Heterodimer antibodies, uses in therapeutic applications and compositions related thereof

Description

1 / 55 “HETERODIMERIC ANTIBODIES, USES IN THERAPEUTIC APPLICATIONS AND RELATED COMPOSITIONS THEREOF CROSS-REFERENCE TO RELATED REQUESTS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 461,078 filed April 21, 2023, and U.S. Provisional Application No. 63 / 578,789 filed August 25, 2023. The entirety of each of these applications is incorporated herein by reference for all purposes. DECLARATION RELATED TO RESEARCH OR DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT

[0002] This invention was made with the support of the government under AI149297 granted by the National Institute of Health. The government has certain rights in the invention. Incorporation by reference of material submitted as an XML file through the office's electronic filing system.

[0003] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference in the descriptive report. The name of the XML file containing the Sequence Listing is 23010PCT.xml. The XML file is 3 KB in size, was created on April 19, 2024, and is being submitted electronically via the USPTO Electronic Patent Filing System. FUNDAMENTALS

[0004] Therapeutic antibodies constitute an important class of drugs for the treatment of a wide range of diseases, such as cancer. Their ability to recruit Petition 870250094951, dated 10 / 17 / 2025, page 8 / 77 2 / 55 and stimulate immune system cells, which are often linked to their clinical efficacy, especially in the immunotherapeutic treatment of cancer, are housed in the constant domains of the heavy chain (Fc domains). Fc domains interact with Fc receptors and complement in order to induce antibody-mediated effector functions that direct cell death in vivo. Although antibody therapies are used successfully in cancer treatment, clinical resistance and disease refractory to these agents remain a significant issue, as cancer recurrence is not uncommon. Thus, there is a need to identify improvements.

[0005] Ridgway et al. report 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996, 9(7):617-621.

[0006] Ha et al. report an Fc heterodimer immunoglobulin platform. Front Immunol, 2016, 7:394.

[0007] Mimura et al. report glycosylation engineering of therapeutic IgG antibodies. Protein Cell 2018, 9(1):47-62.

[0008] Li et al. report that endoglucosidase S2 glucosynthase mutants show transglycosylation activity. J Biol Chem, 2016, 291(32):16508-18.

[0009] Moore et al. report a genetically engineered heterodimeric Fc platform for the efficient development of bispecific antibodies. Methods, 2019, 154: 3850.

[0010] Du et al. report mechanisms and Petition 870250094951, dated 10 / 17 / 2025, page 9 / 77 3 / 55 Specificities of endoglycosidases active against IgG. Glycobiology, 2020, 30(4): 268-279.

[0011] Klontz et al. report a highly efficient α-fucosidase for IgG transfucosylation. Nat Comm, 2020, 11:620.

[0012] The references cited in this document are not an admission of the state of the art. SUMMARY

[0013] This disclosure relates to mutant heavy chain antibody sequences. In certain embodiments, this disclosure covers heterodimeric antibodies with 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 in constructing stabilized antibodies useful in therapeutic applications.

[0014] In certain embodiments, stabilized heterodimeric antibodies allow selective chain synthesis or cleavage of glycosylated substituents, providing asymmetrically glycosylated antibody constructs.

[0015] In certain embodiments, a heterodimeric antibody comprises a first heavy chain with a mutation or combination of mutations disclosed in this document and a second heavy chain.

[0016] In certain embodiments, this disclosure refers to a recombinant antibody heavy chain, or heterodimeric antibody containing the same, with K409S and T411Y mutations, additionally optionally comprising any Petition 870250094951, dated 10 / 17 / 2025, p. 10 / 77 4 / 55 one of the additional mutations disclosed in this document.

[0017] In certain embodiments, this disclosure refers to a recombinant antibody heavy chain, or heterodimeric antibody containing the same, with L368S and D399Y mutations, optionally additionally comprising any of the additional mutations disclosed in this document.

[0018] In certain embodiments, this disclosure refers to a recombinant antibody heavy chain, or heterodimeric antibody containing the same, with D399Y and K447S mutations, optionally additionally comprising any of the additional mutations disclosed in this document.

[0019] In certain embodiments, this disclosure refers to a recombinant antibody heavy chain, or heterodimeric antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first heavy chain comprises any of the mutations disclosed in this document. In certain embodiments, the second heavy chain comprises any of the mutations disclosed in this document.

[0020] In certain embodiments, this disclosure refers to a nucleic acid encoding a recombinant heavy chain, as disclosed in this document, in operational combination with a heterologous promoter.

[0021] In certain modalities, this disclosure is Petition 870250094951, dated 10 / 17 / 2025, p. 11 / 77 5 / 55 refers to a vector comprising a nucleic acid encoding the recombinant heavy chain, as disclosed in this document, in operable combination with a heterologous promoter.

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

[0023] In certain embodiments, this disclosure refers to a heterodimeric or unnaturally occurring antibody comprising chimeric recombinant antibody heavy chain(s), as disclosed in this document.

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

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

[0026] Figure 1 shows a diagram of the potential constructs formed after co-expression of heavy chain, light chain, and Fc plasmids. The relative percent increase in the formation of the Fab1Fc heterodimer using rituximab WT sequences, subsequently detected by MS, was used to determine the stability of specific mutations in combYSelect 1 and combYSelect 2.

[0027] Figure 2A shows data for constructs Petition 870250094951, dated 10 / 17 / 2025, page 12 / 77 6 / 55 with combYSelect mutations 1. The histograms indicate the relative percentage of heterodimerization as determined by intact LC / MS when some or all of the Protomer A mutations are in the fragment containing a Fab and Fc and the Protomer B mutations are in the fragment containing only an Fc (right) or when the orientation is reversed (left).

[0028] Figure 2B shows data for constructs with combYSelect 2 mutations. The histograms indicate the relative percentage of heterodimerization as determined by intact LC / MS when some or all Protomer A mutations are in the fragment containing a Fab and Fc and Protomer C mutations are in the fragment containing only an Fc (right) or when the orientation is reversed (left).

[0029] Figure 3A illustrates the bridging assay using a bispecific antibody in which the Fc of Protomer A (combYSelect 1 & 2) is genetically engineered with an anti-CD20 rituximab Fab and Protomer B (combYSelect 1) or Protomer C (combYSelect 2) of the Fc is linked to an anti-HER2 nanobody. Binding to CFSE-stained Raji cells and Calcein-Violet BT474 cells is evaluated.

[0030] Figure 3B shows flow cytometry density plot data and a scatter plot representing cell cluster formations of Raji and BT474 cells when combYSelect 1, 2, a non-specific IgG1 isotype control, or no antibody was added to the cell mix. Each IgG combYSelect was compared to both no antibody and the isotype control.

[0031] Figure 3C illustrates a trap of Petition 870250094951, dated 10 / 17 / 2025, page 13 / 77 7 / 55 cytokine combYSelect 2 and the cell-based luciferase inhibition assay.

[0032] Figure 3D shows data on the percentage of luciferase response normalized to that when no inhibitor is added, which is plotted as a function of the varying inhibitor concentration in the presence of 5 pM IL1p. IC50 values ​​were determined using a nonlinear least squares fit. DETAILED DESCRIPTION

[0033] Before the present disclosure is described in more detail, it should be understood that this disclosure is not limited to the embodiments described, and as such may, of course, vary. It should also be understood that the terminology used in this document is to describe particular embodiments only, and is not intended to be limiting, since the scope of this disclosure will be limited only by the appended claims.

[0034] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as commonly understood by one skilled in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described in this document may also be used in the practice or testing of the present disclosure, preferred methods and materials are now described.

[0035] All publications and patents cited in this descriptive report are incorporated herein by reference, as if each individual publication or patent were included herein. Petition 870250094951, dated 10 / 17 / 2025, p. 14 / 77 8 / 55 were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in combination with which the publications are cited. The citation of any publication is for its disclosure prior to the date of deposit and should not be construed as an admission that such disclosure does not have the right to precede such publication from the earlier disclosure. Furthermore, the publication dates provided could be different from the actual publication dates, which may need to be independently verified.

[0036] As will be evident to those skilled in the art upon reading this disclosure, each of the individual modalities described and illustrated herein has discrete components and features that can be easily separated from or combined with the features of any of the other various modalities without departing from the scope or spirit of the present disclosure. Any method cited can be performed in the order of events cited or in any other order that is logically possible.

[0037] One embodiment of this disclosure refers to an example and implies that the example is not necessarily limited to the example. The embodiments of this disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology and the like, which are within the skills of the artist. These techniques are fully explained in the literature. Petition 870250094951, dated 10 / 17 / 2025, p. 15 / 77 9 / 55

[0038] It should be noted that, as used in the descriptive report and the appended claims, the singular forms *um uma* and *o, a* include plural referents, unless the context clearly indicates otherwise. In this descriptive report and the claims that follow, reference will be made to numerous terms which will be defined to have the following meanings, unless a contrary intention is apparent.

[0039] As used in this disclosure and in the claim(s), the words comprising (and any form of comprising, such as comprises and comprise), having (and any form of having, such as has and have), including (and any form of including, such as includes and include) or containing (and any form of containing, such as contains and contain) have the meaning ascribed to them in U.S. patent law, to the extent that they are inclusive or open-ended and do not exclude additional elements or steps of method not explicitly stated.

[0040] Consisting essentially of or consisting of or similar, they have the meaning ascribed to them in U.S. patent law in which, when applied to methods and compositions covered by this disclosure, it refers to the idea of ​​excluding certain elements of the prior art as an inventive feature of a claim, but which may contain additional compositional components or method steps, etc., that do not materially affect the basic and novel feature(s) of the compositions or methods, compared with those of the compositions or methods. Petition 870250094951, dated 10 / 17 / 2025, p. 16 / 77 10 / 55 corresponding figures are published in this document.

[0041] As used in this document, an oxazoline refers to a chemical group with a structure of 5-(hydroxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2d]oxazol-6,7-diol optionally substituted with a glycol, saccharide, or polysaccharide, which can be prepared by the procedures presented in Noguchi et al. J. Org. Chem. 2009, 74, 2210-2212. Reactions with glycols, saccharides, and polysaccharides result in N-acetyl-2-amino sugars when catalyzed by the enzyme EndoS2 D184M.

[0042] In certain contexts, an antibody refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule, or that is not recognized by the animal's immune system as self, i.e., recognized by the animal as a foreign molecule, i.e., an antigen to the antibody. The animal's immune system will create an antibody to bind specifically to the antigen and thus direct the antigen for degradation or elimination, or any cell or organism bound to the antigen. It is well recognized by those skilled in the art that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof, providing control over variations in the antibody's amino acid sequences.Thus, the term antibody is intended to include natural antibodies, monoclonal antibodies, or synthetic antibodies produced non-naturally, such as antibodies. Petition 870250094951, dated 10 / 17 / 2025, p. 17 / 77 11 / 55 of specific single-stranded binding, bispecific antibodies or fragments thereof. These antibodies may have chemical modifications. The term monoclonal antibodies refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or another cell line, or by a transgenic mammal, such that each monoclonal antibody will typically recognize the same antigen. The term monoclonal is not limited to any particular method for producing the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

[0043] From a structural point of view, 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; however, the modalities of this disclosure are directed at antibodies that have two heavy chains that are different sequences or have alternative mutation patterns. Similarly, the two light chains typically have the same amino acid sequence; however, light chains with different sequences are contemplated. Frequently, each of the heavy and light chains contains a variable segment that contains amino acid sequences that participate in antigen binding. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. Variable segments are often called antigen-binding domains. The Petition 870250094951, dated 10 / 17 / 2025, page 18 / 77 12 / 55 The antigen and the antibody's variable regions can physically interact with each other in smaller, antigen-specific segments often called the epitope. Epitopes generally consist of surface clusters of molecules, for example, amino acids or carbohydrates. The terms variable region, antigen-binding domain, and antigen-binding region refer to the portion of the antibody molecule that contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigen-binding domain that typically interact with the epitope are also commonly referred to alternatively as complementarity-determining regions, or CDRs.

[0044] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such that the entire molecule does not occur naturally. Examples of chimeric antibodies include those with a variable region derived from a non-human antibody and a constant region from human immunoglobulin. The term is also intended to include antibodies with a variable region derived from a human antibody grafted onto a constant region of immunoglobulin of predetermined sequences or the constant region of another human for which there are allotypic differences residing in the constant regions of any naturally occurring antibody with the variable regions, for example, CDRs 1, 2 and 3 of the light and heavy chain. Human heavy chain genes exhibit structural polymorphism (allotypes) that are inherited as a Petition 870250094951, dated 10 / 17 / 2025, page 19 / 77 13 / 55 haplotype. Serologically defined allotypes differ within and between population groups. See 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 comprise a non-naturally occurring chimeric amino acid sequence, such that there is at least one mutation that is not present in naturally occurring antibodies.

[0045] Smith et al. report a protocol for the production of antigen-specific chimeric human monoclonal antibodies (hmAbs) in which antibody-secreting cells (ASCs) are isolated from whole blood collected after vaccination and sorted by flow cytometry in single-cell plates. 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 transfected into a human cell line. Meijer et al. report methods for isolating human antibody repertoires with preservation of natural heavy and light chain pairing. J Mol Biol, 2006, 358(3):764-72. Wrammert et al. report the use of immunoglobulin variable regions isolated from single-sorted ASCs to produce human monoclonal antibodies (mAbs) that bind with high affinity. Nature, 2008, 453(7195): 667-671.

[0046] Methods for the production of chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods Petition 870250094951, dated 10 / 17 / 2025, p. 20 / 77 14 / 55 125:191-202; and Pat. US Nos. 6,311,415, 5,807,715, 4,816,567 and 4,816,397. Chimeric antibodies comprising one or more CDRs of a non-human species and structural regions of a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; international publication no. WO 91 / 09967; and US pat. nos. 5, 225, 539, 5, 530, 101 and 5,585,089), coating or resurgence (EP 592,106; EP 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 91:969), and chain scrambling (US patent no. 5,565,332).

[0047] The polypeptide sequences of heavy chains and antibodies containing them, as disclosed in this document, can be produced by any commonly used method. Typical examples include recombinant expression in suitable host systems, e.g., cell, mammalian cell, bacterium, or yeast. In general, polypeptides can be produced by living host cells that have been genetically engineered to produce the polypeptide. Methods of genetically engineering cells to produce proteins are well known in the art. See, for example, Ausubel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include the introduction of nucleic acids that encode and allow the expression of the polypeptide into host cells. These host cells can be bacterial cells, fungal cells, or animal cells grown in culture. In one embodiment, the Petition 870250094951, dated 10 / 17 / 2025, page 21 / 77 15 / 55 polypeptides are produced in mammalian cells. Typical mammalian host cells to express the peptide include Chinese Hamster Ovary (CHO cells), lymphocytic cell lines, for example, NS0 myeloma cells, SP2 cells, COS cells.

[0048] In addition to the nucleic acid sequences that encode the peptide, recombinant expression vectors may carry additional sequences, such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, the selectable marker gene typically confers resistance to drugs such as G418, hygromycin, or methotrexate in a host cell into which the vector has been introduced.

[0049] Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect host cells, select for transformants, culture host cells, and recover peptides or peptide-coated cells from the culture medium. For example, peptides or cells can be isolated by affinity chromatography.

[0050] In certain embodiments, this disclosure refers to nucleotide sequences or nucleic acids encoding heavy chain peptides, or antibodies containing them, as disclosed in this document, constructs Petition 870250094951, dated 10 / 17 / 2025, page 22 / 77 16 / 55 genetic materials that include nucleotide sequences or nucleic acids and one or more elements for genetic constructs known per se. In certain embodiments, this disclosure refers to hosts or host cells that contain such nucleotide sequences or nucleic acids and / or that express (or are capable of expressing) the heavy chain peptides disclosed in this document.

[0051] In certain embodiments, this disclosure refers to methods for preparing heavy chain peptides or antibodies containing the same or cells expressing the heavy chain peptide or antibodies containing the same using constructs disclosed in this document, the method comprising cultivating or maintaining a host cell under conditions such that said host cell produces or expresses the heavy chain peptide(s) or antibody constructs thereof, as disclosed in this document.

[0052] As used in this document, a mutation, mutant or similar of an antibody heavy chain sequence refers to the expression of a variant amino acid within a defined antibody heavy chain position compared to base amino acids within the sequence segment, for example, of UNIPROTKB / SWISS-PROT with accession number P01857.1.

[0053] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS Petition 870250094951, dated 10 / 17 / 2025, p. 23 / 77 17 / 55 FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1), in which the N-terminal amino acid serine (S) is at position 119. Mutants can be constructed by building peptide sequences synthetically or, more typically, constructed using recombinant nucleic acid techniques, for example, heavy chain expression in a cell from a template nucleic acid. Due to the translation of three nucleic acid amino acid codons, multiple three-nucleotide codons can express the same amino acid variant. Sometimes the variant is due to a single nucleotide change, and sometimes the variant is due to more than one nucleotide change. Thus, reference to a mutation, mutant, or similar of a heavy chain antibody sequence is not necessarily limited to single nucleotide changes.

[0054] As used in this document, the term cell refers to a biological compartment containing a lipid membrane and cytosol that may contain a nucleus containing genetic material, mitochondria, and other organelles. In certain embodiments, the cell is a somatic cell.

[0055] The terms polypeptide, peptide, and protein are used interchangeably in this document to refer to polymers of amino acids of any length. The polymer may comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other Petition 870250094951, dated 10 / 17 / 2025, page 24 / 77 18 / 55 manipulation or modification, such as conjugation with an identifying component. Also included in the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids and creatine), as well as other modifications known in the art.

[0056] The term nucleic acid refers to a polymer of nucleotides, or a polynucleotide, for example, RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules. Nucleic acids can be single-stranded or double-stranded and may include coding regions and regions of various control elements.

[0057] A heterologous nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or peptide sequence that does not occur naturally, for example, because the entire sequence contains a segment from other plants, bacteria, viruses, other organisms, or a joining of two sequences that occur in the same organism but are joined in a way that does not occur naturally in the same organism or in any natural state.

[0058] The term recombinant, when used in reference to a nucleic acid molecule, refers to a nucleic acid molecule that is composed of nucleic acid segments joined together by means of molecular biological techniques, provided that the entire nucleic acid sequence does not occur in nature, that is, there is at least one mutation in the Petition 870250094951, dated 10 / 17 / 2025, p. 25 / 77 19 / 55 general sequence, so that the entire sequence does not occur naturally, although separate segments may occur in nature. The segments may be joined in an altered arrangement so that the entire nucleic acid sequence from beginning to end does not occur naturally. The term recombinant, when used in reference to a protein or peptide, refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0059] The terms vector or expression vector refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences required for the operationally bound expression of the coding sequence in a particular host organism or expression system, for example, a cell- or cell-free expression system. The nucleic acid sequences required for expression in prokaryotes generally include a promoter, an (optional) operator, 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 contemplates a vector encoding heavy chain peptide(s) or antibodies containing the same as disclosed herein in operational combination with a heterologous promoter.

[0060] In certain embodiments, this disclosure contemplates a peptide or heavy chain antibody containing the same, as reported in this document, conjugated to a marker. A marker refers to a detectable fraction that Petition 870250094951, dated 10 / 17 / 2025, p. 26 / 77 20 / 55 is directly or indirectly conjugated to another molecule, such as an antibody or a protein, to facilitate the detection of that molecule. Specific and non-limiting examples of markers include fluorescent markers, enzymatic linkages, and radioactive isotopes. A marker includes the incorporation of a radiolabeled amino acid or the covalent linkage of biotinyl moieties to a peptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Several methods of labeling peptides and glycoproteins are known in the art and can be used.Examples of peptide markers include, but are not limited to, the following: radioisotopes or radionucleotides (such as 18F, 35S, or 131I), fluorescent markers (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic markers (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined peptide epitopes recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal-binding domains, epitope markers), or magnetic agents such as gadolinium chelates. In some embodiments, the markers are fixed by spacer arms (linking groups) of varying lengths to reduce potential steric hindrance.

[0061] In certain modalities, the disclosure is Petition 870250094951, dated 10 / 17 / 2025, p. 27 / 77 21 / 55 refers to recombinant antibody heavy chain peptides and antibodies containing them, comprising sequences disclosed in this document or variants or fusions thereof, wherein the internal amino acid sequence, the amino terminal end or the carbon terminal end of the amino acid sequence are optionally linked to a heterologous amino acid sequence, marker or reporter molecule.

[0062] In certain embodiments, the disclosure refers to recombinant vectors comprising a nucleic acid encoding an antibody heavy chain peptide or an antibody containing the same as disclosed in this document. In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, virus, bacterial, bacterial plasmid or yeast-associated origin of replication or gene, such as a gene or a retroviral gene or a lentivirus LTR, TAR, RRE, PE, SLIP, CRS and INS nucleotide segment, or a gene 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 optionally comprises a gene vector (nucleic acid) element, such as a selectable marker region, lac operon, a CMV promoter, a chicken β-actin / CMV enhancer (CAG) hybrid promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (ires) sequence, cis-acting marmot post-regulatory element (WPRE), scaffold attachment region (SAR), inverted terminal repeats (ITR), c-tag coding region. Petition 870250094951, dated 10 / 17 / 2025, page 28 / 77 22 / 55 myc, metal affinity tag coding region, streptavidin-binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, COL E1 origin of replication, f1 origin, pBR322 origin or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region or a multiple cloning site, such as having 5, 6 or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

[0063] In certain embodiments, antibody heavy chain peptides or antibodies containing them are conjugated with a therapeutic agent or cytotoxic agent for use in the treatment of diseases or conditions, such as cancer. The term cytotoxic agent, as used in this document, refers to a substance that inhibits or prevents cell function and / or causes cell destruction. The term is intended to include radioactive isotopes, chemotherapeutic agents, for example, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof, such as nucleolytic enzymes, antibiotics and toxins, such as small molecule toxins or enzymatically active toxins of bacterial origin, Petition 870250094951, dated 10 / 17 / 2025, page 29 / 77 23 / 55 fungal, plant or animal, including fragments and / or variants thereof, and the various antitumor or anticancer agents, or growth-inhibiting agents. A growth-inhibiting agent, when used in this document, refers to a compound or composition that inhibits the growth of a cell, either in vitro or in vivo. Examples of growth-inhibiting agents include agents that block cell cycle progression, such as agents that induce G1 arrest and M-phase arrest. Such inhibitory agents include, for example, vinca alkaloids (vincristine, vinorelbine and vinblastine), paclitaxel and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, bleomycin, tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and cytarabine.

[0064] In certain embodiments, this disclosure contemplates antibody heavy chains or antibodies containing them, wherein a desired sugar chain is added to a core fucosylated or non-fucosylated GlcNAc acceptor, including fucosylated or non-fucosylated GlcNAc-IgG acceptor. As such, the present disclosure enables the synthesis and remodeling of therapeutic antibodies, or Fc fragments thereof, to provide certain biological activities, such as prolonged half-life in vivo, reduced immunogenicity, increased in vivo activity, increased targeting capability and / or ability to deliver a therapeutic agent. Petition 870250094951, dated 10 / 17 / 2025, p. 30 / 77 24 / 55 Antibody heavy chains, glycosylations, and mutations

[0065] In certain embodiments, a recombinant antibody heavy chain comprises a first heavy chain with a mutation or combination of mutations disclosed in this document and a second heavy chain.

[0066] In certain embodiments, this disclosure refers to a recombinant antibody heavy chain, or heterodimeric antibody containing the same, with K409S and T411Y mutations, optionally additionally comprising any of the additional mutations disclosed in this document.

[0067] In certain embodiments, this disclosure refers to a recombinant antibody heavy chain, or heterodimeric antibody containing the same, with L368S and D399Y mutations, optionally additionally comprising any of the additional mutations disclosed in this document.

[0068] In certain embodiments, this disclosure refers to a recombinant antibody heavy chain, or heterodimeric antibody containing the same, with D399Y and K447S mutations, optionally additionally comprising any of the additional mutations disclosed in this document.

[0069] In certain embodiments, this disclosure refers to a recombinant antibody heavy chain, or heterodimeric antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first Petition 870250094951, dated 10 / 17 / 2025, p. 31 / 77 The 25 / 55 heavy chain comprises any of the mutations disclosed in this document. In certain embodiments, the second heavy chain comprises any of the mutations disclosed in this document.

[0070] In certain embodiments, this disclosure refers to a nucleic acid encoding the recombinant heavy chain, as disclosed in this document, in operational combination with a heterologous promoter.

[0071] In certain embodiments, this disclosure refers to a vector comprising a nucleic acid encoding the recombinant heavy chain, as disclosed in this document, in operable combination with a heterologous promoter.

[0072] In certain embodiments, this disclosure refers to a cell (e.g., somatic cell) comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0073] In certain embodiments, this disclosure refers to a non-naturally occurring or chimeric heterodimeric antibody comprising recombinant antibody heavy chain(s), as disclosed in this document.

[0074] In certain embodiments, the recombinant antibody of unnaturally occurring heterodimeric or chimeric antibody or first heavy chain is conjugated with a first glycan in Asn297(N297) and the second heavy chain is conjugated with a second glycan in Asn297(N297). In certain embodiments, the first and second glycans are the same glycan or are not the same glycan.

[0075] In certain forms, the antibody Petition 870250094951, dated 10 / 17 / 2025, page 32 / 77 26 / 55 unnaturally occurring heterodimeric or chimeric antibody, or the first heavy chain is conjugated with a first glycan at Asn297(N297) and the second heavy chain is conjugated with a second glycan at Asn297(N297). In certain embodiments, the first and second glycans are the same glycan or are not the same glycan. In certain embodiments, the first glycan and second glycan are the same glycan. In certain embodiments, the asymmetrically glycosylated heterodimeric antibody contains at least one N-acetylglucose amine (GlcNAc) or fucosylated N-acetylglucosamine (Fucal, 6GlcNAc), for example, at N297.

[0076] In certain embodiments, this disclosure includes a single-chain antibody that has one or more of the mutations disclosed in this document.

[0077] In certain embodiments, the mutations are variants. In certain embodiments, the variant comprises conservative amino acid substitutions. In certain embodiments, the variant comprises non-conservative amino acid substitutions. In certain embodiments, the conservative and / or non-conservative amino acid substitutions are within the structure regions. In certain embodiments, the variant does not contain substitutions within CDR1, CDR2, or CDR3 light chains.

[0078] Note that, with reference to SEQ ID NO: 1, it is specifically for IgG1. IgG2, IgG3, and IgG4 will have some alternative amino acids in the same positions. For example, IgG2 contains a V at position 309 instead of L at position 309 for IgG1. IgG4 contains an F at position 234 instead of L at position 309. Petition 870250094951, dated 10 / 17 / 2025, p. 33 / 77 27 / 55 234 for IgG1.

[0079] In certain embodiments, this disclosure refers to reported antibodies in which the constant region comprises a mutation that activates immune responses, enhances ADC by increasing FcyRIIIa binding or decreasing FcyRIIb binding, enhances ADCP by increasing FcYRIIa binding or increasing FcYRIIIa binding, enhances CDC by increasing C1q binding or hexamerization, reduces effector functions by aglycosylation, reducing FcyR and C1q binding, increases co-engagement by increasing FcYRIIb binding, increasing FcYRIIa binding or decreasing FcYRIIIa binding and / or increases half-life. Examples of such mutations are further provided below.

[0080] In certain embodiments, this disclosure contemplates that a heavy chain contains at least one mutation in which both heavy chains are not identical. In certain embodiments, this disclosure contemplates that a heavy chain may have alternative mutations with respect to the opposite heavy chain, that is, one of the two heavy chains contains a mutation that the other sequence does not contain, or one of the two heavy chains contains one or more mutations, and the other heavy chain contains different mutations. Antibodies, bispecific antibodies, and additional mutations

[0081] In certain embodiments, heterodimer heavy chains comprising mutations disclosed in this document are used to produce antibodies or bispecific antibodies with binding specificity for therapeutic targets. In certain embodiments, variable sequences Petition 870250094951, dated 10 / 17 / 2025, p. 34 / 77 28 / 55 heavy and light chain mutations are combined with, grafted onto, or joined with the heavy chain mutations, constant regions / sequences disclosed in this document. In certain embodiments, a first heavy-light chain pair with a first specificity and a second heavy-light chain pair with a second specificity different from the first specificity are produced separately and then mixed for heavy-heavy chain pairing with mutations / mutation combinations disclosed herein.

[0082] In certain embodiments, the heterodimeric antibody comprises recombinant antibody heavy chain(s) as disclosed in this document.

[0083] In certain embodiments, the antibody heavy chain with mutations disclosed in this document preferentially forms a heterodimer with a second heavy chain having mutants in which the second heavy chain comprises a second set of mutations that are not in the first chain.

[0084] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations.

[0085] In certain embodiments, the first or second heavy chain additionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more of the following mutations: G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, S228P, G236R, Petition 870250094951, dated 10 / 17 / 2025, p. 35 / 77 29 / 55 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 mutation refers to positions in the amino acid sequence (SEQ ID NO: 1) (segment of UNIPROTKB / SWISS-PROT: P01857.1), where the N-terminal amino acid serine (S) is at position 119.

[0086] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has L234A or L235A mutations, or both.

[0087] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has L234A, L235A or P329G mutations, or all of them, or combinations thereof.

[0088] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second Petition 870250094951, dated 10 / 17 / 2025, p. 36 / 77 30 / 55 heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has D265A or N297A mutations, or both.

[0089] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has D265A or N297G mutations, or both.

[0090] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has D265A or N297Q mutations, or both.

[0091] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has M252Y, S254T or T256E mutations, or all of them, or combinations thereof. Petition 870250094951, dated 10 / 17 / 2025, p. 37 / 77 31 / 55

[0092] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has F243L, R292P, Y300L, V305I or P396L mutations, or all of them, or combinations thereof.

[0093] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has S239D or I332E mutations, or both.

[0094] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has S239D, I332E or A330L mutations, or all of them, or combinations thereof.

[0095] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or antibody containing the same, with mutations comprising a Petition 870250094951, dated 10 / 17 / 2025, p. 38 / 77 32 / 55 first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has S239D, I332E, G236A, or A330L mutations, or all of them, or combinations thereof.

[0096] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has S298A, E333A or K334A mutations, or all of them, or combinations thereof.

[0097] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has S298A, E333A or K334A mutations, or all of them, or combinations thereof.

[0098] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the Petition 870250094951, dated 10 / 17 / 2025, p. 39 / 77 33 / 55 The first or second heavy chain additionally has the mutations L234Y, L235Q, G236W, S239M, H268D, D270E, or S298A, or all of them, or combinations thereof.

[0099] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has D270E, K326D, A330M or K334E mutations, or all of them, or combinations thereof.

[00100] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has G236A, S239D or I332E mutations, or all of them, or combinations thereof.

[00101] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has K326W or E333S mutations, or both. Petition 870250094951, dated 10 / 17 / 2025, p. 40 / 77 34 / 55

[00102] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has E345R, E430G or S440Y mutations, or all of them, or combinations thereof.

[00103] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has E345R, E430G or S440Y mutations, or all of them, or combinations thereof.

[00104] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has S267E or L328F mutations, or both.

[00105] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second Petition 870250094951, dated 10 / 17 / 2025, p. 41 / 77 35 / 55 heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has S267E, L328F, or P238D mutations, or all of them, or combinations thereof.

[00106] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has N325S or L328F mutations, or both.

[00107] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has S267E, H268F or S324T mutations, or all of them, or combinations thereof.

[00108] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has M428L or N434S mutations, or both. Petition 870250094951, dated 10 / 17 / 2025, p. 42 / 77 36 / 55

[00109] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the L368S and D399Y mutations. In certain embodiments, the first or second heavy chain additionally has M252Y, S254T, T256E, M428L or N434S mutations, or all of them, or combinations thereof.

[00110] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has I253A, H310A, Q311A, N315A, E430A or H435A mutations, or all of them, or combinations thereof.

[00111] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more of the following mutations: G236A, S239D, A330L, I332E, S267E, L328F, P238D, H268F, S324T, Petition 870250094951, dated 10 / 17 / 2025, p. 43 / 77 37 / 55 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 mutation refers to positions in the amino acid sequence. (SEQ ID NO: 1) (segment of UNIPROTKB / SWISS-PROT: P01857. 1), where the N-terminal amino acid serine (S) is at position 119.

[00112] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has L234A or L235A mutations, or both.

[00113] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has L234A, L235A or P329G mutations, or all of them, or combinations thereof.

[00114] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second Petition 870250094951, dated 10 / 17 / 2025, p. 44 / 77 38 / 55 heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has the D265A or N297A mutations, or both.

[00115] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has D265A or N297G mutations, or both.

[00116] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has D265A or N297Q mutations, or both.

[00117] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has M252Y, S254T or T256E mutations, or all of them, or combinations thereof. Petition 870250094951, dated 10 / 17 / 2025, p. 45 / 77 39 / 55

[00118] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has F243L, R292P, Y300L, V305I or P396L mutations, or all of them, or combinations thereof.

[00119] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has S239D or I332E mutations, or both.

[00120] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has S239D, I332E or A330L mutations, or all of them, or combinations thereof.

[00121] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or antibody containing the same, with mutations comprising a Petition 870250094951, dated 10 / 17 / 2025, p. 46 / 77 40 / 55 first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has S239D, I332E, G236A, or A330L mutations, or all of them, or combinations thereof.

[00122] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has S298A, E333A or K334A mutations, or all of them, or combinations thereof.

[00123] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has S298A, E333A or K334A mutations, or all of them, or combinations thereof.

[00124] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the Petition 870250094951, dated 10 / 17 / 2025, p. 47 / 77 41 / 55 The first or second heavy chain additionally has the mutations L234Y, L235Q, G236W, S239M, H268D, D270E, or S298A, or all of them, or combinations thereof.

[00125] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has D270E, K326D, A330M or K334E mutations, or all of them, or combinations thereof.

[00126] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has G236A, S239D or I332E mutations, or all of them, or combinations thereof.

[00127] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has K326W or E333S mutations, or both. Petition 870250094951, dated 10 / 17 / 2025, p. 48 / 77 42 / 55

[00128] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has E345R, E430G or S440Y mutations, or all of them, or combinations thereof.

[00129] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has E345R, E430G or S440Y mutations, or all of them, or combinations thereof.

[00130] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has S267E or L328F mutations, or both.

[00131] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second Petition 870250094951, dated 10 / 17 / 2025, p. 49 / 77 43 / 55 heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has S267E, L328F, or P238D mutations, or all of them, or combinations thereof.

[00132] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has N325S or L328F mutations, or both.

[00133] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has S267E, H268F or S324T mutations, or all of them, or combinations thereof.

[00134] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has M428L or N434S mutations, or both. Petition 870250094951, dated 10 / 17 / 2025, p. 50 / 77 44 / 55

[00135] In certain embodiments, this disclosure refers to a heterodimeric antibody heavy chain, or an antibody containing the same, with mutations comprising a first heavy chain with K409S and T411Y mutations and a second heavy chain. In certain embodiments, the second heavy chain comprises the D399Y and K447S mutations. In certain embodiments, the first or second heavy chain additionally has M252Y, S254T, T256E, or M428L, N434S mutations, or all of them, or combinations thereof. Heterodimeric IgG molecules

[00136] Mutations were made to each of the two heavy chain protomers of IgG to promote the pairing of distinct similar protomers, thus resulting in heterodimerization of the IgG heavy chains. IgG heterodimers can be used therapeutically, for example, in bispecific antibodies that include two distinct antibody-binding fragment (Fab) molecules that recognize distinct antigens.

[00137] Mutations to produce IgG / Fc interface heterodimers were investigated. An in silico approach was used where residues at the CH3-CH3 interface of IgG were mutated to tyrosine (Y) or serine (S) and the change in binding energy (AAG) was estimated using the Rosetta 2.3 'interface' mode. Residues in the CH3 region that were within 4 angstroms of the opposite CH3 domain were selected for screening. A total of 31 residues were found: 347Q, 349Y, 350T, 351L, 352P, 353P, 354S, 355R, 356D, 357E, 360K, 364S, 366T, 368L, 370K, 390N, 392K, 393T, 394T, 395P, 397V, 398L, Petition 870250094951, dated 10 / 17 / 2025, page 51 / 77 45 / 55 399D, 400S, 405F, 406L, 407Y, 409K, 411T, 444S, and 447K. In silico screening was performed by introducing up to two mutations in each chain. Evaluation of IgG heterodimer formation

[00138] The results were filtered for scores where the AAG of the heterodimer was less than or equal to 0.8 kcal / mol and the AAG of the homodimers was equal to or greater than 0 kcal / mol. From this selection, two sets of mutations were chosen for experimental evaluation. In order to assess the effectiveness of these mutations in heterodimer formation, the mutations were introduced into the heavy chain (Hc) and Fc constructs of rituximab. The constructs were then co-transfected into HEK293F cells along with the light chain (Lc) of rituximab. In addition to the mutants that were identified, mutations for knobs-in-holes and electrostatic targeting strategies were also tested.

[00139] Mutations are defined by positions compared to base amino acids within the UNIPROTKB / SWISS-PROT sequence segment with accession number P01857.1.

[00140] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1), wherein the N-terminal amino acid serine (S) is at position 119. IgG heterodimers exhibit heterodimerization rates. Petition 870250094951, dated 10 / 17 / 2025, page 52 / 77 46 / 55 improved without affecting IgG stability or function.

[00141] The combYSelect 1 IgG heterodimers (L368S / D399Y-K409S / T411Y) and combYSelect 2 IgGs (D399Y / K447S-K409S / T411Y) were genetically engineered into a bispecific antibody and a cytokine trap to evaluate therapeutic applications. The L368S / D399Y-K409S / T411Y, D399Y / T411Y-K409S / T411Y, and D399Y / K447S-K409S / T411Y heterodimers were identified as having desirable stability by computer modeling methods.

[00142] A model system was developed involving the co-expression of an Fc region of IgG1 and full-length heavy chain (Hc) of rituximab, each from distinct protomers in a heterodimer design, as well as the light chain (Lc) of rituximab, as a model IgG1 antibody. After purification by protein A affinity chromatography and treatment with the IgG-specific endoglycosidase EndoS2 to remove heterogeneous glycosylation at the Asn297 residue, three potential products with unique masses that are detectable by liquid chromatography-mass spectrometry (LC-MS) due to co-expression were obtained, including: an intact Fc region (approximately 50 kDa), an intact IgG (approximately 150 kDa), and a monovalent IgG molecule (Fab1Fc; approximately 100 kDa) (Figure 1). The formation of intact Fc regions and IgG antibodies each results from the two possible homodimeric pairs, while the formation of the Fab1Fc molecule results strictly from heterodimerization.Thus, the ratio of Fab1Fc to Fc and IgG is a direct measure of heterodimer formation.

[00143] Rituximab was evaluated in this trial. Petition 870250094951, dated 10 / 17 / 2025, page 53 / 77 47 / 55 Fab1Fc formation at 63 percent was observed. For the knobin-hole (KiH) heterodimer (T366Y-Y407T), i.e., Ridgway et al. 1996, heterodimerization was not detected when the Hc containing the T366Y mutation was coexpressed with the Fc containing the Y407T mutation, along with the light chain. However, 57% Fab1Fc formation was observed when the Y407T mutation was on the heavy chain and the T366Y mutation was on the Fc. The electrostatic targeting heterodimer that was tested resulted in 91% Fab1Fc formation, regardless of which chain contained each set of mutations. Compared to the controls above, 97% Fab1Fc formation was observed for both mutation chain combinations for combYSelect 1 having L368S / D399Y-K409S / T411Y. Similarly, combYSelect 2 D399Y / K447S-K409S / T411Y resulted in 96% Fab1Fc formation when the D399Y / K447S mutations were contained in the Hc and 91% when this set of mutations was contained in the Fc.These results indicate that the heterodimers outperform those of other previously reported heterodimers that were tested. Table 1 provides a summary of the experimental results. A-strand mutations B-strand mutations % heterodimer formation Mutation T366Y Y407T 29.0 Knobs-in-holes - Ridgway et al. 1996 E357K / S364K L368D / K370S 91.0 Electrostatic targeting Moore et al. 2019 L3685 / D399Y K409S / T411Y 97.1 New mutations in this disclosure D399Y / K447S K409S / T411Y 93.6 Petition 870250094951, dated 10 / 17 / 2025, p. 54 / 77 48 / 55 Heterodimer mutations do not affect IgG stability or FcRn binding.

[00144] In addition to promoting heterodimer formation, experiments were conducted to evaluate whether mutations introduced into the CH3 domains affect the functional properties of IgG molecules. The thermal stability of each construct was assessed by determining the melting curves of the resulting heterodimeric antibodies in the Fab1Fc form (i.e., the product resulting from the co-expression of the heavy chain, light chain, and Fc fragment). Rituximab had three transition temperatures: 74.9°C, 81.8°C, and 90°C corresponding to the unfolding of the CH2, Fab, and CH3 domains, respectively. Furthermore, the nearly identical transition temperatures between rituximab IgG and rituximab in the Fab1Fc form confirmed that the thermal stability of the latter is representative of full-length IgG.In contrast, the knob-in-hole and electrostatically directed constructs, all of which are in the Fab1Fc form, showed only two transition temperatures corresponding to the CH2 and Fab domains. The first transition temperature ranged from 72.2 °C to 74.8 °C, while the second ranged from 80.8 °C to 81.7 °C. When we evaluated the melting temperatures of combYSelect 1 and 2 Fcs, only one melting temperature at approximately 74 °C was observed, which generally corresponds to the CH2 domain. This suggests that the CH3 domain of the heterodimers splits at the same temperature as the CH2 domain or only undergoes reversible splitting within our tested temperature range. Petition 870250094951, dated 10 / 17 / 2025, page 55 / 77 49 / 55

[00145] The potential effects of these mutations on the overall structure of the CH2-CH3 interface were 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 IgGs and prolong their serum half-life. Biolayer interferometry (BLI) analysis was used to compare the binding affinity of rituximab FcRn to rituximab combYSelect 1 and 2 IgGs with rituximab Fab domains. KD values ​​of 14.3, 17.0, and 8.0 nM were obtained for rituximab, combYSelect1, and combYSelect2, respectively, at pH 6. These IgGs showed minimal binding to FcRn at pH 7.4. Mutations for optimal heterodimerization

[00146] An LC / MS method for evaluating heterodimerization was used to determine if mutations are essential for optimal heterodimerization. The L368S mutation was dispensable when the D399Y mutation was in an Fc, with protomer A mutations in the heavy chain. The observed heterodimerization of 96% for K409S / T411Y-D399Y was not significantly different from combYSelect 1. However, there was no detectable heterodimerization when the D399Y mutation was in the Fc fragment linked to Fab, suggesting that L368S may play a role in heavy chain stabilization (Figure 2A). On the other hand, 96% and 94% Fab1Fc formation for the Hc-Fc constructs K409S / T411Y-D399Y and K409S / T411Y-K447S were observed, respectively, indicating improved heterodimerization compared to combYSelect 2. However, when the individual mutations D399Y and K447S were contained in the heavy chain, they were required for the formation of Petition 870250094951, dated 10 / 17 / 2025, pp. 56 / 77 50 / 55 heterodimers (Figure 2B). Finally, constructs with single mutations on each strand in all 4 possible combinations: K409S-D399Y, T411Y-D399Y, K409S-L368S and T411Y-L368S for combYSelect1 and K409S-D399Y, T411Y-D399Y, K409S-K447S and T411YK447S for combYSelect2 significantly reduced heterodimerization, regardless of mutation strand pairing (Figure 2A-2B). IgG1 heterodimers predicted by combYSelect have therapeutic applications.

[00147] IgG heterodimers are frequently used for the development of bispecific monoclonal antibodies and Fc fusion proteins that exhibit unique and / or enhanced therapeutic properties. To confirm that combYSelect heterodimers can be widely used for potential therapeutic applications, combYSelect-based bispecific antibodies and cytokine traps were designed and tested for their ability to simultaneously bind to two target cell lines expressing distinct antigens and to inhibit cytokine-mediated cell signaling, respectively.

[00148] The bispecific antibody was designed with one arm consisting of rituximab anti-CD20 Fab, while the other was a HER2-specific nanobody (Figure 3A). Both bispecific combYSelect heterodimers were able to simultaneously bind to the two antigens expressed on Raji (CD20+) and BT474 (HER2+) cells, as shown by the formation of cell clusters that were positive for the distinct dyes used to stain each of the two cell types. Petition 870250094951, dated 10 / 17 / 2025, pp. 57 / 77 51 / 55 cells. This was the case regardless of the antigen specificity of each protomer in versions of these bispecific antibodies where the anti-CD20 Fab and the anti-HER2 nanobody were fused to the opposite Fc. On the other hand, cell bridge clusters did not form when using a non-specific IgG1 isotype control or any monospecific heterodimer or homodimer.

[00149] IgG heterodimers are also commonly used in Fc fusion-based therapies. Thus, in a second potential therapeutic scenario, the Fc of combYselect2 was used to construct a cytokine trap composed of the IL-1 cognate cytokine receptor, IL-1 receptor type I (IL-1RI), and the IL-1 receptor accessory protein secondary receptor (IL-1RAcP), to sequester IL-1β (Figure 3C). The ability of the combYSelect2 cytokine trap to sequester IL-1β was measured by inhibiting IL-8 promoter-driven luciferase expression in HEK293T cells, which endogenously express IL-1RI and IL-1RAcP. Inhibition by the combYSelect2 trap was comparable to that of a trap fused to an electrostatic E357Q / S364K-L368D / K370S heterodimer.Both traps showed slightly improved inhibition compared to the IL-1 receptor antagonist (IL-1Ra; Figure 3D), the natural antagonist of IL-1 cytokine signaling, whose recombinant form is Anakinra, which is used to treat rheumatoid arthritis and other chronic inflammatory conditions.

[00150] The combYSelect method is useful for redesigning the interface between CH3 domains in any Petition 870250094951, dated 10 / 17 / 2025, pp. 58 / 77 52 / 55 protomer in the Fc homodimer of IgG1, with an eye toward the development of new heterodimeric Fc regions that could serve as platforms for bispecific antibodies (bsAbs). Commonly, bsAbs are genetically engineered to simultaneously target T-cell-specific antigens and tumor-associated antigens (TAAs), uniting the two cell types and enhancing the immune response to tumor cells.

[00151] Another common use of bsAbs is for targeting two different TAAs simultaneously, which improves specificity and decreases the likelihood of tumors escaping by downregulating specific epitopes or reverting to redundant pathways.

[00152] In certain embodiments, this disclosure contemplates IgG-like bsAbs, which retain long serum half-lives due to physiological binding to FcRn, compared to non-IgG-like bsAbs that lack the Fc region. It is contemplated that combYSelect promotes specific associations between a light chain and its correct heavy chain pair, allowing the production of a bsAb that more closely resembles the functionality, stability, and long half-life of a wild-type antibody, while maintaining the advantages of dual antigen specificity.

[00153] It is contemplated that the combYSelect antibodies disclosed in this document can be used as a platform to design bsAbs of other antibody isotypes, considering that the homodimeric conformations and interfaces of the Fcs of other antibody isotypes are similar to IgG Fcs, for example, using isotypes Petition 870250094951, dated 10 / 17 / 2025, page 59 / 77 53 / 55 alternatives, such as IgA and IgE. Protein and glycogenetic engineering efforts have improved the throughput, stability, and half-life of IgA, as monomeric IgA has shown efficacy in preclinical models. IgA has unique properties that include its ability to interact with neutrophils, which highly express FcsRI, making it potentially effective as an antitumor therapy. Furthermore, IgA exhibits greater stability on mucosal surfaces compared to IgGs, enhancing the potential of mAbs targeting lung or gastrointestinal inflammation. Similarly, monoclonal IgE binds with extremely high affinity to FcsRI, which is expressed on tumor-associated macrophages, and may improve the diagnosis of molecular allergy. Thus, engineered bispecific IgA and IgE antibodies contribute to improving the clinical efficacy of such mAbs.

[00154] In certain embodiments, this disclosure includes a heterodimeric antibody containing heavy chain mutation patterns disclosed in this document, for example, a bispecific antibody. In certain embodiments, the bispecific antibody binds specifically to a specific T-cell antigen. In certain embodiments, the specific T-cell antigen is CD3, CD4, CD8, CXCR3, CCR4, CD4, CD25, CD127 and / or CD152.

[00155] In certain embodiments, the bispecific antibody binds specifically to a tumor-associated antigen (TAA). In certain embodiments, TAA is cluster of differentiation 19 (CD19), cluster of differentiation 10 Petition 870250094951, dated 10 / 17 / 2025, pp. 60 / 77 54 / 55 (CD10), cluster of differentiation 20 (CD20), cluster of differentiation 33 (CD33), cluster of differentiation 38 (CD38), CD70 (tumor necrosis factor ligand superfamily member 7), CD133 (prominin 1), CD171 (cell adhesion molecule L1), (EGFR) epidermal growth factor receptor, (HER2) human epidermal growth factor receptor 2, EGFR VIII (epidermal growth factor receptor variant 3), (MUC1) mucin 1, (MUC16) mucin 16, (EpCAM) epithelial cell adhesion molecule, (AFP) alpha-fetoprotein, (FAP) familial adenomatous polyposis, (CEA) carcinoembryonic antigen, (PSCA) prostate stem cell antigen, (PSMA) prostate-specific membrane antigen, (PSA) prostate-specific antigen, (AXL) Tyrosine kinase receptor AXL, (DLL3) delta-type 3, (EPHA2) EPH A2 receptor, (FRa) Folate receptor alpha, (LMP1) Latent membrane protein 1 of Epstein-Barr virus, (MAGE) Melanoma antigen gene protein, 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) General Secretion Pathway G Protein, (ROR) RAR-Related Orphan Receptors, ROR1 (Tyrosine Kinase-like Receptor 1), IL13RA2 (Interleukin 13 Receptor Alpha 2 Subunit), Wilms Tumor 1 (WT1), Survivin, Tn (aGalNAc-O-Ser / Thr), sialyl-Tn (aNeuAc2,6-aGalNAc-O-Ser / Thr), TF (bGal1,3-aGalNAc-O-Ser / Thr) , CA 19-9 (Neu5Acα2-3Galβ13[Fucα1-4]GlcNAcβ), Telomerase reverse transcriptase (TERT), Petition 870250094951, dated 10 / 17 / 2025, pp. 61 / 77 55 / 55 Beta-hCG (human chorionic gonadotropin), p53, Ras, bladder tumor antigen (BTA), Om5 antibody-specific antigen, GD2 (GD2 ganglioside), alpha-v / beta-6 integrin or mesothelin antigen, BCMA (TNF receptor superfamily member 17 / B cell maturation protein), CD123 (interleukin 3 receptor α subunit CD123 antigen), CD138 (syndecan 1), CD22 (SIGLEC2), CD5 (T1 / Leu-1 lymphocyte antigen), Ig kappa 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 (vascular endothelial growth factor receptor kinase insertion domain / receptor 2).

[00156] In certain embodiments, this disclosure contemplates methods of treating a disease or condition with a heterodimeric antibody containing heavy chain mutation patterns disclosed in this document by administering an effective amount of the heterodimeric antibody to a subject in need thereof. In certain embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with cancer, an autoimmune disorder, cardiovascular disease, diabetes, respiratory disease, or inflammatory disease. Petition 870250094951, dated 10 / 17 / 2025, pp. 62 / 77

Claims

1 / 2 CLAIMS 1. Recombinant antibody, characterized in that it has a heavy chain with K409S and T411Y mutations.

2. Recombinant antibody, according to claim 1, characterized in that it has a second heavy chain with L368S and D399Y mutations.

3. Recombinant antibody, according to claim 1, characterized in that it has a second heavy chain with D399Y and K447S mutations.

4. Recombinant antibody, characterized by having a heavy chain with L368S and D399Y mutations.

5. Recombinant antibody, according to claim 4, characterized in that it has a second heavy chain with K409S and T411Y mutations.

6. Recombinant antibody, characterized by having a heavy chain with D399Y and K447S mutations.

7. Recombinant antibody, according to claim 6, characterized in that it has a second heavy chain with K409S and T411Y mutations.

8. Heterodimeric antibody, characterized by the fact that it comprises a first heavy chain with K409S and T411Y mutations and a second heavy chain.

9. Heterodimeric antibody, according to claim 8, characterized in that the second heavy chain comprises the L368S and D399Y mutations.

10. Heterodimeric antibody, according to claim 8, characterized in that the second heavy chain comprises the D399Y and K447S mutations.

11. Nucleic acid, characterized in that it encodes recombinant heavy chains as defined in any of claims 1 to 10, in operational combination with a heterologous promoter.

12. Vector, characterized in that it comprises a nucleic acid as defined in claim 11.

13. Cell, characterized in that it comprises a nucleic acid as defined in claim 11, or a vector as defined in claim 12.

14. Heterodimeric antibody, characterized in that it comprises recombinant antibody heavy chain(s) as defined in any one of claims 8 to 10.

15. Heterodimeric antibody, according to claim 14, characterized in that it is a bispecific antibody.

16. Bispecific antibody, according to claim 15, characterized in that it binds specifically to a specific T-cell antigen.

17. Bispecific antibody, according to claim 15, characterized in that it binds specifically to a tumor-associated antigen. Petition 870250094951, dated 10 / 17 / 2025, pp. 64 / 77

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