Heterodimer proteins

Abstract

Antibody-based therapies have emerged as a crucial component in the treatment of the growing number of human malignancies in fields such as oncology, inflammatory diseases, and infectious diseases. Indeed, antibodies are one of the top-selling drug classes today, with five of the top ten best-selling drugs being antibodies. Antibody therapy is also increasingly being used in veterinary medicine for the treatment of livestock such as dogs. [Solution] The present invention relates to antibody therapy in veterinary medicine, particularly full-length antibodies and antibody fragments, by providing a method for improving the production of heterodimer antibodies for veterinary medicine, and related products and uses.

Description

Technical Field

[0001] Antibody-based therapeutic agents have emerged as an important component in the treatment of an increasing number of human malignancies in fields such as oncology, inflammatory diseases, and infectious diseases. Indeed, antibodies are one of the largest classes of drugs in terms of current sales, and 5 out of the top 10 selling drugs are antibodies. Antibody therapy is also increasingly being used in veterinary medicine for the treatment of livestock such as dogs.

Background Art

[0002] There is a great need for these therapies in veterinary medicine, and in the United States alone, 6 million cases of cancer are diagnosed in dogs and a similar number in cats each year (Cekanova and Rathore, "Animal models and therapeutic molecular targets of cancer: utility and limitations". Drug Des Devel Ther, 8:1911-1922, 2014). Furthermore, 1 in 4 dogs in the United States is diagnosed with some form of arthritis (Bland, "Canine osteoarthritis and treatments: a review". Veterinary Science Development 5(2), 2015). Therefore, there is potential for the application of antibody therapeutic agents to many chronic veterinary diseases. Monoclonal antibodies can also be beneficial for the detection, prevention, and control of parasitic, bacterial, and viral diseases.

[0003] The first monoclonal antibodies for therapeutic use in humans received commercial approval 25 years ago, and since then, 80 types have been approved, with over 50 in late-stage clinical development. In contrast, the use of antibodies in veterinary medicine is in its early stages, with only a few types under development. The slow progress reflects the fact that the development of species-specific therapeutic antibodies is technically difficult and represents only a relatively recent effort. Therefore, there is a need to develop improved antibodies for veterinary use, as well as methods for producing antibodies for veterinary use.

[0004] Antibody structures have been utilized to design a wide variety of antibody formats that target diseases in humans. One example of such designed antibody formats is the bispecific antibody. A bispecific antibody can bind to two different targets and therefore to two different epitopes simultaneously. One area of ​​interest is T-cell-targeted bispecific antibodies for efficiently killing tumors. Bispecific antibodies have "two-target" functionality, binding to two different surface receptors or ligands, thereby potentially influencing numerous disease pathways. Bispecific antibodies can position two targets in close proximity, thereby supporting the formation of protein complexes on a single cell or inducing intercellular contact. Bispecific antibody formats vary in many respects, including their molecular weight, the number of antigen-binding sites, the spatial relationships between the various binding sites, the valence for each antigen, their ability to support secondary immune function, and their pharmacokinetic half-life. These diverse formats offer a great opportunity to adapt the design of bispecific antibodies to match the proposed mechanism of action and intended clinical application (Kontermann and Brinkmann, "Bispecific Antibodies Drug Discovery Today," Vol. 20, No. 7, 2015).

[0005] The production of IgG-type bispecific antibodies by co-expression of two light chains and two heavy chains in a single host cell can be extremely challenging due to the low yield of the desired bispecific IgG and the difficulty in removing closely related mispaired IgG contaminants. This reflects the so-called heavy chain pairing problem, where the heavy chain forms not only the desired heterodimer but also homodimers. Furthermore, the light chain mispairs with unrelated heavy chains, also known as the light chain pairing problem. As a result, co-expression of two antibodies can produce up to nine undesirable IgG species in addition to the desired bispecific antibody.

[0006] Various approaches are described in this technique to promote heterodimerization, i.e., the formation of specific bispecific antibodies of interest for human therapeutic purposes, thereby reducing the content of undesirable homodimers in the resulting mixture. These approaches have been studied in relation to human antibodies or humanized antibodies designed to target diseases in humans.

[0007] Homodimerization of the two heavy chains in IgG is mediated by non-covalent interactions between individual CH3 domains. In other words, CH3-CH3 interactions are the primary driving force for Fc dimerization.

[0008] Furthermore, it is well known that when two CH3 domains interact with each other, they associate at a protein-protein interface containing "contact" residues (also called contact amino acids, interface residues, or interface amino acids). The contact amino acids of the first CH3 domain interact with one or more contact amino acids of the second CH3 domain. The contact amino acids are typically within 5.5 Å (preferably within 4.5 Å) of each other in the three-dimensional structure of the antibody. Interactions between contact residues from one CH3 domain and contact residues from different CH3 domains can be mediated, for example, by van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, disulfide bonds, or other forces known to those skilled in the art. That is, the primary driving forces are hydrophobic and electrostatic interactions in the core.

[0009] This technology employs approaches that inhibit the dimerization of antibody heavy chains in order to favor the production of heterodimeric antibodies. Specific designs in the CH3 domain have been applied to favor heterodimerization over homodimerization. Examples of such designs of the CH3-CH3 interface include the introduction of complementary protrusion and cavity mutations, also known as the "knob-into-hole" approach, described, for example, by WO9627011 and JB Ridgway et al., “'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerisation,” Protein Eng., 9 (1996), pp. 617-621.

[0010] Generally, this method includes the step of introducing a protrusion at the interface of a first polypeptide and a corresponding cavity at the interface of a second polypeptide, thereby allowing the protrusion to be located within the cavity to promote heteromultimer formation and inhibit homomultimer formation. The “protrusion” or “knob” is constructed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory “cavity” or “hole” of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing a large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine). The protrusion and cavity can be produced by synthetic means such as modification of the nucleic acid encoding the polypeptide or peptide synthesis. Starting with a "knob" mutation (T366W) that makes homodimerization of CH3 unfavorable (Ridgway et al., cited above), the compensating "hole" mutations (T366S, L368A, and Y407V) (Atwell et al., "Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library," J. Mol. Biol., 270, pp. 26-35, 1997) were identified by phage display, which provides efficient pairing with the "knob" and makes homodimerization unfavorable.

[0011] Other successful strategies for heavy chain heterodimerization include electrostatic steering mutations (WO2006 / 106905 and Gunasekaran et al., "Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG," J. Biol. Chem., 285, pp. 19637-19646, 2010). This approach is based on the electrostatic manipulation of contact residues within the naturally charged CH3-CH3 interface. Mutations are introduced into the CH3 domain of the heavy chain, replacing naturally occurring charged amino acid contact residues with amino acid residues bearing the opposite charge (i.e., a charge inversion strategy). This creates a change in charge polarity across the Fc dimer interface, supporting favorable attractive interactions for the co-expression of electrostatically matched Fc chains and promoting the formation of desired Fc heterodimers, while the formation of undesirable Fc homodimers is suppressed by unfavorable repulsive charge interactions.

[0012] It has been described that four specific charged residue pairs within the human CH3-CH3 interface are involved in interdomain interactions. These are D356 / K439′, E357 / K370′, K392 / D399′, and D399 / K409′ (numbering according to Kabat, 1991). Here, residues in the first chain are separated from residues in the second chain by " / ", and prime (′) indicates the residue numbering of the second chain. Since the CH3-CH3 interface exhibits twofold symmetry, each specific charge pair is presented twice in the original IgG (i.e., the charge interactions of K439 / D356′, K370 / E357′, D399 / K392′, and K409 / D399′ also exist at the interface). Taking advantage of this bifold symmetry, it was demonstrated that a single charge reversal, such as K409D in the first chain or D399′K in the second chain, reduces homodimer formation due to the repulsion of identical charges. This repulsive effect was further enhanced by combining various charge reversals. It was demonstrated that the expression of various CH3 domains, including various complementary charge reversals, promotes heterodimerization and leads to an increase in the proportion of bispecific species in the mixture (for reviews, see Kontermann and Brinkmann, cited above; Brinkmann and Kontermann: "The making of bispecific antibodies", MABS, vol. 9, no. 2, 2017, pp. 182-212; Ha et al., "Frontiers in Immunology Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications", Therapeutic Antibodies and Proteins, vol. 7, paper 394, 2016). [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] WO9627011 [Patent Document 2] WO2006 / 106905 [Patent Document 3] WO 2019 / 035010 [Non-patent literature]

[0014] [Non-Patent Document 1] Cekanova and Rathore "Animal models and therapeutic molecular targets of cancer: utility and limitations". Drug Des Devel Ther, 8:1911-2, 2014) "Animal models and therapeutic molecular targets of cancer: utility and limitations" Drug design, development and therapy 8:1911-1922 [Non-Patent Document 2] Bland “Canine osteoarthritis and treatments: a review” Veterinary Science Development 5(2)), 2015 [Non-Patent Document 3] Kontermann and Brinkmann, "Bispecific Antibodies: Drug Discovery Today," Vol. 20, No. 7, 2015. [Non-Patent Document 4] JB Ridgway et al., "'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerisation," Protein Eng., 9 (1996), pp. 617-621. [Non-Patent Document 5] Atwell et al., "Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library," J. Mol. Biol., 270, pp. 26-35, 1997. [Non-Patent Document 6] Gunasekaran et al., “Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG,” J. Biol. Chem., 285, pp. 19637–19646, 2010 [Non-Patent Document 7] Brinkmann and Kontermann: "The making of bispecific antibodies," MABS, Vol. 9, No. 2, 2017, pp. 182-212. [Non-Patent Document 8] Ha et al., “Frontiers in Immunology Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications,” Therapeutic Antibodies and Proteins, Volume 7, Paper 394, 2016 [Non-Patent Document 9] Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012). [Non-Patent Document 10] Therapeutic Monoclonal Antibodies: From Bench to Clinic, edited by Zhiqiang An, Wiley, (2009) [Non-Patent Document 11] Antibody Engineering, 2nd Edition, Volumes 1 and 2, edited by Ontermann and Duebel, Springer-Verlag, Heidelberg (2010)

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Non-Patent Document 19

[0015] There is a need to develop improved antibodies for veterinary medicine, as well as methods for producing antibodies for veterinary medicine. This invention aims to address this need by providing a method for improving the production of heterodimer antibodies, particularly for veterinary medicine, and by providing related products and uses. [Means for solving the problem]

[0016] This invention is based on the finding that mutating a specific residue in the canine CH3 domain promotes the generation of heterodimers for the production of veterinary bispecific antibodies.

[0017] The inventors also identified a conserved canine charge pair in the canine IgG CH3 domain that is remarkably highly conserved across all canine IgG isotypes and is uncharged at the corresponding position in all human IgG. The inventors showed that heterodimerization can be promoted by modifying this charge pair.

[0018] The inventors modified the canine IgG CH3 domain interface in the Fc region by mutations in charge pairs so that the manipulated CH3-containing protein preferentially forms heterodimers. This minimizes the formation of homodimer contaminants for the production of bispecific antibodies. While not wanting to be bound by theory, the inventors believe that the mutations alter the charge polarity across the Fc dimer interface, thereby supporting favorable attractive interactions through co-expression of electrostatically matched Fc chains, promoting the formation of desired Fc heterodimers, while unfavorable repulsive charge interactions suppress the formation of undesirable Fc homodimers.

[0019] Typically, in various embodiments of the present invention, the first canine IgG CH3 domain and the second canine IgG CH3 domain are both manipulated in a complementary manner, so that each CH3 domain (or polypeptide containing it) does not homodimerize substantially by itself, or homodimerizes at a slower rate, but is forced heterodimerized with the other complementaryly manipulated CH3 domain. In other words, the first and second CH3 domains heterodimerize, and little homodimer is formed between the two first CH3 domains or the two second CH3 domains.

[0020] Accordingly, the method of the present invention comprises the steps of substituting at least one amino acid in the canine IgG CH3 domain of a first polypeptide and / or substituting at least one amino acid in the canine IgG CH3 domain of a second polypeptide, wherein the amino acid in the CH3 domain of the first polypeptide faces at least one amino acid in the CH3 domain of the second heavy chain in the tertiary structure of a heterodimer protein, such as an antibody, and each amino acid in the CH3 domains of the first and second heavy chains is substituted such that an amino acid having the opposite side chain charge is introduced into the opposite polypeptide. The present invention also relates to polypeptides obtained or to be obtained by such a method.

[0021] The present invention also relates to a modified CH3 domain, and a heterodimer protein or polypeptide containing the modified CH3 domain, such as an isolated CH3 domain, an isolated polypeptide, or a heterodimer protein containing a modified CH3 domain. The heterodimer protein comprises a CH3 domain in a first polypeptide and a CH3 domain in a second polypeptide, wherein the first CH3 domain in the first polypeptide and the second CH3 domain in the second polypeptide each associate at an interface including the original interface between the CH3 domains, and the interface is modified to promote the formation of the heterodimer protein.

[0022] There are several isoforms of canine IgG, namely IgG-A, IgG-B, IgG-C, and IgG-D. The embodiments of this invention relate to all isoforms. Specifically, various embodiments are presented for IgG-D and IgG-B. However, the embodiments are also extended to embodiments that include other isoforms in which residues at corresponding positions (corresponding to the positions described for IgG-D and / or IgG-B) are substituted. Figure 6 shows alignments of various isoforms in canine isoforms. Those skilled in the art can identify corresponding positions and residues in various canine IgG isoforms by aligning the sequences.

[0023] Therefore, in the first aspect, the present invention is a) A first polypeptide containing a first canine IgG CH3 domain, and b) A second polypeptide containing a second canine IgG CH3 domain With respect to a heterodimer protein containing the above, the first and second canine IgG CH3 domains contain one or more charge pair substitutions.

[0024] One or more charge pair substitutions promote heterodimerization.

[0025] In a second aspect, the present invention is a) A first polypeptide containing a first canine IgG CH3 domain, and b) A second polypeptide containing a second canine IgG CH3 domain Regarding heterodimer proteins containing IgG, one of the canine IgG CH3 domains contains one or more charge pair substitutions, while the other does not. For example, the first canine IgG CH3 domain has a substitution, but the second canine IgG CH3 domain does not. In another example, the second canine IgG CH3 domain has a substitution, but the first canine IgG CH3 domain does not.

[0026] In a third aspect, the present invention relates to a heterodimer protein comprising a first polypeptide containing a first canine IgG CH3 domain and a second polypeptide containing a second canine IgG CH3 domain, a) The first canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more positions 424, 423, 377, 378, and 382, ​​and the second canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more positions 414, 416, 433, 392, 377, and 393, an amino acid substitution with a charged amino acid or an amino acid of opposite charge at 463, and / or an amino acid substitution with N at 425, b) The first canine IgG CH3 domain contains amino acid substitutions at 383, 393, and / or 382, ​​and the second canine IgG CH3 domain does not contain the corresponding mutations, or c) The second canine IgG CH3 domain contains an amino acid substitution at 463 or 425, while the first canine IgG CH3 domain does not contain the corresponding mutation.

[0027] IgG may be IgG-A, B, C, or D. The above numbering refers to IgG-D, but the corresponding position in another isoform may be substituted.

[0028] One or more amino acid substitutions promote heterodimerization.

[0029] For example, a) The first canine IgG CH3 domain includes an amino acid substitution with an oppositely charged amino acid at one or more of the E424, D423, D / K377, and E378 positions, and the second canine IgG CH3 domain includes an amino acid substitution with an oppositely charged amino acid at one or more of the K414, H / R416, K433, and K392 positions and / or an amino acid substitution with a charged amino acid if the residue is L and a negatively charged amino acid if the residue is K at L / K463, b) The first canine IgG CH3 domain contains amino acid substitutions at D383, D393, and / or S382, and the second canine IgG CH3 domain does not contain the corresponding mutations, or c) The second canine IgG CH3 domain contains an amino acid substitution at L463 or 425, while the first canine IgG CH3 domain does not contain the corresponding mutation.

[0030] IgG may be IgG-A, B, C, or D. The above numbering refers to IgG-D, but the corresponding position in another isoform may be substituted.

[0031] In a further embodiment, the present invention relates to a heterodimer protein comprising a first polypeptide containing a first canine IgG CH3 domain and a second polypeptide containing a second canine IgG CH3 domain, a) The first canine IgG CH3 domain contains an amino acid substitution with an amino acid of opposite charge at one or more positions 428, 427, 382, ​​and 383, and the second canine IgG CH3 domain contains an amino acid substitution with an amino acid of opposite charge at one or more positions 420, 418, 437, 467, and 396, or b) The second canine IgG CH3 domain contains an amino acid substitution at position 429, while the first canine IgG CH3 domain does not contain the corresponding mutation.

[0032] For example, the first canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more of the following positions: E428, D427, E382, E383, and K386, and the second canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more of the following positions: R420, K418, K437, K467, ​​K396, D397, and D429.

[0033] IgG may be IgG-A, B, C, or D. The above numbering refers to IgG-B, but the corresponding position in another isoform may be substituted.

[0034] In a further embodiment, the present invention relates to a polypeptide comprising a canine IgG CH3 domain, wherein the polypeptide comprises an amino acid substitution with an amino acid of opposite charge at one or more of the above positions (e.g., with respect to IgG-B or IgG-D). In a further embodiment, the present invention relates to nucleic acids encoding the heterodimer proteins or polypeptides described herein.

[0035] In a further embodiment, the present invention relates to a vector comprising nucleic acid or a host cell comprising the vector.

[0036] In a further embodiment, the present invention is a) A step of transforming host cells with nucleic acids or vectors described herein, b) A step of culturing host cells and expressing first and second IgG CH3, and c) A step of recovering heterodimer proteins or polypeptides from host cell cultures. This invention relates to a method for producing heterodimer proteins or polypeptides having amino acid substitutions in the canine IgG CH3 domain, including [specific component].

[0037] In a further embodiment, the present invention relates to a pharmaceutical composition comprising a heterodimer protein or polypeptide and a pharmaceutical carrier as described herein.

[0038] In a further embodiment, the present invention relates to a kit comprising a heterodimer protein or polypeptide as described herein and optionally instructions for use.

[0039] In the description of the diagram, all mutation IDs mentioned below are related to IgG-B, as shown in Table 2. The term "combination / combination ID" also refers to the mutation ID, as shown in the table. [Brief explanation of the drawing]

[0040] [Figure 1] These are the conserved KiH positions in canine and human IgG isoforms. [Figure 2]This residue forms a conserved CH3 charge-pair interaction between humans and dogs. [Figure 3] Canine charge pair mutations enhance heterodimer formation. [Figure 4] This is an evaluation of a heterodimerization method for human IgG4PE KiH. [Figure 5] These are the sequences of canine IgG-A, -B, -C, and -D (sequences 1, 2, 3, and 4, respectively). [Figure 6] This is an alignment of canine IgG-A, -B, -C, and -D sequences (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4). The highlighted residues are those described herein. [Figure 7] Evaluation of heterodimerization by SDS-PAGE (ratio 1:1), 1st of 3 trials. Combinations / mutations IDs 2-5 and 10-12 are expressed and form heterodimers. Mutations IDs 6-9 are not expressed. Regions corresponding to the MW of the heterodimer are highlighted with dotted rectangles. The lanes are as follows: 1: Molecular weight ladder (MW), 2: Combination 1, 3: Combination 2, 4: Combination 3, 5: Combination 4, 6: Combination 5, 7: Combination 6, 8: Combination 7, 9: Combination 8, 10: Combination 9, 11: Combination 10, 12: Combination 11, 13: Combination 12, 14: MW. [Figure 8] Evaluation of heterodimerization by SDS-PAGE (ratio 1:1), second of three trials. Mutations IDs 13, 14, and 16-24 are expressed and form heterodimers. Mutation ID 15 is not expressed. The region corresponding to the MW of the heterodimer is highlighted with a dotted rectangle. The lanes are as follows: 1:MW, 2:combination 13, 3:combination 14, 4:combination 15, 5:combination 16, 6:combination 17, 7:combination 18, 8:combination 19, 9:combination 20, 10:combination 21, 11:combination 22, 12:combination 23, 13:combination 24. [Figure 9]Evaluation of heterodimerization by SDS-PAGE (ratio 1:1), 3rd of 3 trials. Mutations IDs 25, 26, and 28-29 are expressed and form heterodimers. Mutation ID 27 is not expressed. The lanes are as follows: 1: combination 25, 2: combination 26, 3: combination 27, 4: combination 28, 5: combination 29, 6: sample protein, 7: MW. [Figure 10] This is a density analysis of heterodimerization (1:1 ratio) evaluated by SDS-PAGE. The value corresponding to the heterodimer is shown as a percentage of the total protein. Mutation ID 1 (WT) is shown for comparison. The plot shows that mutations IDs 3, 4, 10, 11, 12, 17, 19, 20, 21, 23, and 29 increased the proportion of heterodimers. [Figure 11] Evaluation of heterodimerization by SDS-PAGE (ratio 1:3), first of two trials. All selected mutations except mutation ID 17 are expressed and form heterodimers. The region corresponding to the MW of the heterodimer is highlighted with a dotted rectangle. The lanes are as follows: 1:MW, 2:combination 1, 3:combination 4, 4:combination 11, 5:combination 12, 6:combination 13, 7:combination 16, 8:combination 17, 9:combination 22, 10:combination 23, 11:combination 24, 12:combination 25, 13:combination 26, 14:Fc-wt only, 15:scFv-wt only. [Figure 12] Evaluation of heterodimerization by SDS-PAGE (ratio 1:3), second of two trials. All selected mutations were expressed and formed heterodimers. The gel regions corresponding to the MW of the heterodimer are highlighted with dotted rectangles. The lanes are as follows: 1: MW, 2: combination 1, 3: combination 28, 4: combination 29. [Figure 13] This is a density analysis of heterodimerization (ratio 1:3) evaluated by SDS-PAGE. The value corresponding to the heterodimer is shown as a percentage of the total protein. Mutation ID 1 (WT) is shown for comparison. The plot shows that an increase in the proportion of heterodimers was observed for all combinations except mutation ID 17. [Figure 14]Evaluation of heterodimerization by SDS-PAGE (ratio 1:6), first of two trials. All selected mutations are expressed and form heterodimers. The region corresponding to the MW of the heterodimer is highlighted with a dotted rectangle. The lanes are as follows: 1:MW, 2:combination 1, 3:combination 4, 4:combination 11, 5:combination 12, 6:combination 13, 7:combination 16, 8:combination 17, 9:combination 22, 10:combination 23, 11:Fc-wt only, 12:scFv-wt only. [Figure 15] Evaluation of heterodimerization by SDS-PAGE (ratio 1:6), second of two trials. All selected mutations are expressed and form heterodimers. The region corresponding to the MW of the heterodimer is highlighted with a dotted rectangle. The lanes are as follows: 1: combination 1, 2: MW, 3: combination 24, 4: combination 25, 5: combination 26, 6: combination 28, 7: combination 29. [Figure 16] This is a density analysis of heterodimerization (ratio 1:6) evaluated by SDS-PAGE. The values ​​corresponding to heterodimers are shown as a percentage of total protein. Mutation ID 1 (WT) is shown for comparison. The plot shows that the proportion of heterodimers increased in all combinations except IDs 17 and 24. [Figure 17a] This figure shows the retention times of HPLC SEC for both homodimers (highlighted by dotted lines) and heterodimers. Peaks corresponding to heterodimers are detected and quantified for all mutations. [Figure 17b] This figure shows the HPLC SEC quantification of heterodimer peaks, expressed as a percentage of total protein. [Figure 17c] This figure shows the HPLC SEC quantification of heterodimer peaks, indicated as total area (C). [Figure 18] This table summarizes the results obtained from a 1:3 ratio. Combination / mutation ID 17 is highlighted in gray because it showed the lowest expression of the heterodimer. [Modes for carrying out the invention]

[0041] The present invention is described further here. The following sections define various aspects of the present invention in more detail. Each of the aspects thus defined may be combined with any other aspect unless otherwise expressly indicated. In particular, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

[0042] In general, the nomenclature and techniques used in relation to cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, as described herein, are well known and commonly used in this art. Unless otherwise indicated, the methods and techniques disclosed herein are carried out in accordance with conventional methods generally known in this art, as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, edited by Zhiqiang An, Wiley, (2009); and Antibody Engineering, 2nd edition, Vols. 1 and 2, edited by Ontermann and Duebel, Springer-Verlag, Heidelberg (2010).

[0043] Enzymatic reactions and purification procedures are carried out according to the manufacturer's specifications, as is commonly performed in this technique, or as described herein. The nomenclature and experimental procedures and methods used in relation to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well-known and commonly used in this technique. Standard procedures are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as patient treatment.

[0044] The present invention provides biological therapeutic agents for veterinary use, particularly in the treatment of dogs. In particular, the present invention relates to heterodimers, such as multispecific molecules, for targeting various disease-modifying molecules, and to methods for producing such molecules. The present invention is based on the manipulation of residues that form the interface of dimer proteins, specifically residues of the canine IgG CH3 domain. By introducing paired electrostatic steering amino acid mutations into the canine IgG CH3 domain, a change in charge polarity is created across the Fc dimer interface, thereby enabling heterogeneous heavy chains derived from different parent antibodies to interact with each other more strongly and specifically, while the repulsive charge generated by the electrostatic steering mutation minimizes the formation of homodimers from the same type of heavy chain.

[0045] Therefore, in the first aspect, the present invention is a) A first polypeptide containing a first canine IgG CH3 domain, and b) A second polypeptide containing a second canine IgG CH3 domain With respect to a heterodimer protein containing the above, the first and second canine IgG CH3 domains contain one or more charge pair substitutions.

[0046] One or more charge pair substitutions promote heterodimerization.

[0047] In a second aspect, the present invention is a) A first polypeptide containing a first canine IgG CH3 domain, and b) A second polypeptide containing a second canine IgG CH3 domain Regarding heterodimer proteins containing the following, one of the canine IgG CH3 domains contains one or more charge pair substitutions, while the other does not.

[0048] One or more charge pair substitutions promote heterodimerization.

[0049] The term IgG CH3 domain refers to the constant heavy chain 3 of the immunoglobulin (Ig) molecule.

[0050] "First polypeptide" is any polypeptide to associate with the second polypeptide, and is also referred to herein as "chain A". The first and second polypeptides meet / associate with each other at an "interface". "Second polypeptide" is any polypeptide to associate with the first polypeptide via the "interface", and is also referred to herein as "chain B". The "interface" includes "contact" amino acid residues in the first polypeptide that interact with one or more "contact" amino acid residues at the interface of the second polypeptide. The canine IgG CH3 domain includes contact residues at such an interface.

[0051] As used herein, the interface comprises a canine IgG CH3 domain, i.e., a residue of the CH3 domain of the Fc region derived from a canine or canine IgG antibody.

[0052] A heterodimer, or heterodimer protein, generally refers to a protein consisting of two similar but not identical subunits. An example of a heterodimer protein is a bispecific antibody.

[0053] As used herein, the term “antibody” means any immunoglobulin (Ig) molecule consisting of four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains, or its antigen-binding portion or fragment, or any functional fragment, variant, variant, or derivative thereof that retains the essential epitope-binding properties of the Ig molecule. Such variant, variant, or derived antibody formats are known in this art. As used herein, the term “antibody” does not encompass only the original polyclonal or monoclonal antibodies.

[0054] In a full-length antibody, each heavy chain consists of a heavy chain variable region or domain (abbreviated as HCVR herein) and a heavy chain constant region. The heavy chain variable region consists of three constant heavy chain domains, namely CH1, CH2, and CH3. Each light chain consists of a light chain variable region or domain (abbreviated as LCVR herein) and a light chain constant region. The light chain constant region consists of one domain, CL.

[0055] Each light chain (L) is linked to a heavy chain (H) by a single covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain at the other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain and the heavy chain variable domain. The pairing of VH and VL forms a single antigen-binding site.

[0056] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domain of the antibody’s heavy or light chain. The variable domains of the heavy and light chains are referred to as “VH” and “VL,” respectively. These domains are generally the most variable parts of an antibody (compared to other antibodies of the same class) and contain the antigen-binding site. The term “variable” refers to the fact that certain segments of the variable domain vary significantly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody to that particular antigen. However, variability is not uniformly distributed across the entire span of the variable domain. Instead, variability is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The native heavy and light chain variable domains are each linked by three HVRs and contain four FR regions that primarily take a beta-sheet configuration, forming loops that link the beta-sheet structure and, in some cases, form part of it. The HVRs of each chain are held in close proximity by the FR region and, together with HVRs from other chains, contribute to the formation of the antibody's antigen-binding site. The constant domain is not directly involved in antibody binding to the antigen, but exhibits various effector functions, such as the involvement of the antibody in antibody-dependent cytotoxicity.

[0057] The variable regions of the heavy and light chains can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are scattered alongside more conserved regions called framework regions (FRs). Each variable region of the heavy and light chains consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0058] The immunoglobulin molecule may generally be of any isotype, class, or subclass. The CH3 domain in various aspects of the present invention is the CH3 domain of canine IgG subtypes, such as IgG-A, IgG-B, IgG-C, and IgG-D.

[0059] In dogs, there are four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, and are referred to as IgG-A, IgG-B, IgG-C, and IgG-D. The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. (Vet. Immunol. Immunopathol. 80: pp. 259-270 (2001)). The amino acid and DNA sequences of these heavy chains are also available from the GenBank database (IgGA: accession number AAL35301.1, IgGB: accession number AAL35302.1, IgGC: accession number AAL35303.1, IgGD: accession number AAL35304.1). The canine antibody contains two types of light chains, namely kappa and lambda (GenBank accession numbers: kappa light chain amino acid sequence ABY 57289.1, GenBank accession number ABY 55569.1). The amino acid sequences are shown in Figures 5 and 6.

[0060] In other words, the present invention specifically encompasses embodiments that include modifications of the CH3 domain of canine IgG-A, IgG-B, IgG-C, or IgG-D. As described above, the residues at the defined positions may vary between isoforms. Also, the corresponding positions in other isotypes may be numbered differently, as shown in Figure 6.

[0061] The term "CDR" refers to the complementarity-determining region within the antibody variable sequence. Each of the heavy and light chain variable regions has three CDRs, designated as CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" refers to a group of three CDRs occurring within a single variable region capable of binding an antigen. The precise boundaries of these CDRs may be defined differently depending on the various systems known in this technology.

[0062] Kabat complementarity-determining regions (CDRs) are based on sequence variability and are one of the most commonly used numbering systems for human antibodies (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391, and Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Chothia instead refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The terms “Kabat numbering,” “Kabat definition,” and “Kabat labeling” are used interchangeably herein. As recognized in this technique, these terms refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions or antigen-binding sites of the heavy and light chains of an antibody.

[0063] The numbering used herein when referring to canine residues relates to the canine IgG isoforms, specifically IgG-D and IgG-B shown in Figure 6. The first residue shown (M) is numbered 1, and consecutive amino acid residues are given consecutive numbers. However, as stated above, the present invention is not limited to these isoforms, and aspects of the present invention also encompass the IgG-A and IgG-C isoforms. In these isoforms, certain residues at modification sites (e.g., 377, 416, 463) may differ from those found in IgG-D (see Figure 6).

[0064] Proteolytic digestion of antibodies releases various fragments, referred to as Fv (variable fragment), Fab (antigen-binding fragment), and Fc (crystalline fragment). The Fc fragment contains the carboxyl terminus of both H chains, held together by a disulfide. The constant domain of the Fc fragment is involved in mediating the effector function of the antibody.

[0065] Antigen-binding fragments are also intended according to aspects and embodiments of the present invention. Antigen-binding fragments include, for example, Fab, Fab', F(ab')2, Fd, Fv, single-domain antibodies (sdAb), such as VH single-domain antibodies, fragments containing complementarity-determining regions (CDRs), single-chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, as well as polypeptides containing at least a portion of immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

[0066] "Fv" is the smallest antibody fragment containing a complete antigen recognition site and an antigen binding site. This fragment consists of a dimer of one heavy chain and one light chain variable region domain that are tightly non-covalently associated. The folding of these two domains gives rise to six hypervariable loops (three from each of the H and L chains), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site. "Single-stranded Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain.

[0067] The term "antigen-binding site" refers to the portion of an antibody or antibody fragment that contains a region that specifically binds to an antigen. The antigen-binding site may be provided by one or more antibody variable domains. Preferably, the antigen-binding site is contained within the associated VH and VL of the antibody or antibody fragment.

[0068] A "chimeric antibody" is a recombinant protein that contains a variable domain, including the complementarity-determining region (CDR), of an antibody derived from one species, while the constant domain of the antibody molecule is derived from an antibody molecule of another species, such as a canine antibody. An example of a chimeric antibody is a chimeric human-dog antibody.

[0069] A "humanized antibody" is a recombinant protein in which the CDR of an antibody derived from a single species (e.g., a rodent antibody) is transferred from the variable heavy and light chains of the rodent antibody to human variable heavy and light domains (e.g., framework region sequence). The constant domain of the antibody molecule is derived from the constant domain of the human antibody. In certain embodiments, a limited number of framework region amino acid residues derived from the parent (rodent) antibody are substituted with the human antibody framework region sequence.

[0070] As used herein, the term “canine-like antibody” means a form of recombinant antibody containing sequences from both canine and non-canine (e.g., mouse) antibodies. Generally, a canine-like antibody will contain substantially all of at least one, typically two, variable domains in which all or substantially all of the hypervariable loop corresponds to the hypervariable loop of a non-canine immunoglobulin, and all or substantially all of the framework (FR) region (and typically all or substantially all of the remaining frame) is the framework region of a canine immunoglobulin sequence. A canine-like antibody may contain both three heavy chain CDRs and three light chain CDRs derived from a mouse or human antibody, along with a canine frame or a modified canine frame. A modified canine frame contains one or more amino acid changes that can further optimize the efficacy of the canine-like antibody, for example, by increasing its binding to its target.

[0071] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and have a directivity to a single antigenic site. In contrast to polyclonal antibody preparations, which typically contain various antibodies with directivity to various determinants (epitopes), each monoclonal antibody has directivity to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized by culturing hybridomas and being free from contamination by other immunoglobulins.

[0072] The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody, or antibody fragment specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. This term specifically includes linear epitopes and conformational epitopes. Epitopes within protein antigens are formed from both adjacent amino acids (usually linear epitopes) or non-adjacent amino acids juxtaposed by the three-dimensional folding of the protein (usually conformational epitopes). Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, though not always, while epitopes formed by three-dimensional folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids within a specific spatial conformation. Methods for determining which epitopes a given antibody or antibody fragment binds to (i.e., epitope mapping) are well known in this technique and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or adjacent peptides are tested for reactivity with a given antibody or antibody fragment. When two antibodies recognize the same or sterically overlapping epitopes, the antibodies bind to the "essentially the same epitope" as the reference antibody. The most widely used rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is the competition assay, which can be constructed in various formats using labeled antigens or labeled antibodies.

[0073] The term “isolated” protein or polypeptide means a protein or polypeptide substantially free from other proteins or polypeptides and having different antigen specificity. Furthermore, the protein or polypeptide is substantially free from other cellular materials and / or chemicals. That is, the proteins, nucleic acids, and polypeptides described herein are preferably isolated. That is, as used herein, “isolated” protein, heterodimer protein, heteromultimer, or polypeptide means a heterodimer protein, heteromultimer, or polypeptide identified, isolated, and / or recovered from components of its natural cell culture environment. Contaminating components of its natural environment are materials that may interfere with the diagnostic or therapeutic use of the heterodimer protein, heteromultimer, or polypeptide and may include enzymes, hormones, and other protein or non-protein solutes.

[0074] In this specification, “amino acid” means one of the 20 naturally occurring amino acids or any non-natural analog that may exist at a specific defined position. Amino acids encompass both naturally occurring and synthetic amino acids. In most cases, however, when proteins are produced by recombinant DNA, only naturally occurring amino acids are used.

[0075] As used herein, “substitution of an amino acid residue” by another amino acid residue in the amino acid sequence of a heterodimer protein or polypeptide (e.g., an antibody) described herein is equivalent to “replacing an amino acid residue” with another amino acid residue, and represents that a specific amino acid residue at a specific position in the original (e.g., wild-type / germline) amino acid sequence has been replaced (or substituted) with a different amino acid residue. This can be done using standard techniques available to those skilled in the art, for example, by recombinant DNA technology. The amino acids are modified from the naturally occurring (wild-type / germline) sequence found in the wild-type (wt), but may also be done in an IgG molecule that includes other modifications to the natural sequence. In this specification, “wild-type,” “WT,” or “natural” means a naturally occurring amino acid sequence or nucleotide sequence, including allele variants. WT proteins, polypeptides, antibodies, immunoglobulins, IgG, and others have an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0076] In other words, the present invention provides a variant CH3 domain that is different from the parent CH3 domain. As used herein, “parent polypeptide,” “parent protein,” “precursor polypeptide,” or “precursor protein” means an unmodified polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or manipulated version of a naturally occurring polypeptide. The parent polypeptide may mean the polypeptide itself, a composition containing the parent polypeptide, or an amino acid sequence encoding it.

[0077] In one embodiment of the heterodimer protein, the first and / or second canine IgG CH3 domain contains one or more amino acid substitutions, for example, one, two, or three.

[0078] In this specification, “position” means a location in the amino acid sequence of a protein, as shown, for example, in Figure 5 or Figure 6. The corresponding position is generally determined through alignment with other isoform sequences, as outlined. A residue is an amino acid that occurs at a specified position.

[0079] For example, in canine IgG CH3, amino acids are substituted so that amino acids with opposite side chain charges are introduced into the opposing CH3 domain. That is, one or more substitutions replace an amino acid with an amino acid of the opposite charge.

[0080] As described above, substitution can be performed in any canine IgG isoform, and those skilled in the art can identify the corresponding position in another isoform.

[0081] In this specification, “variant” or “mutant” means a polypeptide sequence that differs from the parent sequence, e.g., the wild-type sequence, due to a modification of at least one amino acid. As used herein, “amino acid residue substitution” by another amino acid residue in the amino acid sequence of a protein or polypeptide described herein is equivalent to “replacing an amino acid residue” with another amino acid residue, and represents that a specific amino acid residue at a specific position in the original (e.g., wild-type / germline) amino acid sequence has been replaced (or substituted) with a different amino acid residue. This can be done using standard methods available to those skilled in the art, e.g., recombinant DNA technology. The amino acids are modified from the natural (wild-type / germline) sequence found naturally in the wild-type (wt), but may also be modified in an IgG molecule that includes other modifications to the natural sequence.

[0082] In one particular embodiment, the heterodimer protein contains the identified amino acid substitution and may have further mutations in the CH3 domain or at other positions in the amino acid sequence. In another embodiment, the heterodimer protein has only the identified amino acid substitution in the CH3 domain and does not contain any other amino acid substitutions and / or other mutations in the CH3 domain. In yet another embodiment, the heterodimer protein has only the identified amino acid substitution in the CH3 domain and does not have any other mutations in the protein sequence. That is, in one embodiment, the provided amino acid substitution consists of the cited amino acid substitution.

[0083] Modifications in IgG-D The following describes modifications related to the numbering of residues in the IgG-D amino acid sequence. This sequence is shown in Figures 5 and 6. The IgG-D sequence is sequence number 4. That is, the numbering is related to sequence number 4.

[0084] In another aspect, the present invention relates to a heterodimer protein comprising a first polypeptide containing a first canine IgG CH3 domain and a second polypeptide containing a second canine IgG CH3 domain, a) The first canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more positions 424, 423, 377, 378, and 382 of IgG-D, or at the corresponding positions of IgG-A, B, or D; the second canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more positions 414, 416, 433, 392, 377, and 393 of IgG-D, or at the corresponding positions of IgG-A, B, or D, an amino acid substitution with a charged amino acid or an amino acid of opposite charge at 463, and / or an amino acid substitution with N at 425. b) The first canine IgG CH3 domain contains amino acid substitutions at 383, 393, and / or 382 of IgG-D, or at the corresponding positions of IgG-A, B, or D, and the second canine IgG CH3 domain does not contain the corresponding mutations or c) The second canine IgG CH3 domain contains an amino acid substitution at 463 or 425 of IgG-D, or at the corresponding position of IgG-A, B, or D, while the first canine IgG CH3 domain does not contain the corresponding mutation.

[0085] The present invention relates to heterodimer proteins, a) The first canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more positions of IgG-D (E424, D423, D / K377, E378) or the corresponding position of IgG-A, B, or D, and the second canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more positions of IgG-D (K414, H / R416, K433, K392) or the corresponding position of IgG-A, B, or D, and / or an amino acid substitution at L / K463 with a charged amino acid if the residue is L, or with a negatively charged amino acid if the residue is K, b) The first canine IgG CH3 domain contains amino acid substitutions at D383, D393 of IgG-D, or the corresponding position of IgG-A, B, or D, and / or at S382 of IgG-D or the corresponding position of IgG-A, B, or D, and the second canine IgG CH3 domain does not contain the corresponding mutation or c) The second canine IgG CH3 domain contains an amino acid substitution at L463 or 425 of IgG-D, or at the corresponding position of IgG-A, B, or D, while the first canine IgG CH3 domain does not contain the corresponding mutation.

[0086] In one embodiment, with respect to a specific residue in canine IgG-D, the first canine IgG CH3 domain includes an amino acid substitution with an oppositely charged amino acid at one or more of the E424, D423, K377, or E378 positions, and the second canine IgG CH3 domain includes an amino acid substitution with an oppositely charged amino acid at one or more of the K414, H416, K433, or K392 positions and / or an amino acid substitution with a charged amino acid at L463, or the second canine IgG CH3 domain includes an amino acid substitution at L463 and the first canine IgG CH3 domain does not include the corresponding mutation. Those skilled in the art will recognize that different residues may be found at specific positions in other canine IgG isoforms. This is shown in Figure 6. For example, in canine IgG-C, the residue at position 377 is D. In canine isoforms IgG-A, IgG-B, and IgG-C, the residue at position 416 is R.

[0087] Substitutions for specific residues in canine IgG-D may be selected as follows: Substitutions at E424 are E424K, E424R, or E424H; substitutions at D423 are D423K, D423R, or D423H; substitutions at E378 are E378K, E378R, or E378H; substitutions at K377 are K377E or K377D; substitutions at D377 are D377K, D377R, or D377H; and substitutions at K414 are K The substitutions are 414E or K414D, H / R416 is H / R416D or H / R416E, K433 is K433D or K433E, L463 is L463K, L463R, L463H, L463E, or L463D, K463 is K463D or K463E, and K392 is K392E or K392D.

[0088] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the E424 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K414 position.

[0089] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the E424 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K414 position and an amino acid substitution with a negatively charged amino acid at the H / R416 position. In one embodiment, the substitution at E424 is E424K, the substitution at K414 is K414E, and the substitution at H / R416 is H / R416D.

[0090] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D423 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K433 position. In one embodiment, the substitution at D423 is D423K, and the substitution at K433 is K433D.

[0091] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D423 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K433 position and an amino acid substitution with a negatively charged amino acid at the H / R416 position. In one embodiment, the substitution at D423 is D423K, the substitution at K433 is K433D, and the substitution at H / R416 is H / R416D.

[0092] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D423 and an amino acid substitution by a positively charged amino acid at position E424, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K433 and an amino acid substitution by a negatively charged amino acid at position K414. In one embodiment, the substitution at E424 is E424K, the substitution at K414 is K414E, and the substitution at K433 is K433D.

[0093] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D423 and an amino acid substitution by a positively charged amino acid at position E424, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K433, an amino acid substitution by a negatively charged amino acid at position K414, and an amino acid substitution by a negatively charged amino acid at position H / R416. In one embodiment, the substitution at E424 is E424K, the substitution at K414 is K414E, and the substitution at H / R416 is H / R416D.

[0094] In one embodiment, the second canine IgG CH3 domain contains an amino acid substitution with a positively charged amino acid at position L463, while the first canine IgG CH3 domain does not contain the corresponding mutation.

[0095] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E378, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K392. In one embodiment, the substitution at E378 is E378K, and the substitution at K392 is K392E.

[0096] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E424, the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K414, an amino acid substitution with a negatively charged amino acid at position H / R416, and an amino acid substitution with a charged amino acid at position L / K463 if the residue is L, or a negatively charged amino acid if the residue is K. In one embodiment, the substitution at E424 is E424K, the substitution at K414 is K414E, the substitution at H / R416 is H / R416D, and the substitution at L463 is L463K.

[0097] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D423 position, the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K433 position, an amino acid substitution with a negatively charged amino acid at the H / R416 position, and an amino acid substitution with a charged amino acid at the L / K463 position if the residue is L, or a negatively charged amino acid if the residue is K. In one embodiment, the substitution at D423 is D423K, the substitution at K333 is K433E, the substitution at H / R416 is H / R416D, and the substitution at L463 is L463K.

[0098] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E424, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K414 and an amino acid substitution with a positively charged amino acid at position L463. In one embodiment, the substitution at E424 is E424K, the substitution at K414 is K414E, and the substitution at L463 is L463E.

[0099] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution at position D423 with a positively charged amino acid, and the second canine IgG CH3 domain includes an amino acid substitution at position K433 with a negatively charged amino acid and an amino acid substitution at position L / K463 with a charged amino acid if the residue is L, and with a negatively charged amino acid if the residue is K. In one embodiment, the substitution at D423 is D423K, the substitution at K333E is K433E, and the substitution at L463 is L463K.

[0100] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E424 and an amino acid substitution by a positively charged amino acid at position D423, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K414, an amino acid substitution by a negatively charged amino acid at position K433, and an amino acid substitution at position L / K463, where the residue is L and the residue is K. In one embodiment, the substitution at E424 is E424K, the substitution at D423 is D423K, the substitution at K414 is K414E, and the substitution at L463 is L463K.

[0101] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution at position E424 with a positively charged amino acid and an amino acid substitution at position K / D377 with a negatively charged amino acid if the residue is K, or with a positively charged amino acid if the residue is D. The second canine IgG CH3 domain includes an amino acid substitution at position K414 with a negatively charged amino acid and an amino acid substitution at position L / K463 with a charged amino acid if the residue is L, or with a negatively charged amino acid if the residue is K. In one embodiment, the substitution at E424 is E424K, the substitution at K377 is K377E, the substitution at K414 is K414E, and the substitution at L463 is L463K.

[0102] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution at position D423 with a positively charged amino acid, and an amino acid substitution at position K / D377 with a negatively charged amino acid if the residue is K, or with a positively charged amino acid if the residue is D. The second canine IgG CH3 domain includes an amino acid substitution at position K433 with a negatively charged amino acid, and an amino acid substitution at position L / K463 with a charged amino acid if the residue is L, or with a negatively charged amino acid if the residue is K. In one embodiment, the substitution at D423 is D423K, the substitution at K / D377 is K / D377E, the substitution at K333E is K433E, and the substitution at L463 is L463K.

[0103] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution at position E424 with a positively charged amino acid, an amino acid substitution at position D423 with a positively charged amino acid, and an amino acid substitution at position K / D377 with a negatively charged amino acid if the residue is K, or with a positively charged amino acid if the residue is D. The second canine IgG CH3 domain includes an amino acid substitution at position K414 with a negatively charged amino acid, an amino acid substitution at position K433 with a negatively charged amino acid, and an amino acid substitution at position L / K463 with a charged amino acid if the residue is L, or with a negatively charged amino acid if the residue is K. In one embodiment, the substitution at D423 is D423K, the substitution at E424 is E424K, the substitution at K377 is K377E, the substitution at K414 is K414E, the substitution at K433 is K433D, and the substitution at L463 is L463K.

[0104] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E378, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K392. In one embodiment, the amino acid substitution at position E378 is E378K, and the amino acid substitution at position K392 is K392E.

[0105] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D393, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K377. In one embodiment, the amino acid substitution at position D393 is D393K, and the amino acid substitution at position K377 is K377D.

[0106] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E378 and an amino acid substitution by a positively charged amino acid at position D393, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K392 and an amino acid substitution by a negatively charged amino acid at position K377. In one embodiment, the amino acid substitution at position E378 is E378K, the amino acid substitution at position K392 is K392E, the amino acid substitution at position D393 is D393K, and the amino acid substitution at position K377 is K377D.

[0107] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution at position K377 with a negatively charged amino acid, and the second canine IgG CH3 domain includes an amino acid substitution at position L / K463 with a charged amino acid if the residue is L, and with a negatively charged amino acid if the residue is K. In one embodiment, the amino acid substitution at position K377 is K377E, and the amino acid substitution at position L463 is L463K.

[0108] In one embodiment, the first canine IgG CH3 domain contains an amino acid substitution with a positively charged amino acid at position S382, while the second canine IgG CH3 domain does not contain the mutation. In one embodiment, the amino acid substitution at position S382 is S382K.

[0109] In one embodiment, the first canine IgG CH3 domain contains an amino acid substitution with a negatively charged amino acid at position D383, while the second canine IgG CH3 domain does not contain the mutation. In one embodiment, the amino acid substitution at position D383 is D383N.

[0110] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position S382 and an amino acid substitution with N at position D393, while the second canine IgG CH3 domain does not contain any mutations. In one embodiment, the amino acid substitution at position S382 is S382K, and the amino acid substitution at position D393 is D393N.

[0111] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position S382, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position D393. In one embodiment, the amino acid substitution at S382 is S382D, and the amino acid substitution at position D393 is D393K.

[0112] In one embodiment, the first canine IgG CH3 domain does not contain amino acid substitutions, while the second canine IgG CH3 domain contains an amino acid substitution with N at the D425 position.

[0113] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position S382, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position D393 and an amino acid substitution with N at position D425. In one embodiment, the amino acid substitution at position S382 is S383D. In one embodiment, the amino acid substitution at position D393 is D393K, and the amino acid substitution at position D383 is D383N.

[0114] That is, in one embodiment, the substitution in E424 is E424K, E424R, or E424H; the substitution in D423 is D423K, D423R, or D423H; the substitution in E378 is E378K, E378R, or E378H; the substitution in K377 is K377E or K377D; the substitution in D377 is D377K, D377R, or D377H; the substitution in K414 is K414E or K414D; the substitution in H / R416 is H / R416D or H / R416E; and the substitution in K433 is K433 The substitution is D or K433E, the substitutions in L463 are L463K, L463R, L463H, L463E, or L463D, the substitution in K463 is K463D or K463E, the substitution in K392 is K392E or K392D, the substitution in E378 is E378K, E378R, or E378H, the substitution in D393 is D393K, D393R, or D393H, the substitution in S382 is S382K, S382R, or S382H, the substitution in D383 is D383N, and the substitution in D425 is N.

[0115] In one embodiment, the mutation in the CH3 domain is selected from one of the mutation IDs 1 to 26 shown in Table 1. The mutation with mutation ID 27 may also be included.

[0116] Modified CH3 domains, such as isolated CH3 domains having amino acid substitutions at one or more positions 428, 427, 382, ​​383, 386, 420, 418, 437, 467, 396, 397, 429, 424, 423, 377, 378, 382, ​​414, 416, 433, 392, 377, 393, 463, 425, 383, 393, and / or 382, ​​are also within the scope of the present invention. The substitutions may be by amino acids with opposite charges and can be selected from those shown in Table 1.

[0117] As described above, substitution can be performed in any canine IgG isoform, and those skilled in the art can identify the corresponding position in another isoform.

[0118] Modifications in IgG-B The following describes modifications related to the numbering of residues in the IgG-B amino acid sequence. This sequence is shown in Figures 5 and 6. The IgG-B sequence is sequence number 2. That is, the numbering is related to sequence number 2.

[0119] In one embodiment, the present invention relates to a heterodimer protein comprising a first polypeptide containing a first canine IgG CH3 domain and a second polypeptide containing a second canine IgG CH3 domain, wherein the IgG is selected from IgG-A, B, C, or D. a) The first canine IgG CH3 domain contains an amino acid substitution with an amino acid of opposite charge at one or more positions 428, 427, 382, ​​and 383 of IgG-B, or at the corresponding positions of IgG-A, C, or D, and the second canine IgG CH3 domain contains an amino acid substitution with an amino acid of opposite charge at one or more positions 420, 418, 437, 467, and 396 of IgG-B, or at the corresponding positions of IgG-A, C, or D, b) The second canine IgG CH3 domain contains an amino acid substitution at 429 of IgG-B, or at the corresponding position of IgG-A, C, or D, and the first canine IgG CH3 domain does not contain the corresponding mutation, or c) The first canine IgG CH3 domain contains an amino acid substitution at IgG-B 387, or at the corresponding position of IgG-A, C, or D, while the second IgG CH3 domain does not contain the corresponding mutation.

[0120] For example, the first canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more positions E428, D427, E382, and E383, and the second canine IgG CH3 domain includes an amino acid substitution with an amino acid of opposite charge at one or more positions R420, K418, K437, K467, ​​and K396.

[0121] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position R420.

[0122] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position R420 and an amino acid substitution with a negatively charged amino acid at position K418.

[0123] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D427 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the R420 position.

[0124] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D427 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the R420 position and an amino acid substitution with a negatively charged amino acid at the K437 position.

[0125] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428K, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K437 and an amino acid substitution by a negatively charged amino acid at position K418.

[0126] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428K, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position R420.

[0127] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428K, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position R420 and an amino acid substitution by a negatively charged amino acid at position K437.

[0128] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428K, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K420 and an amino acid substitution by a negatively charged amino acid at position K418.

[0129] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E382, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K467.

[0130] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E383 and an amino acid substitution with a negatively charged amino acid at position K396.

[0131] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E382 and an amino acid substitution by a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K418 and an amino acid substitution by a negatively charged amino acid at position R420.

[0132] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E382 and an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K418 and an amino acid substitution with a negatively charged amino acid at position R420.

[0133] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E382 and an amino acid substitution with a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K437 and an amino acid substitution with a negatively charged amino acid at position R420.

[0134] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E382 and an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K437 and an amino acid substitution with a negatively charged amino acid at position R420.

[0135] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E383 and an amino acid substitution by a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K418 and a negatively charged amino acid at position R420.

[0136] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E383 and an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K418 and an amino acid substitution with a negatively charged amino acid at position R420.

[0137] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E383 and an amino acid substitution with a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K437 and an amino acid substitution with a negatively charged amino acid at position R420.

[0138] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E383 and an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K437 and an amino acid substitution with a negatively charged amino acid at position R420.

[0139] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K418 and a negatively charged amino acid at position K467.

[0140] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D427 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K437 position and a negatively charged amino acid at the R467 position.

[0141] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K418 and a negatively charged amino acid at position K396.

[0142] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D427 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K437 position and a negatively charged amino acid at the K396 position.

[0143] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D427 and an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position R420 and an amino acid substitution with a negatively charged amino acid at position K467.

[0144] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D427 and an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K396 and an amino acid substitution with a negatively charged amino acid at position K467.

[0145] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D427 and an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K437, a negatively charged amino acid at position K418, and a negatively charged amino acid at position K467.

[0146] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D427 and an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K437, a negatively charged amino acid at position K418, and a negatively charged amino acid at position K396.

[0147] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K418.

[0148] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D427 position, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K437 position.

[0149] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K386, and the second canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D397.

[0150] In one embodiment, the first canine IgG CH3 domain does not contain an amino acid substitution, and the second canine IgG CH3 domain contains an amino acid substitution at position D429.

[0151] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the K386 position, and the second canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D397 position and a substitution at D429.

[0152] In one embodiment, the first canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at the N387 position, while the second canine IgG CH3 domain does not include an amino acid substitution.

[0153] In one embodiment, the substitution at E428 is E428K, E428R, or E428H; the substitution at D427 is D427K, D427R, or D427H; the substitution at E382 is E382K, E382R, or E382H; the substitution at E383 is E383K, E383R, E383H, or E383R; the substitution at R420 is R420E or R420D; and the substitution at K418 is K418E Alternatively, it is K418D, the substitution in K437 is K437D or K437E, the substitution in K467 is K467D or K467E, the substitution in K396 is K396E or K396D, the substitution in K386 is K386E or K386D, the substitution in D397 is D397K, D397R, or D397H, the substitution in D429 is D429N, or the substitution in N387 is N387D.

[0154] In one embodiment, the substitution at E428 is E428K, the substitution at D427 is D427K, the substitution at E382 is E382K, the substitution at E383 is E383K, the substitution at R420 is R420D, the substitution at K418 is K418E, the substitution at K437 is K437D, the substitution at K467 is K467E, the substitution at K396 is K396E, the substitution at K386 is K386D, the substitution at D397 is D397K, and the substitution at D429 is D429N.

[0155] In one embodiment, the mutation in the CH3 domain is selected from one of the mutation IDs shown in Table 2. In one embodiment, the mutation in the CH3 domain is selected from one of the following mutation IDs shown in Table 2: 2, 3, 4, 5, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, or 29. In one embodiment, the mutation in the CH3 domain is selected from one of the combination IDs shown in Figure 18. In one embodiment, the mutation in the CH3 domain is selected from one of the combination IDs 24 and 25.

[0156] Modified CH3 domains, such as isolated CH3 domains having one or more amino acid substitutions at positions 428, 427, 382, ​​383, 386, 420, 418, 437, 467, 396, 397, and 429, are also within the scope of this specification. The substitutions may be by oppositely charged amino acids and can be selected from one of the substitutions shown in Table 2.

[0157] Challenges in bispecific antibodies include assembly and purification issues, and production issues (heterodimerization of heavy chains, pairing of light chains, and purification of bispecificity from monospecific contaminants). As shown in the examples and Figures 7, 8, and 9, under the experimental conditions used, certain combinations of mutations produce molecules that do not express, do not pair, or "blur" on the gel, i.e., molecules that form a number of possible formats rather than producing clearly bispecific molecules. Therefore, in some embodiments, aspects of the present invention relate to mutation IDs 2-5, 10-14, 16-26, 28, and 29, with reference to Table 2.

[0158] In some embodiments, the heterodimer proteins described herein may or may not contain further mutations in the CH3 domain. For example, the CH3 domain may contain 1, 2, 3, 4, 5, or more further mutations. The further mutations may be those that promote heterodimerization. Therefore, those skilled in the art will understand that any of the combinations presented above can be further combined with such further mutations in the CH3 domain.

[0159] In one example, one or more further mutations are introduced to replicate the "knob-in-hole Fc technique" (KiH). Using this method, a larger amino acid, tyrosine, is introduced to replace a smaller amino acid in the CH3 domain of the first polypeptide, forming a "knob." The second polypeptide is then subjected to the opposite method, namely by substituting a larger amino acid with a smaller one, thereby generating a "hole." This creates a steric hindrance effect that facilitates the assembly of the desired heterodimer of the first and second polypeptides having the canine IgG CH3 domain.

[0160] For example, referring to IgG-D numbering, the first canine IgG CH3 domain contains an amino acid substitution at position T388, and the second canine IgG CH3 domain contains amino acid substitutions at positions T388, L390, and Y431.

[0161] In one embodiment, the mutation is a substitution at one or more positions T388, L390, and Y431, with reference to IgG-D numbering, and is selected from T388W, T388S, L390A, and Y431V. With reference to IgG-B numbering, these are T392S, L394A, Y435V, and T392W.

[0162] Those skilled in the art will recognize that various mutations in the CH3 domain, such as mutations that alter the charge at the Fc domain interface and mutations using the "knob-in-hole Fc technique" (KiH), can be combined with the molecules and methods of the present invention.

[0163] When using the modified polypeptide of the present invention, the amount of homodimer formation is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%, as determined, for example, by mass spectrometry.

[0164] The modifications of the present invention include one or more substitutions in the CH3 domain, and any number of further modifications may be included as long as the protein function is still present, as described herein. However, since the goal is often to change the function with minimal modification, one, two, three, four, five, six, seven, eight, nine, or ten modifications in addition to the CH3 modification are commonly used. Some examples have one to five modifications, and in many embodiments, one to two, one to three, and one to four modifications are also applicable. Note that the number of amino acid modifications is included in the functional domain. For example, it is desirable to have one to six modifications in the Fc region of the wild-type or engineered protein, and similarly, one to six modifications in the CH3 region, for example. The variant polypeptide sequence will preferably have at least about 80%, 85%, 90%, 95%, or 98% or 99% identity with the wild-type sequence or the parent sequence. Note that the percentage of identity depends on the number of amino acids, depending on the size of the sequence.

[0165] Among the many platforms for generating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on the fact that it minimizes structural changes to the native Ab and that bsAbs can be simply formed from two parent Abs (Labrijn et al., (2013) "Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange." Proc. Natl. Acad. Sci. USA 110, pp. 5145-5150). Controlled Fab-arm exchange avoids the problem of light chain pairing by using the smallest set of mutations, as it allows for the exchange of half-antibodies without disrupting the precise heavy-light chain interaction. In other words, in addition to the mutations presented above, cFAE can also be used in this invention.

[0166] That is, the present invention also provides modified canine IgG containing a hinge region derived from another isoform instead of the native IgG hinge region, for example, IgG-D containing a hinge region derived from IgG-A, IgG-B, or IgG-C instead of the native IgG-D hinge region, or IgG-B containing a hinge region derived from IgG-A, IgG-C, or IgG-D instead of the native IgG-B hinge region. Such modifications can result in canine IgG-D lacking fab arm exchange. Modified canine IgG can be constructed using standard methods of recombinant DNA technology (e.g., Maniatis et al., Molecular Cloning, A Laboratory Manual (1982)). To construct these variants, the nucleic acid encoding the amino acid sequence of canine IgG, for example, IgG-B or IgG-D, can be modified to encode the modified IgG. The modified nucleic acid sequence is then cloned into an expression plasmid for protein expression.

[0167] In one embodiment of the heterodimer protein of the present invention, the heterodimer protein includes an Fc region, for example, a canine Fc region or an Fc region derived from a canine Fc region.

[0168] In one embodiment of the heterodimer protein of the present invention, the first polypeptide is an antibody heavy chain, and / or the second polypeptide is an antibody heavy chain, e.g., a canine antibody heavy chain, a canine antibody heavy chain, or an antibody heavy chain derived from a canine antibody heavy chain. In one embodiment, both the first polypeptide and the second polypeptide are antibody heavy chains, e.g., a canine antibody heavy chain, a canine antibody heavy chain, or an antibody heavy chain derived from an Fc region or a canine antibody heavy chain.

[0169] In one embodiment of the heterodimer protein of the present invention, the heterodimer protein comprises a first and a second light chain, for example, a canine antibody light chain, a canine-modified antibody light chain, or an antibody light chain derived from a canine antibody light chain.

[0170] In one embodiment, the heterodimer protein is an antibody, such as a canine antibody, a canine antibody light chain, or an antibody light chain derived from a canine antibody.

[0171] In one embodiment, the antibody is a multispecific antibody or a fragment thereof. A multispecific protein, such as a multispecific antibody, binds to at least two different targets, i.e., it is at least bispecific. That is, in one embodiment, the antibody is a bispecific antibody or a fragment thereof. In other embodiments, the multispecific antibody or fragment thereof binds to three, four, or more targets.

[0172] In one embodiment, particularly in the treatment of cancer, the protein targets CD3 and is provided in the format of a bispecific T cell engager (BiTE).

[0173] In one embodiment, the protein is multiparatopic, i.e., it binds to two or more epitopes on the same target.

[0174] A bispecific antibody is specific to no more than two epitopes. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain having binding properties to a second epitope. In one embodiment, the first and second epitopes lie on the same antigen, e.g., the same protein (or subunit of a multimer protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes lie on different antigens, e.g., different proteins (or different subunits of a multimer protein). In another embodiment, the bispecific antibody comprises a heavy-chain variable domain sequence and a light-chain variable domain sequence having binding specificity to a first epitope, and a heavy-chain variable domain sequence and a light-chain variable domain sequence having binding specificity to a second epitope. In further embodiments, the bispecific antibody molecule comprises an antibody having binding specificity to a first epitope and an antibody having binding specificity to a second epitope. In one embodiment, the bispecific antibody molecule comprises an antibody or fragment thereof having binding specificity to a first epitope and a hemiantibody or fragment thereof having binding specificity to a second epitope. In one embodiment, the bispecific antibody or fragment thereof comprises a Fab having binding specificity to a first epitope and a Fab having binding specificity to a second epitope.

[0175] The scFv format is also included in the scope of this invention.

[0176] In another embodiment, a heterodimer protein, such as an Fc polypeptide, or an antibody or a fragment thereof, may be included as a further part.

[0177] For example, heterodimer proteins are Fc receptor fusion proteins.

[0178] The Fc region is involved in a family of receptors called Fcγ receptors (FcγRs). All FcγRs interact with very similar binding sites on Fc. The constant region, particularly the C region within the Fc fragment, is known to be involved in various effector functions of immunoglobulins. On the other hand, the variable region mediates antigen specificity. That is, effector functions are mediated by the Fc fragment, and not by the variable region that provides antigen specificity. Binding to FcγRs induces several responses, such as antibody-dependent cell-mediated cytotoxicity or phagocytosis. Modified Fc regions for veterinary use are described, for example, in WO 2019 / 035010, which is incorporated herein by reference. That is, the modifications of Fc described in WO 2019 / 035010, for example, can be combined with the heterodimerization modifications described herein, and proteins having such combined modifications are included in the scope of the present invention. Such modifications include amino acid alterations to wild-type IgG Fc that result in increased protein A binding (e.g., to facilitate purification), decreased C1q binding (e.g., to reduce complement-mediated immune responses), decreased CD16 binding (e.g., to reduce the induction of antibody-dependent cytotoxicity (ADCC)), increased stability, and / or the ability to form heterodimer proteins.

[0179] There are three classes of human Fcγ receptors (FcγRs) that enable IgG to interact with immune system cells. Furthermore, neonatal FcRn plays a role in the placental transport of IgG and the prevention of IgG degradation, thereby extending the half-life of circulating IgG. The various types of FcγRs have their own characteristics, including diverse intracellular distributions and functionally important polymorphisms. FcγRI (high affinity), FcγRlla (intermediate affinity), and FcγRlla and FcγRlllb (low affinity) are all activating receptors, and the involvement of these receptors can lead to inflammatory responses, including the destruction of target cells. In contrast, the low-affinity FcγRIlb is the only inhibitory FcγR and is therefore of particular interest. FcγRIlb is found on B cells and acts to prevent activation and proliferation, and is found on mast cells and basophils, as well as on macrophages, monocytes, and neutrophils, and can inhibit the destruction of target cells.

[0180] For example, the Fc polypeptide described herein may be combined with a binding domain that can specifically bind to a target molecule. That is, in some embodiments, the present invention relates to a fusion protein comprising the Fc region described herein. In the fusion protein, one or more polypeptides are operably linked to the Fc region of the present invention. For example, the Fc domain may be linked to a Fab binding domain. The binding domain may comprise two or more polypeptide chains associated by, for example, covalent bonds or other means (e.g., hydrophobic interactions, ionic interactions, or linkages via sulfide crosslinks).

[0181] The modifications described herein can also be combined with any other Fc modifications, such as modified effector functions.

[0182] Generally, one or more polypeptides operably linked to the Fc region of the present invention may be any protein or small molecule, for example, a binding domain derived from any molecule that has specificity to another molecule and can bind to said molecule. The binding domain will have the ability to interact with a target molecule, which may be any target (e.g., carbohydrates, lipids (phospholipids, etc.), or nucleic acids), but preferably another polypeptide. Preferably, the interaction will be specific. Typically, the target will be an antigen on a cell or a receptor having a soluble ligand. This is selected as a therapeutic target, and it is desirable that it be bound to a molecule having the properties discussed above. The target may be located on or within a target cell, for example, a target cell in which it is desirable to lyse or in which apoptosis is desirable. That is, protein fusion partners include, but are not limited to, the variable region of any antibody, the target-binding region of a receptor, adhesion molecules, ligands, enzymes, cytokines, antigens, chemokines, or any other protein or protein domain. Small molecule fusion partners include any therapeutic agent that directs Fc fusion to the therapeutic target. Such targets may be any molecule, preferably an extracellular receptor involved in disease.

[0183] The present invention also relates to the use of Fc as defined herein in a method for producing Fc-fusion proteins. For example, this can be used to create heterodimers whose half-lives are extended by having an Fc fusion.

[0184] In another embodiment, the antibody of the present invention comprises further parts, for example, a part that extends the half-life, such as canine or canine serum albumin or a variant thereof. The antibody may be modified to extend its half-life, for example, by chemical modification, particularly PEGylation, or incorporation into liposomes.

[0185] The half-life may be extended by at least 1.5 times, preferably at least 2 times, for example, at least 5 times, for example, at least 10 times, or more than 20 times, compared to the half-life of the corresponding antibody that has not had its half-life extended. For example, the extension of the half-life may be more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example, more than 12 hours, or more than 24, 48, or 72 hours, compared to the corresponding antibody that does not have a portion that extends the half-life.

[0186] In another embodiment, the portion is a therapeutic portion, such as a drug, enzyme, or toxin. In one embodiment, the therapeutic portion is a toxin, such as a cytotoxic radionuclide, chemical toxin, or protein toxin. That is, the present invention also includes immunoconjugates, such as antibodies conjugated to a second molecule, usually a toxin, radioisotope, or label. These conjugates are used in immunotherapy to develop monoclonal antibody therapies as targeted forms of chemotherapy, often known as antibody-drug conjugates (ADCs).

[0187] The toxic payload may be selected from small molecules (e.g., meitansanoids, auristatin), protein toxins (e.g., Pseudomonas exotoxin, diphtheria toxin), cytolytic immunomodulatory proteins for killing target cells (e.g., Fas ligand), bioactive peptides for extending the pharmaceutically active half-life of natural peptides (e.g., GLP-1), enzymes for modifying the biochemistry of the target microenvironment (e.g., urease), or radionuclides for killing or imaging tumor cells (e.g., 90Y, 111In).

[0188] In another embodiment, the antibody is labeled with a detectable or functional label. The label may be any molecule that generates or can induce a signal, including, but is not limited to, fluorescent dyes, fluorescent agents, radiolabels, enzymes, chemiluminescent agents, nuclear magnetic resonance activity labels, or photosensitizers. That is, binding may be detected and / or measured by detecting fluorescence or emission, radioactivity, enzymatic activity, or absorbance.

[0189] The portion can be linked to a heterodimer protein, such as an antibody, using a linker known in this technology, for example, via a chemical linker or a peptide linker. The linkage may be covalent or non-covalent. An exemplary covalent linkage is via a peptide bond. In some embodiments, the linker is a polypeptide linker (L). Preferred linkers include, for example, linkers having a GS residue such as (Gly4Ser)n, where n=1 to 50, for example, 1 to 10. Other linkage / conjugation methods include, for example, cysteine ​​conjugation for cysteine-based site-specific antibody conjugation to a toxic payload.

[0190] In another embodiment, the present invention relates to a polypeptide comprising a canine IgG CH3 domain, wherein the polypeptide comprises an amino acid substitution by a negatively charged amino acid at the K414 position of IgG-D or the corresponding position of IgG-A, B, or C, or an amino acid substitution by a positively charged amino acid at the E424 position of IgG-D or the corresponding position of IgG-A, B, or C. In one embodiment, the polypeptide comprises an Fc domain. In another embodiment, the present invention relates to a polypeptide comprising a canine IgG CH3 domain, wherein the polypeptide includes an amino acid substitution by an amino acid with an opposite charge at one or more positions of IgG-D, such as 424, 423, 378, 414, 416, 433, 392, 383, 393, 383, or 378, or at the corresponding positions of IgG-A, B, or C, and / or an amino acid substitution by a charged amino acid or an amino acid with an opposite charge at position 436 of IgG-D or at the corresponding position of IgG-A, B, or C, and / or an amino acid substitution by N at position 425 of IgG-D or at the corresponding position of IgG-A, B, or C.

[0191] For example, with respect to IgG-B, the polypeptide includes a canine IgG CH3 domain, and the polypeptide includes an amino acid substitution with an oppositely charged amino acid at one or more of the following positions: 428, 427, 382, ​​383, 420, 418, 437, 467, 386, 397, 396, and 429, or a substitution at 429 or 387.

[0192] That is, with respect to IgG-B, in one embodiment of polypeptide, substitution at E428 is E428K, E428R, or E428H; substitution at D427 is D427K, D427R, or D427H; substitution at E382 is E382K, E382R, or E382H; substitution at E383 is E383K, E383R, E383H, or E383R; substitution at R420 is R420E or R420D; substitution at K418 is The substitution in K437 is K418E or K418D, the substitution in K437 is K437D or K437E, the substitution in K467 is K467D or K467E, the substitution in K396 is K396E or K396D, the substitution in K386 is K386E or K386D, the substitution in D397 is D397K, D397R, or D397H, the substitution in K387 is K387D or K387E, and the substitution in D429 is D429N.

[0193] In one embodiment, the substitution at E428 is E428K, the substitution at D427 is D427K, the substitution at E382 is E382K, the substitution at E383 is E383K, the substitution at R420 is R420D, the substitution at K418 is K418E, the substitution at K437 is K437D, the substitution at K467 is K467E, the substitution at K396 is K396E, the substitution at K386 is K386D, the substitution at D397 is D397K, the substitution at K387 is K387D, and the substitution at D429 is D429N.

[0194] The specific substitutions that can be created in polypeptides are shown in Table 1 and Table 2 by their mutation IDs, and are also described above regarding heterodimer polypeptides.

[0195] In one embodiment, the polypeptide is a canine, canine, or canine-derived heavy chain. In one embodiment, the polypeptide contains one or more further mutations in the CH3 domain. Exemplary mutations are described in detail above. The polypeptide is preferably an isolated polypeptide.

[0196] In another aspect, the present invention relates to a nucleic acid molecule encoding a heterodimer protein, or a nucleic acid molecule encoding a polypeptide of the present invention. The nucleic acid is preferably an isolated nucleic acid.

[0197] "Isolated nucleic acid molecules" means genomic DNA or RNA, mRNA, cDNA, or synthetic origin, or any combination thereof, in which the isolated polynucleotide is not associated with all or part of naturally occurring polynucleotides, or is linked to polynucleotides that are not naturally linked.

[0198] Furthermore, the present inventors provide isolated nucleic acid constructs comprising at least one nucleic acid as defined above. The constructs may be in the form of plasmids, vectors, transcription or expression cassettes. That is, the present invention also relates to plasmids, vectors, transcription or expression cassettes comprising the nucleic acids of the present invention. Expression vectors used in the present invention may be constructed from starting vectors such as commercially available vectors. After the vector is constructed and nucleic acid molecules encoding the heavy chain or light chain and heavy chain sequence are inserted into appropriate sites in the vector, the completed vector may be inserted into host cells suitable for amplification and / or polypeptide expression.

[0199] The present invention also relates to isolated recombinant host cells comprising one or more nucleic acid molecules, plasmids, vectors, transcription or expression cassettes as described above. Transformation of selected host cells with an expression vector can be achieved by well-known methods, including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The chosen method will depend in part on the type of host cell used.

[0200] The host cell may be eukaryotic or prokaryotic, e.g., a bacterium, virus, plant, mammal, or other suitable host cell. In one embodiment, the cell is an Escherichia coli (E. coli) cell. In another embodiment, the cell is a yeast cell. In yet another embodiment, the cell is a Chinese hamster (CHO) cell, a HeLa cell, or other cell obvious to those skilled in the art. Mammalian cell lines available as hosts for expression are well known in the Art, but are not limited to immortalized cell lines available from the American Type Culture Collection (ATCC), and the recombinant polypeptide of the present invention can be prepared using any cell line used in expression systems known in the Art.

[0201] Generally, host cells are transformed by a recombinant expression vector containing DNA encoding a desired bispecific antibody. Possible host cells include prokaryotes, yeasts, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli or Bacillus. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include COS-7 cells, L cells, CI27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, or their derivatives, and related cell lines that grow in serum-free medium, HeLa cells, BHK cell lines, CVIIEBNA cell lines, human embryonic kidney cell lines such as 293, 293EBNA, or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues and primary explants, HL-60, U937, HaK, or Jurkat cells. Optionally, mammalian cell lines such as HepG2 / 3B, KB, NIH 3T3, or S49 can be used for polypeptide expression when polypeptide use is desired in various signal transduction or reporter assays.

[0202] Other suitable host cells include insect cells using baculovirus expression systems in insect cells mediated by viruses and nucleic acid vectors, plant cells, transgenic plants, and transgenic animals.

[0203] Alternatively, polypeptides can be produced in fungal cell lines and lower eukaryotes such as yeast, or in prokaryotes such as bacteria. Suitable yeasts include S. cerevisiae, S. pombe, Kluyveromyces strains, Pichia pastoris, Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli, B. subtilis, S. typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. When bispecific antibodies are produced in yeast or bacteria, it is desirable to modify the produced product, for example, by phosphorylation or glycosylation at the appropriate site, in order to obtain a functional product. Such covalent bonding can be achieved using known chemical or enzymatic methods.

[0204] Host cells, when cultured under appropriate conditions, can be used to express bispecific antibodies, which can then be collected from the culture medium (if the host cells secrete the bispecific antibodies into the medium) or directly from the host cells producing them (if the bispecific antibodies are not secreted). The selection of appropriate host cells depends on various factors, such as the desired expression level, the desired or necessary polypeptide modification for activity (glycosylation or phosphorylation, etc.), and the ease of folding into a bioactive molecule.

[0205] Those skilled in the art will know that there are various methods for identifying, obtaining, and optimizing the heterodimer proteins described herein, including in vitro and in vivo expression libraries. Optimization techniques known in this art, such as display (e.g., ribosome and / or phage display) and / or mutagenesis (e.g., error-prone mutagenesis), can be used.

[0206] In such methods, for example, to facilitate screening, a set, collection, or library of amino acid sequences may be displayed on a phage, phagemid, ribosome, or suitable microorganism (such as yeast). Suitable methods, techniques, and host organisms for displaying and screening amino acid sequences (sets, collections, or libraries) will become apparent to those skilled in the art (see, for example, Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press; 1st edition, Brian K. Kay, Jill Winter, John McCafferty, 1996).

[0207] Libraries, such as phage libraries, are generated by isolating cells or tissues that express antibodies or heavy chains, cloning sequences encoding genes derived from mRNA induced from the isolated cells or tissues, and displaying the encoded proteins using the library. Heavy chains can be expressed in mammals, cells, yeast, or other expression systems.

[0208] Therefore, in another aspect, the present invention also relates to an expression library comprising a plurality of polypeptides or antibodies described herein. Phage display library screening involves a large number (typically 10) 9This method is advantageous over several other screening methods because a single phage display library can contain more than 100 different polypeptides. This allows for the screening of highly diverse libraries in a single screening step. Generally, the library contains a repertoire of V genes cloned (e.g., collected from a population of lymphocytes or assembled in vitro) for the display of the variable domains of the associated heavy and light chains on the surface of filamentous bacteriophages. Phages are selected by binding to antigens. Soluble antibodies are expressed from bacteria infected with phages, and the antibodies can be improved, for example, by mutagenesis. Methods for producing antibodies and antibody libraries are established by constructing, screening, and evolving antibodies.

[0209] In a further embodiment, the present invention is a) A step of transforming host cells with nucleic acids or vectors described herein, b) A step of culturing host cells and expressing polypeptides containing first and second IgG CH3, and c) A step of recovering heterodimer proteins or polypeptides from host cell cultures. The present invention relates to a method for producing heterodimer proteins or polypeptides, including [specific components / components].

[0210] The heterodimer proteins or polypeptides obtained by this method are also within the scope of the present invention.

[0211] The bispecific antibodies of the present invention, based on the IgG format consisting of two heavy chains and two light chains, can be produced by various methods known in this art. For example, bispecific antibodies can be produced by fusing two antibody-secreting cell lines to create a new cell line, or by expressing two antibodies in a single cell using recombinant DNA technology. Since each heavy chain from each antibody forms a monospecific dimer (also called a homodimer) containing two identical heavy chain pairs with the same specificity, and a bispecific dimer (also called a heterodimer) containing two different heavy chain pairs with different specificities, these approaches yield a variety of antibody types. Furthermore, the light and heavy chains from each antibody may randomly pair, forming inappropriate, non-functional combinations. This problem is known as heavy-light chain mispairing, but it can be solved by selecting antibodies that share a common light chain for expression as bispecific antibodies. Methods to address the problem of light-heavy chain mispairing include the production of bispecific antibodies using a single light chain. This requires manipulation of heavy and light chains, or a novel antibody library using a single light chain that limits diversity. Furthermore, antibodies with a common light chain have been identified from transgenic mice possessing a single light chain. Another approach involves replacing the CH1 domain of one heavy chain with the CL domain of its associated light chain (crossumab technology). The scFv format is also included.

[0212] In another embodiment, a pharmaceutical composition is provided comprising the heterodimer molecule of the present invention and an optionally pharmaceutically acceptable carrier. The heterodimer proteins or pharmaceutical compositions described herein can be administered, but are not limited to, orally, topically, parenterally, sublingually, transrectally, transvaginally, ophthalmologically, intranasally, transpulmonaryly, intradermally, intravitreally, intratumorally, intramuscularly, intraperitoneally, intravenously, subcutaneously, intracerebrally, transdermally, transmuscularly, by inhalation, or topically, particularly by the ear, nose, eye, or skin, or by convenient routes including inhalation. In another embodiment, delivery is the delivery of a nucleic acid encoding a drug, for example, a nucleic acid encoding the molecule of the present invention is delivered.

[0213] Parenteral administration includes, for example, intravenous, intramuscular, intra-arterial, intraperitoneal, intranasal, transrectal, intrasacral, intradermal, topical, or subcutaneous administration. Preferably, the composition is administered parenterally.

[0214] The pharmaceutically acceptable carrier or vehicle may be particulate, and therefore the composition may be, for example, in the form of a tablet or powder. The term “carrier” means a diluent, adjuvant, or excipient with which the drug-antibody conjugate of the present invention is administered. Such pharmaceutical carriers may be liquids, such as water, and oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and others. The carrier may be saline solution, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and others. Furthermore, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, when administered to animals, the heterodimer protein or composition and the pharmaceutically acceptable carrier of the present invention are sterile. When the drug carrier conjugate of the present invention is administered intravenously, water is a preferred carrier. Salt solution and aqueous dextrose and glycerol solution can also be used as liquid carriers, particularly in injectable solutions. Suitable pharmaceutical carriers include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and other excipients. The composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, if desired.

[0215] The pharmaceutical composition may be in the form of a liquid, such as a solution, syrup, emulsion, or suspension. The liquid may be useful for oral administration or for injection, infusion (e.g., IV infusion), or subcutaneous delivery.

[0216] When intended for oral administration, the composition may be in solid or liquid form, and the forms considered herein as solid or liquid include semi-solid, semi-liquid, suspension, and gel forms.

[0217] As a solid composition for oral administration, the composition can be formulated in the form of a powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, or similar forms. Such a solid composition typically contains one or more inert diluents. Furthermore, it may contain one or more of the following: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, or corn starch; lubricants such as magnesium stearate; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate, or orange flavoring; and colorants. If the composition is in the form of a capsule (e.g., a gelatin capsule), it may contain, in addition to the above types of materials, a liquid carrier such as polyethylene glycol, cyclodextrin, or fatty oil.

[0218] When intended for oral administration, the composition may contain one or more sweeteners, preservatives, dyes / colorants, and flavor enhancers. For administration by injection, the composition may contain one or more surfactants, preservatives, humectants, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0219] The composition may take the form of one or more medication units.

[0220] In certain embodiments, it may be desirable to administer the composition locally to the area requiring treatment, or by intravenous injection or infusion.

[0221] The effective / active amount of the heterodimer proteins or pharmaceutical compositions described herein for the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical methods. Furthermore, in vitro or in vivo assays may be optionally employed to help identify the optimal dosage range. The exact dose to be used in the composition will also depend on the route of administration and the severity of the disease or condition, and should be determined according to the judgment of the clinician and the individual patient's situation. Factors such as age, weight, sex, diet, timing of administration, excretion rate, host condition, drug combination, sensitivity to response, and disease severity should be taken into consideration.

[0222] Typically, the amount is at least about 0.01% of the heterodimer protein of the present invention relative to the mass of the composition. When intended for oral administration, this amount may vary in the range of about 0.1% to about 80% relative to the mass of the composition. A preferred oral composition may contain about 4% to about 50% of the heterodimer protein of the present invention relative to the mass of the composition.

[0223] The composition may be prepared to contain parenteral administration units of the heterodimer protein of the present invention at a concentration of approximately 0.01% to approximately 2% by mass.

[0224] For administration by injection, the composition may typically contain about 0.1 mg / kg to about 250 mg / kg per animal's body weight, preferably about 0.1 mg / kg to about 20 mg / kg per animal's body weight, and more preferably about 1 mg / kg to about 10 mg / kg per animal's body weight. In one embodiment, the composition is administered in doses of about 1 to 30 mg / kg, for example, about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The administration schedule may vary, for example, from once a week to once every two, three, or four weeks.

[0225] The present invention also relates to therapeutic applications of heterodimer proteins, such as antibodies or fragments thereof, as described herein.

[0226] As used herein, “to treat,” “to treat,” or “treatment” means to prevent or alleviate a disease. For example, treatment may include slowing the progression of symptoms associated with a disease and / or reducing the severity of such symptoms that progress with or are expected to progress with the disease. The term includes improvement of existing symptoms, prevention of further symptoms, and improvement or prevention of the underlying cause of such symptoms. That is, the term indicates that a beneficial outcome is conferred to at least some of the mammal being treated, for example, the affected dog. Many medical treatments are effective for some, but not all, of the affected dogs being treated.

[0227] The terms "subject" or "patient" refer to an animal, preferably a companion animal, specifically a dog, that is the subject of treatment, observation, or experimentation.

[0228] The present invention also relates to heterodimer proteins or pharmaceutical compositions described herein for use in the treatment or prevention of diseases.

[0229] In another aspect, the present invention also relates to the use of the heterodimer proteins or pharmaceutical compositions described herein for use in the treatment or prevention of diseases. In another aspect, this disclosure relates to the use of the heterodimer proteins or pharmaceutical compositions described herein in the manufacture of pharmaceuticals for the treatment or prevention of diseases enumerated herein.

[0230] In one embodiment, a heterodimer protein, such as an antibody or a fragment thereof, binds to a therapeutic target. This may be a tumor-associated antigen (TAA). Tumor antigens can be broadly classified into carcinoembryonic antigens (typically expressed only in fetal tissue and cancerous somatic cells), oncoviral antigens (encoded by oncogenic transforming viruses), overexpressed / accumulated antigens (expressed in both normal and neonatal tissues, with significantly elevated expression levels in neonatal tissues), oncotesticular antigens (expressed only in cancer cells and adult reproductive tissues such as the testes and placenta), lineage-restricted antigens (expressed primarily by a single cancer histological type), mutated antigens (expressed only by cancer as a result of gene mutations or transcriptional changes), posttranslationally modified antigens (tumor-associated changes in glycosylation, etc.), or idiotype antigens (highly polymorphic genes in which tumor cells express specific "chronotypes," such as in B-cell or T-cell lymphoma / leukemia, resulting from clonal abnormalities).

[0231] In one embodiment, tumor-associated antigens include PSMA, Her2, Her3, CD123, CD19, CD20, CD22, CD23, CD74, BCMA, CD30, CD33, CD52, EGRF, CECAM6, CAXII, CD24, ETA, MAGE, mesothelin, cMet, TAG72, MUC1, MUC16, STEAP, EphvIII, FAP, GD2, IL-13Ra2, L1-CAM, PSCA, GPC3, gpA33, CA-125, gangliosides G(D2), G(M2) and G(D3), Ep-CAM, CEA, bombesin-like peptide, PSA, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, and VEGFR derivatives. The receptors selected are (e.g., VEGFR3), estrogen receptor, Lewis Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin, IL-2R, TAG-72, and CO17-1A.

[0232] In yet another embodiment, a heterodimer protein, such as an antibody or a fragment thereof, inhibits the growth and / or proliferation of tumor cells through binding to an antigen on the tumor cell.

[0233] In one embodiment, a heterodimer protein, such as an antibody or a fragment thereof, is an inhibitor of an immune checkpoint molecule. This may be selected from inhibitors of one or more of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta. In another embodiment, the antibody may be an activator of a costimulatory molecule selected from, for example, OX40, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4, or an agonist for one or more of ICAM-1.

[0234] In yet another embodiment, a heterodimer protein, such as an antibody or a fragment thereof, binds to cytokines, such as interleukins, including interleukin-17, interleukin-4, interleukin-13, or interleukin-31, which are important cytokines involved in itching and inflammation associated with atopic dermatitis.

[0235] In another embodiment, the target is tumor necrosis factor (TNF), such as TNF-alpha.

[0236] In another embodiment, the target is nerve growth factor (NGF) and / or NGF receptors, which are therapeutic targets in the treatment of acute and chronic pain conditions NGF.

[0237] In one embodiment, the target is GnRH used for immunocastration.

[0238] A bispecific heterodimer protein, such as an antibody or a fragment thereof, that binds to two different targets selected from the above targets, for example, an immune checkpoint molecule and another target, a TAA and another target, or a cytokine and another target. In one embodiment, the heterodimer protein, such as an antibody or a fragment thereof, binds to two different immune checkpoint molecules, two different TAAs, or a TAA and an immune checkpoint molecule.

[0239] Diseases that can be treated with heterodimer proteins, such as antibodies or fragments thereof as described herein, or pharmaceutical compositions, can be selected from cancer, immune diseases, neurological diseases, inflammatory diseases, allergies, transplant rejection, viral infections, immunodeficiency, autoimmune diseases, and other immune system-related diseases.

[0240] In one embodiment, the protein may be used to treat pain by targeting, for example, NGF.

[0241] In one embodiment, the treatment is, for example, immunocastration by targeting anti-GnRH.

[0242] Cancer can be selected from solid or non-solid tumors. For example, cancer can be selected from bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, breast cancer, brain cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, kidney cancer, soft tissue sarcoma, urethral cancer, bladder cancer, kidney cancer, lung cancer, non-small cell lung cancer, thymoma, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphoma, e.g., B-cell lymphoma, Hodgkin's disease, non-Hodgkin's disease, stomach cancer, leukemia, e.g., ALL, CLL, AML, urothelial carcinoma, leukemia, and multiple myeloma.

[0243] In one embodiment, the tumor is a solid tumor. Examples of solid tumors that can be treated accordingly include breast cancer, lung cancer, colorectal cancer, pancreatic cancer, glioma, and lymphoma, such as T-cell lymphoma caused by feline leukemia virus (FeLV). Some examples of such tumors include squamous cell tumors such as epidermal tumors and head and neck tumors, colorectal tumors, prostate tumors, mammary gland tumors, lung tumors including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include CNS, neoplasms, neuroblastoma, hemangioma, meningioma and brain metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma. Examples of angiogenic skin cancers for which the antagonist of the present invention is effective include squamous cell carcinoma, basal cell carcinoma, and skin cancers that can be treated by inhibiting the growth of malignant keratinocytes such as malignant keratinocytes in the veterinary field.

[0244] In one embodiment, the tumor is a non-solid tumor. Examples of non-solid tumors include leukemia, multiple myeloma, and lymphoma.

[0245] In one embodiment, the cancer is locally advanced, unresectable, metastatic, or recurrent cancer.

[0246] Cancers whose growth can be inhibited using the antibodies of the present invention typically include cancers that respond to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer).

[0247] In one embodiment, the protein may target CD3 for cancer treatment. Optionally, the protein may be provided in the format of a bispecific T cell engager (BiTE).

[0248] In one embodiment, the cancer progresses after another treatment, such as chemotherapy.

[0249] In another embodiment, the disease is selected from autoimmune diseases, inflammatory conditions, allergies and allergic conditions, hypersensitivity reactions, severe infections, and rejection after organ or tissue transplantation. The disease is an indefinite list, namely psoriasis, systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, spondyloarthritis, systemic sclerosis, idiopathic inflammatory myopathy, Sjögren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renal disease, demyelinating diseases of the central and peripheral nervous system such as multiple sclerosis, idiopathic demyelinating polyneuropathy or Guillain-Barré syndrome, and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory bowel disease Intestinal diseases, gluten-sensitive intestinal diseases, and autoimmune or immune-mediated skin diseases including Whipple's disease and bullous skin diseases, allergic diseases such as erythema multiforme and contact dermatitis, asthma, allergic rhinitis, atopic dermatitis, food allergies and urticaria, immune pulmonary diseases such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, autoimmune hematological diseases (including, for example, hemolytic anemia, aplastic anemia, erythrocytic anemia, and idiopathic thrombocytopenia), autoimmune inflammatory bowel diseases (including, for example, ulcerative colitis, Crohn's disease, and irritable bowel syndrome), transplant-related diseases including graft rejection and graft-versus-host disease, or any veterinary equivalent thereof may be selected.

[0250] In one embodiment, the heterodimer protein or pharmaceutical composition described herein is used in combination with an existing therapy or therapeutic agent, such as an anticancer therapy. That is, in another embodiment, the present invention also relates to a combination therapy comprising the administration of the heterodimer protein or pharmaceutical composition of the present invention and an anticancer therapy. The anticancer therapy may include a therapeutic agent or radiotherapy, and may include gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, targeted anticancer therapy, or oncolytic drugs. Other therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancer cells, antigen-presenting cells such as dendritic cells pulsed with tumor antigens, tumor-inducing antigens, or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNa2, GM-CSF), target small molecules and biomolecules (components of signal transduction pathways, e.g., tyrosine kinase modulators and tyrosine kinase receptor inhibitors, and agents that bind to tumor-specific antigens, including EGFR antagonists), anti-inflammatory agents, cytotoxic agents, radiotoxic agents, or cells transfected with genes encoding immunosuppressants and immunostimulatory cytokines (e.g., GM-CSF), and chemotherapy. In one embodiment, heterodimer proteins are used in combination with surgery.

[0251] In one embodiment, the heterodimeric protein or pharmaceutical composition described herein is administered with an immunomodulatory agent, a checkpoint modulator, an agent involved in the activation of T cells, a tumor microenvironment modifier (TME), or a tumor-specific target. For example, the immunomodulatory agent may be an inhibitor of an immune checkpoint molecule selected from inhibitors of one or more of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR beta. In another embodiment, the immunomodulatory agent may be an activator of a costimulatory molecule selected from agonists of one or more of OX40, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4, or ICAM-1.

[0252] In certain embodiments of the invention, the heterodimeric protein or composition is administered simultaneously with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of the composition of the invention, preferably at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, more preferably several months (e.g., up to 3 months) before or after administration of the composition of the invention.

[0253] In some embodiments, the heterodimeric protein or pharmaceutical composition described herein may be administered with two or more therapeutic agents. In some embodiments, the heterodimeric protein or pharmaceutical composition may be administered with two or more therapeutic agents.

[0254] The heterodimeric protein or pharmaceutical composition described herein may be administered simultaneously or at different times, e.g., simultaneously, separately, or sequentially, with other therapies or therapeutic compounds or therapies.

[0255] In another embodiment, the present invention provides a kit comprising the heterodimer protein or pharmaceutical composition of the present invention for the treatment or prevention of a disease, the diagnosis, prognosis, or monitoring of a disease. Such a kit may include other components, packaging, and / or instructions.

[0256] The kit may include a labeled heterodimer protein or pharmaceutical composition as described herein and one or more compounds for detecting the label.

[0257] In another aspect, the present invention provides the heterodimer proteins or pharmaceutical compositions of the present invention as described herein, packaged in a lyophilized form or packaged in an aqueous medium.

[0258] In another embodiment, the heterodimer proteins described herein are used for non-therapeutic purposes, such as diagnostic tests and assays.

[0259] Further aspects and embodiments of the present invention will be apparent to those skilled in the art, given the present disclosure, including the following experimental examples.

[0260] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have the meanings commonly understood by those skilled in the art. While the above disclosure provides a general description of the subject matter encompassed within the scope of the invention, including methods for constructing and using the invention and its best modes, the following examples are provided to further enable those skilled in the art to practice the invention and to provide a complete written specification. However, those skilled in the art will recognize that the details of these examples should not be read as limiting the invention, and that the scope of the invention should be understood from the claims and equivalents attached to this disclosure. Further various aspects and embodiments of the invention will become apparent to those skilled in the art in consideration of this disclosure.

[0261] All records described herein, including any references to gene accession numbers and patent applications, are incorporated herein by reference in their entirety.

[0262] As used herein, “and / or” is considered to be each specific disclosure of two identified features or components, with or without other features or components. For example, “A and / or B” is considered to be each specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were presented separately herein. Unless otherwise indicated by the context, the descriptions and definitions of features presented above are not limited to any particular aspect or embodiment of the Invention, but apply equally to all aspects and embodiments described.

[0263] The present invention is further described in non-limiting embodiments and non-limiting sections. [Examples]

[0264] (Example 1) Identification of CH3 / CH3 interface charge pairs in canine IgG isoforms by modeling. Several methods have been applied to manipulate human IgG to enhance heavy-chain CH3-CH3 heterodimerization against interfering monospecific dimers. These IgG heavy-chain heterodimerization methods have paved the way for the production of bispecific and multispecific antibodies. Two major methods promote heterodimerization using isotype and species-matched IgG heavy chains: "knob-in-hole (kiH)" by Genentech (WO9627011) and electrostatic steering by Chugai and Amgen (WO2006 / 106905).

[0265] Methods for pairing bispecific / multispecific antibodies in dogs have not yet been tested.

[0266] To explore the possibility of such a mechanism in canine IgG, we performed structure-based homology modeling using the human IgG dimer structure (PDB database: 1l6x) as a guide for exploring the canine CH3-CH3 interface. We found that the canine position equivalent to the human KiH modification is conserved among all four canine isotypes and the human isotype. These residues are located in the hydrophobic core in the central part of the CH3-CH3 interface (Figure 1).

[0267] To explore the mechanism based on electrostatic steering, charged residues at the interface of canine IgG dimers are mapped based on an experimentally defined human CH3-CH3 interface (Gunasekaran et al., see above).

[0268] In addition to the finding that many residues forming charge pairs are conserved between humans and dogs, this analysis highlights the existence of canine-specific charge pairs (Figure 2, gray shading). These charge pairs are highly conserved across all canine IgG isotypes and are uncharged at their corresponding positions in all human IgG isotypes (Figure 2).

[0269] Amino acid mutations that reverse the charge at these positions should create charge-based heterodimer attraction and homodimer repulsion, and therefore increase the proportion of heterodimer assemblies (Figure 3). A series of amino acid substitutions in chains A and B of canine IgG, including both electrostatic steering and KiH, are summarized in Tables 1 and 2, and methods for generating these point mutations are described in Example 2.

[0270] (Example 2) Construction of a bispecific chimeric antibody expression vector Canine genomic DNA is extracted from the blood of a Beagle (Envigo). Using the canine genomic DNA as a template, the constant regions of canine IgG-A, IgG-B, IgG-C, and IgG-D are PCR amplified using Q5 high-fidelity DNA polymerase (New England Biolabs). PCR primers are designed based on publicly available sequence information from Basenge dogs (IgG-A, IgG-B, IgG-C, IgG-D; GenBank #SDHF01000364), and where available, WGS sequences from Beagle dogs (GenBank accession numbers AOCS01185922, AOCS01019635, AOCS01019304, AOCS01185926) are used to amplify the corresponding regions derived from Beagle DNA. PCR fragments are cloned into the TOPO cloning vector pCR-Blunt using the Zero Blunt PCR cloning kit (Invitrogen). Individual clones are sequence-validated by Sanger sequencing. Alternatively, canine IgG Fc can be synthesized using GeneArt (Thermo Fisher), which employs a codon-optimized amino acid sequence for CHO cell expression.

[0271] To enable the differentiation of heterodimers from homodimer contaminants based on size, two chimeric heavy chains with different molecular weights were generated. One heavy chain was generated by fusing OKT3-ScFv to the hinge-CH2-CH3 region of canine IgG-B and IgG-D isoforms, while the other chain was generated by Fc alone. Specifically, OKT3-ScFV and the canine IgG-B and IgG-D hinge-CH2-CH3 region were PCR amplified using Q5 high-fidelity DNA polymerase, and then assembled using NEBuilder HIFI DNA Assembly (New England Biolabs) to generate ScFv-Fc. Subsequently, the ScFv-Fc (or "scFv-OKT3+Fc-B") and Fc-only (or "Fc-B") heavy chains were cloned into the mammalian expression vectors PetML5 (hygromycin-resistant) and petML6 (brasticidin-resistant), respectively. Both the PetML5 and PetML6 expression vectors consist of a CAG promoter and bgh-pA that drive the expression of the cloned insert, which in this case is a modified ScFv-Fc or Fc. For cell selection, a pgk-hph-pgk-pA cassette is present in the PetML5 vector to confer hygromycin resistance, while a pgk-bsr-pgk-pA cassette is present in the PetML6 vector to confer brassicidine resistance. Both cassettes for expression and drug resistance are flanked by piggyback reverse terminal repeats (ITRs), and thus sequences flanked by piggyback ITRs can be stably incorporated into mammalian host cells, in this example, CHO cells, upon co-transfection with a piggyback transposase. The plasmid vector backbone includes an ampicillin resistance cassette and a ColE1 bacterial origin for bacterial replication for bacterial selection. Heavy chain and antibiotic resistance gene expression units are adjacent to DNA transposase piggyback terminal reverse repeats, which mediate stable integration into host cells in the presence of piggyback transposases.

[0272] Subsequently, a series of mutations shown in Table 1 and Table 2 are generated by site-directed mutagenesis using the method described by Liu H and Naismith J (BMC Biotechnology 2008, 8:91). All mutations are verified by Sanger sequencing. As a control, the KiH mutation is introduced into human IgG4PE (Figure 1).

[0273] (Example 3) Production of chimeric antibodies CHO-S cells are cultured in suspension in an Erlenmeyer shaking flask in FreeStyle F17 CHO expression medium supplemented with L-glutamine and an anti-aggregation agent (Thermo Fisher Scientific). The suspension cells are cultured with shaking at 150 rpm in a humidified incubator at 37 °C and 8% CO2. Using PEIMax, a heterodimer heavy chain pair containing two plasmid DNAs is co-transfected into CHO-S cells with a plasmid containing the piggyBac transposase at a concentration of 1 mg / ml per 1×10 6 cells, with a ratio of 1 μg of DNA to 3 μl of PEIMax. As shown in the figure, constructs of ScFv-Fc and Fc with various ratios are tested. Stable integration is selected using hygromycin and blasticidin 24 hours after transfection. Six to eight days after drug selection, the cells are harvested into non-selective medium prior to production. For production, 2×10 6 CHO cells per ml of medium are seeded with shaking at 150 rpm at 32 °C in 8% CO2. The cells are fed with L-glucose, Cell Boost 7a, and Cell Boost 7b every three days. The cell viability is monitored to exceed at least 70% during the production period, and the supernatant is collected for protein A purification on day 12.

[0274] (Example 4) Evaluation of heterodimerization To evaluate the optimal ratio of two chimeric heavy chains for transfection, plasmids encoding human IgG4PE Fc or ScFv-Fc with manipulated KiH mutations (Fc-knob and ScFv-hole) were transfected into CHO-S cells in ratios of 1:0, 6:1, 3:1, 1:1, 1:3, 1:6, and 0:1. Human IgG4PE, ScFv-Fc, and Fc chains without KiH mutations, with the same transfection ratios, were used as controls. ScFv-Fc and Fc assemblies purified with protein A were evaluated by unreduced SDS gel electrophoresis. The 1:1 ratio produced the largest heterodimer and the smallest monomer contaminants (Figure 4).

[0275] To test all selected canine IgG-D ScFv-FC and Fc heavy chain pairs (Table 1), both chains containing the DNA plasmid were transfected in a 1:1 ratio, and stable integrations were selected as described. The ScFv-Fc and Fc assemblies, purified with Protein A, were examined using non-reducing SDS gel electrophoresis. The ratio of heterodimers to homodimers was analyzed by ion-exchange chromatography. Thermal stability and aggregation were also examined.

[0276] To test all selected canine IgG-B Fc and ScFv-FC strand pairs (Table 2), both strands containing the DNA plasmid were transfected in a 1:1, 1:3, or 1:6 ratio, and stable integrations were selected as described. Homo / hetero ScFv-Fc and Fc assemblies purified with Protein A were quantified using UV spectroscopy (Nanodrop 1000, Thermo Fisher Scientific), normalized to 0.25 mg / mL, and 2.5 μg of total protein was loaded into both non-reducing SDS-PAGE and HPLC size exclusion chromatography (HSEC). Heterodimers migrated or eluted differently in both SDS-PAGE and HSEC, were quantified, and compared to WT molecules. The MW of "Fc-B" is 28 kDa, thus forming a homodimer of approximately 56 kDa. The molecule "scFv-OKT3+Fc-B" has a density of 55 kDa and therefore forms a homodimer of approximately 110 kDa. The heterodimer "Fc-B:scFv-OKT3-Fc-B" has a density of approximately 83 kDa. A schematic diagram of the formation of homodimers and heterodimers is shown in Figure 4.

[0277] SDS-PAGE was stained using the standard Instant Blue (InstantBlue® Coomassie Protein Stain (ISB1L) (ab119211)) protocol. SDS-PAGE bands were quantified by densitometry using ImageJ software (https: / / imagej.nih.gov / ij / ). Briefly, rectangular selections were generated for each lane, and the band peaks were merged using the executed ImageJ plugin. The area under each peak (homo / heterodimer) was plotted.

[0278] HPLC-SEC chromatography (column: BioResolve SEC mAb 200A, 2.5 μm column, Waters Corporation) was performed using ACQUITY H-class Bio (Waters Corporation) with PBS as the mobile phase at an isotonic flow rate of 0.575 mL / min. Test samples were centrifuged at 20000 g for 5 minutes (using a standard benchtop centrifuge) to remove any precipitates. Each detected chromatographic peak was integrated using Empower software. The percentage and area (indicating antibody concentration) of heterodimer species were determined for each molecule and compared with the WT molecule.

[0279] SDS-PAGE analysis with a 1:1 scFv-Fc:Fc DNA ratio (using the mutations shown in Figures 7, 8, and 9; Table 2) shows that many mutants improved heterodimer formation compared to the WT IgG B sequence. In some combinations, expression was low or absent in one or both strands, suggesting that the introduced mutations may have affected polypeptide chain expression, stability, and / or folding. In addition to the increased percentage of heterodimers, it is also noteworthy that many combinations showed a single band in the MW corresponding to the heterodimer, in contrast to the at least three bands observed in the WT IgG B sequence. This likely indicates a more stable conformation induced by these mutations. Furthermore, the presence of monomeric Fc (which migrates to approximately 28 kDa, i.e., observed in mutants IDs 23 and 26) also indicates homodimer repulsion, which may be useful during further purification steps in the large-scale production of clinical products.

[0280] Figure 10 shows the densitometry analysis to present the percentage of heterodimers among these expressed proteins (mutations are as shown in Table 2).

[0281] Based on the results of SDS-PAGE with an scFv-Fc:Fc DNA ratio of 1:1, transfection was performed on selected candidate cells (IDs 4, 11, 12, 13, 16, 17, 22, 23, 24, 25, 26, 28, and 29) in the CHO cell line using two other sets of scFv-Fc:Fc DNA ratios: 1:3 and 1:6. The results of the 1:3 transfection ratio are shown in Figures 11, 12, and 13. The results of the 1:6 transfection ratio are shown in Figures 14, 15, and 16. The mutations shown in Table 2 were used.

[0282] Under these conditions, the mutants also showed increased heterodimer activity compared to the WT (IgG B) control, exhibiting a similar phenotype to monomer Fc in terms of the presence of a single heterodimer band.

[0283] Figure 17a shows the retention time of the heterodimer peak measured by HSEC analysis. Figure 17b shows the HPLC-SEC quantification of the heterodimer peak as a percentage of total protein. This shows similar results to quantification by SDS-PAGE, with an increased percentage of heterodimers in almost all mutants. Figure 17c shows the HPLC-SEC quantification of the heterodimer peak as a total area. All mutants except IDs 17, 22, and 29 showed an increased percentage of the heterodimer peak. All mutants except mutant IDs (combinations) 12, 13, 16, and 17, which showed a less stable heterodimer, and 24 and 25, which showed increased stability, showed similar stability to the WT control.

[0284] A summary table of data obtained from experiments using an scFv-Fc:Fc DNA ratio of 1:3 was created (Figure 18).

[0285] Further analysis is performed using ion-exchange chromatography (H-SCX (strong cation exchange)) to evaluate the percentage of heterodimers. This method allows for the separation of molecules based on their charge, which is particularly useful when testing the effect of these mutations in a standard IgG format where molecular weight is not useful for distinguishing between heterodimers. This is useful, for example, to guide the purification process during the production of bispecific antibodies.

[0286] The stability of heterodimers in IgG format can also be evaluated by performing accelerated stability tests using both temperature and pH as stressors. Subsequently, HSEC and HSCX are used to identify and quantify the aggregation / disintegration tendencies and the presence of protein variants to select the most stable combination.

[0287] [Table 1]

[0288] [Table 2]

Claims

1. A heterodimer protein comprising a first polypeptide containing a first canine IgG CH3 domain and a second polypeptide containing a second canine IgG CH3 domain, wherein the IgG is selected from IgG-A, B, C, or D. a) The first canine IgG CH3 domain contains an amino acid substitution with an amino acid of opposite charge at one or more positions 428, 427, 382, ​​383, and 386 of IgG-B, or at the corresponding positions of IgG-A, C, or D, and the second canine IgG CH3 domain contains an amino acid substitution with an amino acid of opposite charge at one or more positions 420, 418, 437, 467, 396, 397, and 429 of IgG-B, or at the corresponding positions of IgG-A, C, or D, b) The second canine IgG CH3 domain contains an amino acid substitution at 429 of IgG-B, or at the corresponding position of IgG-A, C, or D, and the first canine IgG CH3 domain does not contain the corresponding mutation, or c) A heterodimer protein in which the first canine IgG CH3 domain contains an amino acid substitution at 387 of IgG-B, or at the corresponding position of IgG-A, C, or D, and the second IgG CH3 domain does not contain the corresponding mutation.

2. a) The first canine IgG CH3 domain contains an amino acid substitution with an amino acid of opposite charge at one or more of the following positions: E428, D427, E382, E383, and K386, and the second canine IgG CH3 domain contains an amino acid substitution with an amino acid of opposite charge at one or more of the following positions: R420, K418, K437, K467, ​​K396, D397, and D429, b) The second canine IgG CH3 domain contains an amino acid substitution at D429, and the first canine IgG CH3 domain does not contain the corresponding mutation, or c) The heterodimer protein according to claim 1, wherein the first canine IgG CH3 domain contains an amino acid substitution at N387 and the second IgG CH3 domain does not contain the corresponding mutation.

3. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain contains an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain contains an amino acid substitution by a negatively charged amino acid at position R420.

4. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position R420 and an amino acid substitution with a negatively charged amino acid at position K418.

5. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at the D427 position, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at the R420 position.

6. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position R420 and an amino acid substitution with a negatively charged amino acid at position K437.

7. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428K, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K437 and an amino acid substitution by a negatively charged amino acid at position K418.

8. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428K, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position R420.

9. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428K, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position R420 and an amino acid substitution by a negatively charged amino acid at position K437.

10. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428K, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K420 and an amino acid substitution by a negatively charged amino acid at position K418.

11. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain contains an amino acid substitution by a positively charged amino acid at position E382, and the second canine IgG CH3 domain contains an amino acid substitution by a negatively charged amino acid at position K467.

12. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain comprises an amino acid substitution by a positively charged amino acid at position E383 and an amino acid substitution by a negatively charged amino acid at position K396.

13. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E382 and an amino acid substitution by a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K418 and an amino acid substitution by a negatively charged amino acid at position R420.

14. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E382 and an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K418 and an amino acid substitution by a negatively charged amino acid at position R420.

15. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E382 and an amino acid substitution by a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K437 and an amino acid substitution by a negatively charged amino acid at position R420.

16. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E382 and an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K437 and an amino acid substitution by a negatively charged amino acid at position R420.

17. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E383 and an amino acid substitution by a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K418 and an amino acid substitution by a negatively charged amino acid at position R420.

18. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E383 and an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K418 and an amino acid substitution by a negatively charged amino acid at position R420.

19. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E383 and an amino acid substitution by a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K437 and an amino acid substitution by a negatively charged amino acid at position R420.

20. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position E383 and an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K437 and an amino acid substitution by a negatively charged amino acid at position R420.

21. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes amino acid substitutions with a negatively charged amino acid at position K418 and a negatively charged amino acid at position K467.

22. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at the D427 position, and the second canine IgG CH3 domain includes amino acid substitutions with a negatively charged amino acid at the K437 position and a negatively charged amino acid at the R467 position.

23. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes amino acid substitutions with a negatively charged amino acid at position K418 and a negatively charged amino acid at position K396.

24. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D427, and the second canine IgG CH3 domain includes amino acid substitutions with a negatively charged amino acid at position K437 and a negatively charged amino acid at position K396.

25. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position R420 and an amino acid substitution by a negatively charged amino acid at position K467.

26. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K396 and an amino acid substitution by a negatively charged amino acid at position K467.

27. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K437, a negatively charged amino acid at position K418, and a negatively charged amino acid at position K467.

28. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution by a positively charged amino acid at position D427 and an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain includes an amino acid substitution by a negatively charged amino acid at position K437, a negatively charged amino acid at position K418, and a negatively charged amino acid at position K396.

29. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain contains an amino acid substitution by a positively charged amino acid at position E428, and the second canine IgG CH3 domain contains an amino acid substitution by a negatively charged amino acid at position K418.

30. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain contains an amino acid substitution by a positively charged amino acid at the D427 position, and the second canine IgG CH3 domain contains an amino acid substitution by a negatively charged amino acid at the K437 position.

31. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain contains an amino acid substitution by a negatively charged amino acid at position K386, and the second canine IgG CH3 domain contains an amino acid substitution by a positively charged amino acid at position D397.

32. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain does not contain an amino acid substitution, and the second canine IgG CH3 domain contains an amino acid substitution at the D429 position.

33. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain includes an amino acid substitution with a negatively charged amino acid at position K386, and the second canine IgG CH3 domain includes an amino acid substitution with a positively charged amino acid at position D397 and a substitution at D429.

34. The heterodimer protein according to claim 1 or 2, wherein the first canine IgG CH3 domain contains an amino acid substitution with a negatively charged amino acid at the N387 position, and the second canine IgG CH3 domain does not contain an amino acid substitution.

35. The substitutions in E428 are E428K, E428R, or E428H; the substitutions in D427 are D427K, D427R, or D427H; the substitutions in E382 are E382K, E382R, or E382H; the substitutions in E383 are E383K, E383R, E383H, or E383R; the substitutions in R420 are R420E or R420D; the substitutions in K418 are K418E or K418D; and the substitutions in K437 are The heterodimer protein according to any one of claims 1 to 34, wherein the substitution at K467 is K467D or K467E, the substitution at K396 is K396E or K396D, the substitution at K386 is K386E or K386D, the substitution at D397 is D397K, D397R, or D397H, the substitution at D429 is D429N, and the substitution at N387 is N387D.

36. A heterodimer protein according to any one of claims 1 to 35, comprising amino acid modifications in chain 1 and chain 2 as presented in Table 2.

37. The heterodimer protein according to any one of claims 1 to 36, comprising one or more further amino acid substitutions in the first canine IgG CH3 domain and / or the second canine IgG CH3 domain.

38. A heterodimer protein according to any one of claims 1 to 37, comprising an Fc region.

39. The heterodimer protein according to any one of claims 1 to 38, wherein the first polypeptide is an antibody heavy chain or a fragment thereof, and / or the second polypeptide is an antibody heavy chain or a fragment thereof.

40. A heterodimer protein according to any one of claims 1 to 39, comprising a first and a second light chain.

41. A heterodimer protein according to any one of claims 1 to 40, which is an antibody or a fragment thereof.

42. The heterodimer protein according to claim 41, wherein the antibody is a canine antibody or a canine-modified antibody, or a fragment thereof.

43. The heterodimer protein according to claim 41 or 42, wherein the aforementioned fragment is an Fc receptor fusion protein.

44. The heterodimer protein according to any one of claims 1 to 43, wherein the antibody is a multispecific antibody.

45. The heterodimer protein according to claim 44, wherein the antibody is a bispecific antibody.

46. A heterodimer protein according to any one of claims 1 to 45, including further parts.

47. The heterodimer protein according to claim 46, wherein the aforementioned portion is the portion that extends the half-life.

48. The heterodimer protein according to claim 47, wherein the portion that extends the half-life is canine serum albumin.

49. The heterodimer protein according to claim 47, wherein the aforementioned portion is a cytotoxic portion.

50. A polypeptide containing a canine IgG CH3 domain, comprising one or more amino acid substitutions of oppositely charged amino acids at positions 428, 427, 382, ​​383, 420, 418, 437, 467, 396, 386, 387, or 397 of IgG-B, or at the corresponding positions of IgG-A, C, or D, or an amino acid substitution of N at D429.

51. A nucleic acid encoding a heterodimer protein according to any one of claims 1 to 48 or a polypeptide according to claim 50.

52. A vector comprising the nucleic acid described in claim 51.

53. A host cell comprising the vector according to claim 52.

54. The host cell according to claim 53, which is a bacterial cell, a mammalian cell, or a yeast cell.

55. d) A step of transforming host cells with the nucleic acid described in claim 51 or the vector described in claim 52; e) A step of culturing the host cells to express first and second IgG polypeptides containing the CH3 domain; and f) A step of recovering the heterodimer protein or polypeptide from the host cell culture. A method for producing a heterodimer protein or polypeptide containing [a specific compound / substance].

56. A pharmaceutical composition comprising a heterodimer protein according to any one of claims 1 to 49 or a polypeptide according to claim 50 and a pharmaceutical carrier.

57. A kit comprising a heterodimer protein according to any one of claims 1 to 49 or a polypeptide according to claim 50, and optionally instructions for use.

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