Variant CH3 domains engineered for preferential CH3 heterodimerization, multispecific antibodies comprising same, and methods for making same

Abstract

Variant CH3 domain polypeptides are provided that preferentially form CH3-CH3 heterodimers over CH3-CH3 homodimers. Such variant CH3 domains can be used to promote desired Fc pairings and thus provide efficient development of bispecific and multispecific antibodies and different types of Fc fusions. Methods for producing bispecific antibodies using such variant CH3 domains and methods for producing libraries that include such variant CH3 domains are also provided.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 298,321, filed January 11, 2022, entitled "CH3 DOMAIN VARIANTS ENGINEERED FOR PREFERENTIAL CH3 HETERODIMERIZATION AND MULTI-SPECIFIC ANTIBODIES COMPRISING THE SAME," the contents of which are incorporated by reference in their entirety herein.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (1160430.003613.xml, size: 494,063 bytes, and creation date: January 10, 2023) are incorporated herein by reference in their entirety.

[0003] The present invention relates to variant CH3 domains that associate with other variant CH3 domains to promote Fc heterodimerization by preferential pairing, as well as polypeptides, molecules, and multispecific antibodies or antigen-binding antibody fragments, and compositions comprising any of the foregoing. The present invention further relates to polynucleotides encoding one or more of such variant CH3 domains, polypeptides, molecules, multispecific antibodies or antigen-binding antibody fragments, and compositions and libraries comprising any of the foregoing. The present invention further relates to methods of generating libraries comprising variant CH3 domains, and methods of using these libraries to identify variant CH3 domains that associate with other variant CH3 domains to promote Fc heterodimerization. The present invention further relates to methods of screening for variant CH3 domain combinations (sets) that promote Fc heterodimerization, methods of producing heteromeric molecules such as multispecific antibodies or antigen-binding antibody fragments that comprise the variant CH3 domain sets, and methods of producing heteromeric molecules such as multispecific antibodies and antigen-binding antibody fragments in which Fc heterodimerization is promoted using the variant CH3 domain sets. [Background technology]

[0004] There is a continuing effort to develop antibody therapeutics with more than one antigen binding specificity, e.g., bispecific antibodies. Bispecific antibodies can be used to block multiple surface receptors associated with cancer, autoimmune disease, inflammation, or other diseases and conditions. Bispecific antibodies can also be used to place targets in close proximity, regulate protein complex formation, or drive cell-cell contact. The production of bispecific antibodies was first reported in the early 1960s (Nisonoff et al., Arch Biochem Biophys 1961 93(2):460-462), and the first monoclonal bispecific antibodies were generated using hybridoma technology in the 1980s (Milstein et al., Nature 1983 305(5934):537-540). Interest in bispecific antibodies has grown significantly in the past decade due to their therapeutic potential, and bispecific antibodies are now being used in the clinic. For example, blinatumomab and emicizumab have been approved for the treatment of certain cancers (for recent reviews of bispecific antibody production methods and characteristics of bispecific antibodies approved for pharmaceutical use, see Sedykh et al., Drug Des Devel Ther 12:195-208 (2018) and Labrijn et al. Nature Reviews Drug Discovery 18:585-608 (2019)).

[0005] While bispecific antibodies have shown significant advantages over monospecific antibodies, their widespread commercial application has been hindered by the lack of efficient / low-cost production methods, the lack of stability of bispecific antibodies, and the lack of a long half-life in humans. Bispecific antibodies can be formed by co-expressing two different heavy chains and two different light chains. However, because the heavy chains bind to the light chains in a relatively promiscuous manner, the co-expression of two heavy chains and two light chains can result in a mixture of 16 possible combinations, representing 10 different antibodies, only one of which corresponds to the desired bispecific antibody (if complete promiscuous conditions exist, the maximum yield in the mixture is 12.5%). Even if a first heavy chain-light chain pair with a first specificity and a second heavy chain-light chain pair with a second specificity different from the first specificity are produced separately and then mixed for heavy chain-heavy chain pairing, three possible combinations are possible, only one of which corresponds to the desired bispecific antibody (if complete promiscuous conditions exist, the maximum yield in the mixture is 50%). This mispairing (also called the linkage problem) poses a major challenge in producing bispecific antibodies, and various techniques have been developed to address this issue.

[0006] One strategy used to reduce heavy-heavy chain mispairing is to design bispecific antibodies with a common heavy chain, i.e., two identical heavy chains and two different light chains (see, e.g., Fischer et al., Nature Commun. 6:6113 (2015)). This eliminates the need to eliminate mispairing of the antibody product. However, this strategy requires the identification of two antibodies with different specificities but the same heavy chain, i.e., differing only in the light chain, which is difficult and tends to compromise the specificity of each binding arm, greatly reducing diversity (see, e.g., Wang et al., MABS 10(8):1226-1235 (2018)). Leucine zippers (see, e.g., Kostelny et al., J. Immunol, 148(5):1547-1553 (1992)).

[0007] Another strategy is to modify the antibody constant region to reduce the occurrence of chain-heavy chain mispairing. Many engineering efforts have been made in the CH3 domain to promote CH3 heterodimerization. Such techniques include, for example, "W-SAV" substitutions (see, e.g., Atwell S. et al., J Mol Biol. 1997 Jul 4;270(1):26-35 and US 5,731,168 (Genentech)); "HA-TF" substitutions (see, e.g., Moore G. et al., mAbs 2011 Nov-Dec;3(6):546-557 and US 10,472,427 (Xencor)); "VYAV-VLLW" substitutions (see, e.g., Von Kreudenstein TSet al., MAbs 2013;5:646-54 and US 9,499,634 (Zymeworks)); "7.8.60" and "20.8.34" designs (see, e.g., Lraver-Fay A. et al., Structure. 2016 April 5;24(4):641-651 and US 10,774,156 (University of North Carolina at Chapel Hill and Eli Lilly); electrostatic complementarity through charge exchange substitutions such as "DD-KK" (see, e.g., Gunasekaran K. et al., J Biol Chem 2010;285:19637-46 and US 8,592,562 (Amgen)); and "knob-into-hole" ("KiH") engineering such as "EW-RVT" substitutions (see, e.g., Choi HJ. et al., Mol Cancer Ther. 2013 Dec;12(12):2748-59 and US 9951145B2 (Ajou University)).Further examples of CH3 modifications include US10,597,464 (Genmab), US16 / 482,137 (Centrymed), US9,562,109 (Zymeworks), US15 / 409,456 (Zymeworks), US9,624,291 (Ramot at Tel Aviv University), PCT / EP2019 / 083638 (Morphosys), US9,605,084 (Xencor), US16 / 062,405 (Alphamab), US15 / 997,222 (Janssen), US14 / 989,648 (Zymeworks), US13 / 892,198 (Zymeworks), US15 / 586,686 (Hoffmann La Roche), US9,308,258 (Amgen), US9,200,060 (Amgen), US15 / 554,022 (Laboratoire Francais), US9,574,010 (Zymeworks), PCT / US2019 / 023382 (Dana-Farber Cancer Institute), US13 / 814657 (MedImmune), US11 / 228,026 (Xencor), PCT / US2017 / 045139 (Merrimack), US16 / 244,378 (Hoffmann La Roche), and those described in Brinkmann U. et al, MAbs. 2017 Feb-Mar, 9(2):182-212 (review).

[0008] Although these CH3 modifications increase the propensity to form CH3 heterodimers, improvements are still needed, especially given the general interest in developing improved multispecific antibodies for use in human therapy. Summary of the Invention

[0009] Provided herein are methods for producing heteromeric molecules, such as multispecific antibodies or antigen-binding antibody fragments. Such methods can be driven by cFAE. Optionally, the heteromeric molecules can include IgG, and optionally IgG1, IgG2, IgG3, or IgG4 constant regions.

[0010] In some embodiments, the heteromeric molecule produced or intended to be produced comprises: (A) a first polypeptide comprising a first variant CH3 domain polypeptide (or an immunoglobulin heavy chain polypeptide comprising said first variant CH3 domain polypeptide), wherein the first variant CH3 domain polypeptide comprises a T366V substitution according to EU numbering; and (B) a second polypeptide comprising a second variant CH3 domain polypeptide (an immunoglobulin heavy chain polypeptide comprising the first variant CH3 domain polypeptide), wherein the second variant CH3 domain polypeptide comprises a Y407V substitution according to EU numbering.

[0011] In such heteromeric molecules, the first and second polypeptides can be linked or paired to each other, optionally via at least one disulfide bond.

[0012] In some embodiments, the method may include (i) incubating under a reducing environment or condition (such as a solution containing a reducing agent) (i-1) a first parent molecule comprising at least two of the first polypeptides bonded or paired to each other, optionally via at least one disulfide bond, and (i-2) a second parent molecule comprising at least two of the second polypeptides bonded or paired to each other, optionally via at least one disulfide bond.

[0013] In this step (i), where the heteromeric molecule is a multispecific antibody or antigen-binding antibody fragment, the parent molecules may be the corresponding monospecific parent antibodies (such as IgG), and the parent antibodies may be incubated under reducing conditions, such that the pairings between the heavy chains (e.g. disulfide bonds) in each of the parent antibodies may dissociate, but not between the heavy and light chains.

[0014] In some embodiments, the method may then include (ii) placing the incubation product of step (i) in a less reducing or non-reducing environment, thereby forming the heteromeric molecule. In some embodiments, this step (ii) may remove the reducing agent if a reducing agent is present in the reducing environment.

[0015] In certain embodiments, the first variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG, and / or the second variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG. In certain embodiments, the T366V substitution may be made to the CH3 domain of human IgG, and / or the Y407V substitution may be made to the CH3 domain of human IgG.

[0016] In certain embodiments, the first variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG1, and / or the second variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG1. In certain embodiments, the T366V substitution may be to SEQ ID NO: 1, 2, 3, or 4, and / or the Y407V substitution may be to SEQ ID NO: 1, 2, 3, or 4.

[0017] In certain embodiments, the first variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG2 and / or the second variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG2. In certain embodiments, the T366V substitution may be relative to SEQ ID NO:722 and / or the Y407V substitution may be relative to SEQ ID NO:722.

[0018] In certain embodiments, the first variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG3 and / or the second variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG3. In certain embodiments, the T366V substitution may be relative to SEQ ID NO:723 and / or the Y407V substitution may be relative to SEQ ID NO:723.

[0019] In certain embodiments, the first variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG4 and / or the second variant CH3 domain polypeptide may be derived from the CH3 domain of human IgG4. In certain embodiments, the T366V substitution may be relative to SEQ ID NO:724 and / or the Y407V substitution may be relative to SEQ ID NO:724.

[0020] In certain embodiments, a heteromeric molecule may comprise one or more of the following features: (A) the first polypeptide further comprises a first antigen binding domain; (B) the second polypeptide further comprises a second antigen binding domain; (C) the heteromeric molecule further comprises a third polypeptide optionally comprising a third antigen binding domain, optionally wherein the third polypeptide is bound or paired to the first polypeptide; and / or (D) the heteromeric molecule further comprises a fourth polypeptide optionally comprising a fourth antigen binding domain, optionally wherein the fourth polypeptide is bound or paired to the second polypeptide.

[0021] In certain embodiments of the production methods, the heteromeric molecule may further comprise: (C) a third polypeptide optionally comprising a third antigen binding domain; and / or (D) a fourth polypeptide optionally comprising a fourth antigen binding domain.

[0022] In certain embodiments of the production methods, the heteromeric molecule may be a multispecific antibody or antigen-binding antibody fragment, which may optionally comprise any of the structures depicted in Figures 2-8, and optionally the heteromeric molecule comprises (a) an IgG, or (b) an IgG and one or more scFvs directly or indirectly conjugated to the IgG, and optionally further comprises an IgG1, IgG2, IgG3, or IgG4 constant region.

[0023] In certain embodiments of the production methods, the heteromeric molecule may be a multispecific antibody or antigen-binding antibody fragment comprising one or more of the following features (I) and (II):

[0024] (I) (I-1-i) the first polypeptide comprises a first antigen-binding domain that forms a first antigen-binding site specific for a first epitope, and / or (I-1-ii) the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain that forms a third antigen-binding site specific for a third epitope, optionally the first epitope being the same as or different from the third epitope, or (I-2) the first polypeptide comprises a first antigen-binding domain and the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain, wherein the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for the first epitope, and / or

[0025] (II) (II-1-i) the second polypeptide comprises a second antigen-binding domain that forms a second antigen-binding site specific for a second epitope, and / or (I-1-ii) the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain that forms a fourth antigen-binding site specific for a fourth epitope, optionally the second epitope being the same as or different from the fourth epitope, or (II-2) the second polypeptide comprises a second antigen-binding domain and the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain, wherein the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for a second epitope.

[0026] In some embodiments, the incubation in step (i) may be carried out at a temperature of about 15°C to about 40°C, about 20°C to about 40°C, about 25°C to about 35°C, about 28°C to about 32°C, or about 29°C to about 31°C, or about 30°C.

[0027] In certain embodiments, the incubation in step (i) may be carried out for about 30 minutes to about 20 hours, about 1 hour to about 15 hours, about 2 hours to about 10 hours, about 3 hours to about 7 hours, or about 4 hours to about 6 hours, or about 5 hours.

[0028] In certain embodiments, the incubation in step (i) may be carried out at about 30° C. for about 5 hours.

[0029] In some embodiments, the reducing environment may include at least one reducing agent, optionally at least one weak reducing agent.

[0030] In certain embodiments, the reducing environment may include at least one reducing agent selected from 2-mercaptoethylamine (2-MEA), β-mercaptoethanol (BME), L-cysteine, dithiothreitol (DTT), or dithionite.

[0031] In certain embodiments, the reducing agent may not be glutathione.

[0032] In certain embodiments, the reducing environment is about 25 to about 125 mM, about 50 mM to about 100 mM, about 70 to about 80 mM, or about 75 mM 2-MEA, about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM BME, about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM BME, The solution may contain at least one reducing agent selected from the group consisting of 0 μM or about 100 μM L-cysteine, about 15 to about 400 μM, about 20 to about 200 μM, about 25 to about 100 μM, about 30 to about 70 μM, or about 50 μM DTT, and 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM dithionite.

[0033] In certain embodiments, the reducing environment may optionally include at least about 75 mM 2-MEA.

[0034] In some embodiments, in the first antibody and / or the second antibody incubated in step (i), at least two of the first polypeptides may be bound or paired with each other via at least one disulfide bond, and / or at least two of the second polypeptides may be bound or paired with each other via at least one disulfide bond.

[0035] In some embodiments, the first antibody and / or the second antibody may be produced in a mammalian cell, a yeast cell, an insect cell, a plant cell, or a bacterial cell.

[0036] In some embodiments, the first antibody and / or the second antibody may be produced in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells.

[0037] In some embodiments of the production method, the placing in step (ii) may be performed by buffer exchange, optionally where the buffer is exchanged into phosphate buffered saline (PBS).

[0038] In some embodiments, the placing in step (ii) may be performed by buffer exchange, optionally into PBS, by desalting.

[0039] In some embodiments, the placing in step (ii) may be performed by buffer exchange by diafiltration, optionally into PBS.

[0040] In some embodiments, the disposing in step (ii) may be carried out by addition of an oxidizing agent.

[0041] In some embodiments, the production method may further comprise (iii) incubating the product of step (ii) in a less reducing or non-reducing environment.

[0042] In certain embodiments, incubation may occur at a temperature of about 1° C. to about 20° C., about 2° C. to about 10° C., about 3° C. to about 5° C., or about 4° C. In certain embodiments, incubation may occur for about 12 hours to about 154 hours, about 24 hours to about 96 hours, about 36 hours to about 72 hours, or about 48 hours. In certain embodiments, incubation may occur at about 4° C. for about 48 hours.

[0043] In some embodiments, the production methods may further comprise (iv) analyzing the amount of multispecific antibodies or antigen-binding antibody fragments in the product of step (ii) and / or step (iii), and / or purifying the multispecific antibodies or antigen-binding antibody fragments from the product of step (ii) and / or step (iii).

[0044] In certain embodiments, the analysis and / or purification is performed via chromatography, optionally LC-MS, IEX, and / or SEC.

[0045] In some embodiments, the heteromeric molecule produced is a multispecific antibody. The first polypeptide may comprise a first antibody heavy chain and the second polypeptide may comprise a second antibody heavy chain, the first antibody heavy chain associated with the first antibody light chain and the second antibody heavy chain associated with the second antibody light chain. The multispecific antibody may comprise a third polypeptide comprising a third antigen-binding domain. The third antigen-binding domain may be associated with the first antibody heavy chain, the second antibody heavy chain, the first antibody light chain, or the second antibody light chain. The multispecific antibody may further comprise a fourth polypeptide comprising a fourth antigen-binding domain. The fourth antigen-binding domain may be associated with the first antibody heavy chain, the second antibody heavy chain, the first antibody light chain, or the second antibody light chain.

[0046] The association of the third and / or fourth antigen binding domains may be via a flexible linker. In certain embodiments, the flexible linker is selected from the group consisting of: (i) an amino acid sequence selected from the group consisting of GGGGS (SEQ ID NO:715), GGGS (SEQ ID NO:716), GGGGGS (SEQ ID NO:717), G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG; (ii) multiple repeats of an amino acid sequence selected from the group consisting of SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG; optionally two, three, four, or five repeats; (iii) a (G5S)n linker, a (G4S)n linker, a (G3S)n linker, a (G2S)n linker, a (GS)n linker, or a (G)n linker, where n is a natural number, optionally selected from 1 to 20, further optionally 2, 3, 4, or 5; and / or (iv) the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 718) or GGGGSGGGGSGGGGS (SEQ ID NO: 719).

[0047] The third and / or fourth antigen binding domain may comprise a Fab or a single chain Fv (scFv). The third and / or fourth antigen binding domain may comprise an scFv, which comprises a heavy chain variable domain and a light chain variable domain, and the heavy chain variable domain and the light chain variable domain are linked by a disulfide bond and / or a linker. In certain embodiments, such a linker is selected from the group consisting of (i) an amino acid sequence selected from the group consisting of GGGGS (SEQ ID NO: 715), GGGS (SEQ ID NO: 716), GGGGGS (SEQ ID NO: 717), G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG, (ii) multiple repeats of an amino acid sequence selected from the group consisting of SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG, or any combination thereof. optionally two, three, four or five repeats, (iii) a (G5S)n linker, a (G4S)n linker, a (G3S)n linker, a (G2S)n linker, a (GS)n linker or a (G)n linker, where n is a natural number, optionally selected from 1 to 20, further optionally selected from 2, 3, 4 or 5, and / or (iv) the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 718) or GGGGSGGGGSGGGGS (SEQ ID NO: 719). The multispecific antibody may be a biparatopic antibody.

[0048] In some embodiments, the first parent molecule comprises a first IgG and the second parent molecule comprises a second IgG. At least two of the first polypeptides of the first IgG may each comprise a first antibody heavy chain comprising a first antigen binding domain forming a first antigen binding site for a first epitope. At least two of the second polypeptides of the second IgG may each comprise a second antibody heavy chain comprising a second antigen binding domain forming a second antigen binding site for a second epitope. The first epitope and the second epitope may be part of different antigens. The first epitope and the second epitope may be part of the same antigen. The heteromeric molecule may be an IgG comprising a first antibody heavy chain and a second antibody heavy chain.

[0049] In some embodiments, the methods may include producing a plurality of multispecific antibodies and / or antigen-binding antibody fragments using the methods for producing heteromeric molecules described above.

[0050] In some embodiments, the T366V substitution is the only substitution in the first variant CH3 domain polypeptide. In certain embodiments, the T366V substitution is the only substitution in the CH3 domain of human IgG. In certain embodiments, the T366V substitution is the only substitution in the CH3 domain of human IgG1, optionally with the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. In certain embodiments, the T366V substitution is the only substitution in the CH3 domain of human IgG2, optionally with the amino acid sequence of SEQ ID NO: 722. In certain embodiments, the T366V substitution is the only substitution in the CH3 domain of human IgG3, optionally with the amino acid sequence of SEQ ID NO: 723. In certain embodiments, the T366V substitution is the only substitution in the CH3 domain of human IgG4, optionally with the amino acid sequence of SEQ ID NO: 724. In some embodiments, the Y407V substitution is the only substitution in the second variant CH3 domain polypeptide. In certain embodiments, the Y407V substitution is the only substitution in the CH3 domain of human IgG. In certain embodiments, the Y407V substitution is the only substitution in the CH3 domain of human IgG1, optionally to the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4. In certain embodiments, the Y407V substitution is the only substitution in the CH3 domain of human IgG2, optionally to the amino acid sequence of SEQ ID NO: 722. In certain embodiments, the Y407V substitution is the only substitution in the CH3 domain of human IgG3, optionally to the amino acid sequence of SEQ ID NO: 723. In certain embodiments, the Y407V substitution is the only substitution in the CH3 domain of human IgG4, optionally to the amino acid sequence of SEQ ID NO: 724.

[0051] In some embodiments, the first and second variant CH3 domain polypeptides may be further modified to include one or more variant CH3 domain sets, optionally any of the variant CH3 domain sets described herein, such as, but not limited to, any of the variant CH3 domain sets in any of the tables disclosed herein.

[0052] In some embodiments, the heteromeric molecule may comprise one or more specific CH2 domains. In certain embodiments, one or more of the CH2 domains may comprise one or more amino acid modifications. In certain embodiments, one or more of the CH2 domains may comprise one or more Fc silencing modifications. In certain embodiments, one or more of the CH2 domains may comprise one or more FcRn affinity enhancing or reducing and / or half-life extending or reducing modifications. In certain embodiments, one or more of the CH2 domains may comprise any of the following modifications according to EU numbering: L234A, L235A, and P329A substitutions, L234A, L235A, and P329G substitutions, L234A and L235A substitutions, D265A and P329A substitutions, N297A substitutions, M252Y, S254T, and T256E substitutions, and / or M428L and N434S substitutions.

[0053] In yet another aspect, provided herein are heteromeric molecules such as multispecific (e.g., bispecific) antibodies and antigen-binding antibody fragments produced by the production methods described herein. Optionally, such multispecific antibodies or antigen-binding antibody fragments may comprise IgG, and further optionally IgG1, IgG2, IgG3, or IgG4.

[0054] In some embodiments, the multispecific antibody or antigen-binding antibody fragment may comprise a structure according to any of the structures described herein or shown in Figures 2-8. [Brief description of the drawings]

[0055] [Figure 1A]1A-1C provide schematic diagrams generally illustrating the advantages of heterodimerized CH3 domains. A contemplated bispecific antibody comprises (a) a half antibody specific for epitope A comprising heavy chain A (comprising VH (solid black)) and light chain A (comprising VL (horizontally striped)), and (b) a half antibody specific for epitope B comprising heavy chain B (comprising VH (checkered)) and light chain B (comprising VL (vertically striped)).

[0056] Figure 1A shows an exemplary production of such a bispecific antibody when heavy chain A, light chain A, heavy chain B, and light chain A all contain wild-type constant domains. When such four chains are co-expressed, co-delivered, or mixed in a ratio of about 1:1:1:1, and there is complete promiscuity in the heavy-light-light chain pairings and the heavy-heavy chain pairings, ten different antibody products can be produced with the respective proportions as shown. Approximately 12.5% ​​of the products will correspond to the bispecific antibody of interest (boxed).

[0057] [Figure 1B] FIG. 1B shows an exemplary production of a bispecific antibody similar to FIG. 1A, except that the CH3 domain of heavy chain A (CH3 domain A (diagonal stripes)) and the CH3 domain of heavy chain B (CH3 domain B (dotted line)) are variant CH3 domains that differ from each other and preferentially form heterodimers (i.e., heterodimers between CH3 domain A and CH3 domain B). Existing heavy chain CH3 heterodimerization technologies include those listed in Table 1, such as the "knobs-into-holes" technology (see, e.g., U.S. Pat. No. 5,731,168). When such heavy chain A, light chain A, heavy chain B, and light chain B are co-expressed, co-delivered, or mixed in an approximately 1:1:1:1 ratio, where CH3 domain A and CH3 domain B exclusively permit heavy heteropairing, four different antibody products can be produced in the respective percentages shown. Approximately 25% of the products correspond to the desired bispecific antibody (boxed).

[0058] [Figure 1C]FIG. 1C provides two schematic diagrams (left and right) generally illustrating the benefit of heterodimerized CH3 domains in producing full-sized antibodies (IgG, IgE, or IgD) from two of the already formed half antibodies. Such production methods include, but are not limited to, methods that rely on Fab arm exchange (FAE) or controlled FAE (cFAE). Bispecific antibodies can be produced by combining a half antibody specific for epitope A with a half antibody specific for epitope B. When both heavy chains A and B contain wild-type CH3 domains (left schematic), only 50% of the products (when there is complete promiscuity in the half antibody pairing) are the desired bispecific antibodies. However, as shown in the right scheme, if the CH3 domain A (diagonal stripes) and CH3 domain B (dotted lines) variant CH3 domains are different from each other and preferentially form CH3-CH3 heterodimers (i.e., heterodimers between CH3 domain A and CH3 domain B), the product is biased toward the desired bispecific antibody. In the most ideal set of CH3 domains that form only heterodimers and no homodimers, 100% of the product will be the desired bispecific antibody. A variant CH3 domain that provides heterodimers at more than 50%, if not 100%, will facilitate efficient production of bispecific antibodies.

[0059] In the exemplary multispecific antibodies, the solid black is the VH (specific for epitope A) of heavy chain A (VH Domain A), the horizontal stripes are the VL (specific for epitope A) of light chain A (VL Domain A), the checkered pattern is the VH (specific for epitope B) of heavy chain B (VH Domain B), the vertical stripes are the VL (specific for epitope B) of light chain B (VL Domain B), the diagonal stripes are the variant CH3 domain in heavy chain A (CH3 Domain A), and the dotted lines are the variant CH3 domain in heavy chain B (CH3 Domain B), where CH3 Domain A and CH3 Domain B preferentially form CH3 heterodimers (i.e., resulting in more than 50% CH3 heterodimers). These definitions apply to all figures unless otherwise stated.

[0060] [Diagram 2]Figures 2-8 provide exemplary, non-limiting embodiments of various multispecific antibody structures in which the variant CH3 domains disclosed herein may be used. In Figures 2-8, unless otherwise indicated, the following applies: (1) each domain is presented as a rectangle with text therein indicating the domain name (e.g., CH3, VH1, etc.); (2) a set of multiple domains linked together represents a polypeptide (e.g., heavy chain polypeptide, light chain polypeptide, etc.); (3) the orientation of the domains within the polypeptide follows the orientation of the text indicating the domain name, from N-terminus to C-terminus; (4) even if linkers, hinges, or disulfide bonds are not explicitly shown in the figures, linkers or hinges may be used between domains as necessary, and disulfide bonds may be present between polypeptides (and / or within domains), presumably to allow correct formation of the antigen binding site; (5) the CH2 and / or CH3 domains shown in the figures may be omitted whenever possible and replaced by hinges or linkers where appropriate; (6) the diagonal stripes and dotted lines may represent variant CH3 domains as disclosed herein, barriers that preferentially form CH3-CH3 heterodimers. (7) rectangles without a pattern (i.e., open) are domains that may individually contain the corresponding wild-type sequence or may contain one or more amino acid substitutions compared to the wild-type sequence; (8) the CH1 domain, CH2 domain, and CH3 domain may individually be of any (heavy chain) isotype; (9) if more than one CH1 domain is present in the structure, the CH1 domains may or may not be of the same isotype, if more than one CH2 domain is present in the structure, the CH2 domains may or may not be of the same isotype, if more than one CH3 domain is present in the structure, the CH3 domains may or may not be of the same isotype; (10) the light chain constant (CL) domain may be a kappa CL domain or a lambda CL domain; (11) if more than one CL domain is present in the structure, all CL domains may be kappa CL;or all CL domains may be lambda CLs, or one CL may be a kappa CL and another CL may be a lambda CL domain; (12) when both kappa and lambda CL domains are present, the CH1 domains paired with the CL domains may in some cases be variant CH1 domains, one of which may be a variant CH1 that preferentially binds kappa CL and another CH1 domain may be a variant CH1 that preferentially binds lambda CL (having kappa and lambda CLs and kappa-preferred and lambda-preferred CH1s in a molecule may result in efficient (13) VH-1 and VL-1 form an antigen-binding site for a first epitope, VH-2 and VL-2 form an antigen-binding site for a second epitope, VH-3 and VL-3 form an antigen-binding site for a third epitope, VH-4 and VL-4 form an antigen-binding site for a fourth epitope, VH-5 and VL-5 form an antigen-binding site for a fifth epitope, and VH-6 and VL-6 form an antigen-binding site for a sixth epitope; (14) all of the first epitope to the sixth epitope may be different from each other, or all of the first epitope to the sixth epitope may not be different from each other; and (15) For a given VH-VL pair, if the VH alone confers sufficient specificity for the cognate antigen (i.e., the nanobody), the VL may be omitted even if not shown in the figure.

[0061] Figure 2 provides exemplary and non-limiting embodiments of various multispecific antibody structures in which the variant CH3 domains disclosed herein may be used. The antibody at the top left (boxed) is an exemplary basic full-size bispecific antibody with no hinges or disulfide bodies explicitly shown. The boxed antibody may include hinges, for example, between CH1-1 and CH2-1 and between CH1-2 and CH2-2, and disulfide bonds (dashed lines) may be present between the hinges (top center). Alternatively, the boxed antibody may include hinges, for example, between CH1-1 and CH2-1 and between CH1-2 and CH2-2, and disulfide bonds (dashed lines) may be present between the hinges, between CL-1 and the hinge, and between CL-2 and the hinge (top right). Hinges and disulfide bonds, for example, those shown in the top center and top right antibody structures, may not be explicitly shown or may be present in any structure shown in the figures and described herein. In some variants of the boxed antibodies, the CH2 domain may be absent (center left), or the CH1 and CH2 domains may be absent (bottom left), and the hinge and disulfide bonds may be present as shown in center center, center right, bottom center, or bottom right. Although not explicitly stated, any of the CH1 and / or CH2 domains may be omitted, as desired, in any of the structures of Figures 3-8 or variations thereof.

[0062] [Figure 3A] Figure 3 provides variations of the antibody structure shown in Figure 2. In Figure 3A, the VH and VL positions have been altered compared to the structure in Figure 2. In Figure 3B, the CH1 and CL positions have been altered compared to the structure in Figure 2. The equivalent variations shown in Figure 3 (swiping the VH-VL positions or the CH1-CL positions) may also be applied to any of the structures shown in Figures 3-8 or variations thereof, as appropriate, even if not explicitly stated. [Figure 3B] Same as above.

[0063] [Figure 4]FIG. 4 provides variations of the boxed antibody structures of FIG. 2. Specifically, a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are added to the N-terminus of the heavy and light chains in different orientations. Although both a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are shown, one pair may be added if desired. The equivalent variations shown in FIG. 4 (addition of one or more VH-VL pairs) may also be applied to any of the structures shown in FIGS. 3-8 or variations thereof, as desired, even if not explicitly shown.

[0064] [Diagram 5] Figure 5 provides additional variations of the boxed antibody structures of Figure 2. Similar to the structures of Figure 4, a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are added in a different orientation, and the order of VH and VL on the light chain differs from that of Figure 4. The equivalent variations shown in Figure 4 (addition of one or more VH-VL pairs) may be further applied to any of the structures shown in Figures 2-8 or variations thereof, as desired, even if not explicitly stated.

[0065] [Figure 6A]FIG. 6 provides further variations of the boxed antibody structures of FIG. 2. Specifically, in FIGS. 6A-6D, an scFv specific for a third epitope and an scFv specific for a fourth epitope are added. Although two scFvs are shown, one scFv may be added if desired. In FIG. 6A, the scFvs are added to the C-terminus of the heavy chain. The four structures in FIG. 6A differ in the VH-VL order within each scFv. In FIG. 6B, the scFvs are added to the C-terminus of the light chain. The four structures in FIG. 6B differ in the VH-VL order within each scFv. In FIG. 6C, the scFvs are added to the N-terminus of the heavy chain. The four structures in FIG. 6C differ in the VH-VL order within each scFv. In FIG. 6D, the scFvs are added to the N-terminus of the light chain. The four structures in FIG. 6D differ in the VH-VL order within each scFv. Although not shown in Figures 6A-6D, two scFvs can be added at different positions (e.g., one at the C-terminus of the heavy chain and one at the N-terminus of the light chain). In Figure 6E, four scFvs are added to the N-terminus of the heavy and light chains. The four structures in Figure 6C differ in the VH-VL order within each scFv. The equivalent modifications depicted in Figure 6 (addition of one or more scFvs) can also be applied to any of the structures shown in Figures 2-8 or modifications thereof, as appropriate, even if not explicitly shown. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above.

[0066] [Figure 7A] Figures 7A-7B provide further variations of the boxed antibody structures in Figure 2. Specifically, a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are added to the C-terminus of the heavy and light chains in different orientations. Although both a third epitope-specific VH-VL pair and a fourth epitope-specific VH-VL pair are shown, only one pair may be added if desired. The equivalent variations shown in Figures 7A-7B (addition of one or more VH-VL pairs) may also be applied to all other structures shown in Figures 3-8 or variations thereof, as appropriate, even if not explicitly shown. [Figure 7B] Same as above.

[0067] [Figure 8A] Figures 8A-8E provide additional exemplary and non-limiting embodiments of various multispecific antibody fragment structures in which variant CH3 domains disclosed herein may be used and which do not include the VH-VL antigen binding sites of a conventional antibody, but rather include one or more of the scFvs. In Figure 8A, the antibody on the left (boxed) is an exemplary basic bispecific antibody fragment comprising a first heavy chain comprising a scFv (comprising VH-1 and VL-1) specific for a first epitope and a second heavy chain comprising a second scFv (comprising VH-2 and VL-2) specific for a second epitope. The light chain may be absent. Also provided are variants thereof lacking the CH2 domain (center) or the CH1 and CH2 domains (right). Figures 8B-8E provide further variations of the antibody structure of Figure 8A, including additional scFvs. In Figure 8B, a third scFv specific for a third epitope (comprising VH-3 and VL-3) and a fourth scFv specific for a fourth epitope (comprising VH-4 and VL-4) are added to the N-terminus of the heavy chain. In Figure 8C, a third scFv specific for a third epitope (comprising VH-3 and VL-3) and a fourth scFv specific for a fourth epitope (comprising VH-4 and VL-4) are added to the C-terminus of the heavy chain. In Figure 8D, a first light chain comprising CL-1, a second light chain comprising CL-2, a third scFv specific for a third epitope (comprising VH-3 and VL-3), and a fourth scFv specific for a fourth epitope (comprising VH-4 and VL-4) are added to the N-terminus of the light chain. In Figure 8E, a fifth scFv (comprising VH-5 and VL-5) specific for a fifth epitope and a sixth scFv (comprising VH-6 and VL-6) specific for a sixth epitope are added to the C-terminus of the heavy chain. Although not shown explicitly, the VH-VL order within the scFvs may be swapped if desired. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above.

[0068] [Figure 9A] 9A-9B show the variant CH3 domain selection proof-of-concept (POC) study in Example 1, where two heterodimer technologies (KiH and EW-RVT) were evaluated as controls. FIG. 9A provides a schematic of the selection of heterodimer-preferred variant CH3 domains by flow cytometry. High FLAG expressors exhibit a more modified Fc containing CH3 heterodimers. A population from the library (knobsHISholeFLAG:EWHISRVTFLAG:WTHIS-WTFLAG=1:1:10,000) that stained exceptionally highly with anti-FLAG antibodies (indicating expression of heterodimer-preferred variant CH3 domains) is selected and sorted. FIG. 9B provides an exemplary flow plot from multiple selection rounds showing enrichment of control heterodimers (KiH and EW-RVT). A 1:1:10,000 knobHISholeFLAG:EWHISRVTFLAG:WTHIS-WTFLAG variant CH3 domain library was sorted for high Flag expressers over two rounds. Sequencing after round 1 (R1) yielded 1 of 91 with a KiH mutation. Sequencing after round 2 (R2) yielded 2 of 91 with KiH mutations and 2 with EW-RVT mutations. [Figure 9B] Same as above.

[0069] [Figure 10A]10A-10D show representative data from cycle 1 of variant CH3 domain selection in Example 2. FIG. 10A provides three library designs in which KiH amino acid positions (position 366 in the first heavy chain and positions 366, 368, and 407 in the second heavy chain) are mutated. In the first library, both the knob and hole positions are mutated, with the strand with hole mutation encoding a FLAG tag and the strand with knob mutation encoding a HIS tag. In the second library, both the knob and hole positions are mutated, with the strand with hole mutation encoding a HIS tag and the strand with knob mutation encoding a FLAG tag. In the third library, the hole position is mutated but the knob position is not mutated. The strand with hole mutation encoding a FLAG tag and the strand with no mutation encoding a HIS tag. Variations in the DNA sequence of each library are provided using site saturation mutagenesis (SSM). An "X" in white text on a black background represents mutation. Figure 10B provides exemplary flow plots from six selection rounds performed using a first library, Figure 10C provides exemplary flow plots from six selection rounds using a second library, and Figure 10D provides exemplary flow plots from six selection rounds using a third library. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above.

[0070] [Figure 11A]11A-11C show exemplary AlphaLISA® analyses of identified variant CH3 domains. FIG. 11A (left) provides a schematic of CH3 heterodimer detection by AlphaLISA®. AlphaLISA® was used to determine the relative degree of heterodimerization of Fc fragments by specifically detecting modified Fc containing heterodimeric CH-CH3 sets by the proximity between HISx6-tagged and FLA-tagged polypeptides. FIG. 11A (right) provides results from several samples in the POC set showing clear differences in photon counts between the existing heterodimerized variant CH3 domain sets (KiH and EW-RVT) and the WT CH3 domain set, regardless of which chain contained the FLAG tag. FIG. 11B provides a graph showing AlphaLISA® values ​​(photon counts, fold over background (FOB) ("buffer only", i.e., no Fc, was used as background)) for variant CH3 domain positive controls (KiH and EW-RVT, indicated by arrows), negative (WT / WT, indicated by arrows) controls, and variant CH3 domains identified in Example 2 (bars without arrows). Several of the identified variant CH3 domains identified herein showed comparable or superior heterodimerization (see left bar for EW-RVT). FIG. 11C provides a graph plotting AlphaLISA® values ​​against anti-FLAG antibody staining during the final round of flow cytometry-based selection, showing good correlation. Anti-FLAG FOB and AlphaLISA® FOB values ​​for "T366V-HIS;T366 L368 Y407V-FLAG" were 622 and 86, respectively. "T366V-HIS;T366 L368 Y407V-FLAG" was also found in the reverse orientation (i.e., "T366 L368 Y407V-HIS T366V-FLAG"). The anti-FLAG FOB value and AlphaLISA® FOB value of "T366 L368 Y407V-HIS T366V-FLAG" were 588 and 39, respectively. [Figure 11B] Same as above. [Figure 11C]Same as above.

[0071] [Figure 12A] Figures 12A-12B show exemplary size exclusion chromatography (SEC) analyses of variant CH3 domains. Figure 12A provides results from a control sample. Figure 12B provides results from identified variant CH3 domains. All CH3 sets tested yielded uniform distributions, implying low aggregation. [Figure 12B] Same as above.

[0072] [Figure 13A] 13A-13C show exemplary ion exchange (IEX) analyses of variant CH3 domains. FIG. 13A provides results from a control sample, showing peaks corresponding to different antibody species. FIG. 13B provides output results from identified variant CH3 domains. CH3 sets VV, LV, LM, IF, and W-SG show chromatograms similar to that of EW-RVT, with a sharp single peak. FIG. 13C provides SEC and IEX data in parallel for samples W-SY and SEL-L, which have low AlphaLISA® values. Low AlphaLISA® values ​​correlated with poor SEC and IEX chromatograms. [Figure 13B] Same as above. [Figure 13C] Same as above.

[0073] [Figure 14A] Figures 14A-14C show the production of bispecific antibodies (BsAbs) comprising variant CH3 domains with 354 / 349 disulfide bond substitutions (S354C and Y349C) in HEK293 cells of Example 6. Figure 14A provides a schematic of the different anti-CD3 / anti-HER2 BsAbs produced. Nivolumab (Nivo) was used as a control. Figure 14B provides an exemplary SEC chromatogram of each BsAb. Figure 14C provides an exemplary IEX chromatogram of each BsAb. [Figure 14B] Same as above. [Figure 14C] Same as above.

[0074] [Figure 15A] Figures 15A-15D show the subsequent library generation and screening. Figure 15A provides an exemplary flow plot from a selection round where heterodimerization variant CH3 domains were enriched from the library. Figure 15B shows the selection criteria applied to the set of 430 variant CH3 domains obtained from cycle 2 step 1 to enrich for variant CH3 domains with improved contact rate across the interface, AlphaLISA® value, and Rosetta score. Sequences were also verified to ensure diversity of mutation positions. Figures 15C and 15D provide t-SNE plots. These plots were used to ensure substitution diversity in the variant CH3 domains selected for further production and characterization. Each point represents a set of substitution positions, and points close to each other on the plot contain similar substitution positions. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above.

[0075] [Figure 16] Figure 16 shows the heterodimerization and stability characterization of 48 variant CH3 domains. In this graph, monomeric full size modified Fc% (measured by SEC, showing non-aggregated modified Fc%) is plotted against heterodimer modified Fc% for 48 variant CH3 domains and controls. Based on these results, a set of five variant CH3 domains ("designated") shown in Table 8 was selected as cycle 2 output. The designated clones showed similar heterodimerization and stability as the control.

[0076] [Figure 17A]Figures 17A-17J show the characterization of exemplary BsAbs containing variant CH3 domains produced in HEK293 cells. Figure 17A shows a schematic of the exemplary antibodies produced. For each variant CH3 domain, several different structures were produced: three anti-CD3 / anti-HER2 BsAbs (one in orientation 1, one in orientation 2, and one in orientation 1 with an additional 354 / 349 substitution added to the CH3 set), two anti-CD20 / anti-CD3 BsAbs (one in orientation 1 and one in orientation 2), and one anti-HEL / anti-BCMA BsAb (where the anti-BCMA binding portion is a nanobody). Sequences are provided in Appendix Tables A-D. The structures within the dotted boxes were compared to assess heterodimerization efficiency. Figure 17B shows that heterodimerization is consistent between CH3 orientations and variable regions. Heterodimer percent (%) values ​​of anti-CD3 / anti-HER2 BsAbs (lacking the 354 / 349 substitution) are provided. Figure 17C shows IEX chromatograms of different BsAbs and compares the heterodimer% values ​​measured by IEX and LC-MS. When the BsAbs contained a nanobody as one of the two antigen binding domains, IEX gave lower resolution (BCMA VHH x HEL chromatogram). Figure 17D compares the heterodimer% values ​​measured by IEX and LC-MS of different BsAbs that do not contain the 354 / 349 substitution. The heterodimer% values ​​by IEX and LCMS show good correlation (data points do not include BsAbs containing a nanobody in one Fab arm). Figure 17E compares the heterodimer% values ​​measured by IEX and LC-MS of different BsAbs that contain the 354 / 349 substitution. Figure 17F compares the % heterodimer values ​​measured by LC-MS between BsAbs with and without the 354 / 349 substitution. The 354 / 349 substitution appears to improve heterodimerization (measured by LC-MS) of most CH3 sets, including the wild-type set. Figure 17G compares the AlphaLISA® values ​​of BsAbs with and without the 354 / 349 substitution (CD3xHER2 BsAb and HELxBCMA Fab-VHH BsAb) with the % heterodimer values ​​measured by LC-MS or IEX.The order of heterodimerization determined by LC-MS and IEX was the same. Figure 17H ​​compares the heterodimer % values ​​measured by IEX, LC-MS and AlphaLISA® for the LWG and / or SIG sets in Orientation 1, Orientation 2 and Orientation 1 with the additional 354 / 349 substitution. Figure 17I compares the stability of the different bsAbs (without bsAbs containing a nanobody in one arm) defined by monomeric intact Ab % measured by SEC on day 0 (HEK production day) and the change in monomeric intact Ab % (monomeric intact Ab Δ%) up to day 14. For all BsAbs tested, monomeric intact Ab % values ​​were very low on day 0 and only a slight increase in monomeric intact Ab % values ​​was observed after day 14, indicating minimal aggregation. Figure 17J compares the production yields of the different BsAbs (with or without 354 / 349 substitution) in HEK293 cells. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Same as above. [Figure 17F] Same as above. [Figure 17G] Same as above. [Figure 17H] Same as above. [Figure 17I] Same as above. [Figure 17J] Same as above.

[0077] [Figure 18A]Figures 18A-18G show a comparison of anti-CD3 / anti-HER2 BsAbs containing different CH3 sets (WT, existing CH3 heterodimerization set, cycle 1 output, cycle 2 output, or a combination thereof, with or without CH3 disulfide bond substitution (i.e., 354 / 349 substitution)). Figure 18A compares the heterodimer% values ​​measured by LC-MS and IEX of different BsAbs without the 354 / 349 substitution showing good correlation. Figure 18B compares the ranking of heterodimerization potential determined by the heterodimer% values ​​measured by LC-MS and IEX of different BsAbs with the 354 / 349 substitution. The rankings determined by these two different methods (LC-MS and IEX) were the same. Figure 18C shows the heterodimer% values ​​measured by LC-MS and IEX for different BsAbs with and without the 354 / 349 substitution, demonstrating that the LWG-SIG set consistently provided higher heterodimer% values ​​regardless of the presence or absence of the 354 / 349 substitution. Figure 18D shows the monomeric intact Ab% values ​​measured by SEC for different BsAbs with and without the 354 / 349 substitution. As shown, some BsAbs, such as DVG-VSY and RG-FG, showed higher monomeric intact Ab% values ​​and exhibited less aggregation compared to existing variant CH3 domains (KiH, EW-RVT, or ZW1). Figure 18E provides a graph of monomeric intact Ab% values ​​measured by SEC plotted against heterodimer% values ​​measured by LC-MS for different BsAbs with and without 354 / 349 substitutions, demonstrating that 354 / 349 substitutions increased monomeric intact Ab% values ​​and heterodimer% values ​​overall. Figure 18F compares the production yield in HEK293 cells for different BsAbs with and without 354 / 349 substitutions, showing that none of the substitution sets appeared to adversely affect the production yield. Figure 18G compares the LWG-SIG set and its variant LWG-IG based on heterodimer% values ​​measured by IEX and LC-MS, monomeric intact Ab% values ​​measured by SEC, and production yield in HEK cells. These profiles were similar between LWG-SIG and LWG-IG and were consistent with the Rosetta heterodimer scores. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 18D] Same as above. [Figure 18E] Same as above. [Figure 18F] Same as above. [Figure 18G] Same as above.

[0078] [Figure 19] FIG. 19 provides a summary of exemplary CH3 domain sets ("CH3 set names") identified herein that preferentially form CH3-CH3 heterodimers over homodimers and thus promote desired Fc pairing.

[0079] The amino acid substitutions (positions and amino acid residues) for each of the CH3 sets listed in FIG. 19 can be found, for example, in Appendix Tables E-G. These amino acid substitutions may be incorporated into any CH3 domain sequence. Exemplary variant CH3 domain sequences in which the CH3 substitution sets listed in FIG. 19 are incorporated into the reference CH3 domain sequence of SEQ ID NO: 1 are also shown in Appendix Tables E-G. These exemplary variant CH3 domain sequences are the sequences used in the Examples herein. The SEQ ID NOs assigned to these exemplary variant CH3 domain sequences are also shown in FIG. 19.

[0080] [Figure 20] Figure 20 provides exemplary Tm2 values ​​of Fc-only constructs containing different CH3 sets (WT, pre-existing, cycle 1 output, or cycle 2 output CH3 heterodimerization sets, with or without CH3 disulfide bond substitutions (354 / 349 substitutions) as shown in Table 15) measured by DSC. Open circles represent constructs that do not contain the 354 / 349 substitution and closed circles represent constructs that do contain the 354 / 349 substitution.

[0081] [Figure 21]Figure 21 provides the ADI-64950 CH3-CH3 interface with its electron density. (a) Representative electron density within the region of interest of the crystal structure of IgG1 Fc-only construct ADI-64950, containing (i) chain A with T366S, L368I, and Y407G, and (ii) chain B with S364L, T366W, and K409G (chain B also contains Fc-III knockout substitutions: M252E, I253A, and Y436A). Chain A carbon atoms are in white, chain B carbon atoms are in light grey, nitrogen atoms are in dark grey, oxygen atoms are in black, and sulfur atoms are in very dark grey. The protein is shown in stick representation. The 2Fo-Fc electron density map is shown as a grey mesh drawn at 1.0σ with 2.0Å resolution. Data for this crystal structure extends to near atomic resolution of 2.70Å. (b) Table comparing interface statistics generated from PISA for ADI-64950 (SIG-LWG) and wild-type IgG1 (WT; PDB ID: 5JII).

[0082] [Figure 22] Figure 22 provides polar contacts at the ADI-64950 CH3-CH3 interface. (a) Polar contacts at the CH3-CH3 interface between chain A and chain B. Chain A carbon atoms are in white, chain B carbon atoms are in light grey, nitrogen atoms are in dark grey, and oxygen atoms are in black. The protein backbone is shown in cartoon representation with residues of interest shown in stick representation. Polar contacts are shown as black dotted lines. (b) Table comparing polar interactions generated from PyMol for ADI-64950 (SIG-LWG) and wild type IgG1 (WT; PDB ID:5JII).

[0083] [Diagram 23]FIG. 23 shows that several residues in potential ADI-64950 homodimer off-products are predicted to sterically clash with each other, reducing the propensity for mispairing. (a-d) Diagram of the pairing interface surrounding the region of interest. Alignment of chain A to chain B (a) and chain B to chain A (b-d) reveals steric clashes at the CH3-CH3 interface of several residues at substituted and unsubstituted positions of these potential off-products, including the orthologous sets of (a) Lys409 and Phe405, (b) Asp356 and Tyr349, (c) T366W and Tyr407, and (d) T366W and Tyr407. Chain A carbon atoms are in white, chain B carbon atoms are in light grey, nitrogen atoms are in dark grey, and oxygen atoms are in black. The protein backbone is shown in cartoon representation, the residues of interest are shown in stick representation, and the side chains involved in the collisions are shown as transparent molecular surfaces.

[0084] [Figure 24A] Figures 24A-24B show a comparison of exemplary results obtained in Example 14 using antibodies comprising (i) two identical CH3 domains belonging to the WT, RL, VV, QR-F, or RG-FG set and (ii) the variable domains of ADI-29235 (white) or ADI-26908 (black). Figure 24A compares the production yields of antibodies produced in CHO cells. Figure 24B provides a graph of the % monomeric full size Ab values ​​measured by SEC. [Figure 24B] Same as above.

[0085] [Figure 25A]Figures 25A-25B show exemplary results comparing the FAE output of Example 15 with the respective FAE input. Figure 25A compares protein recovery following the FAE reaction step to produce the indicated bsAbs containing the WT, RL, VV, or QR-F sets. Figure 25B provides IEX results for the RL, VV, and QR-F sets, with each panel showing an overlay of (i) the chromatograms of the FAE input (blue and red), each containing a monospecific parent antibody with the two same indicated CH3 domains, and (ii) the chromatogram of the corresponding FAE reaction output (green). [Figure 25B] Same as above.

[0086] [Figure 26A] 26A-26B show exemplary results comparing the FAE output of Example 16 with the respective FAE input. FIG. 26A provides exemplary SDS-PAGE results comparing protein quality between the FAE input and the FAE output. FAE reactions were performed to produce the indicated bsAbs containing WT, RL, or VV sets. FIG. 26B provides exemplary LC-MS results, with each panel showing an overlay of (i) chromatograms of the FAE input (blue and red) and (ii) the corresponding FAE reaction output chromatogram (black), each containing a monospecific parent antibody with the two same indicated CH3 domains. FIG. 26C provides exemplary binding kinetics curves comparing binding to either HER2 or CD3 by the indicated monospecific antibodies in the FAE input and the indicated bsAbs in the corresponding FAE output. FIG. 26D provides exemplary binding kinetics curves comparing simultaneous binding to HER2 and CD3 by the indicated monospecific antibodies in the FAE input and the indicated bsAbs in the corresponding FAE output. "HER2→CD3" indicates that the test antibody was exposed to HER2 first, then to CD3. "CD3→HER2" indicates that the test antibody was exposed to CD3 first, then to HER2. [Figure 26B] Same as above. [Figure 26C] Same as above. [Figure 26D] Same as above. [Figure 27A] 27A-27E show exemplary results from a GSH exposure experiment comparing the VV set with the RL set, as described in Example 17. FIG. 27A provides a schematic diagram of the GSH exposure experiment. In step 1, anti-HER2, CH3 hetero-IgG1 with test CH3 set and anti-CD3, CH3 hetero-IgG1 with test CH3 set are generated by FAE using 2-MEA. In step 2, the anti-HER2, CH3 hetero-IgG1 from step 1 is placed in a weakly reducing environment containing GSH with (i) two identical CH3 domains that are the same as one of the test CH3 sets, (ii) two identical CH3 domains that are the same as the other of the test CH3 sets, or (iii) anti-CD3 IgG1 with the test CH3 set (i.e., anti-HCD3, CH3 hetero-IgG1 from step 1), and chain reassociation is evaluated by IEX. FIG. 27B provides exemplary IEX results of FAE in step 1 using the RL set and the VV set. Each graph panel shows an overlay of a chromatogram of the FAE output with the chromatograms of the two FAE input antibodies. Figures 27C-27E provide exemplary IEX results of GSH exposure in step 2 using the RL set and the VV set. Each graph panel shows an overlay of a chromatogram of the GSH exposed product with the chromatograms of the two GSH exposed input antibodies. That is, the input antibodies are an anti-HER2, CH3 heterodimer antibody and an anti-CD3 IgG1 with (i) two identical CH3 domains that are the same as one of the tested CH3 sets (Figure 27C), (ii) two identical CH3 domains that are the same as the other of the tested CH3 sets (Figure 27D), or (iii) a test CH3 set (i.e., an anti-HCD3, CH3 heterodimer IgG1 from step 1 (Figure 27E). [Figure 27B] Same as above. [Figure 27C] Same as above. [Figure 27D] Same as above. [Figure 27E] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0088] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0089] It should be understood that aspects and embodiments of the disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0090] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably fragments exhibiting the desired antigen-binding activity, also referred to as "antigen-binding antibody fragments"). "Full antibody", "full Ab", "full size antibody", "full size Ab", "full length antibody", "intact antibody", or "whole antibody", and the like, encompass molecules having a structure substantially similar to a natural antibody, which in the case of IgG, IgD, or IgE, includes two heavy immunoglobulin chains and two light immunoglobulin chains. "Antigen-binding fragment" or "antigen-binding antibody fragment" refers to a portion of an intact antibody, or a combination of portions derived from an intact antibody, that binds to the antigen bound by the intact antibody.

[0091] An "antigen-binding fragment of an antibody" or an "antigen-binding antibody fragment" includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that contains an antibody domain (e.g., a VH domain or a CH3 domain) and specifically binds to an antigen to form a complex. Exemplary antibody fragments include Fv, fragment antigen-binding ("Fab") fragments, Fab' fragments, Fab' containing a free sulfhydryl group ("Fab'-SH"), F(ab') 2 These include, but are not limited to, fragments, diabodies, linear antibodies, single chain antibody molecules (e.g., single chain variable fragments ("scFv"), nanobodies or VHH, or only VH or VL domains), and monospecific or multispecific compounds formed from one or more of the aforementioned antibody fragments. In some embodiments, the antigen-binding fragment of the bispecific antibody described herein is an scFv or nanobody. In a preferred embodiment, the antigen-binding fragment comprises a set of CH3 domains that preferentially form CH3-CH3 heterodimers.

[0092] Like intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific, trispecific, tetraspecific, etc.). A multispecific antigen-binding fragment of an antibody may comprise at least two different variable domains, each capable of specifically binding to a distinct antigen or to different epitopes of the same antigen.

[0093] A "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for potential variant antibodies (e.g., which contain naturally occurring mutations and / or substitutions or which arise during production of the monoclonal antibody preparation), such variants being generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0094] A "multispecific antibody," which may also be referred to herein as a "multispecific compound," refers to an antibody that comprises at least two different antigen-binding domains that recognize and specifically bind to at least two different antigens and / or at least two different epitopes. In some embodiments, a multispecific antibody comprises (1) a first heavy chain and a first light chain that form a cognate pair and bind to a first antigen, and (2) a second heavy chain and a second light chain that form a cognate pair and bind to a second antigen.

[0095] A "bispecific antibody", also referred to herein as a "bispecific compound", is a type of multispecific antibody and refers to an antibody that contains two different antigen-binding domains that recognize and specifically bind to at least two different antigens or at least two epitopes. The at least two epitopes may or may not be within the same antigen. A bispecific antibody may target, for example, two different surface receptors, two different cytokines / chemokines, receptors and ligands on the same or different cells (e.g., an immune cell and a cancer cell).

[0096] In some embodiments, the at least two different antigens may be selected from the following antigens (or the at least two different epitopes may be epitopes in any of the following antigens): CD3; 0772P (CA125, MUC16; Genbank Accession Number AF36148), adipophilin (perilipin-2, adipose differentiation-related protein, ADRP, ADFP, MGC10598; NCBI Reference Sequence: NP-001113.2), AIM-2 (absent in melanoma 2, PYHIN4, interferon-inducible protein AIM2; NCBI Reference Sequence: NP-004824.1), ALDH1 A1 (aldehyde dehydrogenase 1 family, member A1, ALDH1, PUMB1, retinaldehyde dehydrogenase 1, ALDC, ALDH-E1, ALHDII, RALDH 1, EC 1.2.1.36, ALDH11, HEL-9, HEL-S-53e, HEL12, RALDH1, acetaldehyde dehydrogenase 1, aldehyde dehydrogenase 1, soluble aldehyde dehydrogenase, hepatic cytoplasmic ALDH class 1, epididymal luminal protein 12, epididymal luminal protein 9, epididymal secretory sperm-binding protein Li 53e, retinal dehydrogenase 1, RaIDH1, aldehyde dehydrogenase family 1 member A1, aldehyde dehydrogenase, cytoplasmic, EC 1.2.1;NCBI Reference Number: NP-000680.2);alpha-actinin-4 (ACTN4, actinin, alpha 4, FSGS1, focal segmental glomerulosclerosis 1, non-muscle alpha-actinin 4, F-actin cross-linking protein, FSGS, actinin-4, actinin alpha 4, alpha-actinin-4;NCBI Reference Sequence: NP-004915.2);alpha-fetoprotein (AFP, HPAFP, FETA, alpha-1-fetoprotein, alpha-fetoglobulin, alpha-1-fetoprotein, alpha-fetoglobulin, HP;GenBank: AAB58754.1);amphiregulin (AREG, SDGF, schwannoma-derived growth factor, colorectal cell-derived growth factor, AR, CRDGF;GenBank: AAA51781.1); ARTC1 (ART1, ADP-ribosyltransferase 1, mono(ADP-ribosyl)transferase 1, ADP-ribosyltransferase C2 and C3 toxin-like 1, ART2, CD296, RT6, ADP-ribosyltransferase 2, GPI-linked NAD(P)(+)-arginine ADP-ribosyltransferase 1, EC 2.4.2.31, CD296 antigen; NP); ASLG659; ASPHD1 (Aspartate beta-hydroxylase domain containing 1, Aspartate beta-hydroxylase domain-containing protein 1, EC1.14.11, GenBank: AAI44153.1); B7-H4 (VTCN1, V-Set domain-containing T cell activation inhibitor 1, B7H4, B7 superfamily member 1, immune costimulatory protein B7-H4, B7h.5, T cell costimulatory molecule B7x, B7S1, B7X, VCTN1, H4, B7 family member, PRO1291, B7 family member, H4, T cell costimulatory molecule B7x, V-Set domain-containing T cell activation inhibitor 1, protein B7S1; GenBank: AAZ17406.1) ;BAFF-R (TNFRSF13C, tumor necrosis factor receptor superfamily member 13C, BAFFR, B cell-activating factor receptor, BAFF receptor, BLyS receptor 3, CVID4, BROMIX, CD268, B cell-activating factor receptor, prolixin, tumor necrosis factor receptor superfamily member 13C, BR3, CD268 antigen; NCBI sequence NP-443177.1); BAGE-1; BCLX(L); BCR-ABL fusion protein (b3a2); beta-catenin (CTNNB1, catenin (cadherin-associated protein), beta 1, 88 kDa, CTNNB, MRD19, catenin (cadherin-associated protein), beta 1 (88 kDa), armadillo, catenin beta-1; GenBank: CAA61107.1); BING-4 (WDR46, WD repeat domain 46, C6orf11, BING4, WD repeat-containing protein BING4, chromosome 6 open reading frame 11, FP221, UTP7, WD repeat-containing protein 46; NP); BMPR1 B (bone morphogenetic protein receptor type IB, Genbank accession number NM-00120; NP); B-RAF (brevican (BCAN, BEHAB, GenBank accession number AF22905), brevican (BCAN, chondroitin sulfate proteoglycan 7, brain enriched hyaluronan binding protein, BEHAB, CSPG7, brevican proteoglycan, brevican core protein, chondroitin sulfate proteoglycan BEHAB; GenBank:AAH27971.1); CALCA (calcitonin-related polypeptide alpha, CALC1, calciton nin1, calcitonin, alpha-type CGRP, calcitonin gene-related peptide I, CGRP-I, CGRP, CGRP1, CT, KC, calcitonin / calcitonin-related polypeptide, alpha, katacalcin, NP); CASP-5 (CASP5, caspase-5, apoptosis-related cysteine ​​peptidase, caspase-5, apoptosis-related cysteine ​​protease, protease ICH-3, protease TY, ICE(rel)-111, ICE(rel)III, ICEREL-III, ICH-3, caspase-5, TY protease, EC 3.4.22.58, ICH3, EC 3.4.22; NP); CASP-8; CD19 (CD19 - B lymphocyte antigen CD19 isoform 2 precursor, B4, CVID3 [Homo sapiens], NCBI Reference Sequence: NP-001761.3); CD20 (CD20 - B lymphocyte antigen CD20, transmembrane 4 domains, subfamily A, member 1, B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7; NCBI Reference Sequence: NP-690605.1); CD21 (CD21(CR2 (complement receptor or C3DR (C3d / Epstein-Barr virus receptor) or Hs.73792 GenBank accession number M2600; (CD22 (B cell receptor CD22-B isoform, BL-CAM, Lyb-8, LybB, SIGLEC-2, FLJ22814, GenBank accession number AK02646); CD22; CD33 (CD33 molecule, CD33 antigen (Gp67), sialic acid-binding Ig-like lectin 3, sialic acid-binding Ig-like lectin 3, SIGLEC3, gp67, SIGLEC-3, myeloid cell surface antigen CD33, p67, Siglec-3, CD33 antigen; GenBank: AAH28152.1); CD45; CD70 (CD70 tumor necrosis factor (ligand) superfamily, member 7; surface antigen CD70; Ki-24 antigen; CD27 ligand; CD27-L; tumor necrosis factor ligand superfamily member 7; NCBI reference sequence for species Homo sapiens: NP-001243.1; CD72 (CD72 (B cell differentiation antigen CD72, Lyb-; 359aa, μL: 8.66, MW: 40225, TM: 1 [P] gene chromosome: 9p13.3, GenBank accession number NP-001773.); CD79a (CD79a (CD79A, CD79a, immunoglobulin-related alpha, B cell-specific protein that covalently binds Ig beta (CD79B) to form a complex on the surface with Ig M molecules, transducing signals involved in B cell differentiation), μL: 4.84, MW: 25028 TM:2[P] gene chromosome:19q13.2, GenBank accession number NP-001774.1; CD79b (CD79b (CD79B, CD79b, IGb (immunoglobulin-related beta), B29, GenBank accession number NM-000626 or 1103867); Cdc27 (cell division cycle 27, D0S1430E, D17S978E, anaphase-promoting complex subunit 3, anaphase-promoting complex subunit 3, AN APC3, APC3, CDC27Hs, H-NUC, CDC27 homolog, cell division cycle 27 homolog (S. Cerevisiae), HNUC, NUC2, anaphase promoting complex, protein 3, cell division cycle 27 homolog, cell division cycle protein 27 homolog, Nuc2 homolog; GenBank: AAH11656.1; CDK4 (cyclin-dependent kinase 4, cell division protein kinase 4, PSK-J3, EC 2.7.11.22, CMM3, EC 2.7.11; NCBI reference sequence: NP-000066.1; CDKN2A (cyclin-dependent kinase inhibitor 2A, MLM, CDKN2, MTS1, cyclin-dependent kinase inhibitor 2A (inhibits melanoma, P16, CDK4), cyclin-dependent kinase 4 inhibitor A, multiple tumor suppressor 1, CDK4I, MTS-1, CMM2, P16, ARF, INK4, INK 4A, P14, P14ARF, P16-INK4A, P16INK4, P16INK4A, P19, P19ARF, TP16, CDK4 inhibitor P16-INK4, cell cycle negative regulator beta, p14ARF, p16-INK4, p16-INK4a, p16INK4A, p19ARF; NP; CEA; CLL-1 (CLEC12A, MICL, and DCAL, C It encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune responses. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced ​​transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined. This gene is tightly linked to other CTL / CTLD superfamily members within the natural killer gene complex region on chromosome 12p13 (Drickamer, K Curr.Opin.Struct.Biol.9:585-90

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[2004] ;Marshall AS,et al J.Biol.Chem.279:14792-80, 2004. CLL-1 has been shown to be a type II transmembrane receptor containing a single C-type lectin-like domain (not predicted to bind either calcium or sugar), a stalk region, a transmembrane domain, and a short cytoplasmic tail containing ITIM motifs. );CLPP (caseinolytic mitochondrial matrix peptidase proteolytic subunit, endopeptidase Clp, EC 3.4.21.92, PRLTS3, ATP-dependent protease ClpAP (E. coli), ClpP (caseinolytic protease, ATP-dependent, proteolytic subunit, E. coli) homolog, ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog (E. coli), ClpP caseinolytic protease, ATP-dependent, proteolytic subunit homolog (E. coli), human, proteolytic subunit, ATP-dependent protease ClpAP, proteolytic subunit, human, ClpP caseinolytic peptidase ATP-dependent, proteolytic subunit, ClpP caseinolytic peptidase, ATP-dependent, proteolytic subunit homolog, ClpP caseinolytic protease, AT. P-dependent, proteolytic subunit homolog, putative ATP-dependent Clp protease proteolytic subunit, mitochondrial; NP; COA-1; CPSF; CRIPTO (CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, GenBank accession number NP-003203 or NM-00321); Cw6; CXCR5 (Burkitt lymphoma receptor 1, G protein-coupled receptor activated by CXCL13 chemokine functions in lymphocyte migration and humoral defense and plays a role in HIV-2 infection and possibly in the pathogenesis of AIDS, lymphoma, myeloma, and leukemia); 372 aa, μL: 8.54 MW: 41959 TM: 7 [P] gene chromosome: 11q23.3, GenBank accession number NP-001707); CXORF61 CXORF61 - chromosome X open reading frame 61 [Homo sapiens], NCBI Reference Sequence: NP-001017978.1; cyclin D1 (CCND1, BCL1, PRAD1, D11S287E, B-cell CLL / lymphoma 1, B-cell lymphoma 1 protein, BCL-1 oncogene, PRAD1 oncogene, cyclin D1 (PRAD1:parathyroid adenomatosis 1), G1 / S-specific cyclin D1, parathyroid adenomatosis 1, U21B31, G1 / S-specific cyclin-D1, BCL-1; NCBI Reference Sequence: NP-444284.1); cyclin-A1 (CCNA1, CT146, cyclin A1; GenBank: AAH36346.1); dek-can fusion protein; DKK1 (Dickkopf WNT signaling pathway inhibitor 1, SK, hDkk-1, Dickkopf (Xenopus Laevis) homolog 1, Dickkopf 1 homolog (Xenopus Laevis), DKK-1, Dickkopf 1 homolog, Dickkopf-related protein-1, Dickkopf-1-like, Dickkopf-like protein 1, Dickkopf-related protein 1, Dickkopf-1, Dkk-1; GenBank:AAQ89364.1); DR1 (downregulator of transcription 1, TBP-binding (negative cofactor 2), negative cofactor 2-beta, TATA-binding protein-associated phosphoprotein, NC2, NC2-beta, protein Dr1, NC2-beta, downregulator of transcription 1; NCBI reference sequence: NP-001929.1); DR13 (major histocompatibility complex, class II, DR beta 1, HLA-DR1B, DRw10, DW2.2 / DR2.2, SS1, DRB1, HLA-DRB, HLA class II histocompatibility antigens, DR-1 beta chain, human leukocyte antigen DRB1, lymphocyte antigen DRB1, MHC class II antigen, MHC class II HLA-DR beta 1 chain, MHC class II HLA-DR-beta cell surface glycoprotein, MHC class II HLA-DRw10-beta, DR-1, DR-12, DR-13, DR-14, DR-16, DR-4, DR-5, DR-7, DR-8, DR-9, DR1, DR12, DR13, DR14, DR16, DR4, DR5, DR7, DRB, DR9, DRw11, DRw8, HLA-DRB2, clone P2-beta-3, MHC class II antigen DRB1*1, MHC class II antigen DRB1*10, MHC class II antigen DRB1*11, MHC class II antigen DRB1*12, MHC class II antigen DRB1*13, MHC class II antigen DRB1*14, MHC class II antigen DRB1*15, MHC class II antigen DRB1*16, MHC class II antigen DRB1 *3, MHC class II antigen DRB1*4, MHC class II antigen DRB1*7, MHC class II antigen DRB1*8, MHC class II antigen DRB1*9; NP; E16 (E16(LAT1, SLC7A5, GenBank accession number NM-00348); EDAR (EDAR-Tumor necrosis factor receptor superfamily member EDAR precursor, EDA-A1 receptor; Downless homolog; Ectodysplasin-A receptor; Ectodermal dysplasia receptor; Anhydroectodysplasin receptor 1, DL; ECTD10A; ECTD10B; ED1R; ED3; ED5; EDA-A1R; EDA1R; EDA3; HRM1 [Homo sapiens]; NCBI reference sequence: NP-071731.1);EFTUD2 (elongation factor Tu GTP-binding domain-containing 2, elongation factor Tu GTP-binding domain-containing protein 2, hSNU114, SNU114 homolog, U5 SnRNP-specific protein, 116KDa, MFDGA, KIAA0031, 116KD, U5 SnRNP-specific protein, 116KDa U5 small nuclear ribonucleoprotein component, MFDM, SNRNP116, Snrp116, Snu114, U5-116KD, SNRP116, U5-116KDa; GenBank:AAH02360.1);EGFR (epidermal growth factor receptor, ERBB, proto-oncogene C-ErbB-1, receptor tyrosine-protein kinase ErbB-1, ERBB1, HER1, EC 2.7.10.1, epidermal growth factor receptor (avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog), erythroblastic leukemia virus (V-Erb-B) oncogene homolog (avian), PlG61, avian erythroblastic leukemia virus (V-Erb-B) oncogene homolog, cell proliferation inhibitor protein 40, cell proliferation inducer protein 61, mENA, EC 2.7.10; GenBank: AAH94761.1); EGFR-G719A; EGFR-G719C; EGFR-G719S; EGFR-L858R; EGFR-L861 Q; EGFR-57681; EGFR-T790M; elongation factor 2 (EEF2, eukaryotic translation elongation factor 2, EF2, polypeptidyl-tRNA translocase, EF-2, SCA26, EEF-2; NCBI reference sequence: NP-001952.1); ENAH (hMena) (effective homolog (Drosophila), MENA, mammalian effective, ENA, NDPP1, protein effective homolog; GenBank: AAH95481.1) - yields simply "ENAH", not "ENAH (hMena)"; EpCAM (epithelial cell adhesion molecule, M4S1, MIC18, tumor-associated calcium signal transducer 1, TACSTD1, TROP1, adenocarcinoma-associated antigen, cell surface glycoprotein Trop-1, epithelial glycoprotein 314, major gastrointestinal tumor-associated protein GA733-2, EGP314, KSA, DIAR5, HNPCC8, antigen identified by monoclonal antibody AUA1, EGP-2, EGP40, ESA, KS1 / 4, MK-1, human epithelial glycoprotein-2, membrane component, chromosome 4, surface marker (35 kD glycoprotein), EGP, Ep-CAM, GA733-2, M1S2, CD326 antigen, epithelial cell surface antigen, hEGP314, KS 1 / 4 antigen, ACSTD1; GenBank: AAH14785.1; EphA3 (EPH receptor A3, ETK1, ETK, TYRO4, HEK, Eph-like tyrosine kinase 1, tyrosine-protein kinase receptor ETK1; EK4, EPH-like kinase 4, EC 2.7.10.1, EPHA3, HEK4, ephrin type A receptor 3, human embryonic kinase 1, TYRO4 protein tyrosine kinase, hEK4, human embryonic kinase, tyrosine-protein kinase TYRO4, EC 2.7.10; GenBank: AAH63282.1; EphB2R; epiregulin (EREG, ER, proepiregulin; GenBank: AAI36405.1); ETBR (EDNRB, endothelin receptor type B; HSCR2, HSCR, endothelin receptor nonselective type; ET-B, ET-BR; ETRB, ABCDS, WS4A; ETB, endothelin B receptor; NP); ETV6-AML1 fusion protein; EZH2 (Enhancer of Zeste homolog 2 (Drosophila), Lysine N-methyltransferase 6, ENX-1, KMT6 EC 2.1.1.43, EZH1, WVS, Enhancer of Zeste (Drosophila) homolog 2, ENX1, EZH2b, KMT6A, WVS2, Histone-lysine N-methyltransferase EZH2, Enhancer of Zeste homolog 2, EC 2.1.1; GenBank: AAH10858.1); FcRH1 (FCRL1, Fc receptor-like 1, FCRH1, Fc receptor homolog 1, FcR-like protein 1, immunoreceptor translocation-associated protein 5, IFGP1, IRTA5, hIFGP1, IFGP family protein 1, CD307a, Fc receptor-like protein 1, immunoglobulin superfamily Fc receptor, Gp42, FcRL1, CD307a antigen; GenBank: AAH33690.1); FcRH2 (FCRL2, Fc receptor-like 2, SPAP1, SH2 domain-containing phosphatase anchor protein 1, Fc receptor homolog 2, FcR-like protein 2, immunoglobulin receptor translocation-associated protein 4, FCRH2, IFGP4, IRTA4, IFGP family protein 4, SPAP1A, SPAP1 B, SPAP1C, CD307b, Fc receptor-like protein 2, immune receptor translocation-associated protein 4, immunoglobulin superfamily Fc receptor, Gp42, SH2 domain containing phosphatase anchor protein 1, FcRL2, CD307b antigen; GenBank:AAQ88497.1; FcRH5 (FCRL5, Fc receptor-like 5; IRTA2, Fc receptor homolog 5, FcR-like protein 5, immune receptor translocation-associated protein 2; BXMAS1, FCRH5 , CD307, CD307e, PRO820, Fc receptor-like protein 5, immunoglobulin superfamily receptor translocation associated 2 (IRTA2), FCRL5, CD307e antigen; GenBank: AAI01070.1; FLT3-ITD; FN1 (fibronectin 1, cold insoluble globulin, FN, migration-stimulating factor, CIG, FNZ, GFND2, LETS, ED-B, FINC, GFND, MSF, fibronectin; GenBank: AAI43764.1);G250 (MN, CAIX, carbonic anhydrase IX, carbonic anhydratase, RCC-associated protein G250, carbonic anhydrase IX, membrane antigen MN, renal cell carcinoma-associated antigen G250, CA-IX, P54 / 58N, pMW1, RCC-associated antigen G250, carbonic anhydrase 9; NP);-alias result of "G250" instead of "G250 / MN / CAIX", GAGE-1, 2, 8; GAGE-3, 4, 5, 6, 7; GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1 L; GDNFR-alpha 1; GFR-alpha-; U95847; BC014962; NM-145793 NM-005264);GEDA (GenBank accession number AY26076);GFRA1-GDNF family receptor alpha-1;GDNF receptor alpha-1;GDNFR-alpha-1;GFR-alpha-1;RET ligand 1;TGF-beta-related neurotrophic factor receptor 1 [Homo sapiens];ProtKB / Swiss-Prot:P56159.2;Glypican-3 (GPC3, glypican 3, SDY S, glypican proteoglycan 3, intestinal protein OCI-5; GTR2-2, MXR7, SGBS1, DGSX, OCI-5; SGB, SGBS, heparan sulfate proteoglycan, secreted glypican-3, OCI5; GenBank:AAH35972.1; GnTVf; gp100 (PMEL, pre-melanosomal protein, SILV, D12S53E, PMEL17; SIL, melanocyte protein Pmel 17, melanocyte lineage-specific antigen GP100, melanoma-associated ME20 antigen, silver locus protein homolog, ME20-M, ME20M, P1, P100, silver (mouse homolog)-like, silver homolog (mouse), ME20, SI, melanocyte protein Mel 17, melanocyte protein PMEL, melanosome matrix protein 17, silver, mouse, homolog Of; GenBank: AAC60634.1); GPC; GPNMB (glycotan. protein (transmembrane) Nmb, glycoprotein NMB, glycoprotein Nmb-like protein, osteoactivin, transmembrane glycoprotein HGFIN, HGFIN, NMB, transmembrane glycoprotein, transmembrane glycoprotein NMB; GenBank: AAH32783.1; GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); NP-078807.1; NM-024531.3; GPR19 (G protein-coupled receptor 19; Mm.478; NP-006134.1; NM-006143.2); GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR1; NP-115940.2; NM-032551.4); HAVCR1 (Hepatitis A virus cell receptor 1, T cell immunoglobulin globulin mucin family member 1, kidney injury molecule 1, KIM-1, KIM1, TIM, TIM-1, TIM1, TIMD-1, TIMD1, T cell immunoglobulin mucin receptor 1, T cell membrane protein 1, HAVCR, HAVCR-1, T cell immunoglobulin domain and mucin domain protein 1, HAVcr-1, T cell immunoglobulin and mucin domain containing protein 1; GenBank:AAH13325.1; HER2 (ERBB2, V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2, NGL, NEU, neuro / glioblastoma derived oncogene homolog, metastatic lymph node gene 19 protein, proto-oncogene C-ErbB-2, proto-oncogene Neu, tyrosine kinase cell surface receptor HER2, MLN19, p185erbB2, EC 2.7.10.1, V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2 (neuro / glioblastoma-derived oncogene homolog), CD340, HER-2, HER-2 / neu, TKR1, C-Erb B2 / Neu protein, herstatin, neuroblastoma / glioblastoma derived oncogene homolog, receptor tyrosine-protein kinase ErbB-2, V-Erb-B2 erythroblastic leukemia viral oncogene homolog 2, neuro / glioblastoma derived oncogene homolog, MLN19, CD340 antigen, EC 2.7.10;NP);HER-2 / neu-alternatives of the above;HERV-K-MEL;HLA-DOB (beta subunit of MHC class II molecules (la antigens) that bind peptides and present them to CD4+ T lymphocytes);273aa, μL:6.56, MW:30820.TM:1[P] gene chromosome:6p21.3, GenBank accession number NP-002111);hsp70-2 (HSPA2, heat shock 70 kDa protein 2, heat shock 70 kDa protein 2, HSP70-3, heat shock associated 70 KDa protein 2, heat shock 70 KDa protein 2;GenBank:AAD21815.1);IDO1 (indoleamine 2,3-dioxygenase 1, IDO, INDO, indoleamine-pyrrole 2,3-dioxygenase, IDO-1, indoleamine-pyrrole 2,3 dioxygenase, indoleamine 2,3 dioxygenase, indole 2,3 dioxygenase, EC 1.13.11.52; NCBI Reference Sequence: NP-002155.1; IGF2B3; IL13R alpha 2 (IL13RA2, interleukin 13 receptor, alpha 2, cancer / testis antigen 19, interleukin-13-binding protein, IL-13R-alpha-2, IL-13RA2, IL-13 receptor subunit alpha-2, IL-13R subunit alpha-2, CD213A2, CT19, IL-13R, IL13BP, interleukin-13 binding protein, interleukin-13 receptor alpha 2 chain, interleukin-13 receptor subunit alpha α-2, IL13R, CD213a2 antigen; NP; IL20Rα; intestinal carboxylesterase, IRTA2 (also known as FcRH5); kallikrein 4 (KLK4, kallikrein-related peptidase 4, PRSS17, EMSP1, enamel matrix serine proteinase 1, kallikrein-like protein 1, serine protease 17, KLK-L1, PSTS, AI2A1, kallikrein 4 (prostase, enamel matrix, prostate), ARM1, EMSP, androgen-regulated message 1, enamel matrix serine protease 1, kallikrein, kallikrein-4, prostase, EC 3.4.21.-, prostase EC 3.4.21; GenBank: AAX30051.1); KIF20A (kinesin family member 20A, RAB6KIFL, RAB6 interacting, kinesin-like (rab kinesin 6), mitosis a; LAGE-1; LDLR-fucosyltransferase AS fusion protein; lengusin (LGSN, lengusin, lens protein with glutamine synthetase domain; GLULD1, glutamate-ammonia ligase domain-containing protein 1, LGS, glutamate-ammonia ligase (glutamine synthetase) domain-containing 1 ... LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR6; NP-003658.1, NM-003667.2; LY64 (lymphocyte antigen 64 (RP10, a type I membrane protein of the leucine-rich repeat (LRR) family that regulates B cell activation and apoptosis, and loss of function is associated with increased disease activity in patients with systemic lupus erythematosus); 661aa, μL:6.20, MW:74147 TM:1[P] gene chromosome:5q12, GenBank accession numbers NP-005573; Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-; NP-002337.1; NM-002346.2); Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT; NP-067079.2; NM-021246.2); LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ3522; NP-059997.3; NM-017527.3); LyPD1 -LY6 / PLAUR domain containing 1, PHTS [Homo sapiens], GenBank:AAH17318.1;MAGE-A1 (Melanoma antigen family A, 1 (Direct expression of antigen MZ2-E, MAGE1, Melanoma antigen family A, 1, MAGEA1, Melanoma antigen MAGE-1, Melanoma associated antigen 1, Melanoma associated antigen MZ2-E, Antigen MZ2-E, Cancer / Testis antigen 1.1, CT1.1, MAGE-1 antigen, Cancer / Testis antigen family 1, member 1, Cancer / Testis antigen family 1, member 1, MAGE1A; NCBI reference sequence:NP-004979.3); MAGE-A10 (MAGEA10, melanoma antigen family A, 10, MAGE10, MAGE-10 antigen, melanoma associated antigen 10, cancer / testis antigen 1.10, CT1.10, cancer / testis antigen family 1, member 10, cancer / testis antigen family 1, member 10; NCBI reference sequence: NP-001238757.1); MAGE-A12 (MAGEA12, melanoma antigen family A, 12, MAGE12, cancer / testis antigen 1.12, CT1.12, MAGE12F antigen, cancer / testis antigen family 1, member 12, cancer / testis antigen family 1, member 12, melanoma associated antigen 12, MAGE-12 antigen; NCBI reference sequence: NP-001159859.1); MAGE-A2 (MAGEA2, melanoma antigen family A, 2, MAGE2, cancer / testis antigen 1.2, CT1.2, MAGEA2A, MAGE-2 antigen, cancer / testis antigen family 1, member 2, cancer / testis antigen family 1, member 2, melanoma antigen 2, melanoma associated antigen 2; NCBI reference sequence: NP-001269434.1); MAGE-A3 (MAGEA3, melanoma antigen family A, 3 , MAGE3, MAGE-3 antigen, antigen MZ2-D, melanoma associated antigen 3, cancer / testis antigen 1.3, CT1.3, cancer / testis antigen family 1, member 3, HIPS, HYPD, MAGEA6, cancer / testis antigen family 1, member 3; NCBI reference sequence: NP-005353.1; MAGE-A4 (MAGEA4, melanoma antigen family A, 4; MAGE4, ​​melanoma associated antigen 4, cancer / testis antigen 1.4, CT1.4, MAGE-4 antigen, MAGE-41 antigen, MAGE-X2 antigen, MAGE4A, MAGE4B, cancer / testis antigen family family 1, member 4, MAGE-41, MAGE-X2, cancer / testis antigen family 1, member 4; NCBI reference sequence: NP-001011550.1; MAGE-A6 (MAGEA6, melanoma antigen family A, 6, MAGE6, MAGE-6 antigen, melanoma associated antigen 6, cancer / testis antigen 1.6, CT1.6, MAGE3B antigen, cancer / testis antigen family 1, melanoma antigen family A, 6, member 6, MAGE-3b, MAGE3B, cancer / testis antigen family 1, member 6; NCBI reference sequence: NP-787064.1);MAGE-A9 (MAGEA9, melanoma antigen family A, 9; MAGE9, MAGE-9 antigen, melanoma associated antigen 9; cancer / testis antigen 1.9; CT1.9, cancer / testis antigen family 1, member 9; cancer / testis antigen family 1, member 9; MAGEA9A; NCBI reference sequence: NP-005356.1);MAGE-C1 (MAGEC1, melanoma antigen family C, 1; cancer / testis antigen 7.1; CT7.1, MAGE-C1 antigen, cancer / testis antigen family 7, member 1; CT7, cancer / testis antigen family 7, member 1; melanoma associated antigen C1; NCBI reference sequence: NP-005453.2);MA GE-C2 (MAGEC2, melanoma antigen family C, 2; MAGEE1, cancer / testis antigen 10, CT10; HCA587, melanoma antigen family E, 1, cancer / testis specific; hepatocellular carcinoma associated antigen 587; MAGE-C2 antigen; MAGE-E1 antigen; hepatocellular carcinoma antigen 587; melanoma associated antigen C2; NCBI reference sequence: NP-057333.1); mammaglobin-A (SCGB2A2, secretoglobin, family 2A, member 2; MGB1, mammaglobin 1; UGB2, mammaglobin A; mammaglobin-A; mammaglobin-1; secretoglobin family 2A member 2; NP); MART2 (H HAT, hedgehog acyltransferase, SKI1, melanoma antigen recognized by T cells 2, lean hedgehog protein 1, Skn, melanoma antigen recognized by T cells 2, protein-cysteine ​​N-palmitoyltransferase HHAT, EC 2.3.1.-; GenBank: AAH39071.1); M-CSF (CSF1, colony-stimulating factor 1 (macrophage), MCSF, CSF-1, lanimostim, macrophage colony-stimulating factor 1, lanimostim; GenBank: AAH21117.1); MCSP (SMCP, sperm mitochondrial-associated cysteine-rich protein, MCS, mitochondrial capsule selenoprotein, HSMCSGEN1, sperm mitochondrial-associated cysteine-rich protein; NCBI reference sequence: NP-109588.2);XAGE-1b / GAGED2a;WT1 (Wilms tumor 1; WAGR; GUD; WIT-2;WT33; EWS amino-terminal domain;NPHS4; the last three zinc fingers of the DNA-binding domain of WT1;AWT1; Wilms tumor protein;EWS-WT1;GenBank:AAB33443.1);VEGF;tyrosinase (TYR;OCAIA;OCA1A;tyrosinase;SHEP;NP-000363.1;NM-0 00372.4; GenBank: AAB60319.1); TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, GenBank accession number NM-01763); TRP2-INT2; TRP-2; TRP-1 / gp75 (tyrosinase-related protein 1, 5,6-dihydroxyindole-2-carboxylic acid oxidase, CAS2, CATB, TYR. P, OCAS, catalase B, b-protein, glycoprotein 75, EC 1.14.18., melanoma antigen Gp75, TYRP1, TRP, TYRRP, TRP1, SHEP11, DHICA oxidase, EC 1.14.18, GP75, EC 1.14.18.1; triosephosphate isomerase (triosephosphate isomerase 1, TPID, triosephosphate isomerase, HEL-S-49, TIM, epididymal secretory protein Li 49, TPI, triosephosphate isomerase, EC 5.3.1.1; TRAG-3 (CSAG family member 2, cancer / testis antigen family 24; CSAG3B, member 2; CSAG family member 3B, cancer / testis antigen family 24 member 2, cancer / testis antigen 24.2, chondrosarcoma-associated gene 2 / 3 protein, taxol resistance-associated gene 3 protein, chondrosarcoma-associated gene 2 / 3 protein-like, CT24.2, taxol resistance-associated gene 3, TRAG-3, CSAG3A, TRAG3;); TMEM46 (Schisa homolog 2 (Xenopus); Shisa (SHISA); NP-001007539.1; NM-001007538.1; TMEM118 (Ring finger protein, transmembrane 2;RNFT2;FLJ1462;NP-001103373.1;NM-001109903.1;TMEFF1 (transmembrane protein with EGF-like domain and two follistatin-like domains 1;Tomoregulin-;H7365;C9orf2;C9ORF2;U19878;X83961;NM-080655;NM-003692;TGF-beta RII (TGFBR2, transforming growth factor, beta receptor II (70 / 80 kDa), TGF-beta-RII,MFS2,tbeta R-II,TGFR-2,TGF-beta receptor type IIB,TGF-beta type II receptor,TGF-beta receptor type 2,EC 2.7.11.30, transforming growth factor beta receptor type IIC, AAT3, T beta R-II, transforming growth factor, beta receptor II (70-80 kD), TGF-beta receptor type II, FAA3, transforming growth factor beta receptor type II, LDS1 B, HNPCC6, LDS2B, LDS2, RITC, EC 2.7.11, TAAD2; TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF / heregulin family of growth factors and follistatin); 374aa, NCBI accession: AAD55776, AAF91397, AAG49451, NCBI reference sequence: NP-057276; NCBI gene: 23671; OMIM: 605734; SwissProt Q9UIK5; GenBank accession numbers AF179274; AY358907, CAF85723, CQ782436; TAG-2; TAG-1 (contactin 2 (axonal), TAG-1, AXT, axonin-1 cell adhesion molecule, TAX, contactin 2 (transiently expressed), TAXI, contactin-2, axonal glycoprotein TAG-1, transiently expressed axonal glycoprotein, transient axonal glycoprotein, axonin-1, TAX-1, TAG1, FAMES; PRF:444868); SYT-SSX1 or -SSX2 fusion proteins; survivin; STEAP2 (HGNC 8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, GenBank accession number AF45513; STEAP1 (six transmembrane epithelial antigen of prostate, GenBank accession number NM-01244; SSX-4; SSX-2 (SSX2, synovial sarcoma, X-breakpoint 2, X-breakpoint 2, SSX, X-breakpoint 2B, cancer / testis antigen 5.2, X chromosome-associated 2, tumor antigen HOM-MEL-40, CT5.2, HD21 , Cancer / Testis Antigen Family 5, HOM-MEL-40, Isoform B, Cancer / Testis Antigen Family 5 Member 2a, Member 2a, Protein SSX2, Sarcoma, Sarcoma, Synovium, X-linked 2, Synovium, Synovial Sarcoma, X-breakpoint 2B, Synovial Sarcoma, SSX2A; Sp17; SOX10 (SRY (Sex Determining Region Y)-box 10, Mouse, PCWH, DOM, WS4, WS2E, WS4C, Dominant Megacolon, Mouse, Human Homolog Of, Dominant Megacolon, SRY-related HMG-box Gene 10, Human Homolog Of, Transcription Factor SOX-10; GenBank: CAG30470.1); SNRPD1 (small nuclear ribonucleoprotein D1, small nuclear ribonucleoprotein D1, polypeptide 16 kDa, polypeptide (16 kD), SNRPD, HsT2456, Sm-D1, SMD1, Sm-D autoantigen, small nuclear ribonucleoprotein D1 polypeptide 16 kDa pseudogene, SnRNP core protein D1, small nuclear ribonucleoprotein Sm D1); SLC35D3 (solute carrier family 35, member D3, FRCL1, Fringe connection-like protein 1, bA55K22.3, Frc, Fringe-like 1, solute carrier family 35 member D3; NCBI GenBank: NC-000006.11 NC-018917.2 NT-025741.16);SIRT2 (Sirtuin 2, NAD-dependent deacetylase sirtuin-2, SIRL2, silent signal regulator 2, regulatory protein SIR2 homolog 2, Sir2-related protein type 2, SIR2-like protein 2, sirtuin type 2, sirtuin (silent mating signal regulator 2 homolog) 2 (budding yeast), sirtuin-2, sirtuin (silent mating signal regulator 2, budding yeast, homolog) 2, EC 3.5.1., SIR2; GenBank: AAK51133.1), Sema5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain™ and short cytoplasmic domain, (semaphorin) 5B, GenBank Accession No. AB04087; seserin1 (SCRN1, SES1, KIAA0193, seserin-1; GenBank: EAL24458.1); SAGE (SAGE1, sarcoma antigen 1, cancer / testis antigen 14, CT14, putative tumor antigen; NCBI Reference Sequence: NP-061136.2); RU2AS (KAAG1, kidney-associated antigen 1, RU2AS, RU2 antisense gene protein, kidney-associated antigen 1; GenBank: AAF23613.1); RNF43-E3 ubiquitin-protein ligase RNF43 precursor [Homo sapiens], RNF124; URCC; NCBI Reference Sequence: NP-060233.3; RhoC (RGS5 (regulator of G-protein signaling 5, MSTP032, regulator of G-protein signaling 5, MSTP092, MST092, MSTP106, MST106, MSTP129, MST129; GenBank: AAB84001.1); RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE; NP-066124.1; NM-020975.4); RBAF600 (UBR4, ubiquitin protein ligase E3 component N-recognin 4, zinc finger, UBR1 type 1, ZUBR1, E3 ubiquitin-protein ligase UBR4; RBAF600, 600KDa retinoblastoma protein-associated factor, zinc finger UBR1-type 1 protein, EC 6.3.2., N-recognin-4, KIAA0462, p600, EC 6.3.2, KIAA1307; GenBank: AAL83880.1; RAGE-1 (MOK, MOK protein kinase, renal tumor antigen; RAGE, MAPK / MAK / MRK overlapping kinase, renal tumor antigen 1, renal cell carcinoma antigen, RAGE-1, EC 2.7.11.22, RAGE1; UniProtKB / Swiss-Prot:Q9UQ07.1; RAB38 / NY-MEL-1 (RAB38, NY-MEL-1, RAB38, member RAS oncogene family, melanoma antigen NY-MEL-1, Rab-related GTP-binding protein, Ras-related protein Rab-38, rrGTPbp; GenBank:AAH15808.1); PTPRK (DJ480J14.2.1 (protein tyrosine phosphatase, receptor type, K R-PTP-KAPPA, protein tyrosine phosphatase kappa, protein tyrosine phosphatase kappa), protein tyrosine phosphatase, receptor type, K, protein-tyrosine phosphatase kappa, protein-tyrosine phosphatase, receptor type, kappa, R-PTP-kappa, receptor-type tyrosine protein phosphatase kappa, EC 3.1.3.48, PTPK;GenBank:AAI44514.1);PSMA;PSCA hIg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, GenBank accession number AY358628);PSCA (prostate stem cell antigen precursor, GenBank accession number AJ29743);PRDX5 (peroxiredoxin 5, EC 1.11.1.15, TPx type VI, B166, antioxidant enzyme B166, HEL-S-55, liver tissue 2D-Page spot 71B, PMP20, peroxisomal antioxidant enzyme, PRDX6, thioredoxin peroxidase PMP20, PRXV, AOEB166, epididymal secretory protein Li 55, Alu corepressor 1, peroxiredoxin-5, mitochondrial, peroxiredoxin V, prx-V, thioredoxin reductase, Prx-V, ACR1, Alu corepressor, PLP; GenBank: CAG33484.1); PRAME (Preferentially Expressed Antigen in Melanoma, Preferentially Expressed Antigen in Melanoma, MAPE, 01P-4, OIPA, CT130, Cancer / Testis Antigen 130, Melanoma Antigen Preferentially Expressed in Tumors, Opa-Interacting Protein 4, Opa-Interacting Protein 01P4; GenBank: CAG30435.1); pml-RAR alpha fusion protein; PMEL17 (Silver Homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp10 BC001414;BT007202;M32295;M77348;NM-006928;PBF(ZNF395, zinc finger protein 395, PRF-1, Huntington's disease regulation, HD gene regulatory region binding protein, region binding protein 2, protein 2, papillomavirus regulatory factor 1, HD regulatory factor 2, papillomavirus regulatory factor, PRF1, HDBP-2, Si-1-8-14, HDBP2, Huntington's disease gene regulatory region binding protein 2, HDRF-2, papillomavirus regulatory factor PRF-1, PBF;GenBank:AAH01237.1); PAX5 (paired box 5, paired box homeotic gene 5, BSAP, paired box protein Pax-5, B cell lineage-specific activator; paired domain gene 5, paired box gene 5 (B cell lineage-specific activator protein), B cell-specific transcription factor, paired box gene 5 (B cell lineage-specific activator); PAP (REG3A, regenerating islet-derived 3 alpha, INGAP, PAP-H, hepatic enteropancreatic protein, PBBCGF, human proislet peptide, REG-Ill, pancreatitis-associated protein 1, Regi, Reg III-alpha, hepatocellular-intestinal-pancreatic, regenerating islet-derived protein III-alpha, pancreatic beta cell growth factor, HIP, PAP homologous protein, HIP / PAP, proliferation-inducing protein 34, PAP1, proliferation-inducing protein 42, REG-3-alpha, regenerating islet-derived protein 3-alpha, pancreatitis-associated protein; GenBank:AAH36776.1; p53 (TP53, tumor protein P53, TPR53, P53, cell tumor antigen P53, antigen NY-CO-13, mutated tumor protein 53, phosphoprotein P53, P53 tumor suppressor, B. CC7, transformation-associated protein 53, LFS1, tumor protein 53, Li-Fraumeni syndrome, tumor suppressor P53; P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel regulated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, and its deficiency may be involved in the pathophysiology of idiopathic detrusor instability); 422aa), μL:7.63, MW:47206 TM:1[P] gene chromosome:17p13.3, GenBank accession number NP-002552; OGT (O-linked N-acetylglucosamine (GlcNAc) transferase, O-GlcNAc transferase P110 subunit, O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyltransferase, UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110KDa subunit, UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyltransferase, uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase, O-GlcNAc transferase subunit P110, EC 2.4.1.255, 0-linked N-acetylglucosamine transferase 110 KDa subunit, EC 2.4.1, HRNT1, EC 2.4.1.186, 0-GLCNAC; GenBank:AAH38180.1); 0A1 (Osteoarthritis QTL1, OASD; GenBank:CAA88742.1); NY-ESO-1 / LAGE-2 (Cancer / Testis Antigen 1B, CTAG1 B, NY-ESO-1, LAGE-2, ESO1, CTAG1, CTAG, LAGE2B, Cancer / Testis Antigen 1, Autoimmune Cancer / Testis Antigen NY-ESO-1, Cancer (Ancer) Antigen 3, Cancer / Testis Antigen 6.1, New York Esophageal Squamous Cell Carcinoma 1, L Antigen Family Member 2, LAGE2, CT6.1, LAGE2A; GenBank:AAI30365.1); NY-BR-1 (ANKRD30A, ankyrin repeat domain 30A, breast cancer antigen NY-BR-1, serologically defined breast cancer antigen NY-BR-1, ankyrin repeat domain-containing protein 30A; NCBI reference sequence: NP-443723.2); N-ras (NRAS, neuroblastoma RAS viral (V-Ras) oncogene homolog, NRAS1, transforming protein N-Ras, GTPase NRas, ALPS4, N-Ras protein part 4; NS6, oncogene homolog, HRAS1; GenBank:AAH05219.1; NFYC (nuclear transcription factor Y, gamma; HAP5, HSM, nuclear transcription factor Y subunit C; transactivator HSM-1 / 2; CCAAT-binding factor subunit C; NF-YC, CCAAT transcription binding factor subunit gamma; CAAT box DNA-binding protein subunit C; histone H1 transcription factor large subunit 2A; CBFC, nuclear transcription factor Y subunit gamma; CBF-C, transactivator HSM-1; H1TF2A, transcription factor NF-Y, C subunit; neo-PAP (PAPOLG, poly(A) polymerase gamma; Neo-Poly(A) polymerase; nuclear poly(A) polymerase gamma; polynucleotide adenylyltransferase gamma; SRP RNA 3'adenylation enzyme / Pap2, PAP-gamma, Neo-PAP, SRP RNA 3'-adenylation enzyme, PAP2, EC 2.7.7.19, PAPG; NCBI reference sequence: NP-075045.2); NCA (CEACAM6, GenBank accession number M1872); Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, GenBank accession number NM-00642); myosin class I; MUM-3; MUM-2 (TRAPPC1, transport protein particle complex 1, BETS, BETS homolog, M UM2, melanoma ubiquitous mutated 2, multiple myeloma protein 2, transport protein particle complex subunit 1; MUM-1f; mucins (MUC1, mucin 1, cell surface associated; PEMT; PUM; CA15-3; MCKD1, ADMCKD, medullary cystic kidney disease 1 (autosomal dominant); ADMCKD1, mucin 1, transmembrane; CD227, breast cancer associated antigen DF3; MAM6, cancer antigen 15-3; MCD, cancer associated mucin; MCKD, Krebs Von Den Lungen-6, MUC-1 / SEC, peanut-reactive urinary mucin, MUC1 / ZD, tumor-associated epithelial membrane antigen, DF3 antigen, tumor-associated mucin, episialin, EMA, H23 antigen, H23AG, mucin-1, KL-6, tumor-associated epithelial mucin, MUC-1, episialin, PEM, CD227 antigen; UniProtKB / Swiss-Prot:P15941.3);MUCSAC (mucin SAC, oligomeric mucus / gel forming, tracheopulmonary mucin, MUC5, TBM, mucin 5, subtypes A and C, tracheobronchial / gastric, leB, gastric mucin, mucin SAC, oligomeric mucus / gel forming pseudogene, Lewis B blood group antigen, LeB, major airway glycoprotein, MUC-SAC, mucin-5 subtype AC, tracheobronchial;MUC1 (mucin 1, cell surface associated, PEMT, PUM, CA15-3, MCKD1, ADMCKD, medullary cystic kidney disease 1 (autosomal dominant), ADMCKD1, mucin 1, transmembrane, CD227, breast cancer associated antigen DF3, MAM6, cancer antigen 15-3, MCD, cancer associated mucin, MCKD, Krebs Von Den Lungen-6, MUC-1 / SEC, peanut-reactive urinary mucin, MUC-1 / X, polymorphic epithelial mucin, MUC1 / ZD, tumor-associated epithelial membrane antigen, DF3 antigen, tumor-associated mucin, episialin, EMA, h23 antigen, H23AG, mucin-1, KL-6, tumor-associated epithelial mucin, MUC-1, episialin, PEM, CD227 antigen; MSG783 (RNF124, hypothetical protein FLJ20315, GenBank accession number NM-01776; MRP4-multidrug Resistance-associated protein 4 isoform 3, MOAT-B; MOATB [Homo sapiens]; NCBI Reference Sequence: NP-001288758.1; MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, GenBank Accession Number NM-00582); MMP-7 (MMP7, matrilysin, MPSL1, matrine, matrix metalloproteinase 7 (matrilysin, uterine), uterine matrilysin, matrix metalloproteinase-7, EC 3.4.24.23, Pump-1 protease, matrine, uterine metalloproteinase, PUMP1, MMP-7, EC 3.4.24, PUMP-1; GenBank: AAC37543.1); MMP-2 (MMP2, matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kD type IV collagenase), MONA, CLG4A, matrix metalloproteinase 2 (gelatinase A, 72 kD gelatinase, 72 kD type IV collagenase), CLG4, 72 kDa gelatinase, 72 kD type IV collagenase), matrix metalloproteinase-2, MMP-II, 72 KDa gelatinase, collagenase type IV-A, MMP-2, matrix metalloproteinase-II, TBE-1, neutrophil gelatinase, EC 3.4.24.24, EC 3.4.24; GenBank: AAH02576.1); and Meloe. .

[0097] In some embodiments, the at least two different antigens may be selected from the following antigens (or the at least two different epitopes may be epitopes on any of the following antigens): 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, Anti-Id, Aspartic acid, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, Cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCLl, CCLll, CCL12, CCL13, CCL14, CCL15, CCL16, CCLl7, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDl lb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxinl, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, F ZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin Tegrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta l, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta l, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta l, integrin beta 2, interferon gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase Ze, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Mucl), MUC18, Muellerian inhibitor, Mug, MuSK, NAIP, NAP, NCA D, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV)F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, T fR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta l, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid Stimulating Hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB(TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3 DcRl, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R), TNFRSFllB(OPG OCI F、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55~60)、TNFRSFIB(TNF RII). CD120b, p75~80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LINK, LTg), TNFSF15(TL1A / VEGI), TNFSF18 (GITR LINK, AITR LINK, TL6), TNFSFIA(TNF-a diphtheria, DIF, TNFSF2, TNFSF1B(TNF-b LTa, TNFSF1), TNFSF3(LTb TNFC, p33, TNFSF4 (OX40p34, TXGP1), TNFSF5 (CD4). 0. CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6(FasApo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-lBB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, Transfer Receptor, TRF, Trk, TROP-2, TSG, TSLP, Tumor Associated Antigen CA125, Tumor Associated Antigen Expressing Lewis Y-Related Carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Viral Antigen, VLA, VLA-1, VLA-4, VNR Integrin, von Willebrand Factor, WIF-1, WNT1, WNT 2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), receptors for hormones, and growth factors.

[0098] In certain embodiments, a multispecific (e.g., bispecific) antibody according to the present disclosure may have a first antigen binding domain with specificity for CD3 and a second binding domain with specificity for a second antigen selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1 ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, aspartic acid, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, Cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCLl, CCLll, CCL12, CCL13, CCL14, CCL15, CCL16, CCLl7, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDl lb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl, CXCL2, CX CL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 , CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin (eptaxinl), EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, F ZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin Tegrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta l, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta l, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta l, integrin beta 2, interferon gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase Ze, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Mucl), MUC18, Muellerian inhibitor, Mug, MuSK, NAIP, NAP, NCA D, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV)F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, serine, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, T fR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta l, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid Stimulating Hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB(TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C(TRAIL R3 DcRl, LIT, TRID), TNFRSF10D(TRAIL R4 DcR2, TRUNDD), TNFRSF11A(RANK ODF R, TRANCE R) TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55~60)、TNFRSFIB(TNF RII). CD120b, p75~80, NFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM LINK, LTg), TNFSF15(TL1A / VEGI), TNFSF18 (GITR LINK, AITR LINK, TL6), TNFSFIA(TNF-a diphtheria, DIF, TNFSF2, TNFSF1B(TNF-b LTa, TNFSF1), TNFSF3(LTb TNFC, p33, TNFSF4(OX40functional gp34, TXGP1), TNFSF5(CD40 functional CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6(Fas Liquid Apo-1ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-lBB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, Transfer Receptor, TRF, Trk, TROP-2, TSG, TSLP, Tumor Associated Antigen CA125, Tumor Associated Antigen Expressing Lewis Y-Related Carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Viral Antigen, VLA, VLA-1, VLA-4, VNR Integrin, von Willebrand Factor, WIF-1, WNT1, WNT 2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), receptors for hormones, and growth factors.

[0099] In certain embodiments, combinations of antigens that can be targeted by bispecific (or multispecific) antibodies include CD3 and Her2, CD3 and Her3, CD3 and EGFR, CD3 and CD19, CD3 and CD20, CD3 and EpCAM, CD3 and CD33, CD3 and PSMA, CD3 and CEA, CD3 and gp100, CD3 and gpA33, CD3 and B7-H3, CD64 and EGFR, CEA and HSG, TRAIL-R2 and LTbetaR, EGFR and IGFR, VEGFR2, and VEGFR3, VEGFR2 and PDGFR alpha, PDGFR alpha and PDGFR beta, EGFR and MET, EGFR and EDV-miR16, EGFR and CD64, EGFR and Her2, EGFR and Her3, Her2 domain ECD2 and Her2 domain ECD4, Her2 and Her3, IGF-1R and HER3, CD19 and CD22, CD20 and CD22, CD30 and CD16A, FceRI and CD32B, CD32B and CD79B, BCMA and HEL These may include, but are not limited to, MP65 and SAP-2, IL-17A and IL-23, IL-1 alpha and IL-1 beta, IL-12 and IL-18, VEGF and osteopontin, VEGF and Ang-2, VEGF and PDGFR beta, VEGF and Her2, VEGF and DLL4, FAP and DR5, FcgRII and IgE, CEA and DTPA, CEA and IMP288, and LukS-PV and LukF-PV.

[0100] "Different antigens" can refer to different and / or distinct proteins, polypeptides, or molecules, as well as different and / or distinct epitopes that may be contained within one protein, polypeptide, or molecule. As a result, a bispecific antibody can bind to two epitopes on the same polypeptide.

[0101] The term "epitope" is used herein in the broadest sense to encompass the region of an antigen that interacts with a corresponding paratope. A protein or peptide epitope may include amino acid residues that directly interact with the paratope (e.g., via hydrogen bonds or hydrophobic interactions) and those that do not (e.g., residues that contribute generally to the conformation of the epitope). Epitopes may be defined as structural or functional. A functional epitope is generally an epitope that has residues that directly contribute to some function of the antigen (e.g., affinity for another protein or enzymatic activity). A structural epitope is an epitope that has residues that contribute to the antigen structure that may not contribute significantly to the antigen function. Epitopes may also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, an epitope may have specific three-dimensional structural characteristics and / or specific charge characteristics. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. A multispecific antibody may contain multiple antigen binding sites that bind to different epitopes of the same antigen. Bispecific antibodies that bind to different epitopes of the same antigen are referred to herein as "biparatopic" antibodies. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody.

[0102] In some cases, a full-sized antibody comprises four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chains each comprise a variable region, such as a heavy chain variable region ("VH") (also referred to as a heavy chain variable domain), and a heavy chain constant region ("CH"). In the case of an intact antibody, the CH comprises domains CH1, CH2, and CH3. In the case of an antibody fragment, the CH may comprise a CH1 domain, a CH2 domain, and / or a CH3 domain, and in some preferred embodiments, the CH comprises at least a CH1 domain. The variant CH3 domains disclosed herein may be used in combination with one or more wild-type CH2 and / or CH3 domains, or with a CH2 and / or CH3 domain that comprises one or more amino acid substitutions, e.g., amino acid substitutions that alter or improve the stability and / or effector function of the antibody. The light chains each comprise a variable region, such as a light chain variable region ("VL") (also referred to as a light chain variable domain), and a light chain constant region ("CL"). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each of the VH and VL comprises three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or an antigen-binding fragment thereof) may be identical to a human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a comparative analysis of two or more CDRs. Thus, the CDRs in the heavy chain are designated "CDRH1", "CDRH2", and "CDRH3", respectively, and the CDRs in the light chain are designated "CDRL1", "CDRL2", and "CDRL3", respectively. In other cases, the antibody may comprise a multimer thereof (e.g., IgM) or an antigen-binding fragment thereof.

[0103] Numbering of amino acid residues in antibody variable and constant domains may be performed according to the EU index or EU numbering system as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., (1991). Unless otherwise specified, the EU numbering system is used herein.

[0104] According to IMGT (International ImMunoGeneTics Information System for Immunoglobulins or Antibodies, T-cell Receptors, MH, Immunoglobulin Superfamilies IgSF, and MhSF), the CH1 domain is amino acid positions (or simply referred to herein as "positions") 118-215 (EU numbering), and the hinge region is amino acid positions 216-230 (EU numbering). The term "CH1 domain" is used broadly herein to refer to a heavy chain region that includes at least seven contiguous amino acid positions of heavy chain positions 118-215 (EU numbering), and in some cases also includes a portion of the hinge region (a portion of heavy chain positions 216-230 (EU numbering)) (e.g., up to position 218). The CH1 domain reference sequence corresponding to amino acid positions 118 to 220 according to EU numbering is provided herein as SEQ ID NO: 6, which corresponds to the CH1 domain sequence of human IgG1 allotypes "IGHG1*01 (J00228)," "IGHG1*04 (JN582178)," or "IGHG1*07," and is an exemplary amino acid sequence of a wild-type (WT) CH1 domain.

[0105] CH1 domain reference sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (positions 118 to 220 according to EU numbering) (SEQ ID NO: 6).

[0106] Alternative CH1 domain reference sequences for human IgG1 include, but are not limited to, SEQ ID NO: 5, which corresponds to the CH1 domain sequence of human IgG1 allotypes "IGHG1*03(Y14737)" or "IGHG1*08".

[0107] Alternative CH1 domain reference sequence (relative to SEQ ID NO:6) 214R ):ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK R VEPKSC (positions 118 to 220 according to EU numbering) (SEQ ID NO: 5).

[0108] These CH1 domain reference sequences are intended to be exemplary, as it is intended by applicants that the "CH1 domain" sequence includes any naturally occurring CH1 domain allotype or allelic variant.

[0109] According to IMGT, the CH2 domain is amino acid positions (or simply referred to herein as "positions") 231-340 (EU numbering). The term "CH2 domain" is used broadly herein to refer to a heavy chain region that includes at least seven consecutive amino acid positions of heavy chain positions 231-340 (EU numbering). A CH2 domain reference sequence corresponding to amino acid positions 231-340 according to EU numbering is provided herein as SEQ ID NO: 7, which is an exemplary amino acid sequence of a wild-type (WT) CH2 domain.

[0110] CH2 domain reference sequence: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 7).

[0111] Again, the listed CH2 domain reference sequences are intended to be exemplary, as it is intended by the applicant that the "CH2 domain" sequence includes any naturally occurring CH2 domain allotypes or allelic variants.

[0112] According to IMGT, the CH3 domain is amino acid position (or simply referred to herein as "position") 341-446 (EU numbering). The term "CH3 domain" is used broadly herein to refer to a heavy chain region comprising at least seven consecutive amino acid positions of heavy chain positions 341-446 (EU numbering). The CH3 domain reference sequence corresponding to amino acid positions 341-446 according to EU numbering is provided herein as SEQ ID NO: 1, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*01 (J00228)" or "IGHG1*08" and is an exemplary amino acid sequence of a wild-type (WT) CH3 domain.

[0113] CH3 domain reference sequence: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1).

[0114] Alternative CH3 domain reference sequences for alternative human IgG1 may include, but are not limited to, SEQ ID NO: 2, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*03(Y14737)", SEQ ID NO: 3, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*04(JN582178)", and SEQ ID NO: 4, which corresponds to the CH3 domain sequence of human IgG1 allotype "IGHG1*07".

[0115] Alternative CH3 domain reference sequences (relative to SEQ ID NO: 1) 356E and 358M ):GQPREPQVYTLPPSR E E MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2).

[0116] Alternative CH3 domain reference sequences (relative to SEQ ID NO: 1) 422I ):GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN I FSCSVMHEALHNHYTQKSLSLSPG (sequence number 3).

[0117] Alternative CH3 domain reference sequences (relative to SEQ ID NO: 1) 431G ):GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE G LHNHYTQKSLSLSPG (sequence number 4).

[0118] Further, CH3 domain reference sequences for human IgG2, IgG3, and IgG4 include, but are not limited to, SEQ ID NOs: 722, 723, and 724, respectively.

[0119] Human IgG2 CH3 domain reference sequence: GQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPM LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 722)

[0120] Human IgG3 CH3 domain reference sequence: GQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQGNIFSHMHEALHNRFTQKSLSLSPG (SEQ ID NO: 723)

[0121] Human IgG4 CH3 domain reference sequence: GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 724)

[0122] These CH3 domain reference sequences are intended to be exemplary, as it is intended by applicants that the "CH3 domain" sequence includes any naturally occurring CH3 domain allotype or allelic variant.

[0123] Thus, amino acid modifications in a variant CH3 domain polypeptide according to the present disclosure may be compared to and / or incorporated into any parent CH3 domain polypeptide, for example, but not limited to, a wild-type sequence such as SEQ ID NO:1, or any allelic variant such as SEQ ID NO:2, 3, or 4, or 722, 723, or 724.

[0124] There are two major CL isotypes, kappa ("k") and lambda ("λ"), and such CL domains are referred to herein as kappa CL domains ("CLκ" domains) and lambda CL domains ("CLλ" domains).

[0125] According to IMGT, the CLκ domain is amino acid positions 108-214 (EU numbering). The term "CLκ domain" is used broadly herein to refer to a light chain region comprising at least seven consecutive amino acid positions of the kappa light chain positions 108-214 (EU numbering). A CLκ domain reference sequence corresponding to amino acid positions 108-214 (EU numbering) is provided herein as SEQ ID NO:8, which is an exemplary amino acid sequence of a wild-type (WT) CLκ domain.

[0126] CLκ domain reference sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (positions 108 to 214 according to EU numbering) (SEQ ID NO: 8).

[0127] According to IMGT, the CLλ domain is amino acid positions 107-215 (EU numbering). The term "CLλ domain" is used broadly herein to refer to a light chain region that comprises at least seven consecutive amino acid positions of lambda light chain positions 107-215 (EU numbering). A CLλ domain reference sequence corresponding to amino acid positions 107-215 (EU numbering) is provided herein as SEQ ID NO: 9, which is an exemplary amino acid sequence of a wild-type (WT) CLλ domain.

[0128] CL lambda domain reference sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (positions 107 to 215 according to EU numbering) (SEQ ID NO: 9).

[0129] Various standard sequences of the constant domains of human IgG1, IgG2, IgG3, and IgG4 (corresponding to different allotypes) are known in the art and can be found, for example, in Vidarsson et al., Front Immunol. 2014 Oct 20;5:520 and U.S. Patent No. 9,150,663, the disclosures of which are incorporated herein by reference in their entirety. Again, these reference sequences are intended to be exemplary, as the human IgG1, IgG2, IgG3, and IgG4 sequences are intended by the applicant to include all naturally occurring human IgG1, IgG2, IgG3, and IgG4 allotypes.

[0130] As used herein, the term "cognate pair" or "cognate pairing" refers to a pair or association of two antibody chains (e.g., heavy and light chains) that each comprise a variable region (e.g., VH and VL, respectively) and whose combination of variable regions provides the intended binding specificity for a given epitope or antigen. As used herein, the term "non-cognate pair" or "non-cognate pairing" refers to a pair or association of two antibody chains (e.g., heavy and light chains) that each comprise a variable region (e.g., VH and VL, respectively) and whose combination of variable regions does not provide the intended binding specificity for a given epitope or antigen.

[0131] Provided herein are engineered variant CH3 domains that contain at least one amino acid substitution that prevents or reduces the formation of CH3-CH3 homodimers and preferentially forms CH-CH3 homodimers.

[0132] The term "CH3 domain set" or "CH3 set" is used interchangeably and refers to a combination of two CH3 domains. When a CH3 set contains two non-wild-type CH3 domains (i.e., two variant CH3 domains), such a CH3 set can also be referred to as a "variant CH3 domain set" (or a "CH3 domain variant set") or a "variant CH3 set" (or a "CH3 variant set"). A "CH3 set name" is given to each "CH3 set" based on the amino acid substitutions contained in the CH3 domains of the set. A set of substitutions contained in the CH3 domains of a set can be referred to as a "CH3 substitution set".

[0133] The "CH3 set names" used herein are named by the amino acid positions (according to EU numbering) substituted in the CH3 domain of each chain, with dashes separating the heavy chains. For example, the "W-SG" set has a W in the CH3 domain (position 366) of the first heavy chain (referred to as chain A in FIG. 19 and the appendix table), plus an S and a G in the CH3 domain (positions 366 and 407) of the second heavy chain (referred to as chain B in FIG. 19 and the appendix table). These two chains are referred to as chain A and chain B in FIG. 19 and the appendix table, but the chain names are interchangeable. For example, the "W-SG" set may have a W in the CH3 domain (position 366) of the second heavy chain (or "chain B"), plus an S and a G in the CH3 domain (positions 366 and 407) of the first heavy chain (or "chain A"). "(349 / 354)" and "(354 / 349)" refer to substitutions that allow for CH3 interdomain disulfide bonds. For example, "(349 / 354)" indicates the presence of a Y349C substitution in CH3 of chain A and a S354C substitution in CH3 of chain B. Similarly, "(354 / 349)" indicates the presence of a S354C substitution in CH3 of chain A and a Y349C substitution in CH3 of chain B. Thus, "W-SG(354 / 349)" means that an S354C substitution is present in CH3 with a "W" substation (position 366) and a Y349C substitution is present in CH3 with an "SG" substitution (positions 366 and 407).

[0134] As used herein, the term "variant CH3 domain" (also referred to as "variant CH3 domain polypeptide", "CH3 domain variant", or "CH3 domain variant polypeptide") is used interchangeably and refers to a CH3 domain having an amino acid sequence in which one or more amino acid substitutions are made to the CH3 domain sequence. The CH3 sequences in which amino acid substitutions are made include, but are not limited to, the reference CH3 domain sequence SEQ ID NO:1. In the library screened to identify the described variant CH3 domains, the nucleic acid sequence encoding SEQ ID NO:1 was mutated. As used herein, the term "Fab arm exchange" or "FAE" refers to the process in which a half molecule (i.e., one heavy and one light chain pair, also referred to as half antibody or half IgG if the antibody is IgG) of an Ig molecule (e.g., IgG, IgE, or IgD) recombines with another half molecule of another Ig molecule. It has been found that FAEs occur naturally in human IgG4 molecules and that FAEs can be mimicked in vitro by the addition of mild reducing agents (van der Neut Kolfschoten et al. Science. 2007 Sep 14;317(5844):1554-1557). Site-directed mutagenesis studies substituting IgG4 amino acid residues with their IgG1 counterparts identified that FAE in humans may be driven by residues S228 located in the IgG4 core hinge (van der Neut Kolfschoten et al. Science. 2007 Sep 14;317(5844):1554-1557, Labrijn et al. Nat Biotechnol. 2009 Aug;27(8):767-771.) and R409 located in the IgG4 CH3 domain (Labrijn et al. J Immunol. 2011 Sep 15;187(6):3238-46.).It was later discovered that an IgG1 CH3 domain in which position 409 is substituted with R and another in which position 405 is substituted with L preferentially pair with each other, and the use of such CH3s may be useful for the production of bispecific IgG1 molecules (Labrijn et al. Proc Natl Acad Sci US A. 2013 Mar 26;110(13):5145-50).

[0135] The term "controlled FAE" or "cFAE" as used herein refers to a FAE artificially promoted by a set of engineered CH3 domains that preferentially form heterodimers. cFAEs can be particularly useful for efficient production of bispecific antibodies. For example, if an antibody of interest comprises (a) a half antibody specific to epitope A comprising heavy chain A (comprising VH) and light chain A (comprising VL), and (b) a half antibody specific to epitope B comprising heavy chain B (comprising VH) and light chain B (comprising VL), (a) antibody A comprising two half antibodies specific to epitope A and (b) antibody B comprising two half antibodies specific to epitope B can be first produced. Then, antibody A and antibody B can be placed together under mild reducing conditions that allow reduction of disulfide bonds between the heavy chains, resulting in half antibody molecules. If heavy chain A contains an engineered CH3 domain A and heavy chain B contains an engineered CH3 domain B, and CH3 domain A and CH3 domain B preferentially form CH3-CH3 heterodimers, then upon removal of the mildly reducing conditions, cFAE will preferentially form heterodimers between heavy chain A and heavy chain B over heavy chain A and heavy chain B homodimers, resulting in the desired bispecific antibody being more than antibody A and antibody B.

[0136] The term "half molecule" or "half antibody" when referring to IgG, IgE, or IgD, which may also be referred to as "half IgG," "half IgE," or "half IgD," respectively, refers to one heavy chain and one light chain set of the referenced antibody.

[0137] "Preferentially" form heterodimers or "preferentially" form heterodimers when referring to CH3 domains means that the formation of heterodimers with different non-identical CH3 domains occurs more (i.e., more frequently or with higher probability) than the formation of homodimers with different identical CH3 domains. When referring to a set of two different CH3 domains (a first CH3 domain and a second CH3 domain), it means that more heterodimers (of the first CH3 domain and the second CH3 domain) are formed than homodimers (dimers of the first CH3 domain and dimers of the second CH3 domain). For example, when a first CH3 domain and a second CH3 domain different from the first CH3 domain are mixed, co-expressed, or co-provided in a ratio of approximately 1:1, the percentage of dimers formed between the first CH3 domain and the second CH3 domain among the CH3 dimers is greater than 50%. The CH-CH3 heterodimer % (e.g., also referred to as "heterodimer %" or "heterodimer %") or degree of heterodimerization may be quantified by any available assay, such as, but not limited to, AlphaLISA®, liquid chromatography mass spectrometry (LC-MS), ion exchange chromatography (IEX), or flow cytometry. The heterodimer % may be about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% when the CH3 domain comprises a set of CH3 substitutions disclosed herein. In some preferred embodiments, the heterodimer % may be about 70% or more. In some more preferred embodiments, the heterodimer % may be about 75% or more. In some more preferred embodiments, the heterodimer % may be about 80% or more. In some more preferred embodiments, the heterodimer % may be about 85% or greater. In some more preferred embodiments, the heterodimer % may be about 90% or greater. In some more preferred embodiments, the heterodimer % may be about 95% or greater.In some more preferred embodiments, the heterodimer % may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some more preferred embodiments, the heterodimer % may be about 100%.

[0138] The CH3 substitution set or CH3 sets, and / or antibodies comprising such CH3 sets, may be characterized by the degree of aggregation (e.g., the presence of multimers of full antibodies) and / or the amount of half antibodies (i.e., one CH3 per molecule or one heavy chain per molecule), both of which may be quantified, for example, by chromatography, such as size exclusion chromatography (SEC), or electrophoresis, such as SDS-PAGE; the melting temperature (Tm), which may be measured, for example, by differential scanning fluorimetry (DSF); the production yield in an appropriate cell type (e.g., HEK293 cells or yeast cells); the "pI", isoelectric point ("pI"); the level of interaction with multispecific reagents ("PSR"), which may be measured as in WO 2014 / 179363; see, e.g., Estep P, et al. MAbs. MAbs. 2015 The antibody may be further evaluated based on additional properties, such as, but not limited to, hydrophobic interaction of the antibody, which may be measured by hydrophobic interaction chromatography ("HIC"), as measured as described in May-Jun;7(3):553-561; solubility; production cost and / or time; stability; shelf life; in vivo half-life; and / or immunogenicity. Any of these or other properties may be used in addition to the heterodimer % value when evaluating a given variant CH3 domain set or CH3 set. Thus, a variant CH3 domain or CH3 set that results in a relatively low heterodimer % may be as ideal as another CH3 set with a relatively high heterodimer % value, if the variant CH3 domain or CH3 set provides a good profile for one or more properties. For example, a CH3 set that results in 80% heterodimers with 3% aggregation (3% of the expression product is a multimer of a full antibody) may be as ideal as a CH3 set that results in 90% heterodimers with 10% aggregation.

[0139] There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The constant domain according to the present disclosure can be of any antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. As used herein, the CH3 domain can be derived from the CH3 of an antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. The CH3 substitution according to the present disclosure can be made to any CH3 domain sequence, such as, but not limited to, the CH3 reference sequence SEQ ID NO:1. When a CH1 domain and / or a CH2 domain are used together with a variant CH3 domain of the present disclosure, the CH1 domain and / or CH2 domain can be derived from any antibody isotype, and the CH1 domain isotype and / or CH2 domain isotype do not necessarily have to be the same as the CH3 domain isotype.

[0140] "Library" is used herein to encompass any collection of biological material, such as nucleic acids, peptides, proteins, and their sequence information. For example, a "CH3 domain-encoding polynucleotide library" refers to a collection of polynucleotides encoding different CH3 domain polypeptides or their polynucleotide sequences, and a "CH3 domain polypeptide library" refers to a collection of different CH3 domain polypeptides or their amino acid sequences.

[0141] The term "linker" refers to a construct of variable length that connects two or more domains or portions of a polypeptide or connects two or more polypeptides. In some cases, linkers are used to impart flexibility, improved spatial organization, proximity, and the like, and in such cases may be referred to as flexible linkers. Exemplary linkers may include one or more amino acids, optionally 1-50 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the linker may be composed primarily or entirely of G, S, and / or A amino acid residues. In some embodiments, the linker may comprise an amino acid sequence that comprises or consists of an amino acid sequence selected from the group consisting of GGGGS (SEQ ID NO:715, which may also be referred to as "G4S"), GGGS (SEQ ID NO:716, which may also be referred to as "G3S"), GGGGGS (SEQ ID NO:717, which may also be referred to as "G5S"), G, GG, GGG, GS, SG, GGS (which may also be referred to as "G2S"), GSG, SGG, GSS, SGS, and SSG. In some embodiments, the linker may comprise an amino acid sequence that comprises or consists of multiple repeats (e.g., 2, 3, 4, 5, or more repeats) of an amino acid sequence selected from the group consisting of SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, G, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG. When the linker comprises or consists of multiple repeats of GG5S, G4S, G3S, G2S, GS, or G, the linker may optionally be referred to as a (G5S)n linker, (G4S)n linker, (G3S)n linker, (G2S)n linker, (GS)n linker, or (G)n linker, where n is a natural number, optionally selected from 1 to 20, e.g., 2, 3, 4, 5, etc. In certain embodiments, the linker may comprise two or three repeats of SEQ ID NO: 101, i.e., have the sequence GGGGSGGGGS (SEQ ID NO: 718) or GGGGGSGGGGSGGGGS (SEQ ID NO: 719), respectively, and optionally each of (G4S) 2 Linker or (G4S) 3 It may also be called a linker.

[0142] As used herein, "pharmaceutical carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral, intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the carrier can be a liquid in which the active therapeutic agent is formulated. Excipients generally do not provide any pharmacological activity to the formulation, but may provide chemical and / or biological stability, and release characteristics. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, Gennaro, A. editor, 19th edition, Philadelphia, PA: Williams and Wilkins (1995), incorporated by reference.

[0143] The term "scFv", "single-chain Fv", or "single-chain variable fragment" refers to a fusion protein comprising at least one heavy chain variable region (VH) and at least one light chain variable region (VL) of an antibody, where the VH and VL are linked contiguously, and the scFv retains the specificity of the antibody from which it is derived (the antibody from which the VH and VL are derived). Unless specified, as used herein, an scFv can have the VH and VL in any order, e.g., with respect to the N-terminus and C-terminus of the polypeptide. For example, the VH and VL may be linked via a linker, such as a synthetic and / or flexible polypeptide linker, and the scFv can be expressed as a single-chain polypeptide. When a linker connects the VH and VL, the scFv can include the structure VL-linker-VH or VH-linker-VL. When a linker connects the VH and VL, the linker is not limited to any of the linkers described herein, but may be any such suitable linker. In some cases, the VH and VL are additionally or alternatively connected by one or more disulfide bonds. In certain cases, the VH and / or VL sequences may be modified (e.g., one or more amino acids may be substituted) to include cysteine ​​residues that allow for such disulfide bonds (see, e.g., Weatherill et al., Protein Eng Des Sel. 2012 Jul;25(7):321-9).

[0144] "Conservative amino acid substitutions" are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic / negatively charged polar amino acid for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain for another nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic / positively charged polar amino acid for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain for another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain for another β-branched side chain (e.g., Ile, Thr, and Val), an amino acid with an aromatic side chain for another aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0145] Variant CH3 domains and CH3 domain sets As described herein, it has been discovered that certain positions within the CH3 domain, as well as certain amino acid substitutions and sets of substitutions, affect CH3 domain pairing or the formation of CH3-CH3 dimers (or Fc heterodimers).

[0146] In some embodiments, a variant CH3 domain described herein may comprise an amino acid substitution at one or more of the following amino acid positions according to EU numbering: 364, 366, 368, 370, 399, 400, 405, 407, and 409. In some embodiments, a variant CH3 domain described herein may comprise an amino acid substitution at any of the positions listed in Table 7, or any combination of positions listed in Table 7.

[0147] The parent CH3 domain into which such amino acid substitutions may be incorporated may comprise a wild-type or naturally occurring CH3 domain sequence, or a variant or engineered version thereof. Exemplary sequences of such parent polypeptides include, but are not limited to, the reference CH3 sequence SEQ ID NO: 1, which corresponds to amino acid positions 341-446 according to EU numbering.

[0148] The resulting variant CH3 domain preferentially forms CH3-CH3 heterodimers over CH3-CH3 homodimers. Such variant CH3 domains may be useful in producing heterodimeric (or multimeric) polypeptides and molecules containing such polypeptides. Such variant CH3 domains may be useful in producing multispecific antibodies and antibody fragments by improving the fidelity of heterologous Fc pairing while maintaining the native IgG structure of bispecific antibodies, which are preferred due to their well-established properties as therapeutic molecules, including long in vivo half-life and ability to elicit effector functions. These domain CH3 variants can be used to fully or partially resolve chain mispairing in producing multispecific antibodies, e.g., bispecific antibodies, by promoting proper heavy chain-heavy chain pairing. More specifically, multispecific antibodies containing these variant CH3 domains form fewer undesired product-related contaminants, i.e., molecules containing mispaired domains or chains, which may be difficult to eliminate during manufacturing.

[0149] In some embodiments, the amino acid substitutions in the variant CH3 domain are at: (i) 366, (ii) 368, (iii) 407, (iv) 366 and 407, (v) 366 and 368, (vi) 366 and 409, (vii) 368 and 370, (viii) 368 and 407, (ix) 399 and 405, (x) 400 and 409, (xi) 364, 366, and 409, (xii) 364, 407, and 409, (xiii) 366, 368, and 370, (xiv) 366, 368, and 407, (xv) 366, 399, and 405, (xvi (xvii) 366, 400, and 409; (xviii) 368, 400, and 409; (xix) 399, 405, and 407; (xx) 400, 407, and 409; (xxi) 366, 399, 405, and 407; (xxii) 366, 399, 405, and 409; (xxiii) 366, 400, 407, and 409; (xxiv) 366, 368, 399, 405, and 407; or (xxv) 366, 368, 400, 407, and 409 amino acid substitutions.

[0150] In some embodiments, the variant CH3 domain may further comprise a Y349C or S354C substitution, which allows disulfide formation with another variant CH3 domain that contains a S354C or Y349C substitution, respectively. In some embodiments, the variant CH3 domain may comprise one or more of the following amino acid substitutions: S364D; S364L; T366Q; T366R; T366S; T366V; T366W; L368A; L368F; L368S; L368I; K370G; K370Y; D399Q; S400T; F405L; Y407V; Y407G; K409R; K409L; and / or K409G. In some embodiments, the variant CH3 domain may optionally further comprise Y349C or S354C.

[0151] In some embodiments, the amino acid substitutions in the variant CH3 domain are the following substitution combinations: T366W; T366S and Y407G; T366V; Y407V; T366Q and K409R; L368F; T366R and K409G; L368F and K370G; S400T and K409L; D399Q and F405L; S364D, Y407V, and K409G; T366V, L368S, and K370Y; S364L, T366W, and K409G; T366S, L368I, and Y407G; T366W, S400T, and K409L; T366S, L368A, Y407V, D399Q, and F405L; T366W, S400T, and K409L; T366S, Y407G, D399Q, and F40 5L;T366W, D399Q, and F405L;T366S, L368A, Y407V, S400T, and K409L;T366W, D399Q, and F405L;T366S, Y407G, S400T, and K409L;Y407V, S400T, and K409L;T366V, D399Q, and F405L;Y407V, D399Q, and F405L; It may comprise or consist of any one of: T366V, S400T, and K409L; T366Q, K409R, D399Q, and F405L; L368F, S400T, and K409L; Y407V, T366Q, and K409R; T366V and L368F; S364L, T366W, and K409G; or L368I and Y407G. In some embodiments, the amino acid substitutions in the variant CH3 domain may comprise or consist of any one of the following substitution combinations: T366W; T366S and Y407G; T366V; Y407V; T366Q and K409R; L368F; T366R and K409G; L368F and K370G; S400T and K409L; D399Q and F405L; S364D, Y407V, and K409G; T366V, L368S, and K370Y; S364L, T366W, and K409G; T366S, L368I, and Y407G; or L368I and Y407G. In some embodiments, an S354C or Y349C substitution may be further added to any of the substitution combinations.

[0152] In some embodiments, these substitutions are made relative to the reference CH3 domain sequence of SEQ ID NO: 1. In such cases, the amino acid sequence of a variant CH3 domain according to the present disclosure may comprise or consist of the sequence in any one of SEQ ID NOs: 11-16, 21-26, 31-36, 41-46, 51-56, 61-66, 71-76, 81-86, 91-96, 101-106, 111-116, 121-126, 131-136, 141-146, 151-156, and 161-166. In some embodiments, a variant CH3 domain according to the present disclosure may comprise or consist of the sequence in any one of SEQ ID NOs: 11-16, 21-26, 31-36, 41-46, 51-56, 61-66, 71-76, and 161-166. In some preferred embodiments, the variant CH3 domain may comprise an amino acid sequence according to any one of SEQ ID NOs: 11-16 and 71-76.

[0153] In some embodiments, a variant CH3 domain according to the present disclosure may be paired with or form a heterodimer with another variant CH3 domain according to the CH3 domains disclosed herein.

[0154] The variant CH3 domain sets according to the present disclosure that preferentially form CH3-CH3 heterodimers are not identical to those previously identified as heterodimerization-preferred CH3 domain sets, such as existing CH3 technologies listed in Table 1. However, any of the inventive CH3 substitution sets described herein may be combined with existing CH3 technologies, such as those in Table 1. [Table 1]

[0155] In some embodiments, such CH3 heterodimers may comprise any of the following CH3 sets: W-SG, VV, QR-F, RG-FG, TL-QL, DVG-VSY, LWG-SIG, WTL-SAVQL, WTL-SGQL, WQL-SAVTL, WQL-SGTL, VTL-VQL, VQL-VTL, QRQL-FTL, VQR-VF, or LWG-IG. In some embodiments, such CH3 heterodimers may comprise any of the following CH3 sets: W-SG, VV, QR-F, RG-FG, TL-QL, DVG-VSY, LWG-SIG, or LWG-IG. In some embodiments, the CH3 sets may be further supplemented with CH3 disulfide bondable substitutions ("354 / 349" or "349 / 354" substitutions). In such embodiments, such CH3 heterodimers are selected from the following CH3 sets: W-SG(349 / 354), VV(349 / 354), QR-F(349 / 354), RG-FG(349 / 354), TL-QL(349 / 354), DVG-VSY(349 / 354), LWG-SIG(349 / 354), WTL-SA VQL(349 / 354), WTL-SGQL(349 / 354), WQL-SAVTL(349 / 354), WQL-SGTL(349 / 354), VTL- VQL(349 / 354), VQL-VTL(349 / 354), QRQL-FTL(349 / 354), VQR-VF(349 / 354), or LWG-IG (349 / 354), or W-SG(354 / 349), VV(354 / 349), QR-F(354 / 349), RG-FG(354 / 349), TL-QL (354 / 349), DVG-VSY(354 / 349), LWG-SIG(354 / 349), WTL-SAVQL(354 / 349), WTL-SGQL( 354 / 349), WQL-SAVTL(354 / 349), WQL-SGTL(354 / 349), VTL-VQL(354 / 349), VQL-VTL(354 / 349), QRQL-FTL(354 / 349), VQR-VF(354 / 349), or LWG-IG(354 / 349).In some embodiments, such CH3 heterodimers can include any of the following CH3 sets: W-SG(349 / 354), VV(349 / 354), QR-F(349 / 354), RG-FG(349 / 354), TL-QL(349 / 354), DVG-VSY(349 / 354), LWG-SIG(349 / 354), LWG-IG(349 / 354), W-SG(354 / 349), VV(354 / 349), QR-F(354 / 349), RG-FG(354 / 349), TL-QL(354 / 349), DVG-VSY(354 / 349), LWG-SIG(354 / 349), or LWG-IG(354 / 349). Details of the amino acid positions and residues of the substitutions in these sets are shown in Appendix Tables E-G.

[0156] In some preferred embodiments, the CH3 set according to the present disclosure may be W-SG, LWG-SIG, W-SG(349 / 354), LWG-SIG(349 / 354), W-SG(354 / 349), or LWG-IG(354 / 349).

[0157] In further embodiments, any of the substitution sets described herein can be combined with another CH3 heterodimerization-preferring CH3 substitution or substitution set, such as any one of the inventive CH3 substitution or substitution sets described herein, or any one of the existing CH3 heterodimerization-preferring substitutions or substitution sets, such as those listed in Table 1, to further enhance or promote CH3 heterodimerization.

[0158] Furthermore, for each of the specific amino acid substitutions in the CH3 domains provided herein for CH3 heterodimerization preference, the resulting amino acids can be further replaced by conservative amino acid substitutions to obtain another variant CH3 domain that provides the same preference for CH3 heterodimerization. Alternatively, for each of the variant CH3 domains, one or more amino acid positions that did not affect the variant CH3 domain compared to the wild-type sequence can be modified by conservative substitutions to obtain another variant CH3 domain that provides the same preference for CH3 heterodimerization.

[0159] Provided below is a summary of some of the CH3 sets that have been identified as shown in the Examples and that provide at least one superior property, such as higher heterodimerization, than existing CH3 heterodimerization variant CH3 domain sets. For example, as shown in the Examples, when expressed as a "modified Fc" on yeast cells, sets (1)-(7) all exhibit superior heterodimerization as measured by flow cytometry. Some additional superior properties (non-inclusive) of each of (1)-(7) are also provided below.

[0160] (1) “W-SG” set with or without CH3 disulfide substitution The "W-SG" set contains T366W in one CH3 domain and T366S and Y407G in the other CH3 domain.

[0161] For example, the "W-SG" set shows higher heterodimer% values ​​than the controls tested (EW-RVT and KiH) as measured by AlphaLISA® (see FIG. 11B). The "W-SG" set with CH3 disulfide substitution further shows much higher heterodimer% values ​​(100%) than the controls tested (EW-RVT and KiH with CH3 disulfide substitution) as measured by LC-MS when expressed as BsAbs in HEK cells (see Table 13). Furthermore, the "W-SG" set with and without CH3 disulfide substitution shows less aggregation compared to the respective control existing technology tested (EW-RVT and KiH with and without CH3 disulfide substitution) as measured by SEC (see Table 6). In addition, the "W-SG" set with CH3 disulfide substitutions showed higher yields than the existing technologies tested (EW-RVT and KiH with CH3 disulfide substitutions) (see Table 6).

[0162] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 11 and 12, respectively, the amino acid sequences of SEQ ID NOs: 13 and 14, respectively, or the amino acid sequences of SEQ ID NOs: 15 and 16, respectively.

[0163] (2) “VV” set with or without CH3 disulfide substitution The "VV" set contains T366V in one CH3 domain and Y407V in the other CH3 domain.

[0164] For example, the "VV" set exhibits higher % heterodimer values ​​than the existing technologies tested (EW-RVT and KiH) as measured by AlphaLISA® (see FIG. 11B). The "VV" set also exhibits higher yields than the controls tested (EW-RVT and KiH) when produced in HEK293 cells (see Table 6). Furthermore, the "VV" set with and without CH3 disulfide substitutions exhibits less aggregation compared to the respective control existing technologies tested (EW-RVT and KiH, respectively, with and without CH3 disulfide substitutions) as measured by SEC (see Table 6). In addition, the "VV" set provides less aggregation and much higher yields (207 mg / L) than the existing technologies tested (EW-RVT and KiH) as measured by SEC when expressed as BsAbs in HEK293 cells (see Table 13 and FIG. 18F).

[0165] Furthermore, when bsAbs containing the "VV" set were generated using the cFAE-mediated production method, 100% of the products were the intended bsAbs and no mismatches were observed (see Table 18), indicating that the "VV" set is superior to the existing "RL" set in terms of bsAb production efficiency. In addition, it was further found that bsAbs containing the "VV" set were resistant to glutathione exposure (see Figures 28A-28E).

[0166] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO:1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs:21 and 22, respectively, the amino acid sequences of SEQ ID NOs:23 and 24, respectively, or the amino acid sequences of SEQ ID NOs:25 and 26, respectively.

[0167] (3) “QR-F” set with and without CH3 disulfide substitution The "QR-F" set contains T366Q and K409R, as well as L368F in one CH3 domain.

[0168] For example, the "QR-F" set, when expressed as a modified Fc, shows (i) a higher heterodimer % value (100%) as measured by LC-MS, and (ii) a higher Tm (64°C) than the existing technology (KiH) tested (see Table 8). The "QR-F" with CH3 disulfide substitution, when expressed as a BsAb in HEK293 cells, shows a much higher heterodimer % (100%) as measured by LC-MS than the KiH control (see Table 10). In addition, the "QR-F" set with or without CH3 disulfide substitution provides less aggregation as measured by SEC than the relevant existing technology controls tested (EW-RVT and KiH with CH3 disulfide substitution) (see Table 13 and Figure 18D).

[0169] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO:1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs:31 and 32, respectively, the amino acid sequences of SEQ ID NOs:33 and 34, respectively, or the amino acid sequences of SEQ ID NOs:35 and 36, respectively.

[0170] (4) “RG-FG” set with or without CH3 disulfide substitution The "RG-FG" set contains T366R and K409G, as well as L368F and K370G in one CH3 domain.

[0171] For example, the "RG-FG" set, when expressed as a modified Fc, exhibits (i) a higher % heterodimer value (90%) as measured by LC-MS, and (ii) a higher Tm (64 degrees Celsius) than the existing technology (KiH) tested (see Table 8). The "RG-FG" set with CH3 disulfide substitution, when expressed as a BsAb in HEK cells, further exhibits a much higher % heterodimer value (100%) as measured by LC-MS than the controls tested (EW-RVT and KiH with CH3 disulfide substitution) (see Table 13).

[0172] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO:1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs:41 and 42, respectively, the amino acid sequences of SEQ ID NOs:43 and 44, respectively, or the amino acid sequences of SEQ ID NOs:45 and 46, respectively.

[0173] (5) “TL-QL” set with or without CH3 disulfide substitution The "TL-QL" set contains S400T and K409L, as well as D399Q and F405L in one CH3 domain.

[0174] For example, the "TL-QL" set, when expressed as a modified Fc, exhibits a higher Tm (65 degrees Celsius) than the existing technology tested (KiH) (see Table 8).

[0175] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO:1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs:51 and 52, respectively, the amino acid sequences of SEQ ID NOs:53 and 54, respectively, or the amino acid sequences of SEQ ID NOs:55 and 56, respectively.

[0176] (6) “DVG-VSY” set with or without CH3 disulfide substitution The "DVG-VSY" set contains S364D, Y407V, and K409G, as well as T366V, L368S, and K370Y in one CH3 domain.

[0177] For example, the "DVG-VSY" set with CH3 disulfide substitutions, when expressed as BsAbs in HEK293 cells, shows less aggregation as measured by SEC than the existing technology tested (KiH) (see Table 11). In addition, the "DVG-VSY" set with CH3 disulfide substitutions provides less aggregation as measured by SEC than the relevant existing technology controls tested (EW-RVT and KiH with CH3 disulfide substitutions) (see Table 13 and Figure 18D).

[0178] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 61 and 62, respectively, the amino acid sequences of SEQ ID NOs: 63 and 64, respectively, or the amino acid sequences of SEQ ID NOs: 65 and 66, respectively.

[0179] (7) “LWG-SIG” set with or without CH3 disulfide substitution The "LWG-SIG" set contains S364L, T366W, and K409G in one CH3 domain of the set, and T366S, L368I, and Y407G in the other CH3 domain of the set.

[0180] For example, the "LWG-SIG" set, when expressed as a modified Fc, exhibits a higher Tm (62.5 degrees Celsius) than the existing technology tested (KiH) (see Table 8). Furthermore, the "LWG-SIG" set, with or without CH3 disulfide bond substitution, when expressed as an anti-CD3 / anti-HER2 BsAb, exhibits a higher % heterodimer as measured by LC-MS or IEX than the existing technology tested (KiH) (see Figure 18C and Table 13).

[0181] When such substitutions are made to the reference CH3 domain sequence of SEQ ID NO: 1, the set of variant CH3 domain sequences includes the amino acid sequences of SEQ ID NOs: 71 and 72, respectively, the amino acid sequences of SEQ ID NOs: 73 and 74, respectively, or the amino acid sequences of SEQ ID NOs: 75 and 76, respectively.

[0182] Any of the variant CH3 domains described above or herein may be part of a polypeptide, such as a heavy chain polypeptide. Such polypeptides, such as heavy chain polypeptides, are also encompassed by the present invention.

[0183] Polypeptides, molecules, and multispecific antibodies A variant CH3 domain according to the present disclosure may be present within a polypeptide such as an immunoglobulin polypeptide, molecule, and / or a multispecific antibody.

[0184] As used herein, an "immunoglobulin polypeptide" refers to a polypeptide that includes at least one domain of an immunoglobulin (e.g., a CH3 domain). In certain cases, a first CH3 domain can be present in a first polypeptide. In certain cases, a second CH3 domain can be present in a second polypeptide. If the first CH3 domain and the second CH3 domain are CH3 domains that preferentially form CH3-CH3 heterodimers, a heteromeric (e.g., dimeric) molecule can be formed between the first and second polypeptides. Such heteromeric molecules can be multispecific antibodies having structures such as, but not limited to, those disclosed in Figures 2-8.

[0185] In some embodiments, such a heterodimer-preferred CH3 domain set can be any one of the following: W-SG, VV, QR-F, RG-FG, TL-QL, DVG-VSY, LWG-SIG, WTL-SAVQL, WTL-SGQL, WQL-SAVTL, WQL-SGTL, VTL-VQL, VQL-VTL, QRQL-FTL, VQR-VF, or LWG-IG. In some embodiments, such a heterodimer-preferred CH3 domain set can be any one of the following: W-SG, VV, QR-F, RG-FG, TL-QL, DVG-VSY, LWG-SIG, or LWG-IG. In some embodiments, such heterodimer-preferred CH3 domain sets include those with CH3 disulfide bond substitutions ("345 / 354" or "354 / 345"), i.e., W-SG(349 / 354), VV(349 / 354), QR-F(349 / 354), RG-FG(349 / 354), TL-QL(349 / 354), DVG-VSY ... 9 / 354), LWG-SIG(349 / 354), WTL-SAVQL(349 / 354), WTL-SGQL(349 / 354), WQL-SAVTL(349 / 354 ), WQL-SGTL(349 / 354), VTL-VQL(349 / 354), VQL-VTL(349 / 354), QRQL-FTL(349 / 354), VQR-VF( 349 / 354), or LWG-IG(349 / 354), or W-SG(354 / 349), VV(354 / 349), QR-F(354 / 349), RG-FG(354 / 349), TL-QL(354 / 349), DVG-VSY(354 / 349), LWG-SIG(354 / 349), WTL-SAVQL(354 / 349), WTL- It may be any of the foregoing, further including SGQL(354 / 349), WQL-SAVTL(354 / 349), WQL-SGTL(354 / 349), VTL-VQL(354 / 349), VQL-VTL(354 / 349), QRQL-FTL(354 / 349), VQR-VF(354 / 349), or LWG-IG(354 / 349).In some embodiments, such CH3 heterodimers can include any of the following CH3 sets: W-SG(349 / 354), VV(349 / 354), QR-F(349 / 354), RG-FG(349 / 354), TL-QL(349 / 354), DVG-VSY(349 / 354), LWG-SIG(349 / 354), LWG-IG(349 / 354), W-SG(354 / 349), VV(354 / 349), QR-F(354 / 349), RG-FG(354 / 349), TL-QL(354 / 349), DVG-VSY(354 / 349), LWG-SIG(354 / 349), or LWG-IG(354 / 349). The amino acid substitution positions in these CH3 sets are specified in Appendix Tables E–G).

[0186] Such immunoglobulin polypeptides may further comprise one or more antigen binding domains (such as a VH, VL, scFv, or nanobody), a CH1 domain, and / or a CH2 domain. An additional CH3 domain (with or without amino acid substitutions) may further be included. Such polypeptides may be part of a multispecific antibody molecule. In some embodiments, the peptide may comprise an antigen binding domain (such as a VH, VL, scFv, or nanobody) and a variant CH3 domain. In some embodiments, the peptide may comprise an antigen binding domain (such as a VH, VL, scFv, or nanobody), a CH1 domain, and a variant CH3 domain. In some embodiments, the peptide may comprise an antigen binding domain (such as a VH, VL, scFv, or nanobody), a CH2 domain, and a variant CH3 domain. In some embodiments, the peptide may comprise an antigen binding domain (such as a VH, VL, scFv, or nanobody), a CH1 domain, a CH2 domain, and a variant CH3 domain.

[0187] Alternatively, an immunoglobulin polypeptide may not include a VH, VL, CH1, or CH2 domain. For example, a first polypeptide may further include a first domain in addition to a first CH3. If a second polypeptide further includes a second domain in addition to a second CH3 that preferentially forms a heterodimer with the first CH3, and it is desired to form a heterodimer between the first domain and the second domain, the preferential heterodimerization between the first CH3 domain and the second CH3 domain promotes heterodimerization between the first domain and the second domain.

[0188] In one embodiment, such polypeptides can optionally utilize other variants outside the CH3 domain in combination with the variant CH3 domain to further promote preferential pairing between two polypeptides that differ from each other.

[0189] In one embodiment, such polypeptides may optionally utilize a variant CH1 domain in combination with a variant CH3 domain that promotes preferential lambda pairing or preferential kappa pairing. In another embodiment, such polypeptides may optionally utilize a variant CH1 domain in combination with a variant CH3 domain that preferentially pairs with a variant kappa CL domain over another CL domain, such as a wild-type kappa CL domain. In another embodiment, such polypeptides may optionally utilize a variant CH1 domain in combination with a variant CH3 domain that preferentially pairs with a variant lambda CL domain over another CL domain, such as a wild-type lambda CL domain. In yet another embodiment, such polypeptides may optionally utilize a variant kappa or lambda CL domain in combination with a variant CH3 domain that preferentially pairs with a variant CH1 domain over another CH1 domain, such as a wild-type CH1 domain. Such polypeptides may be used to facilitate the generation of antibodies specific for three or more antibodies (e.g., tetraspecific antibodies).

[0190] Any such polypeptide can be present in a molecule having a first polypeptide comprising a first variant CH3 domain and a second polypeptide comprising a second variant CH3 domain that preferentially forms a CH3-CH3 heterodimer with the first CH3. In some cases, the first and second polypeptides can be further linked, for example, via one or more disulfide bonds, linkers, etc.

[0191] Such molecules can be multispecific antibodies or antigen-binding fragments having structures such as, but not limited to, those disclosed in Figures 2-8. Multispecific antibodies according to the present disclosure can be bispecific, trispecific, tetraspecific, or specific for five, six, or more epitopes. Multispecific antibodies according to the present disclosure can be bivalent, trivalent, or tetravalent, or have five, six, or more valencies.

[0192] Polynucleotides, Vectors, Cells, and Compositions The polypeptides, molecules, and / or multispecific antibodies comprising the variant CH3 domains described herein may be encoded by a polynucleotide. Such a polynucleotide may be DNA or RNA, or a combination thereof.

[0193] Any of the polypeptides described herein can be present in a vector.

[0194] Any CH3 domain, polypeptide, molecule, multispecific antibody, polynucleotide, and / or vector may be present in a cell, e.g., a eukaryotic cell. In some embodiments, such polypeptides may be expressed in mammalian cells, such as HEK293 cells or Chinese Hamster Ovary (CHO) cells. In some embodiments, the variant CH3 domain is expressed in yeast (e.g., Saccharomyces cerevisiae). In some embodiments, the yeast strain co-expresses one or more polypeptides, such as one or more light chains.

[0195] Any CH3 domain, polypeptide, molecule, multispecific antibody, polynucleotide, vector, and / or cell may be present in a composition. If the composition is a therapeutic composition, the composition may further comprise a pharma- ceutically acceptable carrier.

[0196] CH3 domain library and variant CH3 domain screening / selection Methods for generating CH3 domain libraries are also contemplated by the present disclosure, which can be used to screen for CH3 sequences and sets that preferentially form CH3 heterodimers.

[0197] In some embodiments, at least one nucleic acid position within a codon encoding any of the amino acid positions of a CH3 where an amino acid substitution is present in any of the invention CH3 sets may be mutated. For example, such predetermined amino acid positions may be 364, 366, 368, 370, 399, 400, 405, 407, and / or 409 according to EU numbering, or any combination thereof.

[0198] In some embodiments, any amino acid position listed in Table 7 can be varied.

[0199] In some embodiments, any of the amino acid positions considered to be CH3-CH3 "interface positions" may also be varied.

[0200] In certain embodiments, some of the CH3 domains expressed by the library may contain CH3 disulfide bond substitutions (i.e., S354C / Y349C) in addition to the substitutions caused by mutating.

[0201] In some embodiments, degenerate codons, optionally degenerate RMW codons representing the six naturally occurring amino acids (D, T, A, E, K, and N), or degenerate NNK codons representing all 20 naturally occurring amino acid residues, may be used to induce variability at a given position.

[0202] Also provided herein are methods for identifying one or more variant CH3 domains and CH3 sets that preferentially form CH3 heterodimers.

[0203] In some embodiments, the method may include at least three steps. The first step may be co-expressing in a cell (e.g., yeast cell, mammalian cell) or ex vivo (1) a first polypeptide comprising a first variant CH3 domain expressed from a first library according to any of the libraries described herein, and (2) a second polypeptide comprising a second variant CH3 domain expressed from a second library according to any of the libraries described herein. The second step may be quantifying the amount of CH3 heterodimers and CH3 homodimers. The third step may be selecting one or more CH3 sets that provide the desired heterodimer %.

[0204] In certain embodiments, the first library and the second library may differ at at least one predefined amino acid position.

[0205] In some embodiments, the predetermined position in the first library and the predetermined position in the second library may comprise or consist of any of the positions or sets of positions substituted in the CH3 sets identified herein as preferential heterodimerization.

[0206] The variability can be performed on any available CH3 sequence, i.e. wild type or modified CH3 sequence. In some embodiments, the variability can be performed on the reference CH3 sequence of SEQ ID NO:1.

[0207] In certain embodiments, the desired heterodimer % can be greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or about 100%.

[0208] In certain embodiments, the desired % heterodimer can be compared to a reference CH3 set, for example, an existing CH3 heterodimerization technology (e.g., Table 1).

[0209] In some embodiments, the first polypeptide may comprise or be expressed with a first tag and the second polypeptide may comprise or be expressed with a second tag that is different from the first tag, which may allow specific identification of CH3 heterodimers by techniques such as AlphaLISA®.

[0210] In some embodiments, the second step of quantifying heterodimers and homodimers may use, for example, liquid chromatography-mass spectrometry (LC-MS), AlphaLISA®, ion exchange chromatography (IEX), and / or flow cytometry.

[0211] In certain embodiments, the identification method may further comprise selecting one or more sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides based on one or more antibody properties. Exemplary properties include (i) (i-1) production yield, optionally assessed in one or more cell types, optionally mammalian cells, e.g., CHO and HEK cells, yeast cells, insect cells, and / or plant cells, and / or (i-2) suitability for one or more antibody purification methods, optionally including Protein A affinity purification, (ii) degree of aggregation, optionally quantified using chromatography, optionally SEC or electrophoresis, optionally SDS-PAGE, and optionally the presence of multimers of full-sized antibodies, (iii) percentage of correct pairing, optionally correct pairing between CH1 domains and / or between CH1 domain and CL domain, optionally assessed using LC-MS, (iv) optional DS-PAGE, and (v) percentage of correct pairing between CH1 domain and / or between CH1 domain and CL domain, optionally assessed using LC-MS, and (vi) optional DS-PAGE, and optionally the presence of ... These may include, but are not limited to, Tm and / or Tagg, optionally Tagg266, measured using F and / or DSC and / or instrumentally, optionally using Uncle®, (v) pI, (vi) level of interaction with PSR, (vii) hydrophobic interactions of the antibody, optionally measured using HIC, (viii) self-interactions, optionally measured by (viii-1) AC-SINS or (viii-2) DLS, (ix) stability against high or low pH stress, (x) solubility, (xi) production costs and / or time, (xii) other stability parameters, (xiii) shelf life, (xiv) in vivo half-life, and / or (xv) immunogenicity.

[0212] Such properties may depend, at least in part, on (a) the particular structure of the molecule or multispecific antibody or antigen-binding antibody fragment structure incorporating the variant CH3 domain set, and / or (b) the variable domains that provide the particular binding specificity. Suitability may be tested in the particular context of the antibody structure and antigen specificity of interest.

[0213] Thus, also provided herein are methods of screening for a set of first and second variant CH3 domain polypeptides suitable for a multispecific antibody or antigen-binding antibody fragment with a given antigen specificity (e.g., having any of the structures described herein).

[0214] In some embodiments, the method comprises: (a) expressing a plurality of multispecific antibodies and / or antigen-binding antibody fragments comprising different sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides; and (b) selecting one or more sets of first variant CH3 domain polypeptides and second variant CH3 domain polypeptides based on one or more antibody properties of the plurality of multispecific antibodies and / or antigen-binding antibody fragments expressed in step (a).

[0215] In some embodiments, one or more antibody properties may be selected from properties (i)-(xv) above.

[0216] Methods for producing cFAE-mediated multispecific antibodies or antigen-binding antibody fragments, and multispecific antibodies and antigen-binding antibody fragments produced by such methods Also contemplated by the present disclosure are methods of producing heteromeric molecules that include a CH3 set (which in some embodiments can be any of the CH3 sets described herein) that preferentially form CH3 heterodimers. The heteromeric molecules can be any of the heteromeric molecules or multispecific antibodies and antigen-binding antibody fragments described herein, optionally having a structure as shown in any one of Figures 2-8.

[0217] In some embodiments, a heteromeric molecule of interest (which the method is intended to produce) may comprise: (A) a first polypeptide (e.g., a first heavy chain) comprising a first variant CH3 domain polypeptide; and (B) a second polypeptide (e.g., a second heavy chain) comprising a second variant CH3 domain polypeptide, wherein the first and second polypeptides may optionally be linked or paired with each other via at least one disulfide bond.

[0218] The examples herein demonstrated that the "RL" set provides superior bsAb production efficiency without causing any mismatching, as shown in Table 18. In addition, it was further found that the bsAb containing the "VV" set was resistant to glutathione exposure (see Figures 28A-28E).

[0219] Thus, in certain embodiments, the CH3 set incorporated into the heteromeric molecule or multispecific antibody or antigen-binding antibody fragment produced by the method can be a "VV" set and comprises a Y407V mutation in one CH3 domain of the set and a T366V mutation in a second CH3 domain of the set. In certain embodiments, the CH3 set can comprise additional substitutions in addition to the "VV" set substitutions, such as, but not limited to, disulfide modifications at positions 349 and 354 described herein (i.e., the VV(349 / 354) set or the "VV(354 / 349)" set).

[0220] In some embodiments, the method may comprise (i) incubating under a reducing environment (i-1) a first antibody (which may also be referred to as a first parent antibody or a first monospecific parent antibody) comprising at least two of the first polypeptides bound or paired to each other, optionally via at least one disulfide bond, and (i-2) a second antibody (which may also be referred to as a second parent antibody or a second monospecific parent antibody) comprising at least two of the second polypeptides bound or paired to each other, optionally via at least one disulfide bond. The first and second parent antibodies may be IgG (e.g., IgG1, IgG2, IgG3, and IgG4).

[0221] In some embodiments, the first polypeptide may further comprise a first antigen binding domain. In some embodiments, the second polypeptide may further comprise a second antigen binding domain. In some embodiments, the heteromeric molecule may further comprise a third polypeptide comprising a third antigen binding domain, which may optionally be bound to or paired with the first polypeptide. In some embodiments, the heteromeric molecule may further comprise a fourth polypeptide optionally comprising a fourth antigen binding domain, which may optionally be bound to or paired with the second polypeptide.

[0222] In certain embodiments, the first polypeptide may comprise a first antigen-binding domain that forms a first antigen-binding site specific for a first epitope, and / or the heteromeric molecule may comprise a third polypeptide that comprises a third antigen-binding domain that forms a third antigen-binding site specific for a third epitope. Optionally, the first epitope may be the same as or different from the third epitope. In alternative embodiments, the first polypeptide may comprise a first antigen-binding domain and the heteromeric molecule may comprise a third polypeptide that comprises a third antigen-binding domain, where the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for the first epitope.

[0223] In certain embodiments, the second polypeptide may comprise a second antigen binding domain that forms a second antigen binding site specific for a second epitope, and / or the heteromeric molecule may comprise a fourth polypeptide that comprises a fourth antigen binding domain that forms a fourth antigen binding site specific for a fourth epitope. Optionally, the second epitope may be the same as or different from the fourth epitope. In alternative embodiments, the second polypeptide may comprise a second antigen binding domain and the heteromeric molecule may comprise a fourth polypeptide that comprises a fourth antigen binding domain, where the second antigen binding domain and the fourth antigen binding domain form a second antigen binding site specific for a second epitope.

[0224] The first and second antibodies may be produced in any suitable cell type. Exemplary cells include, but are not limited to, mammalian cells, yeast cells, insect cells, plant cells, or bacterial cells, more specifically, Chinese Hamster Ovary (CHO) cells or Human Embryonic Kidney (HEK) cells.

[0225] In certain embodiments, the first antibody and the second antibody may be incubated at a temperature of about 15° C. to about 40° C., about 20° C. to about 40° C., about 25° C. to about 35° C., about 28° C. to about 32° C., or about 29° C. to about 31° C., or about 30° C. In certain embodiments, the first antibody and the second antibody may be incubated for about 30 minutes to about 20 hours, about 1 hour to about 15 hours, about 2 hours to about 10 hours, about 3 hours to about 7 hours, or about 4 hours to about 6 hours, or about 5 hours. In certain embodiments, the first antibody and the second antibody may be incubated at about 30° C. for about 5 hours.

[0226] In certain embodiments, the first and second antibodies may be incubated in the presence of at least one reducing agent, optionally at least one weak reducing agent. Ideally, the at least one reducing agent or reducing environment is capable of reducing the disulfide bond between the two heavy chains (or between the first and second polypeptides), but not between the heavy and light chains. Various reducing agents have been shown to provide this reducing function in the context of FAEs (see, for example, van der Neut Kolfschoten et al. Science. 2007 Sep 14; 317(5844): 1554-1557). Exemplary reducing agents include, but are not limited to, 2-mercaptoethylamine (2-MEA), β-mercaptoethanol (BME), L-cysteine, dithiothreitol (DTT), or dithionite.

[0227] In certain embodiments, the at least one reducing agent is about 25 to about 125 mM, about 50 mM to about 100 mM, about 70 to about 80 mM, or about 75 mM 2-MEA, about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM BME, about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, or about 90 to about 120 μM, or about 100 μM BME, The reducing environment may be selected from about 0 μM, about 90 to about 120 μM, or about 100 μM L-cysteine, about 15 to about 400 μM, about 20 to about 200 μM, about 25 to about 100 μM, about 30 to about 70 μM, or about 50 μM DTT, or about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM dithionite. In certain embodiments, the reducing environment may include at least about 75 mM 2-MEA.

[0228] In some embodiments, the method may then include (ii) placing the product of the incubation of step (i) in a less reducing or non-reducing environment.

[0229] In certain embodiments, this step (ii) may allow pairing between the first and second variant CH3 domains, and thus pairing between the first and second polypeptides.

[0230] In certain embodiments, the disposition may be via buffer exchange, allowing for the removal of reducing conditions, such as reducing agents. For example, the buffer may be exchanged with PBS.

[0231] In certain embodiments, buffer exchange may be performed by desalting or diafiltration.

[0232] In another embodiment, the disposing may be performed by adding an oxidizing agent.

[0233] In some embodiments, the product of step (ii) may be incubated in a low reducing or non-reducing environment. In certain embodiments, the incubation may be performed at a temperature of about 1° C. to about 20° C., about 2° C. to about 10° C., about 3° C. to about 5° C., or about 4° C. In certain embodiments, the incubation may be performed for about 12 hours to about 154 hours, about 24 hours to about 96 hours, about 36 hours to about 72 hours, or about 48 hours. In certain embodiments, the incubation may be performed at about 4° C. for about 48 hours.

[0234] In some embodiments, the product of step (ii) and / or step (iii) may be analyzed for the amount of multispecific antibody or antigen-binding antibody fragment of interest in the product of step (ii) and / or step (iii). In some embodiments, the product of step (ii) and / or step (iii) may be purified to obtain purified multispecific antibody or antigen-binding antibody fragment. In certain embodiments, such analysis and / or purification may be performed by chromatography, such as, but not limited to, LC-MS, IEX, and / or SEC. In certain embodiments, mispairings may not be observed by LC-MS.

[0235] In some embodiments, the heteromeric molecules produced include multispecific antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies). The polypeptides of such multispecific antibodies may include an antibody heavy chain that may be associated with an antibody light chain, the antigen-binding domain of which may form an antigen-binding site. The multispecific antibody may include additional polypeptides that may include additional antigen-binding domains and / or form additional antigen-binding sites. Such additional antigen-binding domains or sites may be associated with the antibody heavy or light chains of the multispecific antibody. Such association may be via a linker. Such linkers may include flexible linkers, including polypeptides with multiple glycine and / or serine residues. In some embodiments, the additional antigen-binding domain may include a Fab antibody fragment or a single-chain Fv (scFv) fragment that may be stabilized by disulfide bonds. In some embodiments, the multispecific antibody may be a biparatopic antibody.

[0236] In some production methods, a first IgG and a second IgG are used to prepare a heteromeric molecule. The first polypeptide of the first IgG may comprise a first antibody heavy chain comprising a first antigen binding domain forming a first antigen binding site for a first epitope. The second polypeptide of the second IgG may comprise a second antibody heavy chain comprising a second antigen binding domain forming a second antigen binding site for a second epitope. The first epitope and the second epitope may be part of different antigens. The first epitope and the second epitope may be part of the same antigen. The heteromeric molecule may be an IgG comprising a first antibody heavy chain and a second antibody heavy chain.

[0237] Any multispecific antibodies and antigen-binding antibody fragments produced by the methods of production are further encompassed by the present disclosure.

[0238] Examples are provided below to illustrate the invention. These examples are not intended to limit the invention to any particular application or theory of operation. EXAMPLES

[0239] In the examples described herein, the CH3 domain reference sequence (SEQ ID NO: 1) was used as the wild type CH3 domain sequence for IgG1, and various amino acid substitutions were incorporated into the reference sequence to test for heterodimerization potential. Some of the sequences used in the examples are provided in Appendix Tables A-G and in the Sequence Listing. Although SEQ ID NO: 1 was used as the CH3 domain reference sequence in the examples, the invention regarding CH3 domain sequence modifications may also be applied to other CH3 domain reference sequences, such as, but not limited to, SEQ ID NO: 2, 3, or 4 (for IgG1), or another standard CH3 sequence for IgG1, IgG2, IgG3, or IgG4.

[0240] Unless otherwise stated, CH1 and CH2 reference sequences (SEQ ID NO:6 and SEQ ID NO:7, respectively) were used in the examples where applicable.

[0241] Example 1: Evaluation of flow cytometry-based selection of modified Fc-displaying yeast libraries as a CH3 domain dimerization readout using existing CH3 heterodimerization technology (proof-of-concept study). To screen for heterodimer-preferring variant CH3 domains, we designed a yeast library system in which each cell displays a "modified Fc" (a portion of the Fc encompassing positions D221 to K447 (EU numbering), with a hinge (SPPS instead of CPPC), a modified CH2 domain (N297A), and a portion of the CH3 domain with either the wild-type or variant sequence) and first analyzed by flow cytometry to enrich for cell populations containing CH3 heterodimers.

[0242] To initially evaluate this system as a method to identify variant CH3 domains that preferentially form CH3 heterodimers, the system was tested using existing variant CH3 domain sets, W-SAV (i.e., KiH) and EW-RVT (see Table 1) as controls. These two CH3 sets were confirmed to exhibit a high preference for CH3 heterodimer formation when used in bispecific antibodies, as measured by AlphaLISA® (using methods described herein) and liquid chromatography-mass spectrometry (LC-MS).

[0243] A library of variant CH3 domains was constructed and expressed in engineered yeast strains. Specifically, two heavy chain expression plasmids (pAD6234 and pAD6233) were constructed, each containing a "modified Fc". pAD6234 contained a FLAG tag, and URA and pAD6233 contained a HIS tag and TRP.

[0244] Three sets of pAD6234 and pAD6233 were generated. The first set contained the KiH substitution set (knob HIS hole FLAG ), and the second set encodes the EW-RT set (EW HIS RVT FLAG ), and the third set encodes a WT-WT set (also referred to as the "WT set") (WT HIS -WT FLAG ) encoding a CH3 domain containing

[0245] Yeast proof-of-concept (POC) libraries were generated in which yeast cells were transfected with a 1:1:10,000 mix of the first, second, and third plasmid sets, respectively, and grown as previously described (see, e.g., WO2009036379, WO2010105256, WO2012009568, Xu et al., Protein Eng Des Sel. 2013 Oct;26(10):663-70) and subjected to flow cytometry-based selection of high FLAG expressers (representing CH3 heterodimer expressers) (schematic diagram in Figure 9A).

[0246] Briefly, after expression of the modified Fc, engineered yeast cells (approximately 10 7 ~10 8 ) were stained with anti-HIS FITC (Invitrogen, Carlsbad, California, Catalog No. MA1-81891) diluted 1:100 and anti-FLAG APC (BioLegend, San Diego, California, Catalog No. 637308) diluted 1:500 in PBSF for 15 min at 4°C. After washing twice with ice-cold wash buffer, cell pellets were resuspended in 0.4 mL PBSF and transferred to strainer-capped sort tubes. Sorting was performed using a FACS ARIA sorter (BD Biosciences) to determine sort gates and enrich for heterodimers. Libraries were selected over two rounds. Clones with both HIS and FLAG tags were sequenced from the fourth and fifth rounds and analyzed for sequence uniqueness. Cells were plated on medium lacking uracil and tryptophan to generate single isolates and sequence identified.

[0247] As expected, cells expressing the CH3 heterodimer (KiH or EW-RVT) were significantly enriched after two selection rounds (Figure 9B), indicating that the modified Fc library coupled with flow cytometry-based selection can indeed be used to identify CH3 heterodimer-preferring variant CH3 domains.

[0248] Example 2: Cycle 1 selection using a variant CH3 domain library saturating one or more of the four KiH positions. In this example, the variant CH3 domain selection system described in Example 1 was used to identify novel variant CH3 domains with amino acid substitutions at one or more of the KiH substitution positions (KiH has a W at position 366 (the "knob" position) in one CH3 and an S, A, and V at positions 366, 368, and 407 (the "hole" positions) in another CH3, see Table 1).

[0249] Specifically, two pools of variant CH3 domain DNA fragments were generated and inserted into an expression plasmid (Figure 10A). The first pool was generated by allowing substitution of all 20 amino acids at positions T366 in the first CH3 domain and T366, L368, and Y407 in the second CH3 domain. Two variations were made in the first pool, one with hole position variability in the strand with the Flag tag (knob SSM -HIS Hall SSM -FLAG) (Library 1 in FIG. 10A), and the other is a knob position variable in a strand with a Flag tag (Hole SSM -HIS knob SSM -FLAG) (Library 2 in FIG. 10A). The second pool was generated by fixing the constant T336W position of the first CH3 domain for all 20 amino acids and varying the T366, L368, and Y407 positions of the second CH3 domain (Knob-HIS-Hole). SSM -FLAG) (Library 3 in FIG. 10A) The library was constructed using a DNA shuffling method as previously described (Stemmer, Proc. Natl. Acad. Sci., 91 (1994), pp. 10747-10751). The library was expanded and subjected to six selection rounds, and the selection products were sequenced, as described in Example 1 (FIGS. 10B-10D).

[0250] As shown in Table 2, selection did not result in any collapse of order. [Table 2]

[0251] At the end of the sixth round, 86 unique CH3 heterodimers were found. The top substitution combinations at the knob-into-hole (KiH) positions are provided in Table 3 along with their frequency of occurrence (repeats). [Table 3]

[0252] In the following examples, 86 unique CH3 heterodimer sequences were generated in yeast and characterized by AlphaLISA®, ion exchange chromatography (IEX), and size exclusion chromatography (SEC). Melting temperatures were also determined.

[0253] Example 3: AlphaLISA® analysis of the CH3 set identified in Example 2. The 86 unique CH3 heterodimers identified in Example 2 were analyzed by AlphaLISA® (Figure 11A left). AlphaLISA® was used to determine the relative degree of heterodimerization of Fc fragments. Briefly, 5 μl of fragments were added as a 0.5 nM final test concentration solution together with 10x biotin-a-Flag (5 μL, 20 nM final test concentration) in Perkin Elmer AlphaLISA immunoassay buffer (10x) and placed in a 384-well AlphaPlate (Perkin Elmer). Then, 10x acceptor bead solution with a-His (5 μL) was added, the plate was covered with a black cover and incubated at room temperature for 1 hour. Next, 10x (5 μL) donor bead (SA coated) solution was added to the assay in the dark and incubated at room temperature for 30 minutes. The plate was read using the EnSpire Alpha program (Perkin Elmer).

[0254] Data on existing CH3 heterodimerization substitution sets demonstrated that AlphaLISA® can be used to measure CH3 heterodimerization (Figure 11A right). These results are provided in Figure 11B. Five CH3 sets showed AlphaLISA® values ​​higher than the positive controls (KiH and EW-RT) (Table 4). AlphaLISA® values ​​(fold over background) were further plotted against α-Flag APC values ​​(fold over background) on the x-axis in Figure 11C. The "VV" set was found in both orientations (366V in FLAG, 407V in FLAG). [Table 4]

[0255] Example 4: Size Exclusion Chromatography (SEC) analysis of the CH3 set selected in Example 3. Expression and quality of purified antibodies containing one of the five CH3 sets ("VV") identified in Example 3 were tested in both orientations, resulting in a total of six variants) were assessed by size exclusion chromatography (SEC). A higher main peak % represents higher quality. Briefly, column chromatography (TSKgel Super SW3000 column) was monitored using an Agilent 1 100 HPLC. Prior to use, the column was preconditioned with highly glycosylated and aggregated IgG to minimize potential antibody-column interactions and equilibrated with wash buffer (200 mM sodium phosphate, 250 mM sodium chloride, pH 6.8). Approximately 2-5 μg of protein sample was injected onto the column and the flow rate was adjusted to 0.400 mL / min. Protein migration was monitored at a wavelength of 280 nm. Total assay time was approximately 11 min. Data was analyzed using ChemStation software.

[0256] The SEC chromatographs of the WT and control CH3 sets (W-SAV (i.e., KiH) and EW-RVT) are shown in FIG. 12A, and the SEC chromatographs of the CH3 sets selected in Example 3 are shown in FIG. 12B.

[0257] Example 5: Ion exchange (IEX) chromatography analysis of cycle 1 output Ion exchange chromatography (IEX) was performed on a subset of clones. All chromatographic separations were performed on a computer-controlled AKTA Avant 150 preparative chromatography system equipped with an integrated pH electrode allowing in-line pH monitoring and a Mono S 5 / 50 GL column. The cation exchange buffer consisted of 15.6 mM CAPS, 9.4 mM CHES, 4.6 mM TAPS, 9.9 mM HEPPSO, 8.7 mM MOPSO, 11.0 mM MES, 13.0 mM acetate, 9.9 mM formate, 10 mM NaCl, and the pH was adjusted to 4.0 (buffer A) or 11.0 (buffer B) using NaOH. 500 ug of protein was buffer exchanged into 25% buffer B and filtered through a 0.2 mm filter. Prior to each separation, the column was equilibrated with 10 column volumes of 25% buffer B. The protein was then loaded onto the column via a capillary loop, followed by a 10 column volume wash with 25% Buffer B, a 20 column volume linear pH gradient from 25% to 100% Buffer B, and a 10 column volume hold at 100% B.

[0258] IEX chromatographs of the WT and control CH3 sets (W-SAV (i.e., KiH) and EW-RVT) are shown in Figure 13A, and SEC chromatographs of the CH3 sets selected in Example 3 are shown in Figure 14B. Several CH3 sets with low AlphaLISA® values ​​exhibited poor quality as measured by SEC and IEX.

[0259] Example 6: Production of cycle 1 output in HEK293 The impact of the identified variant CH3 domains on a control bispecific common light chain antibody in an IgG-like format (two Fab regions are linked to the N-terminus of a dimeric Fc molecule) was also evaluated. The W-SG substitution set (containing T366W in one CH3 and T366S and Y407G in the other CH3) and the VV substitution set (containing T366V in one CH3 and Y407V in the other CH3) were selected as exemplary test sets and produced in HEK293 cells as anti-Her2 / anti-CD3 bispecific antibodies. The VH-CH1 sequences derived from two antibodies, ADI-29235 (anti-HER2) and ADI-26908 (anti-CD3), were used. The wild-type CH3 set, the W-SAV set (i.e., KiH), and the EW-RVT set were included as controls. In addition, CH3 domain substitutions (S354C / Y349C) were introduced to promote the desired heterodimeric pairing of the heavy chains. The CH3 sets tested are summarized in Table 5.

[0260] The DNA plasmids were verified by Sanger sequencing and then transfected into HEK293 cells by standard protocols. The transfected HEK cells were cultured in CD optiCHO medium (Invitrogen), and the supernatants were harvested 6 days after transfection and subjected to Protein A-based affinity purification. [Table 5]

[0261] Anti-Her2 / anti-CD3 bispecific antibodies produced in HEK293 cells, including control antibodies (WT or containing CH3 sets containing W-SAV(KiH) or EW-RVT substitutions) are summarized in FIG. 14A.

[0262] The percentage of heterodimers (HC1 / HC2 hetero) and homodimers (HC1 homo and HC2 homo) among the full-sized antibodies, as well as the presence of half antibodies ("1 / 2 Ab", i.e., containing only one heavy chain (HC)), were analyzed by LC-MS. To assess CH3 heterodimerization using LC-MS, antibody samples were digested with PNGaseF glycosidase to remove N-linked glycans and then injected onto an Acquity ultra-performance liquid chromatography (UPLC) system (Waters) equipped with a Thermo Scientific MabPac RP® 4 μM column (2.1×100 mm) maintained at 80° C. After injection, the sample was eluted from the column using a 13 min gradient of 20-55% acetonitrile (mobile phase A: 0.1% formic acid in HO, mobile phase B: 0.1% formic acid in acetonitrile) at a flow rate of 0.3 mL / min. Species eluted from the column were detected by a Q Exactive mass spectrometer (Thermo) in positive electrospray ionization mode. Instrument parameters were set as spray voltage of 3.5 kV, capillary temperature of 350 °C, sheath gas flow rate of 35, and auxiliary gas flow rate of 10, and S-lens RF level of 90. MS spectra were acquired with a scan range of 750-4000 m / z. The acquired MS data were analyzed using Biopharma Finder software (Thermo Scientific) followed by manual inspection to ensure correct assignment and relative quantification accuracy. Relative quantification of each of the heterodimeric and homodimeric species was calculated based on the intensity of the peak relative to the sum of all heterodimeric and homodimeric peak intensities.

[0263] The HEK products were also analyzed by Protein A-based size-exclusion chromatography (SEC) and ion-exchange (IEX) chromatography, and the chromatographic profiles from SEC and IEX are shown in Figures 14B and 14C. The LC-MS results, SEC results, and resulting titers are summarized in Table 6. "(354 / 349)" means that HC1 contained Y349C and HC2 contained S354C. [Table 6]

[0264] Example 7: Cycle 2 library generation based on CH3-CH3 interface position and cycle 1 output. To further explore variant CH3 domains that preferentially form CH3-CH3 heterodimers, we designed a new CH3 domain library based on amino acid positions at the CH3-CH3 interface (interface positions) and KiH positions (including cycle 1 output substitutions). First, to identify interface positions, 32 high-resolution aligned wild-type CH3 crystal structures were constructed from the Protein Data Bank (PDB) and used in a structure-guided approach to identify CH3 interface residues for diversification. Interface residues for variability were defined as those that 1) had a side chain SASA (solvent exposed surface area) in the monomer of 15% or more, 2) had adjacent atom contact distances of 8.2 Å or less (a distance set to capture the distances between known knob-in-hole mutations), and 3) were not pointing away from the partner strand or solvent exposed (as determined by manual inspection). By applying these rules, 24 positions for variability at the CH3 interface were identified.

[0265] Next, a library was designed to test one or two "anchor" mutations on one side of the interface (on strand A) against one, two, or three "neighbor" mutations on the other side of the interface (on strand B). For each anchor position A on strand A, a set of neighbor positions B was identified as the subset of interface positions on strand B that contact position A (with interstrand Cb-Cb distances less than 8.2 Å (Cb=beta carbon). In the case of glycine, the C-alpha atom was used since glycine does not have a Cb atom). Then, a combination of all possible singlet, doublet, and triplet mutations within the set of neighbors (B) was generated. Dimers and trimers containing pairs of residues that were not within an intrastrand Cb-Cb distance cutoff of 8.0 Å or less were filtered out. The resulting set identified the following neighbor mutations to test: 24 singlets, 39 doublets, and 16 triplets.

[0266] The set of neighbor / anchor pairing positions was divided into 14 library pools for screening by the following steps: 1) the neighbor / anchor pairing positions were sorted by increasing diversity (singlets, doublets, triplets) and by common position in the protein; and 2) the neighbor / anchor pairs were combined into pools (selecting the closest pool as measured by interstrand contact distance) until a limit of diversity was reached. Each individual library pool contained approximately 10 6 In addition, two pools were constructed on the output obtained in Example 1 (T366V / Y407V ("VV") and T366W / T366S Y407G ("W-SG")). The anchor and neighbor positions and position combinations that were variegated, as well as the DNA and amino acid sequence diversity possible by variegation in some of the pools, are summarized in Table 7. DNA sequence diversity was calculated as follows: Diversity == ((N アンカーモノマー * +N アンカーダイマー * )*(N ネイバーモノマー * +N ネイバーダイマー * +N ネイバートリマー * )) [Table 7]

[0267] Example 8: Cycle 2 Selection Step 1: Selection using modified Fc displayed on yeast. Library DNA was synthesized with the BioXp system and transformed into yeast as described above. Clones were selected using anti-His and anti-Flag reagents for high His and high Flag signals. The library was selected over five rounds (Figure 15A). Clones were sequenced from rounds 4 and 5 and analyzed for sequence uniqueness. A set of 430 unique variant CH3 domains was obtained.

[0268] The 430 CH3 set was characterized by IEX (subset) and AlphaLISA as previously described. Rosetta scoring was also used to characterize the 430 CH3 set. ΔΔG, defined as the change in interface binding energy (predicted by Rosetta), was determined as described in Barlow et al, J Phys Chem B (2018), pp. 5389-5399. Briefly, ΔΔG was calculated for the input PDB crystal structure and averaged (1l6x, 2iwg, 4wi2, 5gsq). The ΔΔG score of the heterodimer state was then calculated along with the ΔΔG scores of both possible homodimer states. Finally, the Rosetta heterodimerization score (RHS) was calculated, where RHS = ΔΔG ヘテロ二量体 -min (ΔΔG ホモ二量体A , ΔΔG ホモ二量体B ).

[0269] Based on the uniqueness, IEX, AlphaLISA®, and Rosetta characterization data, 48 CH3 sets were selected for further production in HEK293 cells in Example 9. A comparison of the three variables IEX (percent interchain contacts), AlphaLISA® values, and Rosetta scores between the selected and unselected CH3 sets is provided in Figure 15B.

[0270] The set was further characterized by sequence uniqueness using t-SNE (t-distributed stochastic neighbor embedding) visualization. t-SNE plots were constructed to visualize the position space in a 2d plot, with each point representing a set of mutation positions, and points close to each other on the plot contained similar mutation positions. The clones selected to be carried forward spanned a wide range of the design space visualized in the t-SNE plot (Figure 15C and Figure 15D). The clones selected to be carried forward spanned a wide range of the design space visualized in the t-SNE plot.

[0271] Example 9: Cycle 2 Selection Step 2: Selection using modified Fc production in HEK293 cells. The set of 48 variant CH3 domains selected in Example 8 were cloned as CH2-CH3 constructs, produced in HEK293 cells, and further characterized using LCMS (as described above), melting temperature, and 14-day stability.

[0272] Melting temperatures (Tm) were measured by differential scanning fluorimetry (DSF). Twenty microliters of samples at 0.1-1 mg / ml were mixed with 10 μL of 20× Sypro orange (Sigma-Aldrich) before undergoing a controlled temperature ramp from 40 to 95°C at 0.5°C intervals in a C1000 thermocycler (BioRad) and the Fret signal was collected. Melting temperatures were obtained by taking the negative of the first derivative of the raw signal.

[0273] For accelerated stability testing, samples were incubated in HBS at 40° C. for 14 days and samples were taken on days 0, 1, 2, 7, and 14. Samples were then analyzed for aggregation by SEC. For SEC analysis, the running buffer composition was 200 mM sodium phosphate, 250 mM sodium chloride, pH 7.0. The accelerated stability slope was calculated from the percent aggregation measured by SEC.

[0274] Based on the additional characterization data, five CH3 sets were selected as cycle 2 outputs and further produced as IgG-like bispecific antibodies (BsAbs) in Example 10. Figure 16 provides plots showing the heterodimer % values ​​(measured by LC-MS) and stability (measured by SEC) of the tested CH3 sets, with the filled data points representing the variant CH3 domain sets designated for bispecific antibody production in HEK293 cells. Table 8 summarizes the five cycle 2 outputs along with the controls (wild type and W-SAV (i.e., KiH)) with their respective heterodimer % values ​​measured by LC-MS and melting temperatures Tm measured by differential scanning fluorimetry (DSF). [Table 8]

[0275] Example 10: Characterization of cycle 2 output produced as bispecific antibodies (BsAbs) in HEK293 cells. The five cycle 2 output variant CH3 domain sets selected in Example 9 were produced as BsAbs with three different Fv sets to evaluate heterodimerization efficiency in an IgG-like format. As shown in Figure 17A, three Fv sets were used in different orientations (orientation 1 or orientation 2) using a total of five different structures per output variant CH3 domain set: anti-CD3 / anti-HER2 (Adimab), anti-CD20 / anti-CD3 (Regeneron), or anti-HEL / anti-BCMA (Nanjing Legend Bio / Janssen). The anti-BCMA antigen binding domain is a nanobody (VHH).

[0276] The wild-type CH3 domain set (i.e., dimers of reference sequence SEQ ID NO:1) of either the bispecific or monospecific antibodies was used as a negative control, and the W-SAV (i.e., KiH) substitution set used for the anti-CD3 / anti-HER2 bispecific antibody was used as a positive control. In addition, anti-CD3 / anti-HER2 bispecific antibodies were also produced that contained either the wild-type CH3 set in orientation 1 or the cycle 2 output variant CH3 domain set, which additionally incorporated the S354C / Y349C substitutions.

[0277] In total, 41 different antibodies were produced, as summarized in Table 9. The heavy and light chain sequences of the antibodies in Table 9 are provided in Appendix Tables A-D. [Table 9]

[0278] The antibodies in Table 9 produced in HEK293 cells were analyzed by LC-MS (% heterodimer and % homodimer of full size antibody), IEX (% heterodimer), and AlphaLISA® as described above. The results are summarized in Table 10. [Table 10]

[0279] The antibodies in Table 9 produced in HEK293 cells were further analyzed by accelerated stability by SEC, i.e., % intact antibody on day 0 (the day of production) ("% monomeric intact Ab") and change in % intact antibody by day 14 ("Δ% monomeric intact Ab") on Protein A purified samples. The SEC results and process yields from production in HEK293 cells are summarized in Table 11. [Table 11] Heterodimer % data, monomeric full size antibody % data, and yields were further compared between specific sets of antibodies out of the 41 antibodies (Figures 17B-J). In these comparisons, when comparing "anti-CD3 / anti-HER2 orientation 1", "anti-CD3 / anti-HER2 orientation 2", "anti-CD20 / anti-CD3 orientation 1", "anti-CD20 / anti-CD3 orientation 2", and "anti-HEL / anti-BCMA orientation 1", the heterodimer % measured by LC-MS was comparable between BsAbs with the same CH3 substitutions (Figure 17B). When comparing the heterodimer % measured by LC-MS and the heterodimer % measured by IEX in BsAbs, LC-MS and IEX provided different heterodimer % values ​​(e.g., 54% and 41%, respectively) for anti-HEL / anti-BCMA antibodies (BsAbs containing a nanobody in one Fab arm) where the anti-BCMA binding moiety is a nanobody ("VHH") instead of a VH / VL pair (Figure 17C). In contrast, the heterodimer % values ​​by LC-MS and IEX correlated well for other antibodies with two VH / VL pairs (Figure 17D). The correlation between the heterodimer % values ​​by LC-MS and the heterodimer % values ​​by IEX was not evident for some of the anti-CD3 anti-HER2 BsAbs with 354 / 349 disulfide bonds (Figure 17E).

[0280] When comparing the % heterodimer values ​​measured by LC-MS in the CH3 sets with and without the 354 / 349 substitution, most of the CH3 sets with the 354 / 349 substitution (except "TL-QL") showed higher % heterodimer values ​​than the corresponding CH3 sets without the 354 / 349 substitution (Figure 17F), indicating that the disulfide bond between S354C and Y349C enhances heterodimerization in most of the sets (except "TL-QL").

[0281] The results show that when the % heterodimer values ​​measured by AlphaLISA® were compared to those measured by LC-MS or IEX, the order (i.e., rank) of the CH3 sets with respect to heterodimerization potential was maintained even if there were some discrepancies in the % values ​​(Figure 17G).

[0282] Overall, regardless of the method used to determine the heterodimer %, "LWG-SIG" in orientation 1 or orientation 2, with or without the 354 / 349 substitution, appears to provide the highest heterodimer % among the various CH3 sets tested (Figure 17H).

[0283] The stability of the BsAbs was also compared based on % monomeric intact Ab at day 0 and the change in % monomeric intact Ab (Δ% monomeric intact Ab) by day 14 as measured by SEC. As shown in Figure 17I, regardless of whether the antibody was "anti-CD3 / anti-HER2 orientation 1", "anti-CD3 / anti-HER2 orientation 2", "anti-CD20 / anti-CD3 orientation 1", "anti-CD20 / anti-CD3 orientation 2", or "anti-HEL / anti-BCMA orientation 1", the % monomeric intact Ab values ​​were low (i.e., less aggregation) at day 0 and only little aggregation occurred by day 14.

[0284] Finally, the production yields of BsAbs in HEK293 cells were compared. As shown in FIG. 17J, the production yields were similar whether the antibodies were "anti-CD3 / anti-HER2 orientation 1", "anti-CD3 / anti-HER2 orientation 2", "anti-CD3 with 354 / 349 substitution / anti-HER2 orientation 1", "anti-CD20 / anti-CD3 orientation 1", "anti-CD20 / anti-CD3 orientation 2" or "anti-HEL / BCMA orientation 1".

[0285] Example 11: Simultaneous characterization of BsAbs containing cycle 1 output substitutions, cycle 2 output substitutions, or a combination of cycle 1 and cycle 2 output substitutions. In Example 11, anti-CD3 anti-HER2 BsAbs containing cycle 1 output substitutions (W-SG or VV) and cycle 2 output substitutions (QR-F, RG-FG, TL-QL, DVG-VSY, or LWG-SIG) were compared side-by-side with and without the 354 / 349 substitution. In addition, BsAbs containing some combination of cycle 2 output substitutions with cycle 1 output substitutions or KiH substitutions (WTL-SAVQL, WTL-SGQL, WQL-SAVTL, WQL-SGTL, VTL-VQL, VQL-VTL, QRQL-FTL, or VQR-VF) were tested side-by-side, along with a modified version of the cycle 2 output substitution (LWG-IG).

[0286] The BsAbs used in Example 11 are summarized in Table 12. All BsAbs were produced in HEK293 cells. [Table 12]

[0287] The BsAbs in Table 12 produced in HEK293 cells were analyzed for % heterodimer by LC-MS and IEX. The results are summarized in Table 13. [Table 13]

[0288] The bsAbs in Table 12 produced in HEK293 cells and Protein A purified were further analyzed for % monomeric intact Ab by SEC. The SEC results and process yields from production in HEK293 cells are summarized in Table 14. [Table 14]

[0289] The heterodimer % data, monomeric full size antibody % data, and yields were further compared between certain antibody sets of the 31 antibodies. This comparison revealed that when the heterodimer % values ​​measured by LC-MS and IEX were compared for BsAbs containing wild-type or cycle 1 or 2 output CH3 domains without the 354 / 349 substitution, a good correlation was observed between the LC-MS and IEX values ​​(Figure 18A). Furthermore, when the same comparison was performed for BsAbs containing wild-type or cycle 1 or 2 output CH3 domains further containing the 354 / 349 substitution, the order (i.e., rank) of the CH3 sets in terms of heterodimerization potential was maintained even if there were some discrepancies in the % values ​​(Figure 18B).

[0290] As observed in Example 10, among the CH3 sets identified in cycle 1 and cycle 2 selections (i.e., cycle 1 outputs (W-SG and VV) and cycle 2 outputs (QR-F, RG-FG, TL-QL, DVG-VSY, and LWG-SIG) with and without the 354 / 349 substitution, and the existing heterodimerization technologies tested (i.e., KiH, EW-RVT, and ZW1), LWG-SIG consistently provided the highest heterodimerization rate with and without the 354 / 349 substitution (Figure 18C). W-SG, RG-FG, and QR-F also exceeded the heterodimerization rates of the existing heterodimerization technologies tested (i.e., KiH, EW-RVT, and ZW1) when combined with the 354 / 349 substitution.

[0291] Interestingly, several of the cycle 1 and cycle 2 output CH3 sets, such as VV and QR-F (with or without the 354 / 349 substitution), showed less aggregation compared to existing heterodimerization technologies tested (i.e., KiH, EW-RVT, and ZW1) as measured by SEC in Protein A purified product (Figure 18D).

[0292] When comparing CH3 sets with and without the 354 / 349 substitution, the CH3 set with the 354 / 349 substitution showed overall higher heterodimer values ​​as measured by LC-MS and higher monomeric intact Ab values ​​as measured by SEC (Figure 18E), indicating that the addition of the 354 / 349 substitution promoted CH3 heterodimerization and improved stability.

[0293] The production yield was compared between different CH3 sets with and without the 354 / 349 substitution. As shown in FIG. 18F, none of the CH3 substitution sets appeared to reduce the production yield. Furthermore, it was observed that certain CH3 sets, such as VV, appeared to have higher yields than the existing heterodimerization techniques tested (i.e., KiH, EW-RVT, and ZW1). In addition, as can be seen from Table 13, many of the combination substitution sets (combinations of cycle 1 substitution and cycle 2 substitution or combinations of cycle 2 substitution and KiH substitution), such as WTL-SAVQL, WTL-SGQL, WQL-SGTL, and VQL-VTL, especially the LWG-IG substitution, provided better production yields than the existing heterodimerization techniques tested (i.e., KiH, EW-RVT, and ZW1).

[0294] The new CH3 set "LWG-IG", which is a variant of cycle 2 output LWG-SIG, showed similar properties overall to LWG-SIG, except that LWG-IG provided higher production yields than LWG-SIG, as shown in Figure 18G. This similarity between LWG-SIG and LWG-IG is consistent with the Rosetta heterodimer scores of LWG-SIG and LWG-IG.

[0295] The invention CH3 domain substitution sets described in the Examples, e.g., cycle 1 output and cycle 2 output, with or without CH3 disulfide bond substitutions (which may be 354 / 349 substitutions or 349 / 354 substitutions), as well as additional variant CH3 domains tested in Example 11 (i.e., combinations of cycle 1 output substitutions and cycle 2 output substitutions, combinations of cycle 2 output substitutions and cycle 2 output substitutions, combinations of cycle 2 output substitutions and KiH substitutions, and LWG-IG), and many of their associated variations, are provided in Appendix Tables E-G. Exemplary variant CH3 domain sequences incorporating such CH3 substitution sets into the reference CH3 domain sequence of SEQ ID NO:1 are also provided in Appendix Tables E-G. The exemplary sequences are those used in the Examples herein. However, it should be noted that these sequences are exemplary and the same CH3 substitution sets may be incorporated into any CH3 domain sequence, i.e., may not be limited to SEQ ID NO:1. A summary of the SEQ ID NOs assigned to these exemplary variant CH3 domain sequences is provided in FIG. 19.

[0296] Example 12: Simultaneous evaluation of the effect on Tm of cycle 1 and cycle 2 output sets. In Example 12, two variant CH3 domain sets from cycle 1 (W-SG and VV), with or without the 354 / 349 substitution, and four variant CH3 domain sets from cycle 2 (QR-F, RG-FG, DVG-VSY, and LWG-SIG), as well as existing CH3 sets (W-SAV (also referred to as KiH), VYAV-VLLW (also referred to as ZW1), and EW-RVT), were produced in HEK293 cells as CH2-CH3 constructs (i.e., Fc-only constructs) and the effect of the CH3 substitution on melting temperature (Tm) measured by differential scanning calorimetry (DSC) was analyzed.

[0297] method

[0298] Protein Expression:

[0299] The heterodimer fc-only construct of the CH3 mutation set contained both a HIS tag and a FLAG tag, so that the heterodimer was expressed as a HIS tag and a FLAG purification tag. Proteins were transiently transfected in HEK cells as described above.

[0300] Primary acquisition:

[0301] Transiently transfected HEK cultures were harvested by centrifugation at 2400G for 5 min. The supernatant was decanted from the cell pellet and after a second spin at 2400G for 5 min, loaded onto Ni Sepharose 6 Fast Flow resin (Cytiva 1753180) equilibrated with 10 column volumes of 20 mM sodium phosphate, 500 mM NaCl, pH 7.4 buffer. Bound proteins were then washed with 5 column volumes of equilibration buffer containing 2 mM imidazole and eluted with 5 column volumes of equilibration buffer containing 250 mM imidazole. The eluate was immediately desalted to 25 mM HEPES, 150 mM sodium chloride, pH 7.2 using Sephadex G25 medium (Cytiva 1700330).

[0302] Secondary purification:

[0303] Proteins were treated with 10x binding buffer (0.5M Tris, 1.5M sodium chloride, 100mM calcium chloride, pH 7.4) and then loaded onto anti-FLAG M1 resin (Sigma Aldrich A4596) equilibrated with 15 column volumes of 50mM Tris, 150mM sodium chloride, pH 7.4. Bound proteins were washed with 36 column volumes of equilibration buffer containing 1mM calcium chloride and eluted with 4 column volumes of equilibration buffer containing 2mM EDTA. The eluate was buffer exchanged into 25mM HEPES, 150mM sodium chloride, pH 7.2 over 3x5 diafiltration volumes through an Amicon™ Ultra-15 centrifugal filter device. Proteins were normalized to a final target concentration of 1mg / mL and filtered through 0.2um.

[0304] Tm measurement by DSC:

[0305] DSC measurements were performed using a MicroCal VP-Capillary DSC (now Malvern Panalytical). Data were typically collected over the range 15-100 °C at 120 °C / hr, referenced to HBS buffer. 400 μL of sample was used for the DSC test. The software running was VPViewer2000. The analysis software was Microcal LLC Cap DSC Version Origin70-L3, which was used to convert the raw data to molar heat capacity (MHC).

[0306] result

[0307] The resulting first and second Tm values ​​(Tm1 and Tm2) are provided in Table 15 along with the substitutions within each CH3 domain. The Tm2 values ​​are further visualized in FIG. [Table 15]

[0308] As shown in Table 15, all variant CH3 sets exhibited similar Tm1 values. The Tm1 values ​​of VV, QR-F, RG-FG, RG-FG(354 / 349), and LWG-SIG(354 / 349) were slightly higher than the Tm1 of the WT CH3 set. As shown in Table 15 and Figure 20, the CH3 sets with 354 / 349 substitutions exhibited higher Tm2 values ​​than the corresponding CH3 sets without 354 / 349 substitutions. The CH3 sets without 354 / 349 substitutions (W-SAV, QR-F, and RG-FG) that did not result in distinct Tm2 peaks obtained more distinct Tm2 peaks when the 354 / 349 substitution was added.

[0309] Example 13: Structural analysis of IgG1 Fc with LWG-SIG. To analyze the effect of substitutions within CH3 on CH3-CH3 interactions, an Fc-only construct containing the LWG-SIG set, designated ADI-64950, was produced in CHO-K1 cells and the crystal structure was analyzed.

[0310] method

[0311] Crystallization and structure determination of ADI-64950 IgG1 Fc:

[0312] ADI-64950, a human IgG1 Fc dimer containing a variant CH3 (IgG1) domain with T366S, L368I, and Y407G on chain A and S364L, T366W, K409G on chain B, where chain B also contains Fc-III knockout substitutions (M252E, I253A, and Y436A), was concentrated to 10.9 mg / mL in a buffer containing 2 mM Tris-HCl pH 8.0 and 150 mM NaCl. ADI-64950 at 10.9 mg / ml was mixed with 1 mM Fc-III dissolved in DMSO to 25 mM. JCSG+, PACT, BCS, and ProPlex screens were placed using 100+100 nl sitting drops in MRC plates across the reservoirs. The crystals used for data collection were grown in JCSG+screen, well B9, across the reservoir: 0.1 M citrate pH 5.0 and 20% (w / v) PEG (polyethylene glycol) 6000. After adding a freezing solution containing 0.1 M citrate pH 5.0, 20% (w / v) PEG (polyethylene glycol) 6000, and 25% glycerol, the crystals were flash frozen in liquid nitrogen. Data were collected at 100 K (λ = 0.9763 Å) on station BioMAX (MAX IV, Lund, Sweden). 3600 images were collected with an oscillation width of 0.1° per image. The beamline was equipped with an Eiger 16M hybrid pixel detector.Data up to 2.7 Å were processed using autoPROC (Vonrhein, C., Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W., Womack, T. & Bricogne, G. (2011). Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D Biol. Crystallogr. 67, 293-302), which includes the software XDS (Kabsch W. (2010) “XDS” Acta. Crystallogr. D Biol. Crystallogr. 66, 125-132) and Aimless (Evans PR and Murshudov, GN (2013) “How good are my data and what is the resolution” Acta Crystallogr D Biol. Crystallogr. 69, 1204-1214). Crystals consisted of a single molecule in the asymmetric unit (ASU) in the P21 space group. A molecular replacement solution of ADI-64950 was obtained by PHASER (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Stroni, LC, & Read, RJ (2007). Phaser crystallographic software. Journal of Applied Crystallography, 40(4), 658-674) using PDB entry 5DJ6 (Leaver-Fay et al. (2016). Computationally Designed Bispecific Antibodies using Negative State Repertoires. Structure. 24(4):641-651).These structures were manually constructed using COOT (Emsley P., Lohkamp, ​​B., Scott, WG and Cowtan K. (2010) "Features and development of Coot" Acta Crystallogr. D Biol. Crystallogr. 66, 486-501) and refined using Refmac5 (Murshudov, GN, Skubak, P., Lebedev, AA, Pannu, NS, Steiner, RA, Nicholls, RA, Winn, MD, Long, F. and Vagin, AA (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr. D Biol. Crystallogr. 67, 355-367), with final R and R of 20.4% and 25.6%, respectively (Figure 21).

[0313] PDB ID:5JII was used as the WT reference for comparison.

[0314] result

[0315] CH3-CH3 pairing mediated by substitutions present in ADI-64950:

[0316] Based on the free energy gain upon formation of the CH3-CH3 interface calculated by PISA (Protein, Interface, Structure, and Assembly), ADI-64950 was found to have stronger CH3-CH3 interactions than the human IgG1 Fc dimer containing the WT CH3 domain (Figure 21). The pairing between chain A (T366S, L368I, and Y407G) and chain B (S364L, T366W, K409G) (AB heterodimer) was found to be mediated by several novel polar contacts at the CH3-CH3 interface (Figure 22). These contacts include a salt bridge formed between strand A Lys409 and strand B Asp399, as well as hydrogen bonds between strand A Lys409 and strand B Asp399, between strand A Glu357 and strand B Lys370, between strand A Ser364 and strand B Lys370, between strand A Leu398 and strand B Lys392, between strand A T366S and strand B Tyr407, between strand A Lys360 and strand B Tyr349, and between strand A Ser354 and strand B Thr350 (Figure 22).

[0317] Steric clashes at the CH3-CH3 interface of the ADI-64950 off-products AA and BB homodimers are predicted to reduce the propensity for mispairing.

[0318] Potential off-product homodimers "AA" (i.e., a dimer of two chains A) and "BB" (i.e., a dimer of two chains B) were generated by aligning chain A to chain B and vice versa and then probed for PyMol clashes. Several substantial clashes were observed, including (a) chain A Phe405 and chain A Lys409, (b) chain B Tyr349 and chain B Asp356, (c) chain B T366W and chain B Tyr407, and orthogonal (d) chain B T366W and chain B Tyr407 (Figure 23). These clashes are predicted to reduce the propensity for formation of the ADI-64950 mismatched homodimer constructs AA and BB.

[0319] Example 14: cFAE compatibility testing, part 1 - Production of antibodies containing two identical variant CH3 domains. Among the many different methods for making bispecific antibodies, some rely on FAE. Some of the cycle 1 output CH3 domain sets and cycle 2 output CH3 domain sets were tested for their applicability to cFAE-based manufacturing methods using the WT CH3 set as a negative control and the existing CH3 domain set "RL" (Labrijn et al. Proc Natl Acad Sci USA. 2013 Mar 26;110(13):5145-50) known to mediate FEA between IgG1 molecules as a positive control (see Table 16). [Table 16]

[0320] For example, if an antibody of interest comprises (a) a half antibody specific for epitope A comprising heavy chain A (comprising VH) and light chain A (comprising VL), and (b) a half antibody specific for epitope B comprising heavy chain B (comprising VH) and light chain B (comprising VL), then (a) antibody A (Antibody A) comprising two half antibodies specific for epitope A and (b) antibody B (Antibody B) comprising two half antibodies specific for epitope B can be produced. Antibody A and Antibody B can then be placed together under mild reducing conditions to allow for reduction of the disulfide bond between the heavy chains, resulting in the half antibody molecules. If heavy chain A contains a variant CH3 domain A (CH3 domain A) and heavy chain B contains a variant CH3 domain B (CH3 domain B), and CH3 domain A and CH3 domain B preferentially form CH3-CH3 heterodimers, then upon removal of the mildly reducing conditions, cFAE will preferentially form heterodimers between heavy chain A and heavy chain B over heavy chain A and heavy chain B homodimers, resulting in more of the desired bispecific antibody than monospecific antibody A and antibody B (see FIG. 1C).

[0321] Since monospecific antibody A and antibody B are produced first, if the production of antibodies containing two of a given variant CH3 domains (two of CH3 domains A or two of CH3 domains B) is poor, the method of producing such bispecific antibodies may not be performed efficiently. This means that not all CH3 sets that favor heterodimerization may be useful for the generation of bispecific antibodies by cFAE. For example, based on the applicant's experience, the existing CH3 set, KiH (Table 1), is not compatible with cFAE-mediated production because the monospecific parent antibodies are not produced well. For this purpose, antibodies with two of the same CH3 domains belonging to the CH3 sets listed in Table 16, i.e., monospecific parent antibodies, were first tested for their production yield and purity.

[0322] method

[0323] Monospecific IgG1 antibodies containing (i) the variable region sequences of an anti-HER2 antibody named ADI-29235 or an anti-CD3 antibody named ADI-26908, and (ii) the CH3 domains WT, K409R, F405L, Y407V, T366V, T366Q_K409R, L368F, T366R_K409G, or L368F_K370G (i.e., two CH3 domains identical to each other) were produced in CHO cells and subjected to Protein A-based affinity purification. The production yields (mg / L) were compared. The purity of the purified products in % full-sized monomeric IgG molecules was also analyzed by SEC as described above.

[0324] result

[0325] The resulting production yields are summarized in Figure 24A. As shown in Figure 24A, all variant CH3 domains provided sufficient production yields. Some variant CH3 domains (such as ADI-29235 with a T366V CH3 domain, ADI-29235 with a L368F CH3 domain, and ADI-29235 and ADI-26908 with a T366Q_K409R CH3 domain) provided higher yields compared to WT CH3.

[0326] Purity values ​​after Protein A-based purification in full size monomer Ab% are summarized in Figure 24B. As shown in Figure 24B, all variant CH3 domains except the T366R_K409G CH3 domain yielded high purity. Based on this result, the "VV" and "QR-F" sets, along with the control CH3 set, were further tested for cFAE-based production in the following examples. However, it is noted that although the purity of antibodies containing the T366R_K409G CH3 domain is relatively low when produced in CHO cells, it is still possible that such antibodies can achieve high purity when produced and / or purified using different conditions, e.g., using different cell types.

[0327] Overall, these results highlight that even if a bsAb containing a variant CH3 set is efficiently produced when all four chains of the bsAb are expressed in the same cell, this does not mean that parent antibodies each containing only one of the variant CH3 domains of the set, but not the other variant CH3 domain of the set, would not be efficiently produced. That is, the applicability of a variant CH3 set to a FAE-based bsAb production method is not necessarily predictable based on how preferentially the variant CH3 set forms heterodimers.

[0328] Example 15: cFAE suitability testing, part 2 - cFAE-based bsAb production. This example tested whether the "VV" set and / or the "QR-F" set could mediate cFAE to produce bsAbs. The bsAbs of interest in Example 15 include (a) an anti-HER2 half antibody comprising heavy chain A (comprising the VH, WT CH1-CH2 domains of ADI-29235, and the CH3 domain of the test CH3 set listed in Table 17) and light chain A (comprising the VL and WT CL domain of ADI-29235), and (b) an anti-CD3 half antibody comprising heavy chain B (comprising the VH, WT CH1-CH2 domains of ADI-26908, and the other CH3 domain of the test CH3 set) and light chain B (comprising the VL and WT CL domain of ADI-26908). ADI-29235 and ADI-26908 share a common light chain, and therefore light chain A and light chain B are identical to each other.

[0329] method

[0330] Anti-HER2 full size antibody, which contains two anti-HER2 half antibodies, and anti-CD3 full size antibody, which contains two anti-CD3 antibodies (to produce the bsAbs in Table 17), were produced in CHO cells and subjected to Protein A-based affinity purification. In addition, panitumumab, which contains two K409R CH3 domains, and nivolumab, which contains two F405L CH3 domains (to produce bsAb index number 3 in Table 17), were also produced and purified.

[0331] The purified products were then subjected to the following FAE reaction step and the reaction products were analyzed based on protein recovery (protein content in FAE product per mg protein reaction) and bsAb formation assessed by IEX.

[0332] FAE Response:

[0333] A 10x 2-mercaptoethylamine-HCl (2-MEA) stock solution (750 mM) was prepared by dissolving 1.70 g of 2-MEA in 20 mL of PBS. The pH was adjusted to 7.4 using approximately 600-700 µL of 2 N NaOH.

[0334] For each bsAb tested, 500 μg of the corresponding anti-HER2 full size antibody (250 μL 2 mg / mL in PBS) and 500 μg of the corresponding anti-CD3 full size antibody (250 μL 2 mg / mL in PBS) were placed in a well of a deep well plate. For bsAb index number 3, 500 μg of panitumumab with K409R and 500 μg of nivolumab with F405L were used. Then, 400 μL of PBS was added to each well, followed by 100 μL of 10x 2-MEA stock solution, resulting in a final 2-MEA concentration of 75 mM. Samples were incubated stationary at 30° C. for 5 hours.

[0335] Each sample was desalted using Sephadex® G25 plates (1 mL split across three wells), buffer exchanged into PBS, and 2-MEA was removed (to achieve approximately <50 μM). Samples were then incubated at 4° C. for a further 48 hours. [Table 17]

[0336] result

[0337] 1. Protein Recovery

[0338] Protein recovery is provided in Figure 25 A. As shown in Figure 25 A, recovery was approximately 80% and similar among the different bsAb samples.

[0339] 2. BsAb formation assessed by IEX

[0340] Exemplary IEX results for the three tested variant CH3 sets, RL, VV, and QR-F (corresponding to BsAb index numbers 2 and 4-8), are provided in FIG. 25B, where each panel shows an overlay of a chromatogram of the FAE reaction product (denoted as "output") with a chromatogram of the corresponding purified product of the parent antibody (denoted as "input"). As shown in FIG. 25B, the RL set (bsAb index numbers 2 and 4) and the VV set (bsAb index numbers 5 and 6) were successful in obtaining the intended bsAb upon the FAE reaction step. In contrast, much less of the intended bsAb was obtained when the QR-F set was used.

[0341] These results especially emphasize that even though the variant CH3 domain set preferentially forms heterodimers over homodimers and allows preferential production of bsAb when all four chains are expressed in the same cell, this does not mean that this variant CH3 domain set mediates FAE, i.e., its applicability to FAE-based bsAb production methods is unpredictable.

[0342] Example 16: cFAE Conformance Testing, Part 3 - Further Analysis of the VV Set. This example further analyzed the FAE products for bsAb production using the "VV" set (bsAb Index #5-6) along with a negative control (WT) (bsAb Index #1) and a positive control (RL) (bsAb Index #3-4). Specifically, product quality analyzed by SDS-PAGE, bsAb formation efficiency analyzed by LC-MS, and separate or simultaneous binding to cognate antigens analyzed by biolayer interferometry (BLI) were compared between the FAE reaction products (output) and their monospecific parent antibodies (input).

[0343] 1. Quality of FAE products by SDS-PAGE:

[0344] The protein content in the FAE product and purified products (FAE output) of the monospecific parental antibodies (FAE input) were visualized and compared by SDS-PAGE (non-reducing).

[0345] The SDS-PAGE results are presented in Figure 26A. As shown in Figure 26A, similar banding patterns were observed between the input and output samples in all CH3 sets tested. No prominent bands below 60 kDa were observed. That is, the protein quality was consistent between the input and output.

[0346] 2. BsAb formation assessed by LC-MS

[0347] The FAE products of bsAbs containing the RL or VV set (bsAb index numbers 2 and 4-6) and their parental antibodies were analyzed by LC-MS under non-reducing conditions.

[0348] Exemplary LC-MS results are provided in FIG. 26B, where each panel shows an overlay of a chromatogram of the FAE reaction product (shown as "output") with a chromatogram of the corresponding purified product of the parent antibody (shown as "input"). As shown in FIG. 26B, both the RL and VV sets were successful in producing the intended bsAb. The % of each species ("aAAa", "aABa", or "aBba") obtained from the total full-sized antibody product, calculated based on the LC-MS results, is provided in Table 18. In Table 18, "aABa" represents an antibody with one heavy chain A ("A") and one heavy chain B ("B") each paired with a common light chain ("a"), i.e., the intended bsAb, "aAAa" represents parent antibody A with two heavy chains A each paired with a common light chain, i.e., ADI-29235 with the indicated variant CH3, and "aBBa" represents parent antibody B with two heavy chains B each paired with a common light chain, i.e., ADI-26908 with the indicated variant CH3. The % values ​​are % of all full-sized IgG molecules obtained. As shown in Table 18, the VV set achieved excellent bsAb production providing 100% of the intended bsAb, which is even higher than the values ​​achieved using the positive control (RL set). [Table 18]

[0349] 3. Binding kinetics to cognate antigen:

[0350] The binding kinetics of the FAE products (bsAb index numbers 1-2 and 4-6) and their monospecific parental antibodies to their cognate antigens were compared.

[0351] method

[0352] Binding to cognate antigen (HER2 or CD3) was measured by BLI using a ForteBio Octet HTX instrument (Molecular Devices). IgG was captured (1.5 nm) on an anti-human IgG capture (AHC) biosensor (Molecular Devices) and allowed to sit in PBSF (PBS with 0.1% (w / v) BSA) for a minimum of 30 min. After a short (60 s) baseline step in PBSF, the IgG-loaded biosensor chip was exposed to HER2 or CD3 (100 nM in PBSF) (180 s, 1000 rpm orbital shaking) and then immersed in PBSF (180 s, 1000 rpm orbital shaking) to measure any dissociation of antigen from the biosensor chip surface. Data for which the binding response was greater than 0.1 nm were aligned, step-by-step corrected (for the association step), and fitted to a 1:1 binding model using ForteBio data analysis software version 11.1.

[0353] result

[0354] Exemplary binding kinetics curves are provided in Figure 26C. The binding kinetics of the bsAbs to their cognate antigens were consistent with those of their corresponding monospecific parent antibodies. The binding kinetics was not significantly affected by the CH3 substitutions.

[0355] 4. Simultaneous antigen binding:

[0356] Finally, we tested whether the FAE products (bsAb matches 1-2 and 4-6) could bind to the two cognate antigens simultaneously and compared the binding kinetics to that of their parent antibodies.

[0357] method

[0358] Simultaneous antigen binding was tested on a ForteBio Octet HTX instrument (Sartorius, Göttingen, Germany) at 25° C. The binding kinetics of the individual bsAbs and each monospecific parent antibody to HER2 and then CD3, or CD3 and then HER2, were analyzed. All reagents were formulated in phosphate buffered saline (PBSF) with 0.1% (w / w) BSA.

[0359] To test binding to HER2 and then CD3, monomeric HER2-moFc (100 nM) was first loaded onto an anti-mouse Fc IgG capture sensor chip (Sartorius, Göttingen, Germany) and then allowed to sit in PBSF for a minimum of 15 min. These loaded sensor chips were first exposed to wells containing PBSF (60 s) to establish a stable baseline for the assay, followed by exposure to bsAb (100 nM) (180 s) and finally to CD3 (100 nM) (600 s).

[0360] To test binding to CD3 and then HER2, monomeric CD3-moFc (100 nM) was first loaded onto an anti-mouse Fc IgG capture sensor chip (Sartorius, Göttingen, Germany) and then allowed to sit in PBSF for a minimum of 15 min. These loaded sensor chips were first exposed to wells containing PBSF (60 s) to establish a stable baseline for the assay, followed by exposure to bsAb (100 nM) (180 s) and finally to HER2 (100 nM) (600 s).

[0361] BsAbs with sufficient binding responses in the last two steps of the assay were considered as dual binders.

[0362] result

[0363] Exemplary binding kinetic curves are provided in Figure 26D. As shown in Figure 26D, the FAE products from the VV and RL sets showed simultaneous binding to HER2 and CD3, regardless of whether the FAE products were exposed to HER2 or CD3 first.

[0364] Example 17: cFAE suitability study, part 4 - glutathione exposure. This example tested whether antibodies containing a VV set produced by a FAE-based method are stable in the presence of glutathione (GSH). Specifically, Example 17 tested whether CH3 heterodimers generated by FAE under 2-MEA dissociate and reassociate with another CH3 domain generated from another (homo- or hetero-) CH3 set when exposed to GSH. The stability was compared to that of the RL set.

[0365] method

[0366] The following steps 1 and 2 were performed for each RL set and VV set.

[0367] Step 1: First, a first anti-HER2 IgG1 was produced and purified, comprising (i) the ADI-29235 variable domain and (ii) one variant CH3 (i.e., two identical CH3 domains) of the test CH3 set. A second anti-HER2 IgG1 was also produced and purified, comprising (i) the ADI-29235 variable domain and (ii) the other variant CH3 (i.e., two identical CH3 domains) of the test CH3 set. Next, the mixture of the first anti-HER2 IgG1 and the second anti-HER2 IgG1 was subjected to FAE reaction essentially as described in Example 15, using 75 mM 2-MEA and incubation at 30° C. for 5 hours, to obtain an anti-HER2, CH3 hetero-IgG1 comprising (i) the ADI-29235 variable domain and (ii) the test CH3 set.

[0368] Similarly, a first anti-CD3 IgG1 was produced and purified, comprising (i) the ADI-26908 variable domain and (ii) one variant CH3 of the test CH3 set (i.e., two identical CH3 domains). A second anti-CD3 IgG1 was also produced and purified, comprising (i) the ADI-26908 variable domain and (ii) the other variant CH3 of the test CH3 set (i.e., two identical CH3 domains). The mixture of the first anti-CD3 IgG1 and the second anti-CD3 IgG1 was then subjected to FAE reaction essentially as described in Example 15, using 75 mM 2-MEA and incubation at 30° C. for 5 hours, to obtain an anti-CD3, CH3 hetero-IgG1 comprising (i) the ADI-26908 variable domain and (ii) the test CH3 set.

[0369] Step 2: Anti-HER2, CH3 heterodimer antibodies were mixed with (I) a first anti-CD3 IgG1, (II) a second anti-CD3 IgG1, or (III) an anti-CD3, CH3 heterodimer IgG1, placed in a mildly reducing environment containing 0.5 mM GSH, and incubated for 24 hours at 37° C. (This incubation process with GSH is referred to herein as “GDH exposure.”) The GSH exposure products were analyzed by IEX to determine whether further FAEs had occurred.

[0370] The experimental scheme for Example 17 is summarized in FIG. 27A.

[0371] result Figure 27B shows exemplary IEX results for the RL and VV sets during FAE using 2-MEA in step 1. Figures 27C-27E provide exemplary IEX results for the RL and VV sets during GSH exposure in step 2. Each graph panel shows an overlay of the chromatograms of the GSH-exposed product and two GSH-exposed input antibodies (i.e., anti-HER2, CH3 heterodimer, and (I) the first anti-CD3 IgG1 in the case of Fig. 27C, (II) the second anti-CD3 IgG1 in the case of Fig. 27D, or (III) the anti-CD3, CH3 heterodimer in the case of Fig. 27E). As shown in Figs. 27C-27E, GSH exposure did not result in new IEX peaks, indicating that chain reassociation between the GSH-exposed input antibodies did not occur. That is, the CH3 heterodimer generated by FAE under 2-MEA is stable and does not reassociate with another CH3 domain generated from another (homo- or hetero-) CH3 set in the presence of GSH. The stability of the VV set under GSH stress was comparable to that of the RL set.

[0372] Example 18: FAE under 75 mM 2-MEA for 5 hours at 30° C. does not cause dissociation between heavy and light chains. In this example, it was tested whether the cFAE reaction conditions used in Examples 15 to 17 caused dissociation between heavy and light chains. The BsAbs shown in Table 19, each of which contained (i) a half antibody specific to a first antigen containing heavy chain A and light chain A, and (ii) a half antibody specific to a second antigen containing heavy chain B and light chain B, were the bsAbs of interest in this example. The variable sequences used were derived from panitumumab (anti-EGFR), nivolumab (anti-PD-1), or ingatuzumab (anti-EGFR). [Table 19]

[0373] method

[0374] For each bsAb listed in Table 19, the respective monospecific parent antibody (i.e., antibody A specific for a first antigen with the indicated CH3 modifications and antibody B specific for a second antigen) was produced in CHO cells and subjected to Protein A-based affinity purification. The purified products were then subjected to the following FAE reaction steps. The FAE reaction products were digested with GingisKHAN® enzyme to obtain Fab fragments, which were analyzed by LC-MS.

[0375] FAE Response:

[0376] A 10x 2-mercaptoethylamine-HCl (2-MEA) stock solution (750 mM) was prepared by dissolving 1.70 g of 2-MEA in 20 mL of PBS. The pH was adjusted to 7.4 using approximately 600-700 µL of 2 N NaOH.

[0377] For each bsAb tested, 500 μg of the corresponding anti-HER2 full size antibody (250 μL 2 mg / mL in PBS) and 500 μg of the corresponding anti-CD3 full size antibody (250 μL 2 mg / mL in PBS) were placed in a well of a deep well plate. For bsAb index number 3, 500 μg of panitumumab with K409R and 500 μg of nivolumab with F405L were used. Then, 400 μL of PBS was added to each well, followed by 100 μL of 10x 2-MEA stock solution, resulting in a final 2-MEA concentration of 75 mM. Samples were incubated stationary at 30° C. for 5 hours.

[0378] 2-MEA was removed by buffer exchange via diafiltration using 10 kDa MWCO spin tubes (<500 μL + 3 mL x 6). Samples were then incubated at 4° C. for an additional 48 hours.

[0379] result

[0380] The Fab species identified by LC-MS are provided in Table 20. In Table 20, "aA" represents a Fab derived from a half antibody containing one heavy chain A ("A") and one light chain A ("a"); "bA" represents a Fab derived from a half antibody containing one heavy chain A ("A") and one light chain B ("b"); "aB" represents a Fab derived from a half antibody containing one heavy chain B ("B") and one light chain A ("a"); and "bB" represents a Fab derived from a half antibody containing one heavy chain B ("B") and one light chain B ("b"). That is, "aA" and "bB" are cognate pairs, and "bA" and "aB" are non-cognate pairs. The % values ​​are % of the total Fab obtained by digestion of the FAE products. As shown in Table 20, no non-cognate pairs were found in any of the specificity combinations tested. That is, the cFAE reaction conditions do not disrupt the disulfide bonds between the heavy and light chains. [Table 20]

[0381] Example 19: FAEs with alternative variable domains To confirm the broad applicability of FAE to use with various parent antibody combinations, additional FAE reactions were performed using human IgG1 antibodies directed against different targets, each with a different variable domain pair. Parent antibody constructs were prepared using sequences encoding antibody variable domains derived from mosunetuzumab, panitumumab, or nivolumab; CH2 domain Fc silencing mutations (L234A, L235A, and P329A according to EU numbering); and either Y407V or T366V CH3 mutations. The specific pairs tested are shown in the table below. [Table 21]

[0382] Parent antibody constructs were expressed in CHO cells and isolated by protein A-based affinity purification. For the FAE reaction, a 10x 2-mercaptoethylamine-HCl (2-MEA) stock solution (750 mM) was prepared by dissolving 1.70 g of 2-MEA in 20 mL of PBS. The pH was adjusted to 7.4 using approximately 600-700 μL of 2 N NaOH. 500 μg of each parent antibody (250 μL of 2 mg / mL in PBS) was placed into a well of a deep-well plate. Then, 400 μL of PBS, followed by 100 μL of 10x 2-MEA stock solution, was added to each well to obtain a final 2-MEA concentration of 75 mM. Samples were incubated at 30 °C for 5 h.

[0383] Each sample was desalted using a SEPHADEX® G25 plate (1 mL split across three wells), buffer exchanged into PBS, and 2-MEA was removed (to achieve approximately <50 μM). Samples were then incubated at 4° C. for 48 hours.

[0384] The resulting antibody output was tested for (1) percent protein recovered, (2) percent intact antibody monomer by size exclusion chromatography (SEC), and (3) formation of the desired heterodimeric antibody species by comparison of retention times obtained by analytical ion exchange chromatography (IEX) with parental antibody retention times (see table below). [Table 22]

[0385] Results showed greater than 70% protein recovery, with greater than 95% intact, non-aggregated antibody. Furthermore, output retention times were closer to the average parent antibody retention time than to the individual parent antibody retention times, indicating successful heterodimer chain pairing.

[0386] Example 20: FAE generation of biparatopic antibodies Biparatopic antibodies are bispecific antibodies in which each paratope is directed to a different epitope of the same antigen. To evaluate the suitability of FAE for the generation of biparatopic antibodies, FAE was performed using two parent antibody groups, Group 1 and Group 2. The Group 1 antibodies each targeted a different epitope of the same viral antigen. The Group 2 antibodies each targeted a different epitope of the same cell surface antigen. Each Group 1 and Group 2 antibody was expressed in CHO cells as a human IgG1 antibody with either the Y407V (Parent Antibody 1) or T366V (Parent Antibody 2) CH3 mutation.

[0387] Parent antibody constructs were expressed in CHO cells and isolated by protein A-based affinity purification. For the FAE reaction, a 10x 2-mercaptoethylamine-HCl (2-MEA) stock solution (750 mM) was prepared by dissolving 1.70 g of 2-MEA in 20 mL of PBS. The pH was adjusted to 7.4 using approximately 600-700 μL of 2 N NaOH. 500 μg of each parent antibody (250 μL of 2 mg / mL in PBS) was placed into a well of a deep-well plate. Then, 400 μL of PBS, followed by 100 μL of 10x 2-MEA stock solution, was added to each well to obtain a final 2-MEA concentration of 75 mM. Samples were incubated at 30 °C for 5 h.

[0388] Each sample was desalted using a SEPHADEX® G25 plate (1 mL split across three wells), buffer exchanged into PBS, and 2-MEA was removed (to achieve approximately <50 μM). Samples were then incubated at 4° C. for 48 hours.

[0389] The antibody output obtained was examined for the percentage of recovered protein and the formation of the desired heterodimeric antibody species by comparison of retention times obtained by analytical ion exchange chromatography (IEX) with the parental antibody retention times (see table below). [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5] [Table 23-6] [Table 23-7]

[0390] Results showed greater than 60% protein recovery in all FAE reactions. Furthermore, the output retention times were closer to the average parent antibody retention times than to the individual parent antibody retention times, indicating successful heterodimer chain pairing and biparatopic antibody formation.

[0391] Example 21: FAE with conjugated antibody fragments To examine the compatibility of FAE with antibodies with conjugated antibody fragments, FAE was performed using an IgG1 antibody with a combined scFv fragment. The parent antibody was produced by expression of constructs encoding antibody heavy and light chains in CHO cells. The encoded light chain contained an anti-CD3 scFv fragment linked to the light chain C-terminus via a flexible linker (GGGGSGGGGS (SEQ ID NO: 718)). The anti-CD3 scFv variable domain was also linked by a flexible linker (GGGGSGGGGSGGGGS (SEQ ID NO: 719)). The CH3 domain of the corresponding heavy chain contained either a Y407V or T366V mutation to promote favorable pairing.

[0392] For the FAE reaction, a 10x 2-mercaptoethylamine-HCl (2-MEA) stock solution (750 mM) was prepared by dissolving 1.70 g of 2-MEA in 20 mL of PBS. The pH was adjusted to 7.4 using approximately 600-700 μL of 2 N NaOH. 500 μg of each parent antibody (250 μL of 2 mg / mL in PBS) was placed into a well of a deep-well plate. Then, 400 μL of PBS, followed by 100 μL of 10x 2-MEA stock solution, was added to each well to obtain a final 2-MEA concentration of 75 mM. Samples were incubated at 30 °C for 5 h.

[0393] Each sample was desalted using a SEPHADEX® G25 plate (1 mL split across three wells), buffer exchanged into PBS, and 2-MEA was removed (to achieve approximately <50 μM). Samples were then incubated at 4° C. for 48 hours.

[0394] The resulting antibody output was examined by SEC, IEX, and LC-MS for the formation of the desired heterodimeric antibody species. Heterodimeric species with correct chain pairing were evident from the analysis demonstrating the suitability of FAE for bispecific antibody formation from parent antibodies with conjugated antibody fragments.

[0395] Exemplary embodiments Described herein below are several exemplary embodiments according to the present disclosure. Embodiment 1. A first immunoglobulin heavy chain constant region 3 ("CH3") domain variant polypeptide, optionally comprising an amino acid substitution at one or more of the following amino acid positions: 364, 366, 368, 370, 399, 400, 405, 407, and 409 according to EU numbering, such that the CH3 domain variant polypeptide preferentially forms a heterodimer with a second CH3 domain variant polypeptide; the second CH3 domain variant polypeptide comprising: (a) differs from the first CH3 domain variant polypeptide in at least one amino acid; and (b) comprising an amino acid substitution at one or more of the following positions according to EU numbering: 364, 366, 368, 370, 399, 400, 405, 407, and 409; Optionally, (i) the first CH3 domain variant polypeptide further comprises the amino acid substitution S354C and the second CH3 domain variant polypeptide further comprises the amino acid substitution Y349C; or (ii) the first CH3 domain variant polypeptide further comprises the amino acid substitution Y349C and the second CH3 domain variant polypeptide further comprises the amino acid substitution S354C; Further optionally, (i) if the amino acid substitution in the first CH3 domain variant polypeptide consists of T366Y, then the amino acid substitution in the second CH3 domain variant polypeptide does not consist of Y407T; (ii) if the amino acid substitution in the first CH3 domain variant polypeptide consists of Y407T, then the amino acid substitution in the second CH3 domain variant polypeptide does not consist of T366Y; (iii) if the amino acid substitution in the first CH3 domain variant polypeptide consists of T366W, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of T366S, L368A, and Y407V; (iv) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of T366S, L368A, and Y407V, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of T366W; (v) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of S354C and T366W, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of Y349C, T366S, L368A, and Y407V; (vi) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of Y349C, T366S, L368A, and Y407V, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of S354C and T366W; (vii) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of S364H and F405A, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of Y349T and T394F; (viii) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of Y349T and T394F, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of S364H and F405A; (ix) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of T350V, L351Y, F405A, and Y407V, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of T350V, T366L, K392L, and T394W; (x) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of T350V, T366L, K392L, and T394W, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of T350V, L351Y, F405A, and Y407V; (xi) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of K392D and K409D, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of E356K and D399K; (xii) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of E356K and D399K, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of K392D and K409D; (xiii) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of D221E, P228E, and L368E, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of D221R, P228R, and K409R, and the first CH3 domain and the second CH3 domain are derived from a human IgG1 CH3 domain; (xiv) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of D221R, P228R, and K409R, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of D221E, P228E, and L368E, and the first CH3 domain and the second CH3 domain are derived from a human IgG1 CH3 domain; (xv) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of C223E, P228E, and L368E, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of C223R, E225R, P228R, and K409R, and the first CH3 domain and the second CH3 domain are derived from a human IgG2 CH3 domain; (xvi) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of C223R, E225R, P228R, and K409R, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of C223E, P228E, and L368E, and the first CH3 domain and the second CH3 domain are derived from a human IgG2 CH3 domain; (xvii) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of K360E and K409W, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of Q347R, D399V, and F405T; (xviii) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of Q347R, D399V, and F405T, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of K360E and K409W; (xix) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of K360E, K409W, and Y349C, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of Q347R, D399V, F405T, and S354C; (xx) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of Q347R, D399V, F405T, and S354C, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of K360E, K409W, and Y349C; (xxi) if the amino acid substitution in the first CH3 domain variant polypeptide consists of 366K, or consists of 366K and 351K, then the amino acid substitution in the second CH3 domain variant polypeptide does not consist of 351D, does not consist of 349E, does not consist of 349D, does not consist of 368E, does not consist of 368D, does not consist of 349E and 355E, does not consist of 349E and 355D, does not consist of 349D and 355E, or does not consist of 349D and 355D; (xxii) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of 351D, consist of 349E, consist of 349D, consist of 368E, consist of 368D, consist of 349E and 355E, consist of 349E and 355D, consist of 349D and 355E, or consist of 349D and 355D, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of 366K, or do not consist of 366K and 351K; (xxiii) if the amino acid substitution in the first CH3 domain variant polypeptide consists of F405L, then the amino acid substitution in the second CH3 domain variant polypeptide does not consist of K409R; (xxiv) if the amino acid substitution in the first CH3 domain variant polypeptide consists of K409R, then the amino acid substitution in the second CH3 domain variant polypeptide does not consist of F405L; (xxv) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of K360D, D399M, and Y407A, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of E345R, Q347R, T366V, and K409V; (xxvi) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of E345R, Q347R, T366V, and K409V, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of K360D, D399M, and Y407A; (xxvii) if the amino acid substitutions in the first CH3 domain variant polypeptide consist of Y349S, K370Y, T366M, and K409V, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of E356G, E357D, S364Q, and Y407A; and (xxviii) A first CH3 domain variant polypeptide, wherein if the amino acid substitutions in the first CH3 domain variant polypeptide consist of E356G, E357D, S364Q, and Y407A, then the amino acid substitutions in the second CH3 domain variant polypeptide do not consist of Y349S, K370Y, T366M, and K409V. Embodiment 2. A first CH3 domain variant polypeptide according to embodiment 1, comprising: (I) comprises an amino acid substitution at one or more of the following amino acid positions: 364, 366, 400, 407, and 409, and optionally forms a heterodimer with a second CH3 domain variant polypeptide that comprises one or more of the following amino acid positions: 366, 368, 370, 399, 405, and 407; or (II) The first CH3 domain variant polypeptide of embodiment 1, comprising an amino acid substitution at one or more of the following amino acid positions: 366, 368, 370, 399, 405, and 407, and optionally preferentially forming a heterodimer with a second CH3 domain variant polypeptide comprising one or more of the following amino acid positions: 364, 366, 400, 407, and 409. Embodiment 3. A first CH3 domain variant polypeptide according to embodiment 1 or 2, wherein the first CH3 domain is (I) one or more of the following amino acid positions: 364, 366, 400, 407, and 409; or (II) The first CH3 domain variant polypeptide of embodiment 1 or 2, comprising amino acid substitutions only at one or more of the following amino acid positions: 366, 368, 370, 399, 405, and 407. Embodiment 4. A first CH3 domain variant polypeptide according to any one of embodiments 1-2, wherein the amino acid substitution in the first CH3 domain variant polypeptide is (i) 366th, (ii) 366th and 407th, (iii) 364th, 366th, and 409th, (iv) 366th, 368th, and 407th, (v) 368th, (vi) 407th, (vii) 366th and 368th, (viii) 366th and 409th, (ix) 368th and 370th, (x) 368th and 4 07, (xi) 399 and 405, (xii) 400 and 409, (xiii) 364, 407, and 409, (xiv) 366, 368, and 370, (xv) 366, 399, and 405, (xvi) 366, 400, and 409, (xvii) 366, 407, and 4 3. The first CH3 domain variant polypeptide according to any one of embodiments 1 to 2, comprising or consisting of amino acid substitutions at positions 366, 399, 405 and 407, (xviii) 368, 400 and 409, (xix) 399, 405 and 407, (xx) 400, 407 and 409, (xxi) 366, 399, 405 and 407, (xxii) 366, 399, 405 and 409, (xxiii) 366, 400, 407 and 409, (xxiv) 366, 368, 399, 405 and 407, or (xxv) 366, 368, 400, 407 and 409. Embodiment 5. A first CH3 domain variant polypeptide according to any one of embodiments 1 to 4, (i) the amino acid substitution in the first CH3 domain variant polypeptide comprises or consists of an amino acid substitution at position 366, and the amino acid substitution in the second CH3 domain variant polypeptide comprises or consists of amino acid substitutions at positions 366 and 407; (ii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366 and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of an amino acid substitution at position 366; (iii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 364, 366, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 368, and 407; (iv) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 368, and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 364, 366, and 409; (v) the amino acid substitution in the first CH3 domain variant polypeptide comprises or consists of an amino acid substitution at position 366, and optionally the amino acid substitution in the second CH3 domain variant polypeptide comprises or consists of an amino acid substitution at position 407; (vi) the amino acid substitution in the first CH3 domain variant polypeptide comprises or consists of an amino acid substitution at position 368, and the amino acid substitution in the second CH3 domain variant polypeptide comprises or consists of amino acid substitutions at positions 366 and 409; (vii) the amino acid substitution in the first CH3 domain variant polypeptide comprises or consists of an amino acid substitution at position 407, and optionally the amino acid substitution in the second CH3 domain variant polypeptide comprises or consists of an amino acid substitution at position 366; (viii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366 and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 368, or at positions 368 and 370; (ix) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 368 and 370, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366 and 409. (x) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 399 and 405, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 400 and 409; (xi) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 400 and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 399 and 405; (xii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 364, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 368, and 370; (xiii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 368, and 370, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 364, 407, and 409; (xiv) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366 and 368, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 407, and 409; (xv) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366 and 368; (xvi) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 368 and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 364, 366, and 409; (xvii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 364, 366, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 368 and 407; (xviii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 399, and 405, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 400, 407, and 409, 366, 400, 407, and 409, or 366, 368, 400, 407, and 409; (xix) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 400, 407, and 409, 366, 400, 407, and 409, or 366, 368, 400, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 399, and 405; (xx) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 400, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407; (xxi) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 400, and 409; (xxii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 368, 400, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409; (xxiii) the amino acid substitutions in the first CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide comprise or consist of amino acid substitutions at positions 368, 400, and 409; (xxiv) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 366 and 368, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 366, 407, and 409; (xxv) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 366, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 366 and 368; (xxvi) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 368 and 407, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 364, 366, and 409; (xxvii) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 364, 366, and 409, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 368 and 407; (xxviii) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 366, 399, and 405, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 400, 407, and 409, 366, 400, 407, and 409, or 366, 368, 400, 407, and 409; (xxix) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 400, 407, and 409, 366, 400, 407, and 409, or 366, 368, 400, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 366, 399, and 405; (xxx) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 366, 400, and 409, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407; (xxxi) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 399, 405, and 407, 366, 399, 405, and 407, or 366, 368, 399, 405, and 407, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 366, 400, and 409; (xxxii) the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 368, 400, and 409, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409; or (xxxiii) The first CH3 domain variant polypeptide according to any one of embodiments 1 to 4, wherein the amino acid substitutions in the first CH3 domain variant comprise or consist of amino acid substitutions at positions 366, 399, 405, and 409, and optionally the amino acid substitutions in the second CH3 domain variant comprise or consist of amino acid substitutions at positions 368, 400, and 409. Embodiment 6. A first CH3 domain variant polypeptide according to any one of embodiments 1-2 or 4-5, (i) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349 and 366, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, and 407; (ii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354 and 366; (iii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 364, 366, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, 368, and 407; (iv) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, 368, and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 364, 366, and 409; (v) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349 and 366, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354 and 407; (vi) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349 and 368, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, and 409; (vii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349 and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354 and 366; (viii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354 and 368, or at positions 354, 368, and 370; (ix) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 368, and 370, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, and 409; (x) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 399, and 405, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 400, and 409; (xi) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 400, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 399, and 405; (xii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 364, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, 368, and 370; (xiii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, 368, and 370, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 364, 407, and 409; (xiv) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, and 368, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, 407, and 409; (xv) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, and 368; (xvi) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 368, and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 364, 366, and 409; (xvii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 364, 366, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 368, and 407; (xviii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, 399, and 405, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 400, 407, and 409, 354, 366, 400, 407, and 409, or 354, 366, 368, 400, 407, and 409; (xix) the amino acid substitutions in the first CH3 domain variant polypeptide are at positions 349, 400, 407, and 409, or at positions 349, 366, 400, 407, and 409, or at positions 349, 366, 368, 400, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide are at positions 354, 366, 399, and 405; (xx) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, 400, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 399, 405, and 407, 354, 366, 399, 405, and 407, or 354, 366, 368, 399, 405, and 407; (xxi) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 399, 405, and 407, or at positions 349, 366, 399, 405, and 407, or at positions 349, 366, 368, 399, 405, and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, 400, and 409; (xxii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 368, 400, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, 399, 405, and 409; or (xxiii) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, 399, 405, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 368, 400, and 409; (xxiv) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 349, 366, and 368, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, 407, and 409; (xxv) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 366, 407, and 409, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at positions 354, 366, and 368; (xxvi) the amino acid substitutions in the first CH3 domain variant polypeptide consist of amino acid substitutions at positions 349, 368, and 407, and optionally the amino acid substitutions in the second CH3 domain variant polypeptide consist of amino acid substitutions at posit...

Claims

1. 1. A method for producing a heteromeric molecule, the heteromeric molecule comprising: (A) a first polypeptide comprising a first variant CH3 domain polypeptide, wherein the first variant CH3 domain polypeptide comprises a T366V substitution according to EU numbering; and (B) a second polypeptide comprising a second variant CH3 domain polypeptide, wherein the second variant CH3 domain polypeptide comprises a Y407V substitution according to EU numbering; the first polypeptide and the second polypeptide are optionally linked or paired to each other via at least one disulfide bond; The method comprises: (i) incubating under a reducing environment (i-1) a first parent molecule comprising at least two of said first polypeptides bound or paired to each other, optionally via at least one disulfide bond, and (i-2) a second parent molecule comprising at least two of said second polypeptides bound or paired to each other, optionally via at least one disulfide bond; (ii) placing the incubation product of step (i) in a less reducing or non-reducing environment, thereby forming said heteromeric molecules; Optionally, (a) the first variant CH3 domain polypeptide is derived from a CH3 domain of human IgG, and / or the second variant CH3 domain polypeptide is derived from a CH3 domain of human IgG, and optionally the T366V substitution is relative to the CH3 domain of human IgG, and / or the Y407V substitution is relative to the CH3 domain of human IgG; (b) the first variant CH3 domain polypeptide is derived from the CH3 domain of human IgG1, and / or the second variant CH3 domain polypeptide is derived from the CH3 domain of human IgG1, and optionally the T366V substitution is relative to SEQ ID NO: 1, 2, 3, or 4, and / or the Y407V substitution is relative to SEQ ID NO: 1, 2, 3, or 4; (c) the first variant CH3 domain polypeptide is derived from the CH3 domain of human IgG2, and / or the second variant CH3 domain polypeptide is derived from the CH3 domain of human IgG2, and optionally the T366V substitution is relative to SEQ ID NO: 722, and / or the Y407V substitution is relative to SEQ ID NO: 722; (d) the first variant CH3 domain polypeptide is derived from the CH3 domain of human IgG3, and / or the second variant CH3 domain polypeptide is derived from the CH3 domain of human IgG3, and optionally the T366V substitution is relative to SEQ ID NO: 723, and / or the Y407V substitution is relative to SEQ ID NO: 723, and / or (e) the first variant CH3 domain polypeptide is derived from the CH3 domain of human IgG4, and / or the second variant CH3 domain polypeptide is derived from the CH3 domain of human IgG4, and optionally the T366V substitution is relative to SEQ ID NO: 724, and / or the Y407V substitution is relative to SEQ ID NO: 724; Optionally, said heteromeric molecule has the following characteristics: (A) the first polypeptide further comprises a first antigen-binding domain; (B) the second polypeptide further comprises a second antigen-binding domain; (C) the heteromeric molecule further comprises a third polypeptide optionally comprising a third antigen-binding domain, optionally wherein the third polypeptide is bound or conjugated to the first polypeptide; and / or (D) the heteromeric molecule further comprises a fourth polypeptide optionally comprising a fourth antigen-binding domain, optionally wherein the fourth polypeptide is bound or conjugated to the second polypeptide; Further optionally, the heteromeric molecule is a multispecific antibody or antigen-binding antibody fragment, optionally comprising a structure as shown in any one of Figures 2-8, and optionally, the heteromeric molecule comprises (a) an IgG or (b) an IgG and one or more scFvs conjugated to the IgG, and further optionally comprises an IgG1, IgG2, IgG3, or IgG4 constant region.

2. Features include: (I) (I-1-i) the first polypeptide comprises a first antigen-binding domain forming a first antigen-binding site specific for a first epitope, and / or (I-1-ii) the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain forming a third antigen-binding site specific for a third epitope, optionally wherein the first epitope is the same as or different from the third epitope; or (I-2) the first polypeptide comprises a first antigen-binding domain, the heteromeric molecule comprises a third polypeptide comprising a third antigen-binding domain, and the first antigen-binding domain and the third antigen-binding domain form a first antigen-binding site specific for a first epitope; and / or (II) (II-1-i) the second polypeptide comprises a second antigen-binding domain forming a second antigen-binding site specific for a second epitope, and / or (I-1-ii) the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain forming a fourth antigen-binding site specific for a fourth epitope, optionally wherein the second epitope is the same as or different from the fourth epitope; or (II-2) The method of claim 1, comprising one or more of: the second polypeptide comprises a second antigen-binding domain; the heteromeric molecule comprises a fourth polypeptide comprising a fourth antigen-binding domain; and the second antigen-binding domain and the fourth antigen-binding domain form a second antigen-binding site specific for a second epitope.

3. The step (i) has the following characteristics: (a) the incubation is carried out at a temperature of about 15°C to about 40°C, about 20°C to about 40°C, about 25°C to about 35°C, about 28°C to about 32°C, or about 29°C to about 31°C, or about 30°C; (b) the incubation is performed for about 30 minutes to about 20 hours, about 1 hour to about 15 hours, about 2 hours to about 10 hours, about 3 hours to about 7 hours, or about 4 hours to about 6 hours, or about 5 hours; (c) the incubation is carried out at about 30° C. for about 5 hours; (d) the reducing environment comprises at least one reducing agent, optionally at least one weak reducing agent; (e) the reducing environment comprises at least one reducing agent selected from 2-mercaptoethylamine (2-MEA), b-mercaptoethanol (BME), L-cysteine, dithiothreitol (DTT), or dithionite; (f) the reducing environment is about 25 to about 125 mM, about 50 mM to about 100 mM, about 70 to about 80 mM, or about 75 mM 2-MEA; BME of about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM; about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM L-cysteine; about 15 to about 400 μM, about 20 to about 200 μM, about 25 to about 100 μM, about 30 to about 70 μM, or about 50 μM DTT; or at least one reducing agent selected from about 20 to about 500 μM, about 40 to about 250 μM, about 80 to about 150 μM, about 90 to about 120 μM, or about 100 μM dithionite; (g) the reducing environment comprises at least 2-MEA, optionally at about 75 mM; (h) the at least two of the first polypeptides are linked or paired with each other via at least one disulfide bond, and / or the at least two of the second polypeptides are linked or paired with each other via at least one disulfide bond. (i) the first antibody and / or the second antibody is produced in a mammalian cell, a yeast cell, an insect cell, a plant cell, or a bacterial cell; and / or 3. The method of claim 1 or 2, comprising one or more of: (j) the first antibody and / or the second antibody are produced in Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) cells.

4. The step (ii) has the following characteristics: (a) the disposing is optionally performed by buffer exchange into phosphate buffered saline (PBS); (b) the disposing is optionally performed by buffer exchange into PBS by desalting; (c) the disposing is performed by buffer exchange by diafiltration, optionally into PBS; and / or (d) the disposing is effected by the addition of an oxidizing agent.

5. (iii) incubating the product of step (ii) in a reducing or non-reducing environment, optionally at a temperature of about 1° C. to about 20° C., about 2° C. to about 10° C., about 3° C. to about 5° C., or about 4° C., optionally for about 12 hours to about 154 hours, about 24 hours to about 96 hours, about 36 hours to about 72 hours, or about 48 hours; and / or 3. The method of claim 1 or 2, further comprising (iv) analyzing the amount of said multispecific antibody or antigen-binding antibody fragment in the product of step (ii) and / or step (iii) and / or purifying said multispecific antibody or antigen-binding antibody fragment from the product of step (ii) and / or step (iii), optionally wherein said analyzing and / or purifying is performed via chromatography, optionally LC-MS, IEX, and / or SEC.

6. The method of claim 1 or 2, wherein the heteromeric molecule comprises a multispecific antibody.

7. 7. The method of claim 6, wherein the first polypeptide comprises a first antibody heavy chain, the second polypeptide comprises a second antibody heavy chain, the first antibody heavy chain associated with a first antibody light chain, and the second antibody heavy chain associated with a second antibody light chain.

8. 8. The method of claim 7, wherein the multispecific antibody comprises a third polypeptide comprising a third antigen-binding domain and / or a fourth polypeptide comprising a fourth antigen-binding domain.

9. 9. The method of claim 8, wherein the third antigen-binding domain is associated with the first antibody heavy chain, the second antibody heavy chain, the first antibody light chain, or the second antibody light chain, and / or the fourth antigen-binding domain is associated with the first antibody heavy chain, the second antibody heavy chain, the first antibody light chain, or the second antibody light chain.

10. the third antigen-binding domain association and / or the fourth antigen-binding domain association comprises a linker, optionally a flexible linker, further optionally, the flexible linker comprises: (i) an amino acid sequence selected from the group consisting of GGGGS (SEQ ID NO:715), GGGS (SEQ ID NO:716), GGGGGS (SEQ ID NO:717), G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG; (ii) a plurality of repeats, optionally two, three, four, or five repeats, of an amino acid sequence selected from the group consisting of SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG; (iii) a (GS)n linker, a (GS)n linker, a (GS)n linker, a (GS)n linker, or a (G)n linker, wherein n is a natural number, optionally selected from 1 to 20, further optionally selected from 2, 3, 4, or 5; and / or (iv) the method of claim 9, comprising or consisting of the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 718) or GGGGSGGGGSGGGGGS (SEQ ID NO: 719).

11. 9. The method of claim 8, wherein the third antigen-binding domain and / or the fourth antigen-binding domain comprises a Fab or a single-chain Fv (scFv), Optionally, the third antigen-binding domain comprises an scFv comprising a heavy chain variable domain and a light chain variable domain linked by a disulfide bond and / or a linker, optionally a flexible linker, and further optionally, the linker is (i) an amino acid sequence selected from the group consisting of GGGGS (SEQ ID NO:715), GGGS (SEQ ID NO:716), GGGGGS (SEQ ID NO:717), G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG; (ii) a plurality of repeats, optionally two, three, four, or five repeats, of an amino acid sequence selected from the group consisting of SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, G, GG, GGG, GS, SG, GGS, GSG, SGG, GSS, SGS, and SSG; (iii) a (GS)n linker, a (GS)n linker, a (GS)n linker, a (GS)n linker, or a (G)n linker, wherein n is a natural number, optionally selected from 1 to 20, further optionally selected from 2, 3, 4, or 5; and / or (iv) A method comprising or consisting of the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 718) or GGGGSGGGGSGGGGGS (SEQ ID NO: 719).

12. The method of claim 6 , wherein the multispecific antibody comprises a biparatopic antibody.

13. 3. The method of claim 1 or 2, wherein the first parent molecule comprises a first IgG and the second parent molecule comprises a second IgG.

14. each of the at least two of the first polypeptides of the first IgG comprises a first antibody heavy chain comprising a first antigen-binding domain that forms a first antigen-binding site for a first epitope; and 14. The method of claim 13, wherein each of the at least two of the second polypeptides of the second IgG comprises a second antibody heavy chain comprising a second antigen binding domain that forms a second antigen binding site for a second epitope, and optionally the first epitope and the second epitope are part of different antigens or part of the same antigen, and / or the heteromeric molecule comprises an IgG comprising the first antibody heavy chain and the second antibody heavy chain.

15. (A)(I)(i) the T366V substitution is the only substitution in the first variant CH3 domain polypeptide, optionally relative to a human IgG CH3 domain; and optionally (i-1) the CH3 domain of human IgG1, optionally the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4; (i-2) the CH3 domain of human IgG2, optionally the amino acid sequence of SEQ ID NO: 722; (i-3) the CH3 domain of human IgG3, optionally the amino acid sequence of SEQ ID NO: 723; or (i-4) the CH3 domain of human IgG4, optionally corresponding to the amino acid sequence of SEQ ID NO: 724; and / or (ii) the Y407V substitution is the only substitution in the second variant CH3 domain polypeptide, optionally relative to a human IgG CH3 domain; and optionally (i-1) the CH3 domain of human IgG1, optionally the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4; (i-2) the CH3 domain of human IgG2, optionally the amino acid sequence of SEQ ID NO: 722; (i-3) the CH3 domain of human IgG3, optionally the amino acid sequence of SEQ ID NO: 723; or (i-4) the CH3 domain of human IgG4, optionally corresponding to the amino acid sequence of SEQ ID NO: 724; or (II) the first and second variant CH3 domain polypeptides are further modified to comprise one or more variant CH3 domain sets, optionally any of the variant CH3 domain sets described herein, and further optionally any of the variant CH3 domain sets described in any of the Tables; and / or (B) the heteromeric molecule comprises one or more CH2 domains, optionally one or more of the CH2 domains comprises one or more amino acid modifications, optionally wherein the one or more amino acid modifications are: (a) one or more Fc-silencing modifications; (b) one or more FcRn affinity-enhancing and / or half-life-extending modifications, and / or 3. The method of claim 1 or 2, comprising or consisting of any of the following modifications according to EU numbering: L234A, L235A, and P329A substitutions; L234A, L235A, and P329G substitutions, L234A and L235A substitutions, D265A and P329A substitutions, N297A substitutions, M252Y, S254T, and T256E substitutions, and / or M428L and N434S substitutions.

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