Tris column and centrifuged at 1000×g for 4 min to remove non-reactive free IgG. 125 I was removed. 125 I-Ab or 125 The final concentration of I-Ab-oligonucleotide conjugate was determined using the Bradford protein assay and a BSA standard curve. 2 Radioactivity (counts of radioactive events detected per minute, CPM) of the samples, expressed as CPM per μg protein, and free 125 The presence of residual I was measured. For this purpose, 2 μL of 125 I-Ab or 125 The I-Ab-oligonucleotides were diluted in 3% BSA in PBS to a total volume of 1 mL and then measured in a gamma counter. Then, 200 μL of TCA was added (final TCA concentration=17%, v / v) and the samples were vortexed and incubated at room temperature for 15 minutes. The samples were then centrifuged at 2418×g for 5 minutes to obtain a 100% elution volume. 125 I-Ab-oligonucleotides were precipitated and free 125 A 600 μL aliquot of the supernatant (half of the total reaction volume) was then measured in a γ counter and the free I in the sample injected into the mouse was calculated using the following formula: 125 The presence of I was estimated. Free iodine CPM = total CPM - (2 x supernatant CPM).

[0158] Mouse models: Eight-week-old C57BL / 6 wild-type male mice were obtained from Jackson Laboratory (Bar Harbor, ME). Mice were provided with food and water ad libitum and used for experiments at approximately 25 g body weight (BW). Mice were anesthetized intraperitoneally with a mixture of 100 mg / kg·BW ketamine and 10 mg / kg·BW xylazine for each in vivo experiment. All animal experiments were performed in compliance with regulations and under institutionally approved protocols.

[0159] Circulation and Biodistribution: Anesthetized C57BL / 6 mice were 125 Mice were injected with I-labeled antibody or antibody-oligonucleotide conjugate. Blood samples were taken at the indicated time points and sacrificed 60 min after injection, followed by harvesting and weighing of major organs (e.g., brain, heart, kidneys, liver, lungs, and spleen), homogenization of organs, and determination of any potential free radicals in the samples. 125 Sample precipitation with trichloroacetic acid (TCA) to eliminate I was performed. The obtained radioactivity measurements were then utilized to calculate the % injected dose (%ID) using a gamma counter, where the injected dose is the dose measured before injection minus the dose residue in the syringe after injection. The % injected dose per gram of organ (%ID / g) was also calculated to compare relative accumulation in organs with different weights, thus reflecting the organ concentration. The localization ratio (LR=%ID / g in organ:%ID / g in blood) is to represent the organ vs. blood distribution, and the specificity index (SI), calculated as the %ID / g of a species divided by the %ID / g of the other species, is to compare them.

[0160] Figure 1 shows that IgG-oligonucleotides enhance clearance from the bloodstream. Mice were injected intravenously with a non-specific IgG control antibody or the same antibody conjugated to an oligonucleotide (IgG-oligo). These entities also react with iodine. 125The antibodies were radiolabeled with 500 mM NaCl and the circulation was followed up to 60 min after administration. Data were calculated as mean ± standard error of the mean. The parameters shown are % of injected dose (%ID), reflecting absolute biodistribution values ​​in the circulation. Figure 1 demonstrates a faster clearance from the circulation for non-specific IgG-oligos compared to IgG, which may help to reduce the potential for systemic side effects associated with the use of antibodies.

[0161] Figure 2 shows that IgG-oligonucleotides enhance splenic and hepatic clearance. Mice were injected intravenously with a non-specific IgG control antibody or the same antibody conjugated to an oligonucleotide (IgG-oligo). These moieties were iodized. 125 The antibodies were radiolabeled with 5000 ng / mL and the biodistribution was followed 60 min after administration. Data were calculated as mean ± standard error of the mean. The parameters shown are the % of injected dose per gram of organ (%ID / g) (panel A), reflecting the concentration in the organ, and the localization ratio (LR), which is the %ID / g in organ divided by the %ID / g in blood, reflecting the tissue to blood ratio (panel B). Figure 2 demonstrates an increased accumulation of non-specific IgG-oligos in the liver and spleen compared to IgG, which may help clear the antibody-oligos from the body via these clearance organs and attenuate possible side effects in other organs. There is also some reduction in the levels of IgG-oligos in the bladder, kidney, and stomach. This may be due to enhanced clearance in the liver and spleen, or faster excretion through the kidney and stomach routes, so that less amount is found at this time point in these organs, which is enough to clear the antibody from the body and avoid side effects.

[0162] Figure 3 shows that anti-ICAM-oligonucleotides are rapidly cleared from the bloodstream. Mice were injected intravenously with a nonspecific IgG control antibody, an ICAM-1 specific anti-ICAM antibody, or an anti-ICAM-oligo conjugate (Panel A), or an anti-ICAM-oligo or IgG-oligo conjugate (Panel B). In both cases, the solid moiety was iodinated to follow the circulation up to 60 minutes after administration. 125 The antibodies were radiolabeled at 1000 ng / mL. Data were calculated as mean ± standard error of the mean. The parameters shown are % of injected dose (%ID), reflecting absolute biodistribution values ​​in the circulation. Figure 3 demonstrates the faster clearance of specific anti-ICAM antibodies from the circulation when conjugated to oligonucleotides compared to non-oligonucleotide anti-ICAM, which should also help protect against systemic side effects as described above. Furthermore, the faster clearance of anti-ICAM-oligonucleotides compared to IgG-oligonucleotides indicates that this protective effect is more pronounced for antibodies that recognize specific antigens. This is of substantial value since specific antibodies are those that recognize antigens and are therefore intended to be used in research, diagnostic, or therapeutic settings.

[0163] Figure 4 shows that anti-ICAM-oligo enhances pulmonary targeting. Mice were intravenously injected with a nonspecific IgG control antibody, an ICAM-1 specific anti-ICAM antibody, or an anti-ICAM-oligo conjugate (panels A and B), or with an anti-ICAM-oligo or IgG-oligo conjugate (panels C and D). In all cases, the solid moiety was iodinated. 125and followed their body distribution 60 min after administration. Data were calculated as mean ± standard error of the mean. Parameters shown are percent of injected dose per gram of organ (%ID / g), reflecting the concentration in the organ (Panels A and C), and localization ratio (LR), reflecting the tissue-to-blood ratio, %ID / g in organ divided by %ID / g in blood (Panels B and D). Figure 4 demonstrates that specific anti-ICAM antibodies target the lung (the major site of ICAM-1 expression) significantly better than anti-ICAM controls and to a much greater extent than nonspecific IgG (conjugated with oligonucleotides or not), demonstrating enhanced absolute targeting and specificity.

[0164] Figure 5 shows that pulmonary targeting of anti-ICAM-oligonucleotides is highly specific to the lungs compared to other formulations, providing minimal accumulation in lymph nodes. Mice were intravenously injected with a nonspecific IgG control antibody, an ICAM-1 specific anti-ICAM antibody, or the respective antibody conjugated to an oligonucleotide (oligo), and these moieties were iodized. 125 and followed their body distribution 60 min after administration. Data were calculated as mean ± standard error of the mean. The parameter shown is the specificity index (SI) comparing the biodistribution of two species shown in the graph key, calculated as %ID / g of a species divided by %ID / g of the other species, reflecting specificity. Figure 5 shows the enhanced specificity for the main site of ICAM-1 expression (lung) provided by anti-ICAM-oligo compared to all other species injected.

[0165] In addition to those described herein, various modifications of the described subject matter will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including but not limited to journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety.

Claims

1. 1. An antibody-oligonucleotide conjugate composition comprising an antibody or antigen-binding fragment thereof conjugated to an oligonucleotide for use in the treatment or prevention of a disease, wherein the oligonucleotide is not hybridized to a DNA dendrimer, and upon administration of a physiologically acceptable amount of the antibody-oligonucleotide conjugate to a subject: i) the antibody or antigen-binding fragment thereof binds to an antigen to a greater extent or faster than an identical antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide; and / or ii) the antibody or antigen-binding fragment thereof is present to a lesser extent in the blood of the subject during a period following administration than an identical antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide; The antibody-oligonucleotide conjugate composition.

2. 2. The antibody-oligonucleotide conjugate composition for use according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises an IgA antibody or antigen-binding fragment thereof, an IgD antibody or antigen-binding fragment thereof, an IgE antibody or antigen-binding fragment thereof, an IgG antibody or antigen-binding fragment thereof, or an IgM antibody or antigen-binding fragment thereof.

3. the antibody or antigen-binding fragment thereof is further covalently linked to an effector moiety, said covalent linkage not affecting binding of the antibody or antigen-binding fragment thereof to an antigen; the effector moiety is a detectable label, a cytotoxic agent, a chemotherapeutic agent, a nucleic acid molecule, an anti-tumor agent, a drug, a toxin, a biologically active protein, another antibody or antigen-binding fragment thereof, a synthetic or natural polymer, a radionuclide, a radioisotope, a chelated metal, a nanoparticle, a reporter group, a drug moiety, a polypeptide, a signaling molecule, a thrombotic or anti-angiogenic agent, a biological response modifier, or a chemotherapeutic agent; The effector moiety increases the half-life of the composition in vivo or inhibits epithelial burrs.

2. The antibody-oligonucleotide conjugate composition of claim 1 for use in enhancing delivery of the antibody or antigen-binding fragment thereof across the aorta.

4. the antibody or antigen-binding fragment thereof comprises 3 to 5 maytansinoid molecules conjugated to the antibody or antigen-binding fragment thereof by chemical linkers; 2. The antibody-oligonucleotide conjugate composition for use according to claim 1, optionally wherein the chemical linker comprises N-succinimidyl-3-(2-pyridyldithio)propionate, N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), or succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

5. the antibody or antigen-binding fragment thereof is covalently fused to the amino acid sequence of another protein or portion thereof; In some cases, (i) the covalent bond is a peptide bond; (ii) the fusion is via the N-terminus or C-terminus of the antibody or antigen-binding fragment; (iii) the fusion is via an internal portion of the antibody or antigen-binding fragment thereof; (iv) the other protein or portion thereof is linked to the N-terminus of the constant domain of the antibody or antigen-binding fragment thereof; and / or (v) the protein or portion thereof is at least 10, 20, or 50 amino acids in length; 2. An antibody-oligonucleotide conjugate composition for use according to claim 1.

6. 4. The antibody-oligonucleotide conjugate composition for use according to claim 3, wherein the toxin is dlostatin, a dlostatin peptide analog or derivative, abrin, ricin A, saporin, Pseudomonas exotoxin, diphtheria toxin, ethidium bromide, PE40, PE38, gelonin, RNAse, peptide nucleic acid (PNA), ribosome inactivating protein (RIP) type 1 or 2, pokeweed antiviral protein (PAP), bryodin, momordin, a chemotherapeutic agent, or buganin.

7. the antibody or antigen-binding fragment thereof is conjugated to a liposome or a poly(ethylene glycol) (PEG) moiety; Optionally, the PEG moiety is attached to an amino acid side chain or a terminal amino acid functional group; Optionally, the amino acid side chain or terminal amino acid functionality comprises an amino group, an imino group, a thiol group, a hydroxyl group, or a carboxyl group; and / or Optionally, the thiol group is at least one cysteine ​​residue of the antibody or antigen-binding fragment thereof.

2. An antibody-oligonucleotide conjugate composition for use according to claim 1.

8. The antibody-oligonucleotide conjugate composition for use according to claim 1, wherein said antibody or antigen-binding fragment thereof comprises a modification, optionally said modification comprising biotinylation or the addition of horseradish peroxidase.

9. The antibody-oligonucleotide conjugate composition for use according to claim 3, wherein the detectable label is an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, an optical agent, a radioactive material, a positron emitting metal, or a non-radioactive paramagnetic metal ion.

10. The antibody or antigen-binding fragment thereof may be a polyclonal antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof, genetically engineered or otherwise. Antibodies or antigen-binding fragments thereof modified by the method, chimeric antibodies, humanized antibodies, primatized antibodies, heteroconjugate antibodies, Fab, Fab', F(ab') ２ , Fv, scFv, scFv-Fc, diabodies, bispecific diabodies, trispecific triabodies, minibodies, monospecific Fab ２ , bispecific Fab ２ , trispecific Fab ３ , a nanobody, an IgNAR, a V-NAR, a hcIgG, or a VhH fragment.

11. The antibody or antigen-binding fragment thereof is selected from the group consisting of 3F8, 8H9, abagovomab, abciximab, avituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afacevicumab, afelimomab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, amivantamab, anatumomab mafenatox, andecaliximab, anetumab ravtansine, anifrolumab, ansuvimab, anrukinzumab, apolizumab, apurutumab, Ixadotin, arcitumomab, asclinbacumab, acelizumab, atezolizumab, atidortoxumab, atinumab, atortivimab, maftivimab, odesivimab, atorlimumab, avelumab, azintuxizumab Vedotin, bamlanivimab, bapineuzumab, basiliximab, bavituximab, BCD-100, bectumomab, begelomab, belantamab mafodotin, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bermekimab, belsanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, vilutamimab, bivatuzumab, bleselumab, blinatumomab, brontuzumab, brosozumab, bococizumab, brazikumab, brentuximab Vedotin, briakinumab, brodalumab, brolucizumab, broticutuzumab, burosumab, cabilalizumab, camidanlumab tesirin, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, casirivimab, capromab, carumab, carotuximab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, cemiplimab, sergituzumab amnaleukin, certolizumab pegol, cetrelimab, cetuximab, civisatamab, sirmutuzumab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab Tetraxetane, codrituzumab, cofetuzumab peridotin, coltuximabRavtansine, conatumumab, concizumab, cosfrobiximab, crenezumab, crizanlizumab, clotezumab, CR6261, cusatuzumab, dacetuzumab, daclizumab, darotuzumab, dapirorizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, delrotuximab biotin, detumomab, desamizumab, dinutuximab, dinutuximab beta, dilidabumab, domagrozumab, dorlimomab Alitox, dostallimab, drozitumab, DS-8201, durigotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, ersilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab vedotin, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblitzumab, enokizumab, enoticumab, ensituximab, epcolitamab, epitumomab Sitaxetan, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etesevimab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faricimab, faretuzumab, fasinumab, FBTA05, felvizumab , fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, filibumab, framvotumab, fretikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frobocimab, fulnevetomab, fulranumab, futuximab, galcanezumab, galiximab, ganco Tamab, Ganitumab, Gantenerumab, Gatipotuzumab, Gavirimomab, Gedivumab, Gemtuzumab Ozogamicin, Gevokizumab, Gilvetomab, Gimsilumab, Girentuximab, Glenbatumumab Vedotin, Golimumab, Gomiliximab, Goslanemab, Guselkumab, Inalumab, Ibalizumab, IBI308, Ibritumomab Tiuxetan, Icrucumab, Idarucizumab, Ifavotuzumab, Igovomab, Iradatuzumab Vedotin, IMAB362, Imalumab, Imaprelimab, Imusiromab, Imdevimab, Imgatuzumab, Incracumab, Indatuximab Ravtansine, Indusatumab Vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab Ozogamicin, Ipilimumab, Iomab-B, Iratumumab, Isatuximab, Iscalimab, Istiratumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lacnotuzumab, Radiratuzumab Vedotin, Lampalizumab, Lanadelumab, Landgrozumab, Laprituximab emtansine, ralcaviximab, lebrikizumab, remaresomab, lendalizumab, lenbervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, ribivirumab, rifatuzumab vedotin, ligelizumab, roncatuximab tesirin, rosatuxizumab vedotin, rilotomab satetratetan, lintuzumab, lirilumab, roderucizumab, lokivetomab, lorvotuzumab mertansine, lucatumumab, lurizumab Pegol, lumiliximab, lumuletuzumab, rupartumab, rupartumab amadotin, rutikizumab, maftivimab, mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minletumomab, mirikizumab, mirvetuximab soravtansine, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, naratuximab Emtansine, Narutosumab, Natalizumab, Nabicixizumab, Nabivumab, Naxitamab, Nebacumab, Necitumumab, Nemolizumab, NEOD001, Nelerimomab, Nesbacumab, Netakimab, Nimotuzumab, Nirsevimab, Nivolumab, Nofetumomab Merpentan, Oviltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odesivimab, Odulimomab, Ofatumumab, Olaratumab, Olecurumab, Orendalizumab, Olokizumab, Omalizumab, Omburtamab, OMS721, Onartuzumab, Ontuxizumab, Onvatilimab, Opicinumab, Oportuzumab Monatox, oregovomab, olticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placumab, prezalumab, prosalizumab, pogalizumab, polatuzumabVedotin, ponezumab, polgabiximab, prasinezumab, prezarizumab, priliximab, plitoxaximab, pritumumab, PRO140, kirisumab, racotumomab, radletumab, rafivirumab, ralpancizumab, ramucirumab, ranevetomab, ranibizumab, raxibacumab, ravagalimab, ravuturizumab, refanezumab, regavirumab, regdanvimab, relatimab, lemtolumab, reslizumab, rilotumumab, rinukumab, risankizumab, rituximab, rivavazumab pegol, lobatumumab, SII RMAb, loredumab, romilximab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab tesirin, rovelizumab, rozanolixizumab, ruplizumab, SA237, sacituzumab govitecan, samolizumab, samrotamab vedotin, sarilumab, satralizumab, satumomab pendetide, secukinumab, seliclerumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, SGN-CD19A, SHP647, sifalimumab, siltuximab, simtuzumab, siplizumab, siltratumab vedotin, sirukumab, sofit zumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, sputabumab, stimulimab, subizumab, sublatoxumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, tafasitamab, talacuzumab, talizumab, talquetamab, tamtubetomab, tanezumab, tapritumomab paptox, talexuzumab, tabolimab, teclistamab, tefibazumab, terimomab alitox, terisotuzumab, terisotuzumab Vedotin, Tenatumomab, Teneliximab, Teplizumab, Tepositamab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Tiburizumab, Tildrakizumab, Tigatuzumab, Timigtuzumab, Timolumab, Tiragolumab, Tiragotumab, Tislelizumab, Tisotumab Vedotin, TNX-650, Tocilizumab, Tomzotuximab, Toralizumab, Tosatoxumab, Tositumomab, Tobetumab, Tralokinumab, Trastuzumab, Trastuzumab Duocarmazine, Trastuzumab Emtansine, TRBS07, Tregalizumab, Tremelimumab, Treboglumab, Tucotuzumab Celmoleukin, tubilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab talilline, banalimab, bundletuzumab vedotin, vanticutumab, vanucizumab, bapaliximab, balisakumab, varlilumab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, bovalilizumab, volociximab, bonlerolizumab, bopratelimab, borsetuzumab 2. The antibody-oligonucleotide conjugate composition for use according to claim 1, wherein the therapeutic antibody is selected from mafodotin, votumumab, bunakizumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenoctuzumab, diralimumab, zolbetuximab, or zolimomab alitox.

12. the oligonucleotide comprises non-natural nucleotides, modified nucleotides, nucleotide analogs, or nucleotide substitutes; Optionally, the non-natural nucleotide, the modified nucleotide, the nucleotide analog, or the nucleotide substitute comprises a modified base, a modified sugar, or a modified phosphate group; Optionally, non-naturally occurring moieties are incorporated into the structure of the oligonucleotide; Optionally, the non-natural nucleotide comprises a dideoxynucleotide, a biotinylated nucleotide, an amino acid modified nucleotide, a deaminated nucleotide, an alkylated nucleotide, a benzylated nucleotide, or a fluorophore-labeled nucleotide; Optionally, the modified sugar comprises a naturally occurring modification of ribose, a naturally occurring modification of deoxyribose, a synthetic modification of ribose, or a synthetic modification of deoxyribose; Optionally, the modified phosphate group comprises a modification such that the linkage between the first and second nucleotide comprises a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methylphosphonate, alkylphosphonate, phosphinate, phosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, or boranophosphate.

2. An antibody-oligonucleotide conjugate composition for use according to claim 1.

13. said greater degree comprising an increase in affinity or avidity; Optionally, the increased affinity comprises an increased binding constant; Optionally, the binding constant is K Ａ (k ｏｎ / k ｏｆｆ ), 2. An antibody-oligonucleotide conjugate composition for use according to claim 1.

14. The antibody-oligonucleotide conjugate composition for use according to claim 1, wherein said antibody-oligonucleotide conjugate is administered intravenously.

15. The oligonucleotide according to any one of claims 1 to 14, wherein the oligonucleotide has SEQ ID NO:

1. The antibody-oligonucleotide conjugate composition for the above-mentioned use.

16. The antibody-oligonucleotide conjugate composition for use according to any one of claims 1 to 14, wherein said antibody or antigen-binding fragment thereof is an anti-ICAM antibody or antigen-binding fragment thereof.

17. 1. A method for increasing the extent to which an antibody or antigen-binding fragment thereof binds to an antigen, comprising conjugating the antibody or antigen-binding fragment thereof to an oligonucleotide that forms an antibody-oligonucleotide conjugate, wherein the oligonucleotide is not hybridized to a DNA dendrimer, and wherein the antibody-oligonucleotide conjugate binds to the antigen to a greater extent or faster than an identical antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide.

18. 1. A method for reducing non-specific binding of an antibody or antigen-binding fragment thereof, comprising conjugating the antibody or antigen-binding fragment thereof to an oligonucleotide that forms an antibody-oligonucleotide conjugate, wherein the oligonucleotide is not hybridized to a DNA dendrimer, and wherein upon administration of a physiologically tolerable amount of the antibody-oligonucleotide conjugate to a subject, the antibody-oligonucleotide conjugate is present in the blood of the subject to a lesser extent during a period following administration than an identical antibody or antigen-binding fragment thereof that is not conjugated to the same oligonucleotide.

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