Heterodimer antibodies, their use in therapeutic applications, and related compositions

Abstract

This specification discloses variant heavy chain antibody sequences. In certain embodiments, this disclosure considers heterodimer antibodies having a first heavy chain and a second heavy chain, wherein the first and second heavy chains, or fragments thereof, contain different mutations. In certain embodiments, this disclosure relates to nucleic acids and vectors encoding the antibodies and recombinant heavy chains disclosed herein, as well as cells containing them.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Application No. 63 / 461,078, filed on April 21, 2023, and U.S. Provisional Application No. 63 / 578,789, filed on August 25, 2023. The entire contents of each of these applications are incorporated herein by reference for all purposes.

[0002] (Description of Research and Development Funded by the Federal Government) This invention was made with government support under grant AI149297 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0003] (Incorporation by Reference of Materials Submitted as XML Files via the Patent Office's Electronic Filing System) The sequence listing associated with this application is provided in XML format and is incorporated herein by reference. The name of the XML file containing the sequence listing is 23010PCT.xml. The XML file is 3 KB, was created on April 19, 2024, and was electronically submitted via the USPTO Patent Electronic Filing System.

Background Art

[0004] Therapeutic antibodies constitute an important class of drugs for the treatment of a wide range of diseases such as cancer. Their ability to mobilize and stimulate immune system cells is often associated with their clinical effectiveness, particularly in cancer immunotherapy, and is retained in the heavy - chain constant region (Fc region). The Fc region binds to Fc receptors and complement in order to induce antibody - mediated effector functions that direct cell death in vivo. Antibody therapies are successfully used in the treatment of cancer, but clinical resistance and refractory diseases to these drugs remain a major problem because cancer recurrence is not uncommon. Therefore, there is a need to identify improvements.

[0005] Ridgway et al. have reported a knobs-into-holes technique for heavy chain heterodimerization in the antibody CH3 region. (Protein Engineering, 1996, 9(7): 617-621.)

[0006] Ha et al. have reported an immunoglobulin Fc heterodimer platform. (Front Immunol, 2016, 7:394.)

[0007] Mimura et al. have reported a glycosylation technique for therapeutic IgG antibodies. (Protein Cell 2018, 9 (1): 47-62.)

[0008] Li et al. reported that a glycosynthase mutant of endoglycosidase S2 exhibits transglycosylation activity. (J Biol Chem, 2016, 291 (32): 16508-18.)

[0009] Moore et al. report a heterodimer Fc platform designed for the efficient development of bispecific antibodies. (Methods, 2019, 154: 38-50.)

[0010] Du et al. reported the mechanism and specificity of IgG-active endoglycosidase. Glycobiology, 2020, 30 (4): 268-279.

[0011] Klontz et al. have reported an α-fucosidase for highly efficient IgG trans-fucosylation. (Nat Comm, 2020, 11:620.)

[0012] The references cited herein do not constitute prior art. [Overview of the Initiative]

[0013] This disclosure relates to mutant heavy chain antibody sequences. In certain embodiments, this disclosure envisions a heterodimer antibody having a first heavy chain and a second heavy chain, wherein the first and second heavy chains contain different mutations. In certain embodiments, mutant heavy chain antibody sequences are useful for constructing stabilized antibodies useful for therapeutic applications.

[0014] In certain embodiments, a stabilized heterodimer antibody enables the selective synthesis or cleavage of glycosylated substituents, resulting in asymmetrically glycosylated antibody constructs.

[0015] In certain embodiments, the heterodimer antibody comprises a first heavy chain having the mutation or combination of mutations disclosed herein, and a second heavy chain.

[0016] In certain embodiments, the disclosure relates to recombinant antibody heavy chains or heterodimer antibodies containing the same, having mutations K409S and T411Y, and optionally further comprising any of the additional mutations disclosed herein.

[0017] In certain embodiments, the disclosure relates to recombinant antibody heavy chains or heterodimer antibodies containing the same, having mutations L368S and D399Y, and optionally further comprising any of the additional mutations disclosed herein.

[0018] In certain embodiments, the Disclosure relates to recombinant antibody heavy chains or heterodimer antibodies containing the same, having mutations D399Y and K447S, and optionally further comprising any of the additional mutations disclosed herein.

[0019] In certain embodiments, the present disclosure relates to a recombinant antibody heavy chain having mutations or a heterodimeric antibody comprising the same, comprising a first heavy chain having mutations K409S and T411Y and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y. In certain embodiments, the second heavy chain comprises mutations D399Y and K447S. In certain embodiments, the first heavy chain comprises any of the mutations disclosed herein. In certain embodiments, the second heavy chain comprises any of the mutations disclosed herein.

[0020] In certain embodiments, the present disclosure relates to a nucleic acid encoding a recombinant heavy chain disclosed herein, operably combined with a heterologous promoter.

[0021] In certain embodiments, the present disclosure relates to a vector comprising a nucleic acid encoding a recombinant heavy chain disclosed herein, operably combined with a heterologous promoter.

[0022] In certain embodiments, the present disclosure relates to a cell (e.g., a somatic cell) comprising a nucleic acid or a vector disclosed herein.

[0023] In certain embodiments, the present disclosure relates to a non-natural antibody or a heterodimeric antibody comprising a chimeric recombinant antibody heavy chain disclosed herein.

[0024] In certain embodiments, the first heavy chain of the antibody comprises mutations K409S and T411Y. In certain embodiments, the second heavy chain of the antibody comprises mutations L368S and D399Y.

[0025] In certain embodiments, the first heavy chain of the heterodimeric antibody comprises mutations K409S and T411Y. In certain embodiments, the second heavy chain of the antibody comprises mutations D399Y and K447S. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] [Figure 1]Figure 1 shows a schematic diagram of the constructs that may be formed upon co-expression of the heavy chain, light chain, and Fc plasmid. The stability of specific mutations in combYSelect1 and combYSelect2 was determined using the formation rate of Fab1Fc heterodimers, which was relatively increased by using the WT rituximab sequence and subsequently detected by MS.

[0027] [Figure 2A] Figure 2A shows data for constructs with combYSelect1 mutations. The histograms show the relative heterodimerization rates determined by intact LC / MS when some or all of the protomer A mutations are on the fragment containing Fab and Fc, and the protomer B mutations are on the fragment containing only Fc (right panel), or when the arrangement is reversed (left panel).

[0028] [Figure 2B] Figure 2B shows data for constructs with combYSelect2 mutations. The histograms show the relative heterodimerization rates determined by intact LC / MS when some or all of the protomer A mutations are on the fragment containing Fab and Fc, and the protomer C mutations are on the fragment containing only Fc (right panel), or when the arrangement is reversed (left panel).

[0029] [Figure 3A] Figure 3A shows a cross-linking assay using bispecific antibodies where the Fc of protomer A (combYSelect1 and 2) is engineered with anti-CD20 rituximab Fab, and Fc protomer B (combYSelect1) or protomer C (combYSelect2) is conjugated to an anti-HER2 nanobody. Binding to CFSE-stained Raji cells and calcein-violet BT474 cells is evaluated.

[0030] [Figure 3B]Figure 3B shows flow cytometry density and scatter plots illustrating cell cluster formation of Raji and BT474 cells when combYSelect1, 2, or a nonspecific IgG1 isotype control was added to the cell mixture, or when no antibody was added to the cell mixture. Each combYSelectIgG was compared to both the no-antibody and isotype control.

[0031] [Figure 3C] Figure 3C shows cell-based assays for combYSelect2 cytokine trapping and luciferase inhibition.

[0032] [Figure 3D] Figure 3D shows the normalized luciferase response rate as a function of varying inhibitor concentrations in the presence of 5 pM IL1β, relative to the luciferase response rate without inhibitor addition. IC50 was determined using nonlinear least squares fitting. [Modes for carrying out the invention]

[0033] Before proceeding with a more detailed description of this disclosure, please understand that this disclosure is not limited to the embodiments described and is therefore naturally subject to change. Furthermore, please understand that the scope of this disclosure is limited only by the appended claims, and that the terms used herein are intended to describe, and not limit, specific embodiments.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure, but preferred methods and materials are described herein.

[0035] All documents and patents cited herein are incorporated by reference as if each individual document or patent were explicitly and individually incorporated by reference, and are incorporated by reference herein to disclose and explain the methods and / or materials for which the documents are cited in connection. Any citation of a publication is for the purpose of indicating a disclosure prior to the filing date and should not be construed as acknowledging that this disclosure does not grant prior rights to such publication by prior disclosure. Furthermore, the dates of the publications provided may differ from the actual publication dates which may need to be independently verified.

[0036] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with features of any of several other embodiments without departing from the scope or spirit of this disclosure. Any method described may be performed in the order of events described, or in any other logically possible order.

[0037] The “embodiments” in this disclosure refer to examples, and imply that the examples are not necessarily limited to those examples. Unless otherwise expressly stated, the embodiments in this disclosure utilize techniques within the scope of the art, such as medicine, organic chemistry, biochemistry, molecular biology, and pharmacology. Such techniques are all described in the literature.

[0038] When used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context clearly indicates otherwise. In this specification and the following claims, several terms are defined as having the following meanings unless the intention to the contrary is clear.

[0039] When used in this disclosure and claims, the words “comprising” (and any form of “comprising,” such as “comprise” and “comprises”), “having” (and any form of “having,” such as “have” and “has”), “including” (and any form of “including,” such as “include” and “includes”), or “containing” (and any form of “containing,” such as “contains” and “contain”) have the meanings given to them in U.S. patent law that they are inclusive or open-ended and do not exclude additional, undescribed elements or steps of the method.

[0040] When applied to methods and compositions covered by this disclosure, "consisting essentially of" or "consists of" have the meaning given in U.S. patent law to mean that they may include additional compositional components or process steps, etc., that do not include prior art elements that are inventive features of the claims, but do not substantially affect the basic and novel features of the composition or method compared to those of the corresponding composition or method disclosed herein.

[0041] As used herein, “oxazoline” refers to a chemical group having the structure 5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diol, optionally substituted with a glycol, monosaccharide, or polysaccharide, which can be prepared according to the experimental procedure described in Noguchi et al. J. Org. Chem. 2009, 74, 2210-2212. The reaction with glycols, sugars, and polysaccharides yields N-acetyl-2-amino sugars when catalyzed by the EndoS2 D184M enzyme.

[0042] In certain contexts, “antibody” refers to a protein-based molecule that is not recognized as a “self” molecule by the animal’s immune system, i.e., a foreign molecule recognized by the animal as an antigen against the antibody. The animal’s immune system produces antibodies that specifically bind to antigens, thereby targeting the antigen for degradation or removal, or any cell or organism bound to the antigen. It is well known to those skilled in the art that the molecular structure of natural antibodies can be synthesized and modified by laboratory techniques. Recombination techniques can be used to produce fully synthetic antibodies or fragments thereof, providing control over the morphology of the amino acid sequence of the antibody. Therefore, the term “antibody” is intended to include natural antibodies, monoclonal antibodies, or synthetic antibodies that are not produced naturally, such as specifically binding single-chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibody" refers to a group of antibodies encoded by the same nucleic acid molecule, which are produced, optionally by a single hybridoma (or its clone) or other cell line, or by a transgenic mammal, so that each monoclonal antibody typically recognizes the same antigen. The term "monoclonal" is not limited to any particular method for producing antibodies, nor is it limited to antibodies produced in a specific species, such as mice or rats.

[0043] Structurally, an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. The heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence, but embodiments of this disclosure relate to antibodies having two heavy chains with dissimilar sequences or alternative mutation patterns. Similarly, the two light chains typically have the same amino acid sequence, but light chains with dissimilar sequences are also considered. Often, each of the heavy and light chains contains a variable segment containing an amino acid sequence involved in binding to an antigen. The variable segment of the heavy chain does not have the same amino acid sequence as the light chain. The variable segment is often called the antigen-binding domain. The antigen and the variable region of an antibody may physically interact with each other at a specific smaller segment of the antigen, often called an “epitope.” An epitope typically consists of a surface group of a molecule, such as amino acids or carbohydrates. The terms “variable region,” “antigen-binding domain,” and “antigen-binding region” refer to the portion of an antibody molecule containing amino acid residues that interact with the antigen and confer the antibody specificity and affinity to that antigen. A small binding region within an antigen-binding domain that typically interacts with an epitope is generally also called a "complementarity-determining region" or "CDR."

[0044] A "chimeric antibody" is a molecule in which different parts of the antibody originate from different immunoglobulin molecules, such that the entire molecule does not exist in nature. Examples of chimeric antibodies include those having a variable region derived from a non-human antibody and a human immunoglobulin constant region. This term is also intended to include antibodies having a variable region derived from one human antibody, either introduced into the immunoglobulin constant region of a given sequence, or into the constant region of any naturally occurring antibody having a variable region, e.g., CDR1, 2, and 3 of the light and heavy chains, from another human with allotypic differences present in the constant region. Human heavy chain genes exhibit structural polymorphisms (allotypes) that are inherited as haplotypes. Serologically defined allotypes differ within and between populations. Jefferis et al. mAb, 1(2009), pp. 332-338. In certain embodiments, the antibody, antibody heavy chain, antigen-binding fragment, light chain, or heavy chain contains a chimeric amino acid sequence that does not occur in nature, such that there is at least one mutation not present in the naturally occurring antibody.

[0045] Smith et al. reported a procedure for producing antigen-specific chimeric human monoclonal antibodies (hmAbs), in which antibody-secreting cells (ASCs) are isolated from whole blood collected after vaccination and sorted into single-cell plates by flow cytometry. (Nat Protoc. 2009; 4(3): 372-84.) The antibody genes from the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors, and introduced into human cell lines. Meijer et al. reported a method for isolating the human antibody repertoire while preserving native heavy and light chain pairs. (J Mol Biol, 2006, 358 (3): 764-72.) Wrammert et al. reported producing high-affinity bound human monoclonal antibodies (mAbs) using immunoglobulin variable regions isolated from sorted single ASCs. (Nature, 2008, 453 (7195): 667-671.)

[0046] Methods for producing chimeric antibodies are known in the art. See Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., 1989, J.Immunol.Methods 125:191-202; and U.S. Patents Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397. Chimeric antibodies containing one or more CDRs derived from non-human species and framework regions derived from human immunoglobulin molecules can be used, for example, for CDR grafting (European Patent No. 239,400; International Publication No. WO91 / 09967; and U.S. Patents No. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (European Patent No. 592,106; European Patent No. 519,596; Padlan, 1991, Molecular Immunology 28 (4 / 5): 489-498; Studnicka et al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA). It can be produced using various known techniques, including 91:969 and chain shuffling (U.S. Patent No. 5,565,332).

[0047] The heavy chain polypeptide sequences and antibodies containing them disclosed herein can be produced by any commonly used method. Typical examples include recombinant expression in a suitable host system, e.g., cells, mammalian cells, bacteria, or yeast. Generally, polypeptides can be produced by living host cells genetically engineered to produce polypeptides. Methods for genetically engineering cells to produce proteins are known in the art, e.g., Ausubel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods involve introducing nucleic acids that encode polypeptides and enable their expression into host cells. These host cells may be cultured bacterial cells, fungal cells, or animal cells. In one embodiment, polypeptides are produced in mammalian cells. Typical mammalian host cells for peptide expression include Chinese hamster ovary cells (CHO cells), lymphocyte cell lines, e.g., NS0 myeloma cells, SP2 cells, and COS cells.

[0048] In addition to the nucleic acid sequence encoding the peptide, recombinant expression vectors may have further sequences such as sequences that control vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patent No. 4,399,216; U.S. Patent No. 4,634,665; and U.S. Patent No. 5,179,017). Typically, for example, a selection marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced.

[0049] Using standard molecular biology techniques, recombinant expression vectors can be prepared, host cells transfected, transformants selected, host cells cultured, and peptides or peptide-coated cells recovered from the culture medium. For example, the peptides or cells can be isolated by affinity chromatography.

[0050] In certain embodiments, the disclosure relates to nucleotide sequences or nucleic acids encoding heavy chain peptides as disclosed herein, or antibodies containing such peptides, and gene constructs comprising a nucleotide sequence or nucleic acid and one or more elements for known gene constructs. In certain embodiments, the disclosure relates to a host or host cell containing such nucleotide sequences or nucleic acids and / or expressing (or capable of expressing) heavy chain peptides as disclosed herein.

[0051] In certain embodiments, the Disclosure relates to a method for preparing heavy chain peptides or antibodies containing them, or cells expressing heavy chain peptides or antibodies containing them, using the constructs disclosed herein, the method comprising culturing or maintaining host cells under conditions such that the host cells produce or express the heavy chain peptides or antibody constructs disclosed herein.

[0052] As used herein, "mutations," "variants," etc., of an antibody heavy chain sequence refer to the expression of a modified amino acid in a heavy chain antibody, defined by its position, compared to a reference amino acid in a sequence segment such as UNIPROTKB / SWISS-PROT having accession number P01857.1.

[0053] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 1). Here, the N-terminal amino acid serine (S) is at position 119. Mutants may be constructed by synthetically constructing peptide sequences, or more typically, by using recombinant nucleic acid technology, such as the expression of heavy chains from template nucleic acids in cells. Due to the three-codon translation of amino acids from nucleic acids, several sets of three nucleotide codons may express the same amino acid variant. A variant may result from a single nucleotide change, or it may result from two or more nucleotide changes. Therefore, references to "mutations," "mutants," etc., in heavy chain antibody sequences are not necessarily limited to single nucleotide changes.

[0054] As used herein, the term “cell” refers to a biological compartment including a lipid membrane and cytosol, which may include the nucleus containing genetic material, mitochondria, and other organelles.

[0055] In certain embodiments, the cells are somatic cells. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may include modified amino acids. The term also includes amino acid polymers modified naturally or by intervention by any other operation or modification, such as disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or binding with a labeling component. The definition also includes polypeptides containing, for example, one or more analogues of amino acids (including unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), and other modifications known in the art.

[0056] The term "nucleic acid" refers to a polymer of nucleotides, or polynucleotides, such as RNA, DNA, or combinations thereof. This term is used to describe a single molecule or a collection of molecules. Nucleic acids may be single-stranded or double-stranded and may contain coding regions and regions of various regulatory elements.

[0057] A “heterogeneous” nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or peptide sequence that does not exist in nature because, for example, the entire sequence contains segments derived from other plants, bacteria, viruses, or other organisms, or contains the linkage of two sequences that exist in the same organism but are linked in a manner that does not exist naturally in the same organism or in any natural state.

[0058] When the term "recombinant" is used in reference to nucleic acid molecules, it refers to nucleic acid molecules that are segments of nucleic acid linked together by molecular biological techniques so that the entire nucleic acid sequence does not exist naturally, i.e., there is at least one mutation in the entire sequence, and as a result, the entire sequence does not exist naturally, even if individual segments may exist naturally. The segments may be linked together in an altered configuration so that the entire nucleic acid sequence from start to finish does not occur naturally. When the term "recombinant" is used in reference to proteins or peptides, it refers to protein molecules expressed using recombinant nucleic acid molecules.

[0059] The terms “vector” or “expression vector” refer to recombinant nucleic acids containing a desired coding sequence and appropriate nucleic acid sequence required for the expression of an operably linked coding sequence in a specific host organism or expression system, such as a cell or cell-free expression system. Nucleic acid sequences required for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. In certain embodiments, this disclosure considers vectors encoding heavy chain peptides or antibodies containing them, operably combined with heterologous promoters.

[0060] In certain embodiments, this disclosure considers a heavy chain peptide or antibody containing such peptide, as reported herein, conjugated to a label. “Label” refers to a detectable moiety that is directly or indirectly conjugated to another molecule, such as an antibody or protein, to facilitate the detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzyme conjugations, and radioisotopes. Labels include the incorporation of radiolabeled amino acids or the covalent conjugation of a biotinyl moiety to a peptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable optically or colorimetrically). Various methods for labeling peptides and glycoproteins are known and can be used in the art. Examples of peptide labeling include, but are not limited to, radioisotopes or radionucleotides (e.g., 18 F, 35 S or 131 I) fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined peptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding regions, epitope tags), or magnetic agents (e.g., gadolinium chelates). In some embodiments, the labels are linked by spacer arms (linking groups) of varying lengths to reduce potential steric hindrance.

[0061] In certain embodiments, the disclosure relates to recombinant antibody heavy chain peptides and antibodies containing the sequence or variant or fusion thereof disclosed herein, wherein the internal amino acid sequence, amino acid terminus, or carbon terminus of the amino acid sequence is optionally conjugated to a heterologous amino acid sequence, label, or reporter molecule.

[0062] In some embodiments, this disclosure relates to recombinant vectors comprising nucleic acids encoding antibody heavy chain peptides or antibodies containing the same, as disclosed herein. In certain embodiments, the recombinant vector optionally comprises mammalian, human, insect, virus, bacterium, bacterial plasmid, yeast-associated origin of replication, or genes, such as genes or retroviral genes, or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segments, or genes selected from tat, rev, nef, vif, vpr, vpu, and vpx, or structural genes selected from gag, pol, and env. In certain embodiments, the recombinant vector may optionally include, for example, a selection marker region, a lac operon, a CMV promoter, a hybrid chicken B-actin / CMV enhancer (CAG) promoter, a tac promoter, a T7 RNA polymerase promoter, an SP6 RNA polymerase promoter, an SV40 promoter, an internal ribosome entry site (IRES) sequence, a cis-acting woodchuck post-regulatory element (WPRE), a scaffolding binding region (SAR), an inverse terminal repeat (ITR), a c-myc tag coding region, a metal affinity tag coding region, a streptavidin-binding peptide coding region, and a polyH The vector includes gene vector elements (nucleic acids) such as the is tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 replication origin, ColE1 replication origin, f1 origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or multiple clone sites having five, six, or seven or more restriction sites within a continuous segment of less than 50 or 60 nucleotides, or three or four or more restriction sites within a continuous segment of less than 20 or 30 nucleotides.

[0063] In certain embodiments, antibody heavy chain peptides or antibodies containing them are conjugated to therapeutic or cytotoxic agents for use in treating diseases or conditions such as cancer. As used herein, the term “cytotoxic agent” means a substance that inhibits or interferes with the function of cells and / or causes cell destruction. This term is intended to include radioisotopes, chemotherapeutic agents, e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and their fragments, e.g., nucleases, antibiotics, and toxins, e.g., small molecule toxins or enzymatically active toxins, e.g., small molecule toxins or enzymatically active toxins, e.g., fragments and / or variants thereof of bacteria, fungi, plants or animals, and various antitumor agents or anticancer agents or growth inhibitors. As used herein, “growth inhibitor” means a compound or composition that inhibits cell growth, either in vitro or in vivo. Examples of growth inhibitors include drugs that block cell cycle progression, such as those that induce G1 arrest and M phase arrest. Such inhibitors include, for example, vinca alkaloids (vincristine, vinorelbine, and vinblastine), paclitaxel, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, bleomycin, tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and cytarabine.

[0064] In certain embodiments, the Disclosure considers antibody heavy chains or antibodies containing such chains, in which a desired glycan is attached to a core-fucosylated or non-fucosylated GlcNAc receptor, including a fucosylated or non-fucosylated GlcNAc-IgG receptor. Accordingly, the Disclosure enables the synthesis and reconstitution of therapeutic antibodies or their Fc fragments to provide specific biological activities such as extended in vivo half-life, reduced immunogenicity, enhanced in vivo activity, increased targeting ability, and / or the ability to deliver therapeutic agents.

[0065] (Antibody heavy chain, glycosylation, and mutation) In certain embodiments, the recombinant antibody heavy chain comprises a first heavy chain and a second heavy chain having any mutation or combination of mutations disclosed herein.

[0066] In certain embodiments, the disclosure relates to recombinant antibody heavy chains having mutations K409S and T411Y, or heterodimer antibodies containing them, which optionally further include any of the further mutations disclosed herein.

[0067] In certain embodiments, the Disclosure relates to recombinant antibody heavy chains having mutations L368S and D399Y, or heterodimer antibodies containing them, which optionally further include any of the further mutations disclosed herein.

[0068] In certain embodiments, the disclosure relates to recombinant antibody heavy chains having mutations D399Y and K447S, or heterodimer antibodies containing them, which optionally further include any of the further mutations disclosed herein.

[0069] In certain embodiments, the Disclosure relates to a mutant recombinant antibody heavy chain, or a heterodimer antibody containing the same, comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y. In certain embodiments, the second heavy chain comprises mutations D399Y and K447S. In certain embodiments, the first heavy chain comprises any of the mutations disclosed herein. In certain embodiments, the second heavy chain comprises any of the mutations disclosed herein.

[0070] In certain embodiments, this disclosure relates to nucleic acids encoding recombinant heavy chains disclosed herein, operably combined with heterologous promoters.

[0071] In certain embodiments, the disclosure relates to a vector comprising nucleic acids encoding a recombinant heavy chain disclosed herein, operably combined with a heterogeneous promoter.

[0072] In certain embodiments, this disclosure relates to cells (e.g., somatic cells) containing nucleic acids or vectors disclosed herein.

[0073] In one embodiment, this disclosure relates to a non-natural or chimeric heterodimer antibody comprising a recombinant antibody heavy chain as disclosed herein.

[0074] In certain embodiments, the antibody is either a non-natural or chimeric heterodimer antibody recombinant antibody, or the first heavy chain is bound to the first glycan with Asn297(N297) and the second heavy chain is bound to the second glycan with Asn297(N297). In certain embodiments, the first and second glycans are either the same glycan or not the same glycan.

[0075] In certain embodiments, a non-natural or chimeric heterodimer antibody, or in which the first heavy chain is bound to the first glycan at Asn297 (N297) and the second heavy chain is bound to the second glycan at Asn297 (N297). In certain embodiments, the first and second glycans are the same glycan or not the same glycan. In certain embodiments, the first and second glycans are the same glycan. In certain embodiments, the heterodimer asymmetric glycosylated antibody has at least one N-acetylgluconeamine (GlcNAc) or fucosylated N-acetylglucosamine (Fucal,6GlcNAc) at, for example, N297.

[0076] In certain embodiments, the Disclosure considers single-chain antibodies having one or more of the mutations disclosed herein.

[0077] In certain embodiments, a mutation is a variant. In certain embodiments, a variant includes a conserved amino acid substitution. In certain embodiments, a variant includes a non-conserved amino acid substitution. In certain embodiments, the conserved and / or non-conserved amino acid substitutions are located within the framework region. In certain embodiments, a variant does not include substitutions within the light chain CDR1, CDR2, or CDR3.

[0078] Regarding Sequence ID No. 1, please note that it specifically pertains to IgG1. IgG2, IgG3, and IgG4 have several alternative amino acids at the same position. For example, IgG2 contains V at position 309 instead of L at position 309 of IgG1. IgG4 contains F at position 234 instead of L at position 234 of IgG1.

[0079] In certain embodiments, the disclosure relates to reported antibodies in which the constant region contains mutations that enhance ADCs by activating an immune response, increasing FcγRIIIa binding, or decreasing FcγRIIb binding; enhance ADCPs by increasing FcγRIIa binding or FcγRIIIa binding; enhance CDCs by increasing C1q binding or hexamerization; reduce effector function by aglycosylation, decreased FcγR and C1q binding, increased FcγRIIb binding, increased FcγRIIa binding, or increased co-binding due to decreased FcγRIIIa binding, and / or increase half-life. Examples of these mutations are further provided below.

[0080] In certain embodiments, the disclosure considers that the heavy chains comprise at least one mutation and that the two heavy chains are not identical. In certain embodiments, the disclosure considers that one heavy chain may have a different mutation than the other heavy chain, i.e., one of the two heavy chains has a mutation that the other sequence does not have, or one of the two heavy chains has one or more mutations and the other heavy chain has a different mutation.

[0081] (Antibodies, bispecific antibodies, and further mutations) In certain embodiments, heterodimeric heavy chains containing the mutations disclosed herein are used to produce antibodies having binding specificity to a therapeutic target or bispecific antibodies. In certain embodiments, heavy chain and light chain variable sequences are combined, bound, or ligated with the heavy chain mutations, constant regions / sequences disclosed herein. In certain embodiments, a first heavy-light chain pair having a first specificity and a second heavy-light chain pair having a second specificity different from the first specificity are produced separately and then mixed to form a heavy-heavy chain pair having the mutation / mutation combinations disclosed herein.

[0082] In certain embodiments, the heterodimer antibody comprises a recombinant antibody heavy chain as disclosed herein.

[0083] In certain embodiments, an antibody heavy chain having the mutations disclosed herein preferentially forms a heterodimer with a second heavy chain having the mutation, such that the second set of mutations is not present in the first chain.

[0084] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y.

[0085] In certain embodiments, the first or second heavy chain further has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the following mutations: G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, L328R, L234A, L235A, M252Y, S254T, T256E, M428L, N434S, P329G, D265A, N297A, N297G, N297Q, F243L, R292P, Y300L, V305I, P396L, S298A, E333A, K3 34A, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, K326W, E333S, E345R, E430G, S440Y, L235E, N325S, where the mutation refers to the amino acid sequence (Sequence ID 1) (segment of UNIPROTKB / SWISS-PROT:P01857.1), where the N-terminal amino acid serine (S) is at position 119.

[0086] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations L234A or L235A, or both.

[0087] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations L234A, L235A, or P329G, or all of these, or a combination thereof.

[0088] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations D265A or N297A, or both.

[0089] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations D265A or N297G, or both.

[0090] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations D265A or N297Q, or both.

[0091] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations M252Y, S254T, or T256E, or all of these, or a combination thereof.

[0092] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations F243L, R292P, Y300L, V305I, or P396L, or all of these, or a combination thereof.

[0093] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations S239D or I332E, or both.

[0094] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations S239D, I332E, or A330L, or all of these, or a combination thereof.

[0095] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations S239D, I332E, G236A, or A330L, or all of these, or a combination thereof.

[0096] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations S298A, E333A, or K334A, or all of these, or a combination thereof.

[0097] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations S298A, E333A, or K334A, or all of these, or a combination thereof.

[0098] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations L234Y, L235Q, G236W, S239M, H268D, D270E, or S298A, or all of these, or a combination thereof.

[0099] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations D270E, K326D, A330M, or K334E, or all of these, or a combination thereof.

[0100] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations G236A, S239D, or I332E, or all of these, or a combination thereof.

[0101] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations K326W or E333S, or both.

[0102] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations E345R, E430G, or S440Y, or all of these, or a combination thereof.

[0103] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations E345R, E430G, or S440Y, or all of these, or a combination thereof.

[0104] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations S267E or L328F, or both.

[0105] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations S267E, L328F, or P238D, or all of these, or a combination thereof.

[0106] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations N325S or L328F, or both.

[0107] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations S267E, H268F, or S324T, or all of these, or a combination thereof.

[0108] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further has mutations M428L or N434S, or both.

[0109] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations L368S and D399Y. In certain embodiments, the first or second heavy chain further comprises mutations M252Y, S254T, T256E, M428L, or N434S, or all of these, or a combination thereof.

[0110] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further comprises mutations I253A, H310A, Q311A, N315A, E430A, or H435A, or all of these, or a combination thereof.

[0111] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more. G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, L328R, L234A, L235A, M252Y, S254T , T256E, M428L, N434S, P329G, D265A, N297A, N297G, N297Q, F243L, R292P, Y300L, V305I, P396L, S298A, E333A, K334A L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E, K326W, E333S, E345R, E430G, S440Y, L235E, N325S, where the mutations refer to the position in the amino acid sequence (Sequence ID 1) (segment of UNIPROTKB / SWISS-PROT:P01857.1), where the N-terminal amino acid serine (S) is at position 119.

[0112] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations L234A or L235A, or both.

[0113] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain and a second heavy chain having mutations K409S and T411Y. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations L234A, L235A, or P329G, or all of these, or a combination thereof.

[0114] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations D265A or N297A, or both.

[0115] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations D265A or N297G, or both.

[0116] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations D265A or N297Q, or both.

[0117] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations M252Y, S254T, or T256E, or all of these, or a combination thereof.

[0118] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further comprises mutations F243L, R292P, Y300L, V305I, or P396L, or all of these, or a combination thereof.

[0119] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations S239D or I332E, or both.

[0120] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain comprises mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further comprises mutations S239D, I332E, or A330L, or all of these, or a combination thereof.

[0121] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations S239D, I332E, G236A, or A330L, or all of these, or a combination thereof.

[0122] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations S298A, E333A, or K334A, or all of these, or a combination thereof.

[0123] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations S298A, E333A, or K334A, or all of these, or a combination thereof.

[0124] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain and a second heavy chain having mutations K409S and T411Y. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations L234Y, L235Q, G236W, S239M, H268D, D270E, or S298A, or all of these, or a combination thereof.

[0125] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further comprises mutations D270E, K326D, A330M, or K334E, or all of these, or a combination thereof.

[0126] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations G236A, S239D, or I332E, or all of these, or a combination thereof.

[0127] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations K326W or E333S, or both.

[0128] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations E345R, E430G, or S440Y, or all of these, or a combination thereof.

[0129] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations E345R, E430G, or S440Y, or all of these, or a combination thereof.

[0130] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain and a second heavy chain having mutations K409S and T411Y. In certain embodiments, the second heavy chain comprises mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further comprises mutations S267E or L328F, or both.

[0131] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations S267E, L328F, or P238D, or all of these, or a combination thereof.

[0132] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations N325S or L328F, or both.

[0133] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations S267E, H268F, or S324T, or all of these, or a combination thereof.

[0134] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further has mutations M428L or N434S, or both.

[0135] In certain embodiments, the disclosure relates to a mutant heterodimer antibody heavy chain or an antibody comprising a first heavy chain having mutations K409S and T411Y, and a second heavy chain. In certain embodiments, the second heavy chain has mutations D399Y and K447S. In certain embodiments, the first or second heavy chain further comprises mutations M252Y, S254T, T256E, or M428L, N434S, or all of these, or a combination thereof.

[0136] (Heterodimer IgG molecule) Mutations were introduced in each of the two IgG heavy chain protomers to promote the binding of distinct analogous protomers, thereby resulting in heterodimerization of the IgG heavy chain. The IgG heterodimer may be used therapeutically, for example, in bispecific antibodies containing two distinct antibody-binding fragment (Fab) molecules that recognize distinct antigens.

[0137] We investigated mutations that produce heterodimers of the IgG / Fc interface. We used an in silico approach, mutating the CH3-CH3 interface residues of IgG to either tyrosine (Y) or serine (S), and estimating the change in binding energy (ΔΔG) using the "interface" mode of Rosetta 2.3. For screening, we selected residues within the CH3 domain located 4 angstroms from the opposite CH3 domain. A total of 31 residues were identified: 347Q, 349Y, 350T, 351L, 352P, 353P, 354S, 355R, 356D, 357E, 360K, 364S, 366T, 368L, 370K, 390N, 392K, 393T, 394T, 395P, 397V, 398L, 399D, 400S, 405F, 406L, 407Y, 409K, 411T, 444S, and 447K. In silico screening was performed by introducing up to two mutations into each strand.

[0138] (Evaluation of IgG heterodimer formation) Results were filtered based on scores where the heterodimer's ΔΔG was -0.8 kcal / mol or less and the homodimer's ΔΔG was 0 kcal / mol or greater. From this selection, two sets of mutations were chosen for experimental evaluation. To assess the effectiveness of these mutations in heterodimerization, mutations were introduced into the rituximab heavy chain (Hc) and Fc constructs. The constructs were then co-transfected into HEK293F cells along with the rituximab light chain (Lc). In addition to the identified mutants, mutations in knobs-in-holes and electrostatic steering strategies were also tested.

[0139] Mutations are defined by the position of the base amino acid within the sequence segment of UNIPROTKB / SWISS-PROT having accession number P01857.1 compared to the original position. STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 1), where the N-terminal amino acid serine (S) is at position 119.

[0140] (IgG heterodimers exhibit an improved heterodimerization rate without affecting the stability or function of IgG.) The heterodimers combYSelect1IgG (L368S / D399Y-K409S / T411Y) and combYSelect2IgG (D399Y / K447S-K409S / T411Y) were designed as bispecific antibodies and cytokine traps to evaluate their therapeutic applications. The heterodimers L368S / D399Y-K409S / T411Y, D399Y / T411Y-K409S / T411Y, and D399Y / K447S-K409S / T411Y were identified as having the desired stability using computer modeling.

[0141] As a model IgG1 antibody, we developed a model system that involves co-expressing the IgG1Fc region and full-length heavy chain (Hc) of rituximab with the light chain (Lc) of rituximab from separate protomers in heterodimer design. After purification by protein A affinity chromatography and treatment with IgG-specific endoglycosidase EndoS2 to remove heterogeneous glycans at residue Asn297, three possible co-expression products with unique molecular weights, detectable by liquid chromatography-mass spectrometry (LC-MS), include the intact Fc region (approximately 50 kDa), intact IgG (approximately 150 kDa), and a monovalent IgG molecule (Fab1Fc; approximately 100 kDa) (Figure 1). The formation of the intact Fc region and the IgG antibody each arises from a pair of two possible homodimers, while the formation of the Fab1Fc molecule arises strictly from heterodimerization. Therefore, the ratio of Fab1Fc to Fc and IgG is a direct measure of heterodimer formation.

[0142] Rituximab was evaluated using this assay. 63% Fab1Fc formation was observed. For the knob-in-hole (KiH) heterodimer (T366Y-Y407T), i.e., Ridgway et al., 1996, no heterodimerization was detected when the Hc containing the T366Y mutation was co-expressed with the Fc containing the Y407T mutation along with the light chain. However, when the Y407T mutation was on the heavy chain and the T366Y mutation was on the Fc, 57% Fab1Fc formation was observed. The electrostatic steering heterodimers tested resulted in 91% Fab1Fc formation, regardless of which chain contained each set of mutations. Compared to the control group above, 97% Fab1Fc formation was observed for both mutation-chain combinations for combYSelect1 having L368S / D399Y-K409S / T411Y. Similarly, combYSelect2D399Y / K447S-K409S / T411Y resulted in 96% Fab1Fc formation when the D399Y / K447S mutation was present in Hc, and 91% when the set of mutations was present in Fc. These results indicate that the heterodimer surpasses those of other previously reported heterodimers tested.

[0143] Table 1 provides a summary of the experimental results. [Table 1]

[0144] (Heterodimer mutations do not affect IgG stability or FcRn binding.) In addition to promoting heterodimerization, experiments were conducted to evaluate whether mutations introduced into the CH3 region affect the functional properties of the IgG molecule. The thermal stability of each construct was assessed by determining the melting curve of the resulting Fab1Fc heterodimer antibody (i.e., the product resulting from the co-expression of the heavy chain, light chain, and Fc fragment). Rituximab had three transition temperatures corresponding to the unfolding of the CH2, Fab, and CH3 domains, respectively: 74.9°C, 81.8°C, and 90°C. Furthermore, the nearly identical transition temperatures between rituximab IgG and rituximab in the Fab1Fc form confirmed that the thermal stability of the latter represents full-length IgG. In contrast, the knob-in-hole and electrostatic steering constructs, all in the Fab1Fc form, showed only two transition temperatures corresponding to the CH2 and Fab regions. The first transition temperature was 72.2°C to 74.8°C, and the second transition temperature was 80.8°C to 81.7°C. When the melting temperatures of combYSelect1 and 2Fc were evaluated, only one melting temperature of approximately 74°C was observed, which typically corresponds to the CH2 domain. This suggests that the CH3 region of the heterodimer either unfolds at the same temperature as the CH2 region, or undergoes reversible unfolding within the tested temperature range.

[0145] The potential impact of these mutations on the overall structure of the CH2-CH3 interface was also evaluated. This region is of particular interest because it is where the neonatal Fc receptor (FcRn) binds in a pH-dependent manner to recycle endogenous IgG and extend their serum half-lives. Using biolayer interferometry (BLI) analysis, the FcRn binding affinity of rituximab was compared with combYSelect1 and 2IgGs containing the rituximab Fab domain. Rituximab, combYSelect1, and combYSelect2 yielded KD values ​​of 14.3, 17.0, and 8.0 nM, respectively, at pH 6. These IgGs showed minimal binding to FcRn at pH 7.4.

[0146] (Mutations for optimal heterodimerization) To evaluate heterodimerization, LC / MS was used to determine whether the mutations were essential for optimal heterodimerization. The L368S mutation was unnecessary when the D399Y mutation was on the Fc and the protomer A mutation was present on the heavy chain. The 96% heterodimerization observed for K409S / T411Y-D399Y was not significantly different from that of combYSelect1. However, when the D399Y mutation was on the Fc fragment bound to Fab, no detectable heterodimerization was observed, suggesting that L368S may play a role in heavy chain stabilization (Figure 2A). On the other hand, 96% and 94% Fab1Fc formation were observed for the K409S / T411Y-D399Y and K409S / T411Y-K447SHc-Fc constructs, respectively, indicating improved heterodimerization compared to combYSelect2. However, when the individual mutations D399Y and K447S were present in the heavy chain, they were required for heterodimerization (Figure 2B). Finally, constructs with a single mutation in each chain in all four possible combinations: K409S-D399Y, T411Y-D399Y, K409S-L368S, and T411Y-L368S in combYSelect1, and K409S-D399Y, T411Y-D399Y, K409S-K447S, and T411Y-K447S in combYSelect2, significantly reduced heterodimerization regardless of mutation-chain pairing (Figures 2A-2B).

[0147] (IgG1 predicted by combYSelect) Heterodimers, particularly IgG heterodimers with therapeutic applications, are often used for the development of bispecific monoclonal antibodies and Fc fusion proteins exhibiting unique and / or improved therapeutic properties. To confirm the broad potential of combYSelect heterodimers for potential therapeutic applications, combYSelect-based bispecific antibodies and cytokine traps were designed and their ability to simultaneously bind to two target cell lines expressing distinct antigens and inhibit cytokine-mediated cellular signaling was tested.

[0148] The bispecific antibodies were designed so that one arm consisted of rituximab anti-CD20Fab and the other arm was a HER2-specific nanobody (Figure 3A). Both bispecific combYSelect heterodimers could simultaneously bind to the two antigens expressed on Raji(CD20+) and BT474(HER2+) cells, as demonstrated by the formation of cell clusters that were positive for the individual dyes used to stain each of the two cell types. This was true regardless of the antigen specificity of each protomer in versions of these bispecific antibodies where the anti-CD20Fab and anti-HER2 nanobody were fused to the opposite Fc. Conversely, no cell cross-linking clusters were formed when using nonspecific IgG1 isotype controls or any single-specific heterodimer or homodimer.

[0149] IgG heterodimers are also commonly used in Fc fusion-based therapies. Therefore, in a second potential therapeutic scenario, combYselect2Fc was used to construct a cytokine trap that captures IL-1β, composed of the IL-1 cytokine complementary receptor IL-1 receptor I (IL-1RI) and the secondary receptor IL-1 receptor accessory protein (IL-1RAcP) (Figure 3C). The ability of the combYSelect2 cytokine trap to sequester IL-1β was measured by inhibiting luciferase expression driven by the IL-8 promoter in HEK293T cells endogenously expressing IL-1RI and IL-1RAcP. Inhibition by the combYSelect2 trap was equivalent to inhibition by a trap fused to the electrostatic heterodimer E357Q / S364K–L368D / K370S. Both traps exhibited slightly improved inhibition compared to IL-1 receptor antagonists (IL-1Ra; Figure 3D), which are natural antagonists of IL-1 cytokine signaling, and whose recombinant form is anakinra, used to treat rheumatoid arthritis and other chronic inflammatory conditions.

[0150] The combYSelect method is useful for redesigning the interface between CH3 regions on any of the IgG1Fc homodimers and aims to develop novel heterodimeric Fc regions that can function as a platform for bispecific antibodies (bsAbs). Generally, bsAbs are designed to simultaneously target T cell-specific antigens and tumor-associated antigens (TAAs), cross-linking the two cell types together and enhancing the immune response against tumor cells.

[0151] Another common use of bsAbs is to simultaneously target two different TAAs, which improves specificity and reduces the likelihood of tumors escaping by downregulating specific epitopes or returning to redundant pathways.

[0152] In certain embodiments, the disclosure considers IgG-like bsAbs that retain a longer serum half-life due to physiological binding to FcRn compared to non-IgG-like bsAbs lacking the Fc region. It is considered that combYSelect facilitates the specific association between the light chain and its correct heavy chain pair, enabling the production of bsAbs that most closely approximate the functionality, stability, and long half-life of wild-type antibodies while retaining the advantages of biantigen specificity.

[0153] Given that the combYSelect antibodies disclosed herein exhibit conformational and homodimer interfaces similar to IgGFcs for other antibody isotypes, it is considered that they may be used as a platform for designing bsAbs of other antibody isotypes using alternative isotypes, such as IgA and IgE. Since monomeric IgA demonstrated efficacy in preclinical models, attempts at protein and glycosylation have improved the productivity, stability, and half-life of IgA. IgA possesses inherent properties, including its ability to bind to neutrophils highly expressing FcαRI, making it potentially effective as an antitumor agent. Furthermore, IgA exhibits increased stability on mucosal surfaces compared to IgG, improving the potential of mAbs directed towards lung or gastrointestinal inflammation. Similarly, monoclonal IgE binds with extremely high affinity to FcεRI expressed on tumor-associated macrophages, potentially improving molecular allergy diagnostics. Therefore, manipulating bispecific IgA and IgE antibodies contributes to improving the clinical efficacy of such mAbs.

[0154] In certain embodiments, the Disclosure considers heterodimer antibodies containing the heavy chain mutation patterns disclosed herein, for example, bispecific antibodies. In certain embodiments, the bispecific antibody specifically binds to a T cell-specific antigen. In certain embodiments, the T cell-specific antigens are CD3, CD4, CD8, CXCR3, CCR4, CD4, CD25, CD127, and / or CD152.

[0155] In certain embodiments, a bispecific antibody specifically binds to tumor-associated antigens (TAAs). In certain embodiments, the TAAs include differentiation cluster 19 (CD19), differentiation cluster 10 (CD10), differentiation cluster 20 (CD20), differentiation cluster 33 (CD33), differentiation cluster 38 (CD38), CD70 (tumor necrosis factor ligand superfamily member 7), CD133 (prominin 1), CD171 (L1 cell adhesion molecule), (EGFR) epidermal growth factor receptor, (HER2) human epidermal growth factor receptor 2, and EGFRvIII (epidermal growth factor receptor variant). (Ant3) (MUC1) Mucin 1, (MUC16) Mucin 16, (EpCAM) Epithelial cell adhesion molecule, (AFP) α-fetoprotein, (FAP) Familial adenomatous polyposis, (CEA) Carcinoembryonic antigen, (PSCA) Prostate stem cell antigen, (PSMA) Prostate-specific membrane antigen, (PSA) Prostate-specific antigen, (AXL) AXL receptor tyrosine kinase, (DLL3) Delta-like 3, (EPHA2) EPH receptor A2, (FRα) Folate receptor α, (LMP1) Epstein-Barr virus Latent membrane protein 1, MAGE-A1, MAGE-A3, MAGE-A4, (DR5) Death receptor 5, (NKG2D) Natural killer group 2 member D receptor, (CAIX) Carbonic anhydrase IX, (TAG-72) Tumor-associated glycoprotein 72, (GUCY2C) Guanylate cyclase 2C, (ANTXR1) Anthrax toxin receptor 1, (GSPG4) Systemic secretory pathway protein G, (ROR) RAR-related rare receptor, ROR1 (receptor tyrosine kinase-like rare receptor 1), IL13RA2 (interleukin 13 receptor subunit α2), Wilms tumor 1 (WT1), Survivin, Tn(aGalNAc-O-Ser / Thr), Sialyl-Tn(aNeuAc2,6-αGalNAc-O-Ser / Thr), TF(bGal1,3-αGalNAc-O-Ser / Thr), CA19-9(Neu5Acα2-3Galβ1-3[Fucα1-4]GlcNAcβ), telomerase reverse transcriptase (TERT), β-hCG (human chorionic gonadotropin), p53, Ras, bladder tumor antigen (BTA), antibody-specific antigen Om5, GD2 (ganglioside GD2), integrin α-v / β-6, or mesothelin antigen, BCMA (TNF receptor superfamily member 17\B cell maturation protein), CD123 (interleukin 3 receptor subunit α\CD123 antigen), CD138 (syndecane 1), These include CD22 (SIGLEC2), CD5 (lymphocyte antigen T1 / Leu-1), Igκ chain, LeY (fucosyltransferase 3 / Lewis blood group), NKG2D ligand (killer cell lectin-like receptor K1 / CD314), WT1 (Wilms tumor antigen 1), C-Met (MET proto-oncogene), CAIX (carbonic anhydrase 9), GPC3 (glypican 3), HPV16-E6 (human papillomavirus E6 protein), MART1 (Melan-A), NY-ESO-1 (cancer / testicular antigen 1B), PD-L1 (CD274 molecule), PSMA (folate hydrolase 1), or VEGFR2 (kinase insertion region receptor / vascular endothelial growth factor receptor 2).

[0156] In certain embodiments, the Disclosure considers a method of treating a disease or condition using a heterodimer antibody containing the heavy chain mutation pattern disclosed herein, by administering an effective amount of the heterodimer antibody to a subject in need thereof. In certain embodiments, the subject is at risk of, exhibits symptoms of, or has been diagnosed with, cancer, autoimmune disorder, cardiovascular disease, diabetes, respiratory disease, or inflammatory disease.

Claims

1. Recombinant antibody having heavy chains containing mutations K409S and T411Y.

2. The recombinant antibody according to claim 1, having a second heavy chain having mutations L368S and D399Y.

3. The recombinant antibody according to claim 1, having a second heavy chain containing mutations D399Y and K447S.

4. Recombinant antibody having heavy chains containing mutations L368S and D399Y.

5. The recombinant antibody according to claim 4, having a second heavy chain containing mutations K409S and T411Y.

6. Recombinant antibody having heavy chains containing mutations D399Y and K447S.

7. The recombinant antibody according to claim 6, having a second heavy chain containing mutations K409S and T411Y.

8. A heterodimer antibody comprising a first heavy chain containing mutations K409S and T411Y, and a second heavy chain.

9. The heterodimer antibody according to claim 8, wherein the second heavy chain comprises mutants L368S and D399Y.

10. The heterodimer antibody according to claim 8, wherein the second heavy chain comprises mutants D399Y and K447S.

11. A nucleic acid encoding a recombinant heavy chain according to any one of claims 1 to 10, operably combined with a heterologous promoter.

12. A vector comprising the nucleic acid described in claim 11.

13. A cell comprising the nucleic acid described in claim 11 or the vector described in claim 12.

14. A heterodimer antibody comprising a recombinant antibody heavy chain according to any one of claims 8 to 10.

15. A heterodimer antibody according to claim 14, which is a bispecific antibody.

16. A bispecific antibody according to claim 15, which specifically binds to a T cell-specific antigen.

17. A bispecific antibody according to claim 15, which specifically binds to tumor-associated antigens.

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