Method for preparing novel polypeptide by isopeptide bond formation

Abstract

To provide a novel polypeptide comprising an isopeptide bond. In one aspect, provided is a polypeptide comprising an isopeptide bond between first and second polypeptide chains, the polypeptide comprising: a) lysine; b) glutamine, glutamic acid, asparagine or aspartic acid; and c) glutamic acid or aspartic acid, wherein the first and second polypeptide chains each comprise a) and b) in a boundary region in which the first and second polypeptide chains in the three-dimensional structure of the polypeptide are adjacent to each other.

Description

Method for producing novel polypeptides by isopeptide bond formation

[0001] The present disclosure provides polypeptides containing isopeptide bonds and methods for making the same.

[0002] Proteins contain covalent amide bonds within polypeptide chains and sometimes disulfide bonds between cysteine ​​side chains. Inter-polypeptide chain interactions are important for protein conformation, and covalent interactions in particular strongly link polypeptide chains. Therefore, the introduction of additional covalent bonds can be useful for protein engineering.

[0003] A bond called an isopeptide bond is also known as a covalent bond in proteins, but it has only been found in a limited number of proteins, such as the SpyO128 protein of Streptococcus pyogenes (Non-Patent Document 1).

[0004] J Am Chem Soc. 2011 Jan 26;133(3):478-85.

[0005] The present inventors have investigated polypeptide design that forms isopeptide bonds between polypeptide chains and have created novel polypeptides containing isopeptide bonds. Based on this, the present disclosure provides polypeptides containing isopeptide bonds and methods for producing the same.

[0006] Thus, the present invention provides the following: (Item 1) A polypeptide chain having the following formula between a first and a second polypeptide chain: (wherein the dashed lines represent a first or second polypeptide chain, respectively), the method comprising the steps of: constructing a three-dimensional structure of intermediate A comprising the first and second polypeptide chains; identifying an amino acid sequence present in a boundary region where the first and second polypeptide chains are close to each other in the three-dimensional structure; mutating intermediate A by introducing at least one residue of: a) lysine; b) glutamine, glutamic acid, asparagine, or aspartic acid; and c) glutamic acid or aspartic acid into intermediate A to prepare intermediate B, wherein intermediate B comprises a), b), and c), and the first and second polypeptide chains in intermediate B comprise a) and b), respectively, in the boundary region; and obtaining the modified polypeptide by forming the interpeptide linkage in intermediate B. (Item 2) The method of any of the preceding items, wherein a) and / or b) are introduced based on the distance between a first amino acid residue of the first polypeptide chain and a second amino acid residue of the second polypeptide chain present in the boundary region and the types of the first and second amino acid residues. (Item 3) The method of any of the preceding items, wherein b) is asparagine or aspartic acid, and two amino acid positions having an alpha carbon distance of about 7 to 9 Å in the three-dimensional structure of intermediate A are identified as the amino acid positions of a) and b). (Item 4) The method of any of the preceding items, wherein b) is glutamine or glutamic acid, and two amino acid positions having an alpha carbon distance of about 8.5 to 10 Å in the three-dimensional structure of intermediate A are identified as the amino acid positions of a) and b). (Item 5) The method of any of the preceding items, wherein two amino acid positions whose side chains are located inside the three-dimensional structure of intermediate A are identified as the amino acid positions of a) and b). (Item 6) The method according to any one of the preceding items, further comprising a step of constructing the three-dimensional structure of the intermediate B.(Item 7) The method of any of the preceding items, wherein, in the three-dimensional structure of intermediate B, the distance between the nitrogen atom at the ζ-position of residue a) and the oxygen atom of the carboxy group in the side chain of residue c) is about 2 to 5 Å. (Item 8) The method of any of the preceding items, wherein, in the three-dimensional structure of intermediate B, the distance between the oxygen atom of the carboxy group or amide group in the side chain of residue b) and the oxygen atom of the carboxy group in the side chain of residue c) is about 2 to 5 Å. (Item 9) The method of any of the preceding items, wherein, in the three-dimensional structure of intermediate B, the distance between the nitrogen atom at the ζ-position of residue a) and the oxygen atom of the carboxy group or amide group in the side chain of residue b) is about 2 to 5 Å. (Item 10) The method of any of the preceding items, wherein the first polypeptide chain comprises c). (Item 11) The method of any of the preceding items, further comprising the step of determining the hydrophobicity of the interface region. (Item 12) Any of the methods described above, further comprising a step of deleting a hydrophilic amino acid residue in the amino acid sequence present in the boundary region, substituting an amino acid residue with a hydrophobic amino acid residue, or inserting a hydrophobic amino acid residue. (Item 13) Any of the methods described above, further comprising introducing a hydrophobic amino acid having an α-carbon within 12 Å of the nitrogen atom at the ζ-position of residue a) in the three-dimensional structure of intermediate B. (Item 14) Any of the methods described above, further comprising introducing a hydrophobic amino acid having a side chain with a different volume so as to fill the space in the boundary region. (Item 15) Any of the methods described above, wherein the first and second polypeptide chains are contained in different molecules. (Item 16) Any of the methods described above, further comprising a step of deleting or substituting with another amino acid at least one of the two cysteine ​​residues that form a disulfide bond between the first polypeptide chain and the second polypeptide chain in intermediate A. (Item 17) The modified polypeptide and a third polypeptide chain contained in a molecule different from the modified polypeptide have the following formula: 16. The method of claim 15, further producing a secondary modified polypeptide comprising an interpeptide linkage represented by the formula: (wherein the dashed line represents a bond to the polypeptide backbone), the method comprising: constructing a three-dimensional structure of secondary intermediate A comprising the modified polypeptide and the third polypeptide chain; identifying an amino acid sequence present in a boundary region where the modified polypeptide and the third polypeptide chain are adjacent to each other in the three-dimensional structure; mutating the secondary intermediate A by introducing at least one residue of a) lysine, b) glutamine, glutamic acid, asparagine, or aspartic acid, and c) glutamic acid or aspartic acid into the secondary intermediate A to prepare a secondary intermediate B, wherein the secondary intermediate B comprises a), b), and c), and the modified polypeptide and the third polypeptide chain in the secondary intermediate B comprise a) and b), respectively, in the boundary region; and forming the interpeptide linkage in the secondary intermediate B to obtain the secondary modified polypeptide. (Item 18) A compound having the following formula between the first and second polypeptide chains: (wherein the dashed line represents a bond to the polypeptide backbone), comprising: a) lysine, b) glutamine, glutamic acid, asparagine, or aspartic acid, and c) glutamic acid or aspartic acid, wherein the first and second polypeptide chains comprise a) and b), respectively, at the boundary region where the first and second polypeptide chains are adjacent to each other in the three-dimensional structure of the polypeptide. (Item 19) The polypeptide of any of the preceding items, which is an antibody or a T-cell receptor (TCR). (Item 20) The polypeptide of any of the preceding items, wherein the interpeptide linkage is present in a constant region. (Item 21) The polypeptide of any of the preceding items, comprising an interpeptide linkage between the heavy chain constant region and the light chain constant region of an antibody. (Item 22) The polypeptide of any of the preceding items, wherein the first and second polypeptide chains are present on different molecules.

[0007] (Item 23) A CH1 region in which the amino acid at position 183 in the heavy chain is substituted with asparagine in the EU numbering system, or a nucleic acid encoding said region; a heavy chain constant region comprising said CH1 region, or a nucleic acid encoding said region; or a heavy chain comprising said CH1 region, or a nucleic acid encoding said heavy chain. (Item 24) A CL region in which the amino acid at position 133 in the light chain is substituted with glutamic acid and the amino acid at position 176 in the light chain is substituted with lysine in the EU numbering system, or a nucleic acid encoding said region; a light chain constant region comprising said CL region, or a nucleic acid encoding said region; or a light chain comprising said CL region, or a nucleic acid encoding said light chain. (Item 25) An antibody comprising the heavy chain of item 23 and the light chain of item 24. (Item 26) A Fab fragment, Fab' fragment, or F(ab')2 fragment comprising the CH1 region of item 23 and the CL region of item 24. (Item 27) A chimeric antigen receptor having a Fab fragment, a hinge region, a transmembrane domain, and an intracellular domain, the Fab fragment comprising the CH1 region of item 23 and the CL region of item 24. (Item 28) A cell expressing the chimeric antigen receptor of item 27.

[0008] The present invention also provides a technique for forming an isopeptide bond between the CH1 and CL regions of an antibody. Surprisingly, this isopeptide bond can be formed spontaneously in antibody-producing cells by amino acid substitution mutations in the CH1 and CL regions. The antigen-binding properties of an antibody are not substantially impaired by the isopeptide bond, and the antibody can maintain its functionality. Therefore, all applications of existing antibodies can be replaced with antibodies having isopeptide bonds of the present invention. Because isopeptide bonds stabilize Fab fragments and Fab' fragments, various applications that were previously only possible with scFv can also be replaced with Fab fragments and Fab' fragments stabilized by isopeptide bonds.

[0009] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0010] The present disclosure provides novel polypeptides containing isopeptide bonds. The polypeptides containing isopeptide bonds of the present disclosure have one or more of the following useful properties: improved stability, high purity production of a target substance, etc. The present invention is applicable to the production of a wide range of polypeptides, including the production of recombinant antibodies with complex structures, such as multispecific antibodies (particularly bispecific or trispecific antibodies).

[0011] The diagram shows the structures of the side chains of asparagine, lysine, and glutamic acid in the three-dimensional structure of an aggregate composed of isoCH1 and isoCL before isopeptide bond formation. The diagram shows the structure of the isopeptide bond in the three-dimensional structure of an aggregate composed of isoCH1 and isoCL (left) or an aggregate composed of isoCH1v2 and isoCLv2 (right) after isopeptide bond formation. Western blot results demonstrating the formation of the desired aggregate. The left panel shows the results of Coomassie blue staining, the center panel shows the results of staining with an Fc region-binding antibody, and the right panel shows the results of staining with a light chain κ region-binding antibody. The left lane is a molecular weight marker, the center lane (1) is h4D5 IgG, and the right lane (2) is h4D5 iSoMAb. Flow cytometry results demonstrating that h4D5 iSoMAb retains the binding ability of the original antibody. This shows that fluorescently labeled h4D5 IgG and fluorescently labeled h4D5 iSoMAb have equivalent binding ability to HER2-expressing SKBR-3 cells. This figure shows the results of SDS-polyacrylamide gel electrophoresis demonstrating the formation of the desired aggregate. The left panel shows the results for molecular weight markers, the center panel shows the results for h4D5 Fab, and the right panel shows the results for h4D5 iSoMAb Fab. Lane 1 is the culture supernatant sample, lane 2 is the flow-through sample, lane 3 is the wash sample, and lane 4 is the elution sample. This figure shows the results of differential scanning fluorimetry of h4D5 iSoMAb Fab and h4D5 Fab with or without the addition of a reducing agent (TCEP). Both conditions show destabilization at around 75-85°C. This figure shows the results of SDS-polyacrylamide gel electrophoresis demonstrating the formation of the desired iSoMAb based on the anti-CD63 antibody. Lane M shows molecular weight markers, lane 1 shows BSA (control), lanes 2-4 show the original antibody and two iSoMAbs expressed in CHO cells, and lanes 5-6 show two iSoMAbs expressed in Expi293F cells. Flow cytometry results show that iSoMAbs based on anti-CD63 antibodies maintain binding to CD63-expressing cells.The top row shows the results for the original antibody, the middle row shows the results for iSoMAb (v1), and the bottom row shows the results for iSoMAb (v2). This figure shows an outline of the structure of the bispecific HER2 / HER3 iSoMAb constructed in Example 4. This is a bispecific antibody containing the variable regions of h4D5 (anti-HER2) and U1-59 (anti-HER3), with h4D5 containing an isopeptide bond. The results of a binding confirmation test of the h4D5xU1-59 iSoMAb bispecific IgG by sandwich ELISA are shown on the left. The right shows the results, including the results for the monospecific h4D5 IgG and U1-59 IgG, as well as the h4D5xU1-59 iSoMAb bispecific IgG. The vertical axis indicates color intensity, and the horizontal axis indicates antibody concentration. Figure 1 shows the alignment results of various antibody heavy chain sequences (positions 118 to 167). Residue numbers are based on Eu numbering. Figure 2 shows the alignment results of various antibody heavy chain sequences (positions 168 to 217). Residue numbers are based on Eu numbering. Figure 3 shows the alignment results of various antibody heavy chain sequences (positions 218 to 220). Residue numbers are based on Eu numbering. Figure 4 shows the alignment results of various antibody heavy chain sequences (positions 221 to 270). Residue numbers are based on Eu numbering. Figure 5 shows the alignment results of various antibody heavy chain sequences (positions 271 to 320). Residue numbers are based on Eu numbering. Figure 6 shows the alignment results of various antibody heavy chain sequences (positions 321 to 369). Residue numbers are based on Eu numbering. Figure 7 shows the alignment results of various antibody heavy chain sequences (positions 370 to 417). Residue numbers are based on Eu numbering. 1 shows the alignment results of various antibody heavy chain sequences (positions 418 to 447). Residue numbers are based on Eu numbering. 1 shows the alignment results of various antibody light chain kappa chain sequences (positions 108 to 156). Kappa chain residue numbers are based on Eu numbering, and lambda chain residue numbers are based on Kabat numbering. 1 shows the alignment results of various antibody light chain lambda chain sequences (positions 107A to 156). Kappa chain residue numbers are based on Eu numbering, and lambda chain residue numbers are based on Kabat numbering. 1 shows the cancer cytotoxicity of antibody-drug conjugates of anti-CD63 antibody-based WT MAb and iSoMAbv1. The vertical axis indicates cytotoxic activity, and the horizontal axis indicates the concentration of the antibody-drug conjugate.Figure 1 shows the cancer cytotoxicity of antibody-drug conjugates of WT Fab and iSoMAb Fab based on the anti-CD63 antibody Fab format. The vertical axis indicates cytotoxicity, and the horizontal axis indicates the concentration of the antibody-drug conjugate. Figure 2 shows the change in blood concentration after intraperitoneal administration of h4D5 WT MAb and h4D5 iSoMAb to mice. The vertical axis indicates blood concentration, and the horizontal axis indicates time after intraperitoneal administration. Figure 3 shows the binding of WT Fab and iSoMAb Fab to antigen-positive cells based on the anti-CD63 antibody Fab format. The vertical axis indicates cell number, and the horizontal axis indicates fluorescence intensity. Figure 4 shows the results of SDS-PAGE analysis of purified h4D5 iSoMAb mutants. Figure 5 shows the results of SDS-PAGE analysis of purified h4D5 iSoMAb mutants. Figure 6 shows the results of SDS-PAGE and Western blotting analysis of h4D5 iSoMAb mutants. The binding behavior of h4D5 WT Fab and h4D5 iSoMAb Fab to the HER2 extracellular domain is shown by surface plasmon resonance. The vertical axis represents the amount of binding, and the horizontal axis represents the time elapsed since addition of Fab. The left figure shows the electron density of the isopeptide bond introduction site in hCD63 iSoMAb Fab. The right figure shows the iSoMAb Fab crystal structure superimposed on the Fab crystal structure. The isopeptide binding site is also shown as a space-filling model. The results of SDS-PAGE and Western blotting of Nivolumab iSoMAb are shown. The results of SDS-PAGE and Western blotting of Panitumumab iSoMAb are shown. The results of SDS-PAGE of purified 372 WT MAb and 372 iSoMAb are shown. Figure 1 shows the prepared h4D5 iSoMAb Fab Brevibacillus expression plasmid. Figure 2 shows the results of SDS-PAGE and Western blotting analysis of h4D5 iSoMAb Fab produced by Brevibacillus bacteria. Figure 3 shows the results of SDS-PAGE analysis of h4D5 iSoMAb Fab expressed using Brevibacillus bacteria and purified using a His-tag column. Figure 4 shows the prepared CAR expression plasmids (i) and (ii). Figure 5 shows a diagram of an anti-GFP antibody blot of a lysate of HEK293 cells transfected with CAR expression vector (i) and / or CAR expression vector (ii).In cells transfected with CAR expression vector (i) and CAR expression vector (ii), a band containing isopeptide-bonded trastuzumab Fab (indicated by an arrow) was detected on the high molecular weight side. Figure 1 shows fluorescence microscopy of HEK293 cells transfected with CAR expression vector (i) and CAR expression vector (ii). Figure 2 shows HEK293 cells three days after transfection with CAR expression vector (i) and / or CAR expression vector (ii), which were added with culture supernatant containing HER2-mouse Fc and fluorescently stained with anti-mouse IgG-PE and Hoechst 33342. The oval indicates the nucleus. The arrow on the cell membrane surface indicates the presence of HER2-mouse Fc. Figure 3 shows the results of SDS-PAGE and Western blotting analysis of the prepared iSoMAb. Figure 4 shows the results of SDS-PAGE and Western blotting analysis of the prepared iSoMAb.

[0012] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0013] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0014] In this specification, the term "comprise" encompasses "consisting of." "Comprise" means that an unspecified third component may be included as long as it does not defeat the spirit of the invention, whereas "consisting of" means that an unspecified third component is substantially not included. "Substantially not included" means that the product may contain third components that are unavoidable during the manufacturing process or third components (e.g., impurities) below the detection limit.

[0015] As used herein, the term "amino acid" is used in the usual sense in the art and refers to a compound having a carboxyl group and an amino group in one molecule. Typically, amino acids are α-amino acids, but amino acids with a greater distance between the carboxyl group and the amino group, such as β-amino acids and γ-amino acids, may also exist. Unless otherwise specified, amino acids herein refer to L-enantiomers, and D-enantiomers are indicated by adding D (or d) to the beginning (e.g., D-Ala (or d-Ala)) or by a lowercase letter (e.g., a). Representative examples of amino acids are shown below, but other amino acids are also contemplated herein.

[0016] The carbon atom to which the amino group in the amino acid H2N-C(R)-COOH is bonded is specified as the α-position, and the positional designations for representative amino acids relating to isopeptide bond formation in the present disclosure are shown below.

[0017] As used herein, the term "polypeptide" refers to a molecule in which multiple amino acids are linked together. To modify an amino acid in the amino acid sequence of a polypeptide, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR can be appropriately employed. Furthermore, amino acid modification methods in which amino acids other than natural amino acids are substituted can also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which an unnatural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), which is one of the termination codons, is preferably used.

[0018] As used herein, an amino acid or nucleotide in a "corresponding region" or "corresponding position" refers to an amino acid or nucleotide in a polypeptide or polynucleotide molecule that has, or is predicted to have, the same function as an amino acid or nucleotide at a given position in a polypeptide or polynucleotide that is used as a reference for comparison. For example, one skilled in the art can identify corresponding positions by aligning an original molecule with its variant and setting an appropriate threshold. For example, the insertion of a single amino acid can increase the corresponding position of the amino acid residue following the insertion by one. For example, if the alignment results in two regions where at least two consecutive amino acid residues match and the region between them, and the combined sequence shows that 50% or more of the amino acid residues match, the combined sequence can be identified as a corresponding region. For example, if the alignment results in the following sequences, Sequence 1 and Sequence 2, being identified as corresponding regions in two amino acid sequences: Sequence 1: XXXGGXX Sequence 2: XXXA-XX, then within this corresponding region, the "A" in Sequence 2 may be identified as position 4, and the "-" in Sequence 2 may be identified as position 5.

[0019] As used herein, an amino acid "mutation" or "modification" refers to an amino acid insertion, substitution, or deletion. A single amino acid mutation or modification refers to the insertion, substitution, or deletion of a single amino acid. An amino acid addition to the end of an amino acid sequence is also included in an amino acid insertion.

[0020] Herein, an expression indicating an amino acid insertion may be appropriately used, which indicates the position of the inserted amino acid in the sequence after insertion and the single-letter code. For example, a mutation from the amino acid sequence XXXXXXXXX to XXXXXXGGXXX may be expressed as an insertion of 6G and 7G. Herein, an expression indicating an amino acid substitution may be appropriately used, which indicates the single-letter code of the amino acid before and after the substitution, before and after a number indicating a specific position. For example, the substitution G6A, which is used in reference to the amino acid sequence XXXXXXGXXX, indicates the substitution of glycine at position 6 with alanine. Herein, an expression indicating an amino acid deletion may be appropriately used, which indicates the position of the deleted amino acid in the sequence before the deletion (with the single-letter code added if necessary). For example, a mutation from the amino acid sequence XXXXXXGXXX to XXXXXXXXX may be expressed as a deletion at position 6 or a deletion of 6G. Therefore, mutation of the amino acid at position 6 includes the insertion of 6G, the substitution of G6A, and the deletion of 6G in the above examples.

[0021] The position of the mutation does not necessarily have to be expressed with reference to the sequence before the mutation, for example, a mutation of the amino acid sequence XXXXXXXXX to XXXXXGXXX can be expressed as an insertion of 6G into the amino acid sequence XXXXXXXXX before the mutation, and the amino acid sequence XXXXXGXXX after the mutation can be expressed as a sequence containing an insertion at position 6. The position of the mutation does not necessarily have to be expressed with reference to the full-length polypeptide, but may be specified based on the "corresponding region" and "corresponding position" described above.

[0022] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid in a peptide with another amino acid with similar properties, and the properties of the peptide can be expected to be similar before and after the conservative substitution. Specific examples of conservative amino acid substitutions are provided elsewhere in this specification.

[0023] Unless otherwise specified, reference herein to a molecule (e.g., a protein, a nucleic acid) is understood to also refer to variants of that molecule (e.g., variants with modifications in the amino acid sequence) that exhibit biological functions similar to those of the molecule (although not necessarily to the same extent). For example, a heavy chain protein comprising an antibody Fc region may include not only a protein having a specific amino acid sequence, but also conjugates of this protein with a labeling molecule (such as a fluorescent label), isotopic substitutions, variants capable of associating with an antibody light chain and / or with another antibody Fc region, and the like. Such variants may include fragments of the original molecule, molecules that are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identical across the amino acid or nucleic acid sequence of the original molecule of the same size, or when compared to the sequence of the original molecule aligned by computer homology programs known in the art. Variants can include molecules with altered amino acids (eg, modified by disulfide bond formation, glycosylation, lipidation, acetylation, or phosphorylation).

[0024] As used herein, "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, single domain antibodies, and multispecific antibodies (e.g., bispecific or trispecific antibodies).

[0025] As used herein, the term "humanized antibody" refers to an antibody having a structure in which the CDR regions of an antibody produced in a species other than human are incorporated into a human antibody. Additionally, the term "chimeric antibody" refers to an antibody having a structure in which the variable regions of an antibody produced in a species other than human are incorporated into a human antibody.

[0026] As used herein, the term "antigen-binding fragment" refers to a partial structure of an antibody that has the ability to bind to an antigen of interest, and typically comprises at least the heavy chain variable region and light chain variable region of the antibody. Examples of antigen-binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, and single-chain variable region fragment (scFv). In a preferred embodiment, the antigen-binding fragment comprises regions corresponding to the VH and CH1 regions, as well as regions corresponding to the VL and CL regions of an antibody.

[0027] As used herein, the term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) typically each contain four conserved framework regions (FR) and three complementarity-determining regions (CDR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).

[0028] As used herein, "complementarity-determining region" or "CDR" refers to a region in an antibody variable domain that specifically contributes to antigen binding. Typically, antibodies contain six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). There are several methods for determining CDR sequences, including the Kabat numbering system, the IMGT numbering system, the Paratome numbering system, and the Chothia numbering system (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering system) and Kunik, Ashkenazi and Ofran, 2012, "Paratome: an online tool for systematic identification of For antigen-binding regions in antibodies based on sequence or structure, see Nucl. Acids Res., 40:W521-W524 (Paratome numbering system), Lefranc et al., 2003, Dev Comparat Immunol 27:55-77 (IMGT numbering system), and Al-Lazikani et al., 1997, J. Mol. Biol 273:927-948 (Chothia numbering system).

[0029] "Framework" or "FR" refers to the regions of a variable domain other than the complementarity-determining regions (CDRs). The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. The CDR and FR sequences typically appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0030] As used herein, the term "constant region" or "constant domain" refers to a portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical disulfide-bonded light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) and a heavy chain constant region (CH) including a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) and a constant light chain (CL) domain. Two types of constant domains are known in the light chains of natural antibodies, called kappa (κ) and lambda (λ).

[0031] As used herein, "Fc region" refers to a region comprising at least a portion of the CH3, CH2, and hinge regions of an antibody constant domain. In some cases, the Fc region may include the CH4 domain present in some antibody classes. The Fc region may also include the entire hinge region of an antibody constant domain. The Fc region is recognized by T cells and is associated with effector functions. In the case of human IgG1, the heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may be present or absent (the numbering of amino acid residues in the Fc region is according to the EU numbering system (EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991).

[0032] Antibody "classes" are determined by the type of constant domain contained in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0033] As used herein, "antigen" refers to a target molecule or a portion thereof to which an antibody binds. An antigenic determinant present in an antigen is called an epitope, and an antibody binds to the epitope portion of the antigen. Epitopes include linear epitopes and conformational epitopes. A linear epitope in a protein antigen is an epitope whose primary amino acid sequence is recognized by an antibody. A linear epitope in a protein antigen typically consists of at least three, at least five, e.g., 8-10, 6-20 amino acids. In contrast to linear epitopes, a conformational epitope in a protein antigen is an epitope that cannot be defined solely by the primary amino acid sequence. In recognizing a conformational epitope, an antibody recognizes the three-dimensional structure of the protein. Methods for determining the conformation of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-directed spin labeling, and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0034] As used herein, a "chimeric antigen receptor" (CAR) is a chimeric molecule comprising an antigen-binding fragment of an antibody (particularly, an scFv) and an immune cell activation domain. A CAR is generally a molecule comprising an scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signaling domain (e.g., CD3ζ) linked together. A CAR can be introduced into a cell and expressed on the cell surface. A cell expressing a CAR can target a specific antigen. A CAR can be introduced into immune cells, such as T cells or NK cells, to target the immune cells, such as T cells or NK cells, to cancer. While first-generation CARs comprise an scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signaling domain (e.g., CD3ζ), second-generation CARs further comprise a costimulatory molecule signaling domain to activate the immune cells into which the CAR is introduced. Costimulatory factors such as CD28, 4-1BB, OX40, CD27, and ICOS are used as costimulatory molecule signaling domains. Third-generation CARs incorporate multiple costimulatory factors. Fourth-generation CARs are engineered to have the ability to produce cytokines (e.g., inflammatory cytokines such as IL12). Cytokine release can improve the primary microenvironment and promote immune system activation. Fifth-generation CARs are characterized by having a structure that utilizes cytokine receptor-associated signaling pathways (e.g., the JAK-STAT pathway) in addition to the costimulatory molecule signaling domain. Examples of such structures include the intracellular domain of the IL-2 receptor β chain, the intracellular domain of the IL-7 receptor, the intracellular domain of the IL-15 receptor, and the intracellular domain of the IL-21 receptor. Thus, CARs have been improved to enable sustained proliferation of CAR-introduced immune cells in vivo. While regions other than the scFv CDR region are preferably humanized, it is preferable that all domains other than the scFv portion are derived from human proteins. The scFv itself may be derived from a human antibody.In addition, various modified versions of CARs have been developed, and in the present disclosure, the scFv region of all of these CARs can be replaced with an antigen-binding fragment of an antibody having a heavy chain VH-CH1 domain and a light chain VL-CL domain. For example, in the present invention, an antigen-binding fragment of an antibody such as a Fab fragment or a Fab' fragment can be used instead of an scFv, and an isopeptide bond can be formed between the CH1 and CL in a CAR-producing cell, allowing a stabilized CAR having an antigen-binding fragment of an antibody such as a Fab fragment or a Fab' fragment to be expressed on the cell surface.

[0035] Herein, comparison of the identity of amino acid sequences or nucleotide sequences is calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.7.1 (published October 19, 2017). The "identity" value herein generally refers to the value obtained when BLAST is used under default conditions. "Identity" is calculated by calculating the percentage of homologous amino acids or nucleotides between two or more amino acid or nucleotide sequences according to known methods such as those described above. Specifically, before calculating the percentage, the amino acid or nucleotide sequences of the group of amino acid or nucleotide sequences to be compared are aligned, and gaps are introduced into the amino acid or nucleotide sequences, if necessary, to maximize the percentage of identical amino acids or nucleotides. Alignment methods, percentage calculation methods, comparison methods, and related computer programs are well known in the art (e.g., BLAST, as described above). When comparing amino acid or nucleotide sequences using BLAST, the algorithm Blastp can be used with default settings.

[0036] As used herein, the term "pharmaceutical ingredient" refers to any component that can constitute a pharmaceutical, and examples thereof include active ingredients (components that themselves exhibit medicinal effects), additive ingredients (components that are not expected to have medicinal effects themselves, but are expected to play a certain role when included in a pharmaceutical (e.g., excipient, lubricant, surfactant, etc.)), etc. A pharmaceutical ingredient may be a single substance or a combination of multiple substances or agents. It may also include any combination, such as a combination of an active ingredient and an additive ingredient, or a combination of an adjuvant and an active ingredient.

[0037] As used herein, the term "active ingredient" refers to an ingredient that exerts an intended medicinal effect, and may refer to a single ingredient or multiple ingredients.

[0038] As used herein, the term "additive component" refers to any component that is not expected to have a medicinal effect but that plays a certain role when included in a pharmaceutical, and examples thereof include pharmaceutically acceptable carriers, stabilizers, (co)adjuvants, solubility improvers, solubilizers, diluents, excipients, buffers, binders, diluents, flavorings, and lubricants.

[0039] As used herein, the terms "drug," "agent," and "factor" (all of which correspond to the English term "agent") are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, or electricity) that can achieve the intended purpose. Examples of such substances include, but are not limited to, antibodies, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands), etc.), composite molecules thereof, and mixtures thereof.

[0040] As used herein, the term "label" refers to an entity (e.g., a substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) labeling, fluorescence labeling, biotin labeling, and chemiluminescence labeling. When multiple target proteins or factors or means for capturing them are labeled by fluorescence labeling, the labels are labeled with fluorescent substances that have different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more. Any label that does not affect function can be used, and an example of a fluorescent substance is Alexa™Fluor. Alexa™Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that supports a wide range of fluorescent wavelengths. Compared to other fluorescent dyes with corresponding wavelengths, it is very stable, bright, and has low pH sensitivity. Examples of combinations of fluorescent dyes with a fluorescence maximum wavelength of 10 nm or longer include the combination of Alexa™555 and Alexa™633, and the combination of Alexa™488 and Alexa™555. Other fluorescent labels include cyanine dyes (e.g., Cy3, Cy5, etc., of the CyDye™ series), rhodamine 6G reagent, N-acetoxy-N2-acetylaminofluorene (AAF), AAIF (an iodine derivative of AAF), and the like. In the present disclosure, such labels can be used to modify a target substance so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods as appropriate depending on the label and the target substance.

[0041] As used herein, the term "kit" refers to a unit in which the components to be provided (e.g., antibodies, instructions, etc.) are provided, usually separated into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit advantageously includes instructions or manuals describing how to use the provided components or how to handle the reagents. When the kit is used herein as a reagent kit, the kit usually includes instructions describing how to use the antibodies, etc.

[0042] As used herein, "instructions" refers to written instructions for a physician or other user on how to use the present disclosure. The instructions include instructions for administering the medicament or the like of the present disclosure. The instructions may also include instructions for the dosage form. The instructions are prepared in accordance with a format specified by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and clearly state that they have been approved by the regulatory agency. The instructions are so-called package inserts, and are usually provided in paper form, but are not limited thereto and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, email, etc.).

[0043] The term "about" refers to a range of plus or minus 10% of the indicated value. When "about" is used in reference to temperature, it refers to a range of plus or minus 5°C of the indicated temperature, and when "about" is used in reference to pH, it refers to a range of plus or minus 0.5 of the indicated pH.

[0044] As used herein, the "boundary region where the first and second polypeptide chains are adjacent to each other" refers to the region between the backbone chains of the two polypeptides in the first and second polypeptides. The phrase "the first and second polypeptide chains are adjacent to each other" refers to the distance between the α-carbon atoms of a first amino acid in the first polypeptide chain and a second amino acid in the second polypeptide chain being 10 Å or less. The "boundary region where the first and second polypeptide chains are adjacent to each other" refers to the region (a spherical region with a radius of 15 Å) that is 15 Å or less from the midpoint of the line segment connecting the α-carbon atom of the first amino acid and the α-carbon atom of the second amino acid.

[0045] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments can be used alone or in combination.

[0046] In one aspect, the present disclosure provides isopeptide bonds in polypeptides. Any means for achieving this is contemplated as being within the scope of the present disclosure. For example, even if not explicitly stated, a description of a method for producing a polypeptide that forms isopeptide bonds also contemplates embodiments that reflect other means, such as the produced polypeptide, compositions containing the same, and uses thereof.

[0047] (Method for producing a polypeptide that forms an isopeptide bond) The present invention provides a method for producing a polypeptide that forms an isopeptide bond. As used herein, an isopeptide bond refers to a polypeptide having the following structure: where the dashed line represents the first or second polypeptide chain, respectively, and indicates a link between the alpha carbons of both polypeptides, and the interpeptide linkage is formed by bonding the side chain of a lysine in the first or second polypeptide chain to the side chain of an asparagine, aspartic acid, glutamine, or glutamic acid. In an exemplary embodiment, the present disclosure provides a method for producing a modified polypeptide comprising an isopeptide bond interpeptide linkage between first and second polypeptide chains, the method comprising: constructing a three-dimensional structure of intermediate A comprising first and second polypeptide chains; identifying an amino acid sequence present in a boundary region where the first and second polypeptide chains are proximal to each other in the three-dimensional structure; mutating intermediate A by introducing at least one residue of a) lysine, b) glutamine, glutamic acid, asparagine, or aspartic acid, and c) glutamic acid or aspartic acid into intermediate A to prepare intermediate B, wherein intermediate B comprises a), b), and c), and the first and second polypeptide chains in intermediate B comprise a) and b), respectively, in their boundary regions; and forming the interpeptide linkage in intermediate B to obtain the modified polypeptide. Here, intermediate A corresponds to the starting structure, intermediate B corresponds to the structure after mutagenesis and before isopeptide bond formation, and the modified polypeptide corresponds to the structure after mutagenesis and isopeptide bond formation.

[0048] The isopeptide bond formation reaction is thought to proceed without additional factors such as enzymes if the polypeptide is appropriately designed. However, only a limited number of proteins with isopeptide bonds are known, such as the Spy0128 protein of Streptococcus pyogenes (J Am Chem Soc. 2011 Jan 26;133(3):478-85). Therefore, it is important to design a polypeptide that promotes the isopeptide bond formation reaction. An isopeptide bond is formed between lysine (a) and glutamine, glutamic acid, asparagine, or aspartic acid (b), and glutamic acid or aspartic acid (c) plays a particularly important role in promoting this reaction. Residue (c) may be present in the same molecule as the first or second polypeptide chain, or in a separate polypeptide chain. In one embodiment, residue (c) is present in the same molecule as the first polypeptide chain. In one embodiment, residue (c) is present on the first polypeptide chain N-terminal to the lysine residue (a). In one embodiment, c) is located at a position on the first polypeptide chain that is 5 or more residues away from the lysine residue of a).

[0049] A polypeptide chain containing or incorporating a) is referred to herein as a first polypeptide chain, and a polypeptide chain containing or incorporating b) is referred to herein as a second polypeptide chain. The first polypeptide chain and the second polypeptide chain may be present in the same molecule or in different molecules. In one embodiment, the first polypeptide chain and the second polypeptide chain are present in different molecules. In particular, the present disclosure enables the formation of isopeptide bonds between different molecules, which provides advantages in the formation of hetero-associations as described herein.

[0050] As used herein, the term "interface region where the first and second polypeptide chains are adjacent to each other" refers to the region between the backbone chains of the two polypeptides in the first and second polypeptides. As used herein, "the first and second polypeptide chains are adjacent to each other" refers to the presence of at least one combination of a first amino acid and a second amino acid (also referred to as a "proximal amino acid pair") in which the distance between the α-carbon atoms of the first amino acid in the first polypeptide chain and the second amino acid in the second polypeptide chain is 10 Å or less. The term "interface region where the first and second polypeptide chains are adjacent to each other" refers to the region within 15 Å of the midpoint of the line segment connecting the α-carbon atoms of the first amino acid and the α-carbon atom of the second amino acid. When multiple adjacent amino acid pairs exist between the first and second polypeptide chains, the term "interface region where the first and second polypeptide chains are adjacent to each other" refers to the region obtained by overlapping the regions within 15 Å of the midpoint of the line segment connecting the α-carbon atoms of the amino acids in each adjacent amino acid pair for all adjacent amino acid pairs. In the present disclosure, isopeptide bonds do not necessarily have to be formed between adjacent amino acids, but will be formed between amino acids whose side chains are contained within the boundary region. In a representative embodiment, the boundary region may be defined as a region within about 25 Å, about 20 Å, about 15 Å, about 10 Å, about 7 Å, or about 5 Å from the midpoint of the line segment connecting the α-carbon atom of amino acid a) and the α-carbon atom of amino acid b). Since the orientation of amino acid side chains extending from a polypeptide chain is usually not constant, it is particularly preferable to design the amino acid side chains of a) and b) that form an isopeptide bond so that they extend into the boundary region toward the other polypeptide. In one embodiment, the positions of two amino acids whose side chains are located inside the three-dimensional structure are identified as amino acid positions a) and b).

[0051] The construction of the three-dimensional structure may be based on a known three-dimensional structure. For example, known three-dimensional structures can be obtained from the Protein Database, etc. The three-dimensional structure of the starting polypeptide (intermediate A) can be constructed, for example, by downloading coordinate data of the crystal structure of the starting polypeptide registered in the Protein Data Bank (https: / / www.rcsb.org / ) and using PyMOL (https: / / pymol.org / 2 / ). Based on the constructed three-dimensional structure as needed, the three-dimensional structure of the modified polypeptide can be constructed, for example, by constructing a homology model using SWISS-MODEL (https: / / swissmodel.expasy.org / ) and using Coot (https: / / www2.mrc-lmb.cam.ac.uk / personal / pemsley / coot / ). If necessary, default values ​​are used for parameters. The three-dimensional structures described herein may be constructed in this manner. After introducing any mutations, the three-dimensional structure of the polypeptide intermediate can be constructed. In one embodiment, the three-dimensional structure of intermediate B is constructed.

[0052] In one embodiment, a) and / or b) are introduced based on the distance between a first amino acid residue (side chain) of a first polypeptide chain and a second amino acid residue (side chain) of a second polypeptide chain present in the boundary region and the types of the first and second amino acid residues. In one embodiment, b) is asparagine or aspartic acid, and two amino acid positions where the distance between the α-carbons in the three-dimensional structure of intermediate A is about 6 to 10 Å, for example, about 6 Å, about 6.5 Å, about 7 Å, about 7.5 Å, about 8 Å, about 8.5 Å, about 9 Å, about 9.5 Å, about 10 Å, or any two of these values, are identified as the amino acid positions of a) and b). In one embodiment, b) is glutamine or glutamic acid, and in the conformation of intermediate A, two amino acid positions whose α-carbon distance is about 7.5 to 11 Å, for example, about 7.5 Å, about 8 Å, about 8.5 Å, about 9 Å, about 9.5 Å, about 10 Å, about 10.5 Å, about 11 Å, or a range between any two of these values, are identified as the amino acid positions of a) and b).

[0053] When atoms of amino acid residues forming an interaction interface between polypeptide chains are close to each other, but the distances between atoms present in different polypeptide chains are equal to or less than the following: C-C: 4.1 Å, C-N: 3.8 Å, C-O: 3.7 Å, O-O: 3.3 Å, O-N: 3.4 Å, and N-N: 3.4 Å, these atoms are referred to herein as an "interacting atom pair" (see S. Sheriff, W.A. Hendrickson, and J.L. Smith, J. Mol. Biol. 197, 273-296 (1987)). On the other hand, if a pair of an atom present in one polypeptide chain and an atom present in another polypeptide chain that is closest to it is an interacting atom pair, the two polypeptide chains can interact with each other. In one embodiment, in the three-dimensional structure of intermediate B (after mutagenesis but before isopeptide bond formation), the side chains of the amino acids around a) and b) in the first and second polypeptide chains (e.g., amino acids within 7, 6, 5, 4, 3, 2, or 1 of a) or b), including a) and b), have interacting atom pairs. In one embodiment, one, two, three, four, five, or more of the amino acid pairs around a) and b) have interacting atom pairs. In one embodiment, the side chains of the amino acid pairs around a) and b) that have interacting atom pairs have one, two, three, four, five, or more interacting atom pairs. Here, one element (amino acid or atom) can form multiple pairs with multiple different elements. In one embodiment, an amino acid pair within 7, 6, 5, 4, 3, 2, or 1 amino acid on the N-terminal side of a) on the first polypeptide chain and a neighboring amino acid of b) on the second polypeptide chain has an interacting atom pair.

[0054] In one embodiment, in the conformation of intermediate B (after mutagenesis but before isopeptide bond formation), the distance between the nitrogen atom at the ζ-position of residue a) and the oxygen atom of the carboxy group in the side chain of residue c) is about 1 to 6 Å, for example, about 1 Å, about 1.5 Å, about 2 Å, about 2.5 Å, about 3 Å, about 3.5 Å, about 5 Å, about 5.5 Å, about 6 Å, or any two of these values. In one embodiment, in the conformation of intermediate B, the distance between the oxygen atom of the carboxy group or amide group in the side chain of residue b) and the oxygen atom of the carboxy group in the side chain of residue c) is about 1 to 6 Å, for example, about 1 Å, about 1.5 Å, about 2 Å, about 2.5 Å, about 3 Å, about 3.5 Å, about 5 Å, about 5.5 Å, about 6 Å, or any two of these values. In one embodiment, in the conformation of intermediate B, the distance between the nitrogen atom at the ζ-position of the residue a) and the oxygen atom of the carboxyl group or amide group in the side chain of the residue b) is within a range of about 1 to 6 Å, for example, about 1 Å, about 1.5 Å, about 2 Å, about 2.5 Å, about 3 Å, about 3.5 Å, about 5 Å, about 5.5 Å, about 6 Å, or any two values ​​therein.

[0055] In one embodiment, two amino acid positions where the distance between the α-carbons of a) and c) in the three-dimensional structure of intermediate A is about 5 to 10 Å, for example, about 5 Å, about 5.5 Å, about 6 Å, about 6.5 Å, about 7 Å, about 7.5 Å, about 8 Å, about 8.5 Å, about 9 Å, about 9.5 Å, about 10 Å, or any two of these values, are identified as the amino acid positions of a) and c). In one embodiment, two amino acid positions where the distance between the α-carbons of b) and c) in the three-dimensional structure of intermediate A is about 5 to 10 Å, for example, about 5 Å, about 5.5 Å, about 6 Å, about 6.5 Å, about 7 Å, about 7.5 Å, about 8 Å, about 8.5 Å, about 9 Å, about 9.5 Å, about 10 Å, or any two of these values, are identified as the amino acid positions of a) and c). In one embodiment, in the three-dimensional structure of intermediate A, three amino acid positions that satisfy the distance between the α carbons of a) and c) above and the distance between the α carbons of b) and c) above are identified as amino acid positions a), b), and c).

[0056] In one embodiment, in the three-dimensional structure of intermediate B, the angle between the vector oriented from the α carbon to the β carbon of the residue a) and the vector oriented from the α carbon to the β carbon of the residue b) is, but is not limited to, about 180°, about 170°, about 160°, about 150°, about 140°, about 130°, about 120°, about 110°, about 100°, about 90°, about 80°, about 70°, about 60°, or within a range between any two of these values. In one embodiment, in the three-dimensional structure of intermediate B, the angle between the vector oriented from the α carbon of residue a) to the α carbon of residue b) and the vector oriented from the α carbon to the β carbon of residue c) is, but is not limited to, about 130°, about 120°, about 110°, about 100°, about 90°, about 80°, about 70°, about 60°, about 50°, about 40°, about 30°, about 20°, or within a range between any two of these values.

[0057] In one embodiment, in the three-dimensional structure of intermediate B (after mutagenesis but before isopeptide bond formation), one, two, or three of a), b), and c) are not present in the domain. In Spy proteins, isopeptide bonds are formed by reactions between residues present in the domain, but in the present disclosure, isopeptide bonds can be formed independently of domain formation. For example, whether a residue is present in a domain can be determined based on its solvent accessible area. The solvent accessible area of ​​a residue can be calculated using the AREAIMOL program. AREAIMOL is described in J. Mol. Biol. 55, 379-400 (1971) and is available as a program included in the CCP4 program package for protein structure analysis (https: / / www.ccp4.ac.uk / ). Coordinate data for each atom constituting the molecule can be read and calculations can be performed using default parameters. For example, in the Examples of the present application, the calculated result for the isopeptide bond-forming Asn in isoCH1 (SEQ ID NO: 1) was 0.24, the calculated result for the isopeptide bond-forming Lys in isoCL (SEQ ID NO: 2) was 0.24, and the calculated result for the catalytic Glu was 0.25. In one embodiment, when the solvent accessible area of ​​the residue is 0.1 or more, 0.11 or more, 0.12 or more, 0.13 or more, 0.14 or more, 0.15 or more, 0.16 or more, 0.17 or more, 0.18 or more, 0.19 or more, 0.2 or more, 0.21 or more, 0.22 or more, 0.23 or more, 0.24 or more, or 0.25 or more, the residue can be determined to be present in a domain.

[0058] Since the isopeptide bond formation reaction between a) and b) catalyzed by c) can be promoted in a hydrophobic environment, it may be preferable for the interface region to be a hydrophobic environment. In one embodiment, a step of determining the hydrophobicity of the interface region is included. In one embodiment, a mutation is introduced into the amino acid sequence present in the interface region to delete a hydrophilic amino acid residue, to replace an amino acid residue with a hydrophobic amino acid residue, or to insert a hydrophobic amino acid residue.

[0059] In one embodiment, hydrophilic amino acids may include glutamic acid, aspartic acid, lysine, arginine, serine, and threonine. In one embodiment, hydrophilic amino acids may include glutamic acid, aspartic acid, lysine, arginine, serine, threonine, tyrosine, cysteine, and histidine. In one embodiment, hydrophobic amino acids may include phenylalanine, tryptophan, valine, leucine, and isoleucine. In one embodiment, hydrophobic amino acids may include phenylalanine, tryptophan, valine, leucine, isoleucine, methionine, and proline.

[0060] In one embodiment, in the conformation of intermediate B, a hydrophobic amino acid is introduced having an α-carbon within about 20 Å, about 18 Å, about 16 Å, about 14 Å, about 12 Å, about 10 Å, about 8 Å, about 6 Å, or about 4 Å of the nitrogen atom at the ζ position of the residue in a). Optionally, in the conformation of intermediate B, the α-carbon of the introduced hydrophobic amino acid is separated from the nitrogen atom at the ζ position of the residue in a) by about 3 Å or more, about 3.5 Å or more, about 4 Å or more, about 4.5 Å or more, or about 5 Å or more.

[0061] In one embodiment, hydrophobic amino acids with side chains of different volumes are introduced (inserted or substituted) to fill the space in the boundary region. Mutations can be introduced such that the volume ratio of the side chains in the boundary region in the (recalculated) three-dimensional structure of the polypeptide after introduction exceeds the volume ratio of the side chains in the boundary region in the three-dimensional structure of the polypeptide before introduction. Introducing an amino acid with a side chain with a large volume, such as tryptophan, does not necessarily increase the volume ratio of the side chains in the boundary region; in fact, it may decrease due to the increased distance between the polypeptide backbones. The side chain volumes of amino acids can be arranged in ascending order as follows: glycine, proline, alanine, valine, leucine-isoleucine, methionine, phenylalanine, and tryptophan. In one embodiment, in the conformation of intermediate B, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, or at least 84% of the volume of the interface region where the first and second polypeptide chains are adjacent to each other is occupied by residues of the first and second polypeptide chains. Here, for the purpose of calculating the side chain volume, the following atomic radii are assigned to each atom: hydrogen: 0.53 Å, carbon: 0.67 Å, ​​nitrogen: 0.56 Å, oxygen: 0.48 Å, and sulfur: 0.88 Å. If the spherical volumes of atoms overlap, the overlap is subtracted. In one embodiment, in the conformation of intermediate B, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, or at least 84% of the volume of a sphere of radius 15 Å from the epsilon nitrogen atom of lysine in a) is occupied by residues of the first and second polypeptide chains.

[0062] The process of forming interpeptide linkages in intermediate B (the polypeptide after mutagenesis and before isopeptide bond formation) described herein to obtain a modified polypeptide may not require additional components such as enzymes. This process can be performed in vitro or in vivo (in cells). For example, the first and second polypeptide chains can be produced in different cells, and then these polypeptide chains can form isopeptide bonds in vitro. Isopeptide bond formation can be performed in vivo in cells that co-express the first and second polypeptide chains. Isopeptide bond formation can be suppressed or promoted by manipulating the intracellular localization of the first and second polypeptide chains (e.g., by fusing a localization signal molecule).

[0063] Compared to the starting polypeptide (intermediate A), the final polypeptide (altered polypeptide) may have an amino acid sequence that is at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical when compared to the sequence of the original molecule as aligned by computer homology programs known in the art. The final polypeptide (altered polypeptide) may contain any mutations, but in one embodiment, at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the mutations are conservative amino acid substitutions. In one embodiment, conservative amino acid substitutions involve replacing an amino acid in the following group of amino acids with another amino acid in the same group: Group 1: glycine, alanine, valine, leucine, and isoleucine; Group 2: serine and threonine; Group 3: phenylalanine, tyrosine, and tryptophan; Group 4: lysine, hydroxylysine, arginine, and histidine; Group 5: cysteine, methionine, methionine sulfoxide, and homocysteine; Group 6: proline and hydroxyproline; Group 7: glutamic acid and aspartic acid; Group 8: glutamine and asparagine; Group 9: serine and cysteine.

[0064] Polypeptides Comprising Isopeptide Bonds In one aspect, the present disclosure provides polypeptides comprising a first and second polypeptide chain having a bond of the following formula: wherein the dashed line represents a bond to the polypeptide backbone, and the first and second polypeptide chains comprise a) and b), respectively, at a boundary region where the first and second polypeptide chains are adjacent to each other in the three-dimensional structure of the polypeptide.

[0065] Polypeptides comprising isopeptide bonds of the present disclosure may be produced by the method of producing polypeptides that form isopeptide bonds of the present disclosure. In one embodiment, polypeptides comprising isopeptide bonds of the present disclosure have structural differences compared to known or naturally occurring polypeptides due to mutations introduced in the method of producing polypeptides that form isopeptide bonds of the present disclosure. In one embodiment, polypeptides comprising isopeptide bonds of the present disclosure may have the arrangement of a), b), c), and / or hydrophobic residues described above. The arrangement of these residues is generally maintained before and after isopeptide bond formation. In one embodiment, polypeptides comprising isopeptide bonds of the present disclosure may have an amino acid sequence that is at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a known or naturally occurring polypeptide when compared to the sequence of the original molecule aligned by computer homology programs known in the art.

[0066] In one embodiment, a polypeptide comprising an isopeptide bond of the present disclosure is a polypeptide molecule in which different polypeptide molecules are linked by an isopeptide bond, which can be determined by the fact that the two polypeptide chains linked by the isopeptide bond are not linked by a peptide bond.

[0067] In one embodiment, the polypeptide comprising an isopeptide bond of the present disclosure is an antibody or a T cell receptor (TCR). In one embodiment, the isopeptide bond (interpeptide linkage) is present in the constant region. For example, by introducing an isopeptide bond into the constant region of an antibody (such as the constant region involved in the binding between the heavy and light chains), it is possible to provide antibodies with various target specificities while maintaining the ability to form isopeptide bonds by combining them with various variable regions. In one embodiment, the polypeptide comprising an isopeptide bond of the present disclosure is a therapeutic protein, cytokine, or chemokine used in enzyme replacement therapy. In one embodiment, the polypeptide comprising an isopeptide bond of the present disclosure includes, but is not limited to, Fc receptors such as FcRn and cytokine receptors such as TNFα receptors.

[0068] To further illustrate embodiments of antibodies containing isopeptide bonds, Figures 11-12 show alignments of representative antibody sequences. Sequence information is also available for antibodies derived from other species, allowing similar decisions about where to introduce iSoMAb mutations. These sequences were generated and numbered using CLUSTALW (JD Thompson, DG Higgins, and TJ Gibson, Nucleic Acids Res. 22, 4673-4680 (1994)). Residue numbers in the heavy chain constant region and kappa chain constant region follow Eu numbering, while residue numbers in the lambda chain constant region follow Kabat numbering. In this specification, antibody residues may be referred to with the numbers shown in Figures 11-12. Based on the successful examples of antibodies containing isopeptide bonds shown in the examples herein, antibodies containing similar modifications may also form isopeptide bonds. In one embodiment, an antibody comprising an isopeptide bond of the present disclosure comprises an asparagine or aspartic acid at position 183 of the heavy chain, a glutamic acid at position 133 of the light chain, and a lysine at position 176 of the light chain, and optionally one or more of the following: an isoleucine at position 173 of the heavy chain, a leucine at position 181 of the heavy chain, a valine at position 131 of the light chain, a methionine at position 162 of the light chain, a leucine at position 178 of the light chain, and a glutamine at position 180 of the light chain. In one embodiment, an antibody comprising an isopeptide bond of the present disclosure comprises a lysine at position 183 of the heavy chain, a glutamic acid at position 170 of the heavy chain, and an asparagine or aspartic acid at position 176 of the light chain, and optionally one or more of the following: an isoleucine at position 173 of the heavy chain, a leucine at position 181 of the heavy chain, a methionine at position 162 of the light chain, a leucine at position 178 of the light chain, and a glutamine at position 180 of the light chain. It can be understood that the introduction of the above mutations into antibodies results in the formation of isopeptide bonds between the heavy and light chains in antibodies of different subclasses of various animal species (see Figures 11 and 12). In a preferred embodiment, for example, the antibody may be human IgG1, human IgG2, or human IgG4. In a preferred embodiment, the light chain may be a human κ chain or a human λ chain.

[0069] The present invention also provides an antibody comprising a CH1 region in which the amino acid at position 183 in the heavy chain is substituted with asparagine, and a CL region in which the amino acid at position 133 in the light chain is substituted with glutamic acid and the amino acid at position 176 in the light chain is substituted with lysine, wherein an isopeptide bond is formed between the asparagine and glutamic acid. In this embodiment, the substitution of asparagine at position 183 in the heavy chain may be a substitution with glutamine, glutamic acid, or aspartic acid.

[0070] The present invention also provides a CH1 region in which the amino acid at position 183 of the heavy chain has been substituted with asparagine, and a nucleic acid encoding said region. The present invention also provides a heavy chain constant region having a CH1 region in which the amino acid at position 183 of the heavy chain has been substituted with asparagine, and a nucleic acid encoding said region. The present invention also provides a heavy chain having a CH1 region in which the amino acid at position 183 of the heavy chain has been substituted with asparagine, and a nucleic acid encoding said heavy chain. In this embodiment, the substitution of asparagine at position 183 of the heavy chain may be a substitution of glutamine, glutamic acid, or aspartic acid.

[0071] The present invention also provides a CL region in which the amino acid at position 133 of the light chain is substituted with glutamic acid and the amino acid at position 176 of the light chain is substituted with lysine, and a nucleic acid encoding said region. The present invention also provides a light chain comprising a CL region in which the amino acid at position 133 of the light chain is substituted with glutamic acid and the amino acid at position 176 of the light chain is substituted with lysine, and a nucleic acid encoding said light chain.

[0072] The present invention also provides a combination of a CH1 region in which the amino acid at position 183 of the heavy chain is substituted with asparagine, or a nucleic acid encoding said region, and a CL region in which the amino acid at position 133 of the light chain is substituted with glutamic acid and the amino acid at position 176 of the light chain is substituted with lysine, or a nucleic acid encoding said region. The present invention also provides a combination of a heavy chain constant region in which the amino acid at position 183 of the heavy chain is substituted with asparagine, or a nucleic acid encoding said region, and a CL region in which the amino acid at position 133 of the light chain is substituted with glutamic acid and the amino acid at position 176 of the light chain is substituted with lysine, or a nucleic acid encoding said region. The present invention also provides a combination of a heavy chain comprising a CH1 region in which the amino acid at position 183 of the heavy chain is substituted with asparagine, or a nucleic acid encoding said heavy chain, and a light chain comprising a CL region in which the amino acid at position 133 of the light chain is substituted with glutamic acid and the amino acid at position 176 of the light chain is substituted with lysine, or a nucleic acid encoding said light chain. In this embodiment, the substitution for asparagine at position 183 of the heavy chain may be a substitution for glutamine, glutamic acid, or aspartic acid.

[0073] The CH1 region may further have a substitution of a hydrophobic amino acid residue (e.g., isoleucine) at amino acid position 173. The CH1 region may further have a substitution of a hydrophobic amino acid residue (e.g., leucine) at amino acid position 181 (EU numbering). In a preferred embodiment, the CH1 region may further have a substitution of an isoleucine at amino acid position 173 and a substitution of a leucine at amino acid position 181.

[0074] The CL region may further have a substitution of the amino acid at position 131 with a hydrophobic amino acid residue (e.g., valine). The CL region may further have a substitution of the amino acid at position 162 with a methionine. The CL region may further have a substitution of the amino acid at position 178 with a hydrophobic amino acid residue (e.g., leucine). The CL region may further have a substitution of the amino acid at position 180 with a glutamine according to the Kabat numbering system. In a preferred embodiment, the CL region may have a substitution of valine at position 131, a substitution of methionine at position 162, a substitution of leucine at position 178, and a substitution of glutamine at position 180. In a preferred embodiment, for example, the antibody may be human IgG1, human IgG2, or human IgG4. In a preferred embodiment, the light chain may be a human kappa chain or a human lambda chain.

[0075] In certain aspects, antibodies of the present disclosure having isopeptide bonds may have stability equal to or greater than that of an antibody having a heavy chain having the amino acid sequence set forth in SEQ ID NO: 61 and a light chain having the amino acid sequence of SEQ ID NO: 77. In certain aspects, antigen-binding fragments of antibodies of the present disclosure having isopeptide bonds (e.g., Fab, Fab', and F(ab')) may be fragments of the present disclosure having isopeptide bonds. ２The antibody and antigen-binding fragment thereof (such as a fragment thereof in a format of a heavy chain having the amino acid sequence set forth in SEQ ID NO: 61 and a light chain having the amino acid sequence of SEQ ID NO: 77) may have stability equal to or greater than that of an antigen-binding fragment of the corresponding format of an antibody having a heavy chain having the amino acid sequence set forth in SEQ ID NO: 61 and a light chain having the amino acid sequence of SEQ ID NO: 77. In certain aspects, the antibody and antigen-binding fragment thereof of the present disclosure having an isopeptide bond may have a CH1 region in which the amino acid at position 183 of the heavy chain is substituted with asparagine, and a CL region in which the amino acid at position 133 of the light chain is substituted with glutamic acid and the amino acid at position 176 of the light chain is substituted with lysine. In certain aspects, the antibody and antigen-binding fragment thereof of the present disclosure having an isopeptide bond may have a CH1 region in which at least the amino acid at position 183 of the heavy chain is substituted with asparagine, and a CL region in which at least the amino acid at position 133 of the light chain is substituted with glutamic acid and the amino acid at position 176 of the light chain is substituted with lysine. In this aspect, the asparagine substitution at position 183 of the heavy chain may be a substitution with glutamine, glutamic acid, or aspartic acid.

[0076] In all of the above embodiments, the amino acid at position 183 in the heavy chain may be glutamine or glutamic acid, the amino acid at position 133 in the light chain may be asparagine, and the amino acid at position 176 in the light chain may be lysine. Also, in all of the above embodiments, the amino acid at position 170 in the heavy chain may be glutamine or glutamic acid, the amino acid at position 183 in the heavy chain may be lysine, and the amino acid at position 176 in the light chain may be asparagine. In this embodiment, the substitution of asparagine at position 176 in the light chain may be a substitution of glutamine, glutamic acid, or aspartic acid.

[0077] In certain aspects, antibodies of the present disclosure having isopeptide bonds may have stability or yield that is equal to or greater than that of an antibody having a heavy chain having the amino acid sequence set forth in SEQ ID NO: 92 and a light chain having the amino acid sequence of SEQ ID NO: 94. In certain aspects, antigen-binding fragments of antibodies of the present disclosure having isopeptide bonds (e.g., Fab, Fab', and F(ab')) may be prepared. ２The antibody and antigen-binding fragment thereof (such as a fragment thereof) having an isopeptide bond may have a CH1 region in which at least one heavy chain amino acid at position 170 has been substituted with glutamic acid and at least one light chain amino acid at position 183 has been substituted with lysine, and a CL region in which at least one light chain amino acid at position 176 has been substituted with asparagine. In some aspects, the antibody and antigen-binding fragment thereof having an isopeptide bond may have a CH1 region in which at least one heavy chain amino acid at position 170 has been substituted with glutamic acid and at least one heavy chain amino acid at position 183 has been substituted with lysine, and a CL region in which at least one light chain amino acid at position 176 has been substituted with asparagine.

[0078] In one aspect, there are provided heavy chain CH1 regions, heavy chain variable regions, and heavy chains having the amino acid sequence of the CH1 region of a sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 53, 55, 57, 59, 60, 61, 62, 81, 83, 85, 88, 90, 91, and 92 (particularly, the region corresponding to positions 170 to 183 in the EU numbering system), as well as antibodies comprising any of these, or nucleic acids encoding any of these.

[0079] In one aspect, the present invention provides a CL region, a light chain variable region, and a light chain having an amino acid sequence of a CL region selected from the group consisting of SEQ ID NOs: 46, 48, 50, 54, 56, 58, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 82, 84, 86, 87, 90, 93, and 94 (particularly, a region corresponding to positions 176 to 180 (particularly a region corresponding to position 176 of mut3), e.g., a region corresponding to positions 162 to 180, e.g., a region corresponding to positions 131 to 180), as well as antibodies comprising any of these, or nucleic acids encoding any of these.

[0080] In one aspect, a heavy chain CH1 region, a heavy chain variable region, and a heavy chain having an amino acid sequence of a CH1 region (particularly a region corresponding to positions 170 to 183 in the EU numbering system) selected from the group consisting of SEQ ID NOs: 45, 47, 49, 53, 55, 57, 59, 60, 61, 62, 81, 83, 85, 88, 90, 91, and 92, or an antibody comprising any of these, or a nucleic acid encoding any of these; There may be provided a CL region, a light chain variable region, and a light chain having an amino acid sequence of the CL region (particularly the region corresponding to positions 176 to 180) of a sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 54, 56, 58, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 82, 84, 86, 87, 90, 93, and 94, as well as an antibody comprising any of these, or a combination with a nucleic acid encoding any of these.

[0081] In one aspect, a heavy chain CH1 region having an amino acid sequence selected from the group consisting of those described above (particularly, a region corresponding to positions 170 to 183 in the EU numbering system) can be linked to the VH regions of various antibodies.

[0082] In one embodiment, a CL region having an amino acid sequence of the CL region of a sequence selected from the group consisting of those listed above (particularly the region corresponding to positions 176-180) can be linked to various VL regions.

[0083] In one aspect, a heavy chain CH1 region having the amino acid sequence of a CH1 region of a sequence selected from the group consisting of those above (particularly, a region corresponding to positions 170 to 183 in the EU numbering system) can be linked to a hinge or CH2-CH3. In one aspect, a CL region having the amino acid sequence of a CL region of a sequence selected from the group consisting of those above (particularly, a region corresponding to positions 176 to 180), rather than a CH1 region, can be linked to a hinge or CH2-CH3.

[0084] In the present invention, the heavy chain of the antibody may be derived from any one selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4, or an allotype thereof. Human IgG1, human IgG2, human IgG3, and human IgG4 may have, for example, SEQ ID NOs: 11 to 14, respectively. In the present invention, the light chain of the antibody may be derived from any one selected from the group consisting of human κ chain and human λ chain, or an allotype thereof. Human κ chain and human λ chain may have, for example, SEQ ID NOs: 31 and 37, respectively. Thus, the antibody or antigen-binding fragment of the antibody of the present invention may be derived from the above-mentioned human-derived heavy chain and human-derived light chain.

[0085] The heavy and light chain constant regions may each further have additional useful mutations (e.g., mutations selected from the group consisting of insertions, additions, substitutions, and deletions), such as silent Fc mutations, deletions at positions 446 and / or 447 (EU numbering) for regulating the CH / CL interface, and improving C-terminal heterogeneity. Mutations in the heavy chain constant region are permitted as long as they do not significantly inhibit antibody function (particularly, expected antibody function), but may be limited to 15%, 10%, or 5% or less of the number of amino acids in the sequence based on the amino acid sequence of the heavy chain constant region contained in any of SEQ ID NOS: 11 to 14. Mutations in the light chain constant region are permitted as long as they do not significantly inhibit antibody function (particularly, expected antibody function), but may be limited to 15%, 10%, or 5% or less of the number of amino acids in the sequence based on the amino acid sequence of the light chain constant region contained in any of SEQ ID NOS: 31 and 37.

[0086] According to the present invention, there are provided cells (particularly antibody-producing cells, preferably Chinese hamster ovary cells) containing the above nucleic acid, and cells (particularly antibody-producing cells, preferably Chinese hamster ovary cells) containing a combination of the above nucleic acids. According to the present invention, there is provided an antigen-binding fragment of an antibody comprising the above CH1 region and CL region. Examples of antigen-binding fragments include Fab fragments, Fab' fragments, and F(ab')2 fragments, or fragments containing these. The antibody or antigen-binding fragment thereof may be fused to other proteins, etc. The antibody or antigen-binding fragment thereof may be multispecific (e.g., bispecific).

[0087] The present invention provides a chimeric antigen receptor (CAR) comprising a Fab fragment containing the above-mentioned CH1 region and CL region. While CARs typically have an scFv as the antigen-binding domain, the present invention may comprise a Fab fragment containing the above-mentioned CH1 region and CL region instead of the scFv. In these fragments and CARs, an isopeptide bond is preferably formed between the CH1 region and the CL region. The isopeptide bond is formed intracellularly during CAR production, allowing stable CARs containing the Fab fragment or Fab' fragment to be expressed on the cell surface. The present invention includes cells expressing the above-mentioned CAR (e.g., immune cells, such as NK cells, T cells, macrophages, and neutrophils). By using a Fab fragment or Fab' fragment, a CAR with strong antigen-binding affinity can be obtained relatively easily.

[0088] The present invention provides a conjugate (ADC) of an antibody comprising a heavy chain containing a CH1 domain and a light chain containing a CL domain, or an antigen-binding fragment thereof (e.g., a Fab fragment, a Fab' fragment, etc.), with a cytotoxic agent. This ADC preferably contains an isopeptide bond between the heavy chain containing the CH1 domain and the light chain containing the CL domain, thereby stabilizing the bond between the heavy chain and the light chain even when an intermolecular disulfide bond is used to link the drug. The ADC may have a drug-antibody ratio of 1 to 8, for example, 4 to 8, preferably 6 to 8, and more preferably 8. In a preferred embodiment, an ADC containing an isopeptide bond may have higher stability and / or higher cytotoxicity than an ADC without an isopeptide bond.

[0089] According to the present invention, an antigen-binding fragment of an antibody (e.g., Fab fragment, Fab' fragment, F(ab')) comprising a heavy chain comprising the CH1 region and a light chain comprising the CL region is ２ Conjugates (ADCs) of antigen-binding fragments (e.g., Fab fragments, Fab' fragments, F(ab') fragments, etc.) and cytotoxic agents are provided. ２ Similarly, in conjugates (ADCs) of a cytotoxic drug (e.g., a fragment thereof) and a heavy chain, the covalent linkage between CH1 and CL via an isopeptide bond stabilizes the bond between the heavy chain and the light chain, even if an intermolecular disulfide bond is used to link the drug. Therefore, the introduction of an isopeptide bond can be particularly beneficial in ADCs.

[0090] The present invention provides a peptide (first peptide) comprising an amino acid sequence including the amino acid at position 181 of the CH1 region, preferably an amino acid sequence including the amino acids at positions 173 and 181. The present invention also provides a peptide (second peptide) comprising an amino acid sequence including the amino acids at positions 133 and 176 of the CL region. When these peptides coexist under physiological conditions, they can spontaneously form isopeptide bonds between them. Therefore, a stable bond can be formed between the first peptide and the second peptide. By fusing the first peptide and the second peptide as tags to a target protein, multiple target proteins can be allowed to interact with each other or linked by isopeptide bonds. The first peptide and the second peptide may each be at least 5 amino acids long, at least 10 amino acids long, at least 15 amino acids long, at least 20 amino acids long, at least 30 amino acids long, at least 40 amino acids long, at least 50 amino acids long, at least 60 amino acids long, at least 70 amino acids long, at least 80 amino acids long, at least 90 amino acids long, or at least 100 amino acids long, or the entire length of the CH1 or CL1 region. According to the present invention, the peptide may further comprise a heavy chain variable region (VH) or a light chain variable region (VL). In a preferred embodiment, the peptide comprises the VH, CH1, VL, and CL regions and has all or part of its antigen-binding activity.

[0091] Various antibody modifications are known, and any antibody modification may be introduced into an antibody containing an isopeptide bond. For example, according to the EU numbering of the heavy chain shown in Figure 11, mutations that promote Fc heterodimerization include the following: first chain: 354C and 366W; second chain: 349C, 366S, 368A, and 407V; first chain: 356K, 357K, and 399K; second chain: 370E, 409D, and 439E; first chain: 364H and 405A; Examples of such mutations include the mutation combination of 349T and 394F, the mutation combination of first strand: 405L; second strand: 409R, the mutation combination of first strand: 350V, 351Y, 400E, 405A, and 407V, the mutation combination of second strand: 350V, 366L, 390R, 392M, and 394W, and the mutation combination of first strand: 351D and 368E; second strand: 351K and 366K. Mutations that enhance effector function include a combination of 298A, 333A, and 334A mutations, a combination of 239D, 330L, and 332E mutations, a combination of 236A, 239D, and 332E mutations, a combination of 236A, 330L, and 332E mutations, a combination of 236A, 239D, 330L, and 332E mutations, a combination of 243L, 292P, 300L, 305I, and 396L mutations, a combination of 235V, 243L, 292P, 300L, and 396L mutations, and a first chain: 234Y, 235Q, 236W, 239M, 268D, 270E, and 298A;Second chain: Examples of mutation combinations include 270E, 326D, 330M, and 334E. Mutations that enhance CDC activity include combinations of 326W and 333S, 345R, 430G, and 440Y, and 236A, 267E, 268F, 324T, and 332E. Mutations that enhance binding activity to FcγRIIB include combinations of 267E and 328F, and 233D, 237D, 238D, 268D, 271G, and and 330R mutation combinations, and examples of mutations that suppress effector function include a single mutation of 297A, a single mutation of 297Q, a single mutation of 297G, a single mutation of 265A, a mutation combination of 234A and 235A (LALA mutation), a mutation combination of 235A, 237A, and 318A, a mutation combination of 234A and 235A, a mutation combination of 228P and 235E, a mutation combination of 236R and 328R, a mutation combination of 298G and 299A, and 234F, 235E, and 331S. the combination of mutations 233P, 234V, 235A, 236 deletion and 267K; the combination of mutations 234A, 235A and 329G; the combination of mutations 234A, 237A, 238S, 268A, 309L, 330S and 331S; and the combination of mutations 234F, 235E and 265A, as well as combinations of these single mutations and / or combinations of mutations. The variations include, but are not limited to, the 252Y, 254T and 256E mutation combination, the 250Q and 428L mutation combination, the 433K and 434F mutation combination, the 307A, 380A and 434A mutation combination, the 428L and 434S mutation combination, the 308P single mutation, the 252Y, 308P and 434Y mutation combination, the 285D, 307Q and 378V mutation combination, and the 309D, 311H and 434S mutation combination.

[0092] (Performance and Further Development of Polypeptides Comprising Isopeptide Bonds) Isopeptide bonds can be more stable and stronger than other types of inter-peptide chain bonds, such as disulfide bonds, and can therefore confer beneficial effects to polypeptides, such as stability at high temperatures, pH, high concentrations, and / or high osmotic pressures (e.g., reduced aggregate formation), resistance to degradation by enzymes, etc. (e.g., increased plasma half-life), etc. Polypeptide stability can be advantageous not only in the use of the polypeptide but also in its production (e.g., more severe conditions can be used for purification). High concentration stability of a polypeptide can allow the same amount of therapeutic polypeptide to be administered in a smaller volume, thereby reducing the burden on patients.

[0093] Because isopeptide bonds are formed between at least two different amino acids, a) and b), polypeptides with isopeptide bonds have asymmetric heterochains. This reduces the formation of aggregates other than the desired aggregate, which is often a problem when associating different molecules to form heterodimers. In particular, since the formation of isopeptide bonds may require that the paired polypeptide chains have structures that are specific to each other, the desired aggregate can be efficiently produced even when multiple types of polypeptide chains, including a) or b), coexist. By utilizing this advantage, aggregates composed of three or more polypeptide chains (such as trispecific antibodies) can also be efficiently produced. DuetMab technology, which introduces a new disulfide bond at a site other than the original CH1-CL disulfide bond to suppress mispairing between heavy and light chains, is known. However, it has been reported that by-products result from disulfide bond formation with the newly introduced Cys residue (Journal of Pharmaceutical Sciences 110 (2021) 2904-2915). While knob-hole technology is also used to form assemblies of specific pairs, assemblies using the isopeptide bond disclosed herein exhibit stronger binding strength between the pair and can form assemblies with more compact domains. For example, isopeptide bonds may be formed between the constant regions of the two heavy chains of a bispecific antibody.

[0094] In one embodiment, a polypeptide comprising isopeptide bonds of the present disclosure is a polypeptide molecule comprising at least two (e.g., two, three, four, five) isopeptide bonds. In one embodiment, a polypeptide comprising isopeptide bonds of the present disclosure is a polypeptide molecule comprising at least three (e.g., three, four, five, six) different polypeptide molecules linked by isopeptide bonds. In one embodiment, a polypeptide comprising isopeptide bonds of the present disclosure is a polypeptide molecule comprising different polypeptide molecules linked by isopeptide bonds, where one of the different polypeptide molecules is linked to another different polypeptide molecule by at least two (e.g., two, three, four, five) isopeptide bonds.

[0095] In one embodiment, a protein that is naturally expressed as a single molecule can be expressed as different polypeptide molecules by, for example, removing a linker region, and the three-dimensional structure of the native protein can be maintained by isopeptide bonds.

[0096] Disulfide bonds in known polypeptides function to stabilize the polypeptide structure, and it was thought that removing disulfide bonds would result in a loss of proximity between polypeptide chains at disulfide bond formation sites. However, the inventors unexpectedly discovered that isopeptide bonds can be introduced to replace disulfide bonds. In one embodiment, an isopeptide bond-containing polypeptide of the present disclosure has an isopeptide bond introduced to replace the disulfide bond. For example, two cysteine ​​residues that form a disulfide bond in the three-dimensional structure of a known polypeptide are identified, and at least one of these two cysteine ​​residues is deleted. Here, removing the disulfide-bonding cysteine ​​residues can prevent the formation of unintended aggregates through disulfide bonds (which allow symmetric reactions). It is believed that disulfide bond-forming portions of known polypeptides approach each other before disulfide bond formation, with or without a folding-assisting protein such as a chaperone. Therefore, introducing a) and / or b) near the disulfide bond formation site can promote the proximity of a) and b) by a mechanism similar to that of the unmodified polypeptide. Furthermore, the introduction of an isopeptide bond instead of a disulfide bond can maintain the higher-order structure and function of the protein. In one embodiment, a) and / or b) are introduced into the interface region between two domains that are in contact with each other via a disulfide bond. In one embodiment, a) and / or b) are introduced near the disulfide bond formation site (e.g., within 5, 4, 3, 2, or 1 residues of a cysteine ​​involved in disulfide bond formation).

[0097] In one embodiment, a polypeptide comprising an isopeptide bond of the present disclosure is expressed in a cell, and at least one of the different polypeptide molecules linked to each other by an isopeptide bond is expressed from a transgene. For example, a CAR-T cell is provided that expresses a chimeric antigen receptor comprising a Fab-type antibody as the polypeptide comprising an isopeptide bond of the present disclosure.

[0098] In one embodiment, the method of producing a polypeptide comprising an isopeptide bond of the present disclosure comprises creating a bond between the modified polypeptide and a third polypeptide chain contained in a molecule different from the modified polypeptide, the bond having the following formula: (wherein the dashed line represents a bond to the polypeptide backbone), further producing a secondary modified polypeptide comprising an interpeptide linkage represented by the formula: (wherein the dashed line represents a bond to the polypeptide backbone), and constructing a three-dimensional structure of secondary intermediate A comprising the modified polypeptide and a third polypeptide chain; identifying an amino acid sequence present in the boundary region where the modified polypeptide and the third polypeptide chain in the three-dimensional structure are adjacent to each other; mutating secondary intermediate A by introducing at least one residue of a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid into secondary intermediate A to prepare secondary intermediate B, wherein secondary intermediate B comprises a), b) and c), and the modified polypeptide and the third polypeptide chain in secondary intermediate B comprise a) and b), respectively, in the boundary region; and obtaining a secondary modified polypeptide by forming an interpeptide linkage in secondary intermediate B. Similarly, by engineering interpeptide linkages between a fourth polypeptide chain, a fifth polypeptide chain, etc., it is possible to create a polypeptide in which even more polypeptide chains are linked by isopeptide bonds.

[0099] One skilled in the art can use any system to produce polypeptide chains that form polypeptides containing isopeptide bonds as described herein.

[0100] The produced polypeptide can be purified or isolated by any purification method. Examples include immunoglobulin purification methods such as size fractionation, fractionation by salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration, and protein A / G affinity chromatography. In particular, when the monoclonal antibody is mouse IgG, it can be efficiently purified by affinity chromatography using a protein A-bound carrier or an anti-mouse immunoglobulin-bound carrier.

[0101] (Uses) A ​​polypeptide comprising an isopeptide bond of the present disclosure can be used for known uses of polypeptides, for example, when the polypeptide is an antibody, it can be used to detect an antigen and / or treat or prevent a disease associated with the antigen. In one aspect, the present disclosure provides uses of a polypeptide comprising an isopeptide bond of the present disclosure. In one aspect, the present disclosure provides a composition comprising a polypeptide comprising an isopeptide bond of the present disclosure. The composition can be used for known uses of polypeptides.

[0102] (Dosage Forms, etc.) The compositions described herein may be provided in various forms. Examples of the composition may include injections, capsules, tablets, granules, etc. Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers. In addition, aqueous solutions for injection may contain, for example, physiological saline, sugars (e.g., trehalose), NaCl, NaOH, etc.

[0103] In one embodiment, the compositions of the present disclosure include a pharmaceutically acceptable carrier or excipient. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical is administered orally, water is the preferred carrier. When the pharmaceutical composition is administered intravenously, saline and aqueous dextrose are the preferred carriers. Preferably, saline solution and aqueous dextrose and glycerol solution are used as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. The compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can also be formulated as suppositories, using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, USA).In addition to these, the composition may contain, for example, surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, taste masking agents, etc. In one embodiment, the pH of any liquid composition of the present disclosure can be about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, or a range between any two of these values.

[0104] Any component of the compositions of the present disclosure can be provided as a pharmaceutically acceptable salt, such as salts formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; salts formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.; and salts formed with sodium, potassium, ammonium, calcium, and ferric hydroxide.

[0105] In a preferred embodiment, the composition can be formulated as a pharmaceutical composition adapted for administration to humans according to known methods. Such compositions can be administered by injection. Typically, compositions for injection administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active ingredient. If the composition is to be administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0106] When the composition of the present disclosure is applied to a subject, the subject is not particularly limited, and may be a mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, pig, monkey, human, etc.), bird, reptile, amphibian, arthropod, fish, etc.

[0107] The amount of the composition and / or active ingredient of the present disclosure will vary depending on the nature of the disorder or condition to be treated or prevented, but can be determined by one of ordinary skill in the art using standard clinical techniques based on the disclosure herein. In some cases, in vitro assays may be used to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also vary depending on the route of administration and the severity of the disease or disorder, and should be decided according to the judgment of the attending physician and each patient's circumstances. The dosage of the composition and / or active ingredient of the present disclosure is not particularly limited, and may be, for example, 0.00001, 0.0001, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per administration, or within any two of these ranges. The administration interval is not particularly limited, and may be, for example, once or twice per 1, 7, 14, 21, or 28 days, or once or twice within any two of these ranges. The dosage, administration interval, and administration method may be appropriately selected depending on the age and weight of the patient, symptoms, target organ, etc.

[0108] The compositions described herein may be administered, for example, intravenously, intradermally, subcutaneously, intramuscularly, intraperitoneally, intranasally, epidurally, or orally. In one embodiment, the compositions of the present disclosure can be used in conjunction with various delivery systems. These systems include, for example, encapsulation in liposomes, microparticles, and microcapsules; use of receptor-mediated endocytosis; and construction of therapeutic nucleic acids as part of retroviral or other vectors. Medication can be administered by any suitable route, such as by infusion, bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), or by inhaler or nebulizer, optionally with an aerosolizing agent, and can be administered in conjunction with other drugs. Administration can be systemic or local.

[0109] The compositions of the present disclosure can be provided as kits. In one embodiment, the present disclosure provides pharmaceutical packs or kits comprising one or more containers filled with one or more components that can be added to the compositions of the present disclosure. Optionally, such containers can also be associated with information indicating approval by a government agency for the manufacture, use, or sale for human administration, in a manner prescribed by the government agency regulating the manufacture, use, or sale of pharmaceutical or biological products.

[0110] Procedures for formulating the compositions of the present disclosure as pharmaceuticals and the like are known in the art and are described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopoeia, the Pharmacopoeias of other countries, etc. Therefore, given the description herein, one skilled in the art can determine the embodiments, such as the amount to be used, without undue experimentation.

[0111] The antibodies of the present disclosure contain intrachain mutations (e.g., amino acid substitution mutations as described above) to introduce isopeptide bonds, but may also contain various other mutations as long as they do not significantly inhibit or eliminate antibody function (e.g., the desired antibody function). Mutations may be selected and introduced depending on the purpose, but it is desirable to minimize the number of mutations introduced because they may increase the antigenicity of the antibody. Examples of additional mutations that can be introduced into antibodies include mutations to reduce Fcγ receptor binding (binding to any of Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and FcγIIIB) (i.e., silent Fc mutations), mutations at amino acids 297 and 299 to suppress or eliminate glycosylation, and mutations to improve C-terminal heterogeneity (e.g., deletion of G446 and / or K447).

[0112] When the antibody is a bispecific antibody, it is possible to introduce, without particular limitation, mutations for hetero-Fc association (e.g., knobs-into-holes mutations), mutations for controlling the CH / CL interface, and mutations for reducing binding to Protein A.

[0113] In a bispecific antibody having two Fab-type arms, an isopeptide bond can be formed between the CH1-CL of one of the Fab-type arms, which can promote complex formation between the correct heavy and light chains.

[0114] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range" of "two values," the range includes the two values ​​themselves.

[0115] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0116] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0117] The reagents used were specifically the products described in the Examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC., etc.) can also be used.

[0118] (Example 1: Design of isopeptide bond-forming trastuzumab IgG) An attempt was made to form an isopeptide bond between the heavy and light chains of an antibody. The anti-HER2 antibody h4D5 (trastuzumab) was used as the starting protein.

[0119] The coordinate data of the trastuzumab Fab-HER2 complex crystal structure (PDB ID: 1N8Z) registered in the Protein Data Bank (https: / / www.rcsb.org / ) was downloaded, and the three-dimensional structure was constructed using PyMOL (https: / / pymol.org / 2 / ).

[0120] Based on the three-dimensional structure constructed above, the region where the heavy chain constant region and the light chain constant region are adjacent was identified, and asparagine and lysine were introduced into this region. Asparagine was introduced into isoCH1, and lysine was introduced into isoCL. Next, hydrophobic amino acids were introduced to create a hydrophobic environment around the side chains of the introduced asparagine and lysine. In addition, cysteine ​​involved in disulfide bond formation was replaced with serine. Here, a homology model was constructed using SWISS-MODEL (https: / / swissmodel.expasy.org / ) based on appropriate sequence data, and a structural model with an isopeptide bond introduced was created using Coot (https: / / www2.mrc-lmb.cam.ac.uk / personal / pemsley / coot / ).

[0121] As a result, the following sequences were designed. In this example, polypeptides modified for isopeptide bond formation may be referred to as "iso-" or "iSo-" (e.g., isoCH1, iSoMAb). MAb refers to monoclonal antibody.

[0122] The antibody structure after the above mutations was recalculated, and the side chains of asparagine, lysine, and glutamic acid involved in isopeptide bond formation were positioned as shown in Figure 1. The results of recalculation of the three-dimensional structure after isopeptide bond formation are shown in Figure 2.

[0123] (Example 2: Preparation of isopeptide bond-forming trastuzumab IgG and evaluation of binding activity) The polypeptides designed as described above were prepared and evaluated.

[0124] (Preparation of isoCH1 and isoCL iSoMAb) - Preparation of h4D5 iSoMAb IgG expression vector The mutant CH1 (isoCH1) and CL (isoCL) genes were synthesized by Eurofins Genomics. The isoCH1 gene and hinge-Fc gene were ligated by overlap extension PCR and digested with the restriction enzymes Nhe I and Not I. This was inserted into pCAGEN containing the h4D5VH gene previously digested with the same enzymes to produce the expression vector pCA h4D5 isoHC. Furthermore, the h4D5VL gene and isoCL gene were ligated by overlap extension PCR and digested with the restriction enzymes Eco RI and Not I. The vector was inserted into pCAGEN that had been pre-digested with the same enzymes to prepare the expression vector pCA h4D5 isoLC.

[0125] Expression of h4D5 iSoMAb IgG: Expi293F cells (Gibco, Thermo Fisher Scientific) were used as host cells. Expi293F cells were cultured in HE200 medium (Gmep) containing 2 mM L-alanyl-L-glutamine at 37°C, 5% CO2, and 125 rpm. 7.5 x 107 cells were harvested by centrifugation at 1,500 rpm for 5 minutes and suspended in 27 mL of HE400 medium (Gmep) containing 5 mM L-alanyl-L-glutamine. 30 μg of plasmid (15 μg of pCA h4D5 isoHC and 15 μg of pCA h4D5 isoLC) was added to 1.5 mL of Opti-MEM medium (Gibco) and mixed. 120 μL of PEI MAX (1 mg / mL; Polysciences) was added to 1.5 mL of Opti-MEM medium, mixed, and allowed to stand at room temperature for 5 minutes. The diluted plasmid and PEI solutions were mixed and then allowed to stand at room temperature for 20 minutes. This was used as the transfection solution. The transfection solution was added to Expi293F cells suspended in HE400 medium and cultured. After 20 hours, 75 μL of 0.5 M sodium valproate, 120 μL of 1 M sodium propionate, and 750 μL of 20% (w / v) tryptone were added, and the cells were cultured for an additional 6 days.

[0126] Purification of h4D5 iSoMAb IgG Cells were removed from the culture supernatant by centrifugation at 4°C, 6,000 rpm, for 10 minutes. The culture supernatant was filtered using a 0.45 μm pore size filter (Membrane Solutions) and loaded onto a Protein A column (column volume 250 μL; Bipo Resin Protein A; Protein Express) pre-equilibrated with phosphate buffered saline (PBS). After washing with 10 mL of PBS, the target antibody was eluted with 100 mM glycine-HCl (pH 2.5). 200 μL aliquots were collected and neutralized with 10 μL of 100 mM Tris-HCl (pH 9.0) that had been added to the sample tube in advance. Each fraction was analyzed by SDS-PAGE, and the fraction containing the target antibody was dialyzed against PBS. The absorbance at 280 nm was measured using a spectrophotometer (NanoPhotometer NP80; Implen), and the target antibody was quantified by subtracting the background absorbance at 320 nm. The extinction coefficient was calculated as 195,400 M-1 cm-1 / tetramer. The purified antibody was further analyzed by Western blotting. HRP-labeled anti-human Fc antibody (Sigma) and HRP-labeled anti-human kappa chain antibody (Sigma) were used as detection antibodies.

[0127] The results are shown in Figure 3. Because the disulfide bond was cleaved by reduction, no heavy-light chain complex was formed in wild-type h4D5 IgG. On the other hand, in the case of h4D5 iSoMAb, a band was observed in the region corresponding to the target molecular weight of the heavy-light chain complex, suggesting that an isopeptide bond was formed between the heavy and light chains.

[0128] Evaluation of binding activity to HER2-positive cells. Binding activity to the HER2-positive human breast cancer cell line SKBR-3 was evaluated by flow cytometry. SKBR-3 cells were cultured in 10% FBS / D-MEM medium (Wako) under 37°C and 5% CO2 conditions and detached from the culture dish using trypsin / EDTA (0.25% (w / v) trypsin-1 mM EDTA 4Na; Gibco). Cells were collected by centrifugation at room temperature, 1,000 rpm, for 5 minutes, and 5 x 105 cells were dispensed into sample tubes. After washing with 0.1% NaN3 / PBS, the cells were incubated with h4D5 iSoMAb IgG as the primary antibody at a final concentration of 500 nM for 30 minutes on ice. After washing in the same manner, 1 μL of anti-human IgG (Fc-specific)-FITC antibody (2 mg / mL; Sigma) was added as a secondary antibody and 499 μL of 0.1% NaN3 / PBS was added, and the mixture was incubated on ice for 30 minutes. After washing again, the cells were suspended in 500 μL of 0.1% NaN3 / PBS. After filtration through a nylon filter, the cells were dispensed into plastic tubes and analyzed using a flow cytometer (Accuri C6; BD Biosciences).

[0129] The results are shown in Figure 4. The iSoMAb was found to have the same HER2 binding ability as the original antibody.

[0130] (Example 3: Preparation of isopeptide bond-forming antibodies in Fab format) Based on the iSoMAb prepared above, antibody fragments in Fab format were prepared and evaluated.

[0131] Preparation of h4D5 iSoMAb Fab heavy chain expression vector: Using the synthesized isoCH1 gene as a template, a histidine tag sequence was added to the C-terminus of the CH1 region by PCR. The PCR product was digested with restriction enzymes Nhe I and Not I. The PCR product was inserted into pCAGEN containing the h4D5VH gene previously digested with the same enzymes to prepare the expression vector pCA h4D5VH-isoCH1.

[0132] Expression of h4D5 iSoMAb Fab h4D5 iSoMAb Fab was expressed using the same method as above. pCA h4D5VH-isoCH1 and pCA h4D5 isoLC were used as the plasmids for gene transfer.

[0133] Purification of h4D5 iSoMAb Fab: Cells were removed from the culture supernatant by centrifugation at 4°C, 6,000 rpm, for 10 minutes. The culture supernatant was filtered using a 0.45 μm pore size filter (Membrane Solutions) and loaded onto Ni Sepharose Excel (column volume 500 μL; Cytiva) pre-equilibrated with 20 mM sodium phosphate / 500 mM NaCl (pH 7.4). After washing with 10 mL of 20 mM sodium phosphate / 500 mM NaCl (pH 7.4) containing 30 mM imidazole, the target antibody was eluted with 20 mM sodium phosphate / 500 mM NaCl (pH 7.4) containing 500 mM imidazole. After collecting 200 μL of each fraction, each was analyzed by SDS-PAGE, and the fraction containing the target antibody was dialyzed against PBS. The absorbance at 280 nm was measured using a spectrophotometer (NanoPhotometer NP80), and the target antibody was quantified by subtracting the background absorbance at 320 nm. The extinction coefficient was calculated as 64,900 M-1 cm-1 / dimer.

[0134] The results are shown in Figure 5. Because the disulfide bond was cleaved by reduction, no heavy-light chain complex was formed in wild-type h4D5 Fab. On the other hand, in the case of h4D5 iSoMAb Fab, a band was observed in the region corresponding to the target molecular weight of the heavy-light chain complex, suggesting that an isopeptide bond was formed between the heavy and light chains.

[0135] Evaluation of thermal stability by differential scanning fluorometry 17.5 μL of a 50-fold diluted fluorescent dye (SYPRO (registered trademark) dye, protein gel stain; Merck) was added to 70 μL of a 0.1 mg / mL antibody solution and mixed. 25 μL of the antibody-dye mixed solution was dispensed into three wells of a 48-well PCR plate. Using a Real-Time PCR device, the temperature was raised from 25°C to 99°C over approximately 40 minutes, and the fluorescence intensity was measured. Furthermore, to examine thermal stability under reducing conditions, TCEP (tris(2-carboxyethyl)phosphine) was added to the antibody solution to a final concentration of 1 mM, and the solution was left to stand on ice for 3 hours, after which measurements were performed in the same manner.

[0136] The results are shown in Figure 6 and the table below. The h4D5 iSoMAb Fab exhibited a slight decrease in thermal stability compared to the h4D5 Fab, but the decrease in thermal stability was not so great as to prevent purification or in vivo use. Furthermore, unlike the h4D5 Fab, the h4D5 iSoMAb Fab exhibited no decrease in stability under reducing conditions (in the presence of TCEP), confirming that the isopeptide bond is resistant to cleavage under reducing conditions.

[0137] (Example 4: Design of isopeptide bond-forming anti-CD63 antibody) As in the above example based on the anti-HER2 antibody h4D5, an attempt was made to create a modified antibody in which the heavy chain and light chain were linked by an isopeptide bond based on the anti-CD63 antibody (clone No. 2 described in Monoclon. Antib. Immunodiagn. Immunother. 39, 74-76 (2020)).

[0138] A three-dimensional model was constructed in the same manner as in Example 1, and the following sequences were designed based on it. Asparagine was introduced into isoCH1v1, and lysine was introduced into isoCLv1. Conversely, lysine was introduced into isoCH1v2, and asparagine was introduced into isoCLv2. Next, glutamic acid was introduced so that its side chain was adjacent to the side chains of the introduced asparagine and lysine.

[0139] The positional relationships between the atoms of a), b), c) and the hydrophobic mutated residues in the three-dimensional structure are as follows:

[0140] Preparation of hCD63 iSoMAb IgG Expression Vectors We commissioned Eurofins Genomics to synthesize IgG and CL (isoCL) genes containing mutant CH1 (isoCH1). The synthesized genes were inserted into pCEC4.3W by seamless cloning to create pCEC4.3W-isoCH1vC (Afl II) or pCEC4.3W-isoCLvC (BsrG I). These vectors were digested with the restriction enzyme Afl II or BsrG I, and the humanized hCD63 antibody genes for the heavy and light chains were inserted by seamless cloning to create the expression vectors pCEC4.3W-isoCH1vC hCD63 H1 and pCEC4.3W-isoCLvC hCD63 K1.

[0141] Expression of hCD63 iSoMAb IgG: ExpiCHO-S cells (Gibco, Thermo Fisher Scientific) were used as host cells. ExpiCHO-S cells were cultured in ExpiCHO Expression Medium (Gibco, Thermo Fisher Scientific) at 37°C, 5% CO2, and 125 rpm. Culture and transfection methods followed the protocol for the ExpiCHO Expression System (Gibco, Thermo Fisher Scientific). 18.0 × 10 cells were collected by centrifugation at 300 × g for 5 minutes and suspended in 30 mL of ExpiCHO Expression Medium. 24 μg of plasmids (12 μg of pCEC4.3W-isoCH1vC hCD63 H1 and 12 μg of pCEC4.3W-isoCLvC hCD63 K1) were added to 1.2 mL of OptiPRO SFM Complexation Medium (Gibco, Thermo Fisher Scientific) and mixed. Separately, 96 μL of ExpiFectamine CHO Reagent (Gibco, Thermo Fisher Scientific) was added to 1.104 mL of OptiPRO SFM Complexation Medium and mixed. The diluted plasmid solution and the diluted ExpiFectamine CHO Reagent were mixed and then allowed to stand at room temperature for 5 minutes to prepare a transfection solution. The transfection solution was added to CHO-S cells suspended in ExpiCHO Expression Medium and cultured. After 18 hours, 180 μL of ExpiFectamine® CHO Enhancer and 7.2 mL of ExpiCHO® Feed were added, and the cells were cultured for an additional 12 days.

[0142] Purification of hCD63 iSoMAb IgG: Cells were removed from the culture supernatant by centrifugation at 12,000 x g for 10 minutes at 4°C. The culture supernatant was filtered using a 0.45 μm pore size filter (GVS), and 1.0 mL of Protein A Sepharose pre-equilibrated with PBS buffer (PBS) was added to the filtered supernatant. After overnight stirring at 4°C, the gel was loaded onto the column. After washing with 30 mL of PBS, the target antibody was eluted with 0.1 M citric acid eluent (pH 3.0). 1.0 mL fractions were collected, and each fraction was analyzed by SDS-PAGE. The fractions containing the target antibody were eluted with PBS using 2-6 mL of Pierce® Protein Concentrators PES, 10K MWCO. The absorbance at 280 nm was measured using a spectrophotometer (Eppendorf BioPhotometer plus), and the background absorbance at 340 nm was subtracted to quantify the antibody of interest. More accurate concentrations were then determined using Pierce® BCA Protein Assay Kits.

[0143] Evaluation of binding activity to hCD63-positive cells. Binding activity to the hCD63-positive human fetal kidney-derived cell line 293 was evaluated by flow cytometry. Expi293F cells were cultured in Expi293 Expression Medium (Gibco, Thermo Fisher Scientific) at 37°C, 5% CO2, and 125 rpm. The cells were collected by centrifugation at room temperature for 5 minutes at 300 x g, and 5 x 105 cells were dispensed into sample tubes. After washing with 0.2% BSA / PBS, the cells were incubated with hCD63 iSoMAb IgG (primary antibody) at a final concentration of 2 μg / mL for 60 minutes at 4°C. After washing in the same manner, the cells were incubated with a secondary antibody, goat F(ab')2 anti-human IgG-Fc (PE), pre-adsorbed (ab98596, Abcam) at a final concentration of 0.5 μg / mL for 30 minutes at 4°C. After washing again, the cells were suspended in 200 μL of 0.2% BSA / PBS. The cells were dispensed into plastic tubes and analyzed using a flow cytometer (FACS Verse; BD Biosciences).

[0144] Gel electrophoresis (Figure 7) revealed a band at the target molecular weight of the iSoMAb heavy-light chain complex under reducing conditions, suggesting the formation of an isopeptide bond between the heavy and light chains (lanes 3 and 4). Expression of iSoMAb in Expi293F cells was also confirmed (lanes 5 and 6). Flow cytometry (Figure 8) also confirmed that both iSoMAbs maintained their binding to CD63-expressing cells (strain 293). Specifically, when one of the amino acids at position 186 in the heavy chain and the other at position 176 in the light chain was lysine and the other asparagine, an isopeptide bond was formed between these amino acids.

[0145] (Example 5: Preparation of bispecific antibody containing an isopeptide bond) An attempt was made to prepare a bispecific antibody containing one isopeptide bond. An outline of the structure of the constructed bispecific antibody is shown in Figure 9. This is a bispecific antibody containing the variable regions of h4D5 (anti-HER2) and U1-59 (anti-HER3), where h4D5 was modified to form an isopeptide bond according to Examples 1 and 2.

[0146] Preparation of h4D5xU1-59 iSoMAb bispecific IgG expression vector The isoCH1 gene and hinge-Fc (knob) gene were ligated by overlap extension PCR and digested with the restriction enzymes Nhe I and Not I. This was inserted into pCAGEN containing the h4D5VH gene previously digested with the same enzymes to prepare the expression vector pCA h4D5 isoHC (knob). Next, Eurofins Genomics was commissioned to synthesize the U1-59VH and VL genes. A plasmid containing the U1-59VH gene was digested with the restriction enzymes Afl II and Nhe I and then inserted into pCAGEN containing the CH1-Fc (hole) gene pre-digested with the same enzymes to produce the expression vector pCA U1-59 HC (hole). The U1-59VL gene and CL gene were ligated by overlap extension PCR and digested with the restriction enzymes Eco RI and Not I. This was then inserted into pCAGEN pre-digested with the same enzymes to produce the expression vector pCA U1-59 LC.

[0147] Expression of the h4D5xU1-59 iSoMAb bispecific IgG expression vector. Expi293F cells (Gibco, Thermo Fisher Scientific) were used as host cells. Expi293F cells were cultured in HE200 medium (Gmep) containing 2 mM L-alanyl-L-glutamine at 37°C, 5% CO2, and 125 rpm. 7.5 x 10 ７ The cells were collected by centrifugation at 1,500 rpm for 5 minutes and suspended in 27 mL of HE400 medium (Gmep) containing 5 mM L-alanyl-L-glutamine. 30 μg of plasmids (7.5 μg of pCA h4D5 isoHC (knob), 7.5 μg of pCA U1-59 HC (hole), 7.5 μg of pCA h4D5 isoLC, and 7.5 μg of pCA U1-59 LC) were added to 1.5 mL of Opti-MEM medium (Gibco) and mixed. 120 μL of PEI was added to 1.5 mL of Opti-MEM medium. MAX (1 mg / mL; Polysciences) was added, mixed, and allowed to stand at room temperature for 5 minutes. The plasmid and PEI diluted solutions were mixed and allowed to stand at room temperature for 20 minutes, and this was used as the transfection solution. The transfection solution was added to Expi293F cells suspended in HE400 medium and cultured. After 20 hours, 75 μL of 0.5 M sodium valproate, 120 μL of 1 M sodium propionate, and 750 μL of 20% (w / v) tryptone were added, and the cells were cultured for an additional 6 days.

[0148] Purification of h4D5xU1-59 iSoMAb bispecific IgG Cells were removed from the culture supernatant by centrifugation at 4°C, 6,000 rpm, for 10 minutes. The culture supernatant was filtered using a 0.45 μm pore filter (Membrane Solutions) and loaded onto a Protein A column (column volume 250 μL; Bipo Resin Protein A; Protein Express) pre-equilibrated with phosphate buffered saline (PBS). After washing with 10 mL of PBS, the target antibody was eluted with 100 mM glycine-HCl (pH 2.5). 200 μL aliquots were collected and neutralized with 10 μL of 100 mM Tris-HCl (pH 9.0) that had been added to the sample tube beforehand. Each fraction was analyzed by SDS-PAGE, and the fraction containing the target antibody was dialyzed against PBS. The absorbance at 280 nm was measured using a spectrophotometer (NanoPhotometer NP80; Implen), and the target antibody was quantified by subtracting the background absorbance at 320 nm. The extinction coefficient was calculated as 220,500 M-1cm-1 / tetramer.

[0149] Evaluation of bispecificity by sandwich ELISA. 50 μL of 10 μg / mL HER2-ECD-human Fc was added to a 96-well EIA / RIA plate (Corning) and incubated at 37°C for 30 minutes. After removing the solution, 200 μL of PBS containing 0.05% (v / v) Tween® 20 (PBST) was added to each well and washed three times. 100 μL of PBST containing 1% (w / v) BSA as blocking buffer was added to each well. After incubation at 37°C for 1 hour, the blocking buffer was removed, and 200 μL of PBST was added to each well and washed three times. 50 μL of bispecific antibody solution at each concentration diluted with PBST containing 1% (w / v) BSA was added to each well and incubated at 37°C for 30 minutes. After removing the antibody solution, 200 μL of PBST was added to each well and washed three times. 50 μL of 10 μg / mL HER3-ECD-mouse Fc was added to each well. After standing at 37°C for 30 minutes, the solution was removed, and 200 μL of PBST was added to each well, followed by washing three times. 50 μL of 0.25 μg / mL anti-IgG (H+L), Mouse, Goat-Poly, HRP (SeraCare Life Sciences, Inc.) was added as a detection antibody, followed by standing at 37°C for 30 minutes. After removing the antibody solution, 200 μL of PBST was added to each well, followed by washing three times. Chromogenic substrate solution [50 μL of 10 mg / mL TMB, 4 μL of 3% H2O2, Substrate solution A (50 mM Na ２ HPO ４ ・12H ２ 50 μL of a reaction stop solution (1N H O, 25 mM citric acid, pH 5.5) was added to each well, and the mixture was left to stand for 15 minutes. ２ SO ４ ) was added in an amount of 50 μL each. The absorbance at 450 nm was measured using a plate reader Infinite 200 PRO (TECAN).

[0150] The results are shown in Figure 10. While no color development was observed with the monospecific h4D5 IgG and U1-59 IgG, significant color development was observed with the h4D5xU1-59 iSoMAb bispecific IgG. This demonstrates that antibodies containing isopeptide bonds are applicable to bispecific antibody formats. It is believed that bispecific antibodies containing isopeptide bonds in both antigen-binding sites of a bispecific antibody can also be constructed in a similar manner. Note that, in this study, a knob-hole structure was employed to promote pairing between the heavy chains of specific antibodies so that the desired bispecific antibody could be efficiently obtained. However, this knob-hole structure may be replaced with the isopeptide bond of the present disclosure.

[0151] (Example 6: Preparation of variants without hydrophobic mutations) Polypeptides are prepared by introducing only the mutations K, N, and E in the above-mentioned isoCH1v1, isoCLv2, isoCLv1, and isoCH1v2. As a result, it is expected that the formation of isopeptide bonds will be confirmed.

[0152] (Example 7: Preparation of antibodies in various formats containing isopeptide bonds) Based on the sequences into which the above-mentioned isopeptide bond-forming mutations have been introduced, Fab, Fab', or F(ab') ２ Antibodies are produced in various formats. It is expected that isopeptide bonds will still be formed in these different formats.

[0153] Example 8: Measurement of cytotoxic activity of isopeptide-bonded anti-CD63 antibodies Preparation of antibody-drug conjugates (conjugates) Anti-hCD63 iSoMAbv1 (heavy chain having SEQ ID NO: 45, light chain having SEQ ID NO: 46) and non-mutated anti-hCD63 WT (wild-type) MAb (heavy chain having SEQ ID NO: 43, light chain having SEQ ID NO: 44) were prepared according to the method described in Example 4. TCEP (tris(2-carboxyethyl)phosphine) was added to each IgG (1 mg / mL) at a final concentration of 100 μM, mixed, and then allowed to stand at 37°C for 2 hours. This reaction solution was allowed to stand at room temperature for 30 minutes, after which VcMMAE (MedChemExpress) was added at a final concentration of 100 μM, and the mixture was allowed to stand at room temperature for 30 minutes. Next, N-acetylcysteine ​​(NAC) was added to a final concentration of 1 mM, and the mixture was left on ice for 30 minutes to stop the reaction. After that, the mixture was dialyzed against PBS(-) to prepare an antibody-drug conjugate. The drug-antibody ratio of the resulting antibody-drug conjugate was measured by UV absorption, and the drug-antibody ratios of both anti-hCD63 WT MAb-VcMMAE and anti-hCD63 iSoMAbv1-MMAE were approximately 4.

[0154] Measurement of cytotoxic activity Human pancreatic cancer cell line SUIT-2 was plated on a 96-well plate at 4 x 10 cells per well. 3 The next day, cells were seeded, and antibody-drug conjugates were added at final concentrations of 0.1 to 3 μg / mL. The cells were then cultured at 37°C for 96 hours in a CO2 incubator. For the negative control, the same amount of medium was added and the cells were similarly cultured for 96 hours. After the culture, Tween 20 was added to a final concentration of 0.1% for the positive control, and the cells were incubated in a CO2 incubator for 30 minutes. Next, Cell Counting Kit-8 (Dojindo) was added to each well, and the cells were incubated in a CO2 incubator for 1 hour. The absorbance at 450 nm was measured using a plate reader. Cytotoxic activity (%) was calculated according to the following formula: Cytotoxic activity (%) = ((absorbance of negative control - absorbance of antibody-drug conjugate) / (absorbance of negative control - absorbance of positive control)) × 100. The results are shown in Figure 13. At antibody-drug conjugate concentrations of 1 and 3 μg / mL, anti-hCD63 iSoMAbv1-MMAE exhibited significantly higher cytotoxic activity than anti-hCD63 WT MAb-VcMMAE.

[0155] (Example 9: Measurement of cytotoxic activity of isopeptide-bonded anti-CD63 antibody Fab format) Preparation of antibody-drug conjugate An antibody-drug conjugate of a Fab format antibody fragment (heavy chain fragment has SEQ ID NO: 49, light chain fragment has SEQ ID NO: 50) was prepared based on the anti-hCD63 iSoMAbv1 prepared in Example 8. An antibody-drug conjugate of anti-hCD63 WT Fab (heavy chain has SEQ ID NO: 47, light chain has SEQ ID NO: 48) served as a control. The drug-antibody ratio of the obtained antibody-drug conjugate was measured by UV absorption method, and the drug-antibody ratio of both anti-hCD63 WT Fab-VcMMAE and anti-hCD63 iSoMAbv1 Fab-MMAE was approximately 1.

[0156] Measurement of Cytotoxic Activity According to the method described in Example 8, the cytotoxic activity of the antibody-drug conjugate of the Fab format antibody fragment was measured.

[0157] The results are shown in Figure 14. At antibody-drug conjugate concentrations of 1 and 3 µg / mL, anti-hCD63 iSoMAbv1 Fab-MMAE exhibited significantly higher cytotoxic activity than anti-hCD63 WT Fab-VcMMAE.

[0158] Example 10 Measurement of Mouse Blood Concentration of Isopeptide-Bond-Forming Trastuzumab IgG Isopeptide-bond-forming trastuzumab IgG (h4D5 iSoMAb) (heavy chain has SEQ ID NO: 53, light chain has SEQ ID NO: 54) prepared in Example 2 and WT (wild-type) trastuzumab IgG (h4D5 WT MAb) (heavy chain has SEQ ID NO: 51, light chain has SEQ ID NO: 52) that does not form isopeptide bonds were administered to mice, and blood was collected over time to measure the maximum blood concentration and blood half-life.

[0159] Administration and plasma preparation: Five 5-week-old male ICR mice were intraperitoneally administered 10 mg / kg of IgG in PBS (1 mg / mL). Blood was collected from the tail vein 1 hour, 1 day, 2 days, 3 days, 7 days, 10 days, and 15 days after administration. The collected blood was centrifuged at 3,500 rpm at 4°C for 15 minutes to prepare plasma, which was then immediately stored at -30°C.

[0160] Measurement of human IgG concentration in mouse plasma Human IgG concentration in mouse plasma was measured using a Human IgG ELISA Kit (Assaypro).

[0161] The results are shown in Figure 15. The maximum blood concentrations were almost the same: 107.8 ± 10.6 μg / mL (after 1 day) for h4D5 WT MAb and 102.0 ± 10.8 μg / mL (after 2 days) for h4D5 iSoMAb. Meanwhile, the blood half-lives were 6.3 ± 4.1 days for h4D5 WT MAb and 8.2 ± 3.7 days for h4D5 iSoMAb, indicating that h4D5 iSoMAb had a longer blood half-life than h4D5 WT MAb.

[0162] (Example 11: Measurement of binding of isopeptide-bonded anti-CD63 antibody Fab format to antigen-positive cells) Expression of hCD63 iSoMAb Fab hCD63 iSoMAb Fab (heavy chain fragment having SEQ ID NO: 57, light chain having SEQ ID NO: 58) was expressed using the same method as in Example 4. pCEC4.3W-iSoMAb Fab hCD63 H1 and pCEC4.3W-isoCLvC hCD63 K1 were used as plasmids for gene transfer.

[0163] Purification of hCD63 iSoMAb Fab Cells were removed from the culture supernatant by centrifugation at 4°C, 12,000 x g, for 10 minutes. The culture supernatant was filtered using a 0.45 μm pore size filter (GVS) and loaded onto KanCapG (column volume 1.0 mL; KANEKA) pre-equilibrated with PBS. After washing with PBS, the target antibody was eluted with 0.1 M glycine-HCl (pH 2.5) and neutralized with 1 M Tris-HCl (pH 8). The washed fraction (5 mL) and the eluted fraction (1 mL each) were analyzed by SDS-PAGE followed by CBB staining, and the fraction containing the target antibody was analyzed by Pierce. TM The solvent was replaced with PBS using 2-6 ml of Protein Concentrators PES, 10K MWCO. The absorbance at 280 nm was measured using a spectrophotometer (Eppendorf BioPhotometer plus), and the target antibody was quantified by subtracting the absorbance at 340 nm as background.TM A more accurate concentration was determined using a BCA Protein Assay Kit. A purified antibody was similarly prepared for hCD63 WT (wild-type) Fab (heavy chain fragment having SEQ ID NO: 55, light chain having SEQ ID NO: 56).

[0164] Evaluation of binding activity to hCD63-positive cells The binding activity to the hCD63-positive human fetal kidney-derived cell line 293 was evaluated by flow cytometry. Expi293F cells were cultured in Expi293 Expression Medium (Gibco, Thermo Fisher Scientific) at 37°C, 5% CO2, and 125 rpm. The cells were collected by centrifugation at 300 x g for 5 minutes at room temperature, washed with 0.2% BSA / PBS, and placed in a sample tube at 5 x 10 5 Cells were dispensed into the wells. The primary antibody, hCD63 iSoMAb Fab, was incubated at 4°C for 60 minutes at a final concentration of 2 μg / mL. After washing in the same manner, the secondary antibody, mouse type 2 anti-human IgKappa (PE) (TB28-2, BD Biosciences), was incubated at 4°C for 30 minutes at a final concentration of 12.5 μg / mL. After washing again, the cells were suspended in 200 μL of 0.2% BSA / PBS. The cells were dispensed into plastic tubes and analyzed using a flow cytometer (FACS Verse; BD Biosciences).

[0165] The results are shown in Figure 16. The binding of hCD63 iSoMAb Fab and hCD63 WT (wild-type) Fab to antigen-positive cells was comparable.

[0166] Example 12: Analysis of isopeptide bond formation in h4D5 iSoMAbv1 mutants Preparation of h4D5 iSoMAb mutant expression plasmids To investigate the effect of amino acid mutations at positions 173, 181, and 183 in the heavy chain and positions 131, 133, 162, 176, 178, and 180 in the light chain (particularly the amino acid mutations shown in Tables 4 to 6) on isopeptide bond formation, Eurofins Genomics was commissioned to prepare mutant expression plasmids based on the expression plasmids for the h4D5 iSoMAb heavy chain (SEQ ID NO: 53) and light chain (SEQ ID NO: 54) in which amino acids at positions 173, 181, and 183 in the heavy chain and positions 131, 133, 162, 176, 178, and 180 in the light chain were substituted according to Tables 4 to 6. The h4D5 iSoMAb heavy chains mut1-mut4 had SEQ ID NOs: 59-62, respectively, and the h4D5 iSoMAb light chains mut1-mut15 had SEQ ID NOs: 63-77, respectively.

[0167]

[0168] Expression of h4D5 iSoMAb mutants: HEK293T cells were used as host cells. All cultures were performed at 37°C and 5% CO2. First, HEK293T cells were cultured in DMEM containing 10% FBS in a 96-well plate. Transfection was performed when the cells reached 80-90% confluence. Prior to transfection, 2 μL of 10 mg / ml polyethyleneimine solution was mixed with 1100 μL of FBS-free DMEM, and 13 μL of each mixture was dispensed into 96 wells. 0.12 μg of plasmid (heavy chain: 0.065 μg, light chain: 0.065 μg, combinations shown in Table 7) was added.

[0169]

[0170] After standing for 20 minutes, the mixture was added to 200 μL of DMEM containing 2% FBS, which was used as the transfection solution. After removing the medium from the 96-well plate in which the cells were seeded, 200 μL of the transfection solution was poured into each well and cultured for 8 days. The culture supernatant was collected, and expression was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using an anti-human Fc antibody (A0170, SIGMA-ALDRICH). Furthermore, band intensity was calculated using ImageJ to estimate the percentage of isopeptide bond formation. The percentage of formation (%) was calculated according to the following formula:

[0171] Formation rate (%) = ((intensity of the band formed by cross-linking heavy and light chains) / (band intensity of the polypeptide formed by cross-linking heavy and light chains + band intensity of the heavy chain)) x 100

[0172] The results are shown in Table 8. In a mutant (mutant 3-15) in which positions 183 of the heavy chain, 133 of the light chain, and 176 of the light chain were substituted with asparagine, glutamic acid, and lysine, respectively, a polypeptide in which the heavy and light chains were cross-linked by an isopeptide bond was detected.

[0173]

[0174] Example 13: Preparation of h4D5 iSoMAbv1 mutant Expression and purification of h4D5 iSoMAb mutant Using the mutant expression plasmid prepared in Example 12, the mutant was expressed in the same manner as in Example 2, and purified from the culture supernatant using a Protein A column. The purified antibody was analyzed by SDS-PAGE under reducing conditions. In addition, band intensities were calculated using ImageJ to estimate the percentage of isopeptide bond formation. The percentage of isopeptide bond formation (%) was calculated according to the following formula:

[0175] Formation rate (%) = ((intensity of the band formed by cross-linking heavy and light chains) / (band intensity of the polypeptide formed by cross-linking heavy and light chains + band intensity of the heavy chain + band intensity of the light chain)) x 100

[0176] The results are shown in Figures 17 and 18. A mutant in which positions 183 of the heavy chain, 133 of the light chain, and 176 of the light chain were substituted with asparagine, glutamic acid, and lysine, respectively (mutant 3-15, lane 1 of Figure 17) was sufficient for the formation of an isopeptide bond between CH1 and CL under physiological conditions. Furthermore, although the yield was higher than that of h4D5 iSoMAbv1 (lane 4 of Figure 17), the rate of isopeptide bond formation was slightly reduced. It is believed that an isopeptide bond was formed between asparagine at position 183 of the heavy chain and lysine at position 176 of the light chain in these antibodies.

[0177]

[0178] On the other hand, a mutant in which position 183 of the heavy chain, position 133 of the light chain, and position 176 of the light chain were substituted with asparagine, glutamic acid, or lysine, respectively, and position 181 of the heavy chain was substituted with leucine (mutant 1-15, lane 2 in Figure 18) showed a rate of isopeptide bond formation equivalent to that of h4D5 iSoMAbv1. In these antibodies, it is believed that an isopeptide bond was formed between asparagine at position 183 of the heavy chain and lysine at position 176 of the light chain.

[0179]

[0180] Example 14: Analysis of isopeptide bond formation in h4D5 iSoMAbv1 H-S183D mutant Preparation of h4D5 iSoMAbv1 H-S183D mutant expression plasmid We commissioned Eurofins Genomics to prepare a heavy chain mutant expression plasmid in which positions 173, 181, and 183 of the heavy chain were substituted with isoleucine, leucine, and aspartic acid, respectively.

[0181] Expression of h4D5 iSoMAbv1 H-S183D mutant The heavy chain mutant plasmid prepared above was combined with the light chain mutant expression plasmid prepared in Example 12 (a mutant in which positions 133 and 176 of the light chain were substituted with glutamic acid and lysine, respectively) (heavy chain has SEQ ID NO: 45, light chain has SEQ ID NO: 78), and expressed in the same manner as in Example 12, followed by analysis by SDS-PAGE and Western blotting. The proportion of isopeptide bond formation was estimated in the same manner as in Example 12.

[0182] The results are shown in Figure 19. When position 183 of the heavy chain was substituted with aspartic acid, a polypeptide in which the heavy chain and light chain were cross-linked by an isopeptide bond was detected. The rate of isopeptide bond formation was approximately 10%. In these antibodies, it is believed that an isopeptide bond was formed between asparagine or aspartic acid at position 183 of the heavy chain and lysine at position 176 of the light chain.

[0183]

[0184] Example 15: Analysis of binding of h4D5 iSoMAb Fab to HER2 extracellular domain Analysis of interaction between h4D5 Fab and HER2 extracellular domain by surface plasmon resonance The binding of the h4D5 iSoMAb Fab (heavy chain having SEQ ID NO: 80, light chain having SEQ ID NO: 46) prepared in Example 3 to the HER2 extracellular domain was analyzed by surface plasmon resonance. For comparison, h4D5 WT (wild-type) Fab (heavy chain having SEQ ID NO: 79, light chain having SEQ ID NO: 46) was used. Measurement was performed using a molecular interaction analyzer BIACORE3000 (Cytiva). Using a Mouse Antibody Capture Kit (Cytiva), the HER2 extracellular domain fused to a mouse Fc region was captured on a sensor chip (CM5, Cytiva), and then various concentrations of Fab solutions were added to examine the binding behavior. The resulting sensorgrams were fitted with a 1:1 Langmuir binding model using BIAevaluation Software (Cytiva), and the kinetic parameters of the interaction were calculated.

[0185] The results are shown in Figure 20. The binding activity of h4D5 iSoMAb Fab to the HER2 extracellular domain was equal to or greater than that of h4D5 WT (wild-type) Fab.

[0186]

[0187] Example 16: Crystal structure analysis of hCD63 iSoMAb Fab Crystallization of hCD63 iSoMAb Fab Crystallization was performed using the 16.0 mg / mL hCD63 iSoMAb Fab sample prepared in Example 11. The crystallization buffer used was 0.1 M sodium cacodylate pH 6.5, 0.2 M calcium acetate, and 43% PEG300. Crystallization was performed at 20°C by the hanging drop vapor diffusion method.

[0188] Crystallization of hCD63 WT Fab Crystallization was carried out using the 15.8 mg / mL hCD63 WT Fab sample prepared in Example 11. The crystallization buffer used was 0.1 M sodium cacodylate pH 6.5, 0.2 M calcium acetate, and 42% PEG300, and crystallization was carried out at 20°C by the hanging drop vapor diffusion method.

[0189] Structural analysis of hCD63 iSoMAb Fab and hCD63 WT Fab. Diffraction data were collected for the obtained crystals at the Tsukuba KEK Photon Factory BL5A beamline. Molecular replacement was performed using similar molecular structures, and refinement was performed using Phenix Refine. Molecular model construction was performed using Coot.

[0190] The results are shown in Figure 21. Electron density indicating the formation of an isopeptide bond was observed between the side chains of the asparagine residue at position 183 in the heavy chain and the lysine residue at position 176 in the light chain. A glutamic acid residue at position 133 in the light chain was also present nearby. When the structures of the iSoMAb Fab and wild-type Fab were superimposed, the root mean square deviation (RMSD) was 0.34 A, indicating no significant changes in the overall structure.

[0191] Example 17: Formation of isopeptide bonds in human IgG4κ Preparation of expression plasmid Based on the amino acid sequence information of Nivolumab (human IgG4κ), mutations were introduced into sites involved in isopeptide bond formation (positions 183 in the heavy chain, 133 in the light chain, and 176 in the light chain were replaced with asparagine, glutamic acid, and lysine, respectively) to prepare the amino acid sequence of Nivolumab iSoMAb. Eurofins Genomics was commissioned to prepare artificial genes for the heavy chain (SEQ ID NO: 81) and light chain (SEQ ID NO: 82) based on the amino acid sequence, and these were inserted into the expression plasmid.

[0192] Expression of Nivolumab iSoMAb Using the expression plasmid prepared above, the recombinant antibody was expressed in HEK293T cells in the same manner as in Example 12. The culture supernatant was collected, and expression was examined by SDS-PAGE and Western blotting. Anti-human Fc antibody (SIGMA: A0170) and anti-human kappa chain antibody (SIGMA: A7164) were used as detection antibodies. The results are shown in Figure 22. Whether anti-human Fc antibody or anti-human kappa chain antibody was used as the detection antibody, a polypeptide in which the heavy and light chains were cross-linked by an isopeptide bond was detected.

[0193] Example 18: Formation of isopeptide bonds in human IgG2κ Preparation of expression plasmid Based on the amino acid sequence information of panitumumab (human IgG2κ), mutations were introduced into sites involved in isopeptide bond formation (heavy chain position 183, light chain position 133, and light chain position 176 were substituted with asparagine, glutamic acid, and lysine, respectively) to prepare the amino acid sequence of panitumumab iSoMAb. Eurofins Genomics was commissioned to prepare artificial genes for the heavy chain (SEQ ID NO: 83) and light chain (SEQ ID NO: 84) based on the amino acid sequence, and these were inserted into the expression plasmid.

[0194] Expression of Panitumumab iSoMAb Using the expression plasmid prepared above, the recombinant antibody was expressed in HEK293T cells in the same manner as in Example 12. The culture supernatant was collected, and expression was examined by SDS-PAGE and Western blotting. Anti-human Fc antibody (SIGMA: A9544) and anti-human kappa chain antibody (SIGMA: A7164) were used as detection antibodies. The results are shown in Figure 23. When either anti-human Fc antibody or anti-human kappa chain antibody was used as the detection antibody, a polypeptide in which the heavy and light chains were cross-linked by an isopeptide bond was detected.

[0195] Example 19: Formation of isopeptide bonds in human IgG1λ Preparation of expression plasmids The heavy chain (SEQ ID NO: 85) and light chain variable regions (SEQ ID NO: 86) of an anti-BCMA antibody (clone name 372, Human IgG1λ) were synthesized as artificial genes at the request of Eurofins Genomics. The heavy chain expression plasmid for h4D5 iSoMAbv1 prepared in Example 12 was digested with restriction enzymes EcoRI and NheI, and the 372 heavy chain variable region digested with the same enzymes was inserted to prepare a 372 heavy chain expression plasmid. Similarly, a light chain constant region (positions 131, 133, 162, 176, 178, and 180 were substituted with valine, glutamic acid, methionine, lysine, leucine, and glutamine, respectively) synthesized as an artificial gene was ligated to the 372 light chain variable region by overlap extension PCR, followed by digestion with the restriction enzymes EcoRI and NotI. The resulting product was then inserted into an expression plasmid previously digested with the same enzymes to prepare a 372 light chain expression plasmid.

[0196] Expression and purification of 372 iSoMAb: Using the expression plasmid described above, the recombinant antibody was expressed in the same manner as in Example 2, and purified from the culture supernatant by Protein A affinity chromatography. The recombinant antibody after Protein A purification was analyzed by SDS-PAGE under reducing conditions.

[0197] The results are shown in Figure 24. In the purified 372 iSoMAb, a polypeptide in which the heavy and light chains were cross-linked by an isopeptide bond was detected.

[0198] Example 20: Production of h4D5 iSoMAb Fab using Brevibacillus bacteria - Preparation of expression plasmid A plasmid containing the h4D5 iSoMAb light chain sequence (amino acid substitution sequence at positions 133 and 176 in Table 6) (SEQ ID NO: 87) and heavy chain sequence (amino acid substitution sequence at position 183 in Table 4) (SEQ ID NO: 88) was prepared at the request of Eurofins Genomics. Next, using this plasmid as a template, PCR was performed with primers (5'-AGTTCCGCATTCGCTGACATC-3' and 5'-CATCCTGTTAAGCTTAGTGGGTCTTGTCGCTGC-3') to amplify a target sequence having a His tag sequence, which was then inserted into a pBIC3 DNA vector (the vector sequence is shown in Figure 25).

[0199] Purification of h4D5 iSoMAb Fab The expression vector obtained above was introduced into Brevibacillus bacteria to produce transformants, which were then cultured in MT medium at 30°C for 3 days. Ni-NTA resin (Ni Sepharose Excel, Cytiva) was added to the culture supernatant obtained after centrifugation to purify the h4D5 iSoMAb Fab. The culture supernatant was analyzed by SDS-PAGE, CBB staining, and Western blotting. Anti-His tag antibody (MBL: Code No. D291-3) and anti-human kappa chain antibody (SIGMA: A7164) were used as detection antibodies. The purified antibody was subjected to CBB staining. Similarly, purified antibody was prepared for h4D5 WT (wild-type) Fab and subjected to CBB staining.

[0200] The results are shown in Figures 26 and 27. When either the anti-His tag antibody or the anti-human kappa chain antibody was used as the detection antibody, a polypeptide in which the heavy chain and the light chain were cross-linked by an isopeptide bond was detected. Furthermore, a polypeptide in which the heavy chain and the light chain were cross-linked was also detected in the purified h4D5 iSoMAb Fab.

[0201] Example 20: Preparation of chimeric antigen receptor of trastuzumab Fab forming isopeptide bond and measurement of antigen-binding activity) Generally, a chimeric antigen receptor (CAR) is an artificial chimeric protein formed by fusing a single-chain antibody that recognizes a cell surface antigen of a cancer cell with a signal transduction domain that induces T cell activation. However, it is known that it is not easy to prepare a single-chain antibody that recognizes a cell surface antigen. Therefore, a CAR consisting of an antibody that forms an isopeptide bond was prepared and its antigen-binding activity was measured.

[0202] Preparation of a CAR expression vector for isopeptide-bond-forming trastuzumab Fab and confirmation of expression A CAR expression vector (i) was prepared comprising human CD28, CD3zeta, and enhanced green fluorescent protein (EGFP) downstream of the h4D5 iSoMAb Fab light chain prepared in Example 3. A CAR expression vector (ii) was also prepared comprising the h4D5 iSoMAb Fab heavy chain (shown in Figure 28). The obtained CAR had SEQ ID NO:89.

[0203] HEK293 cells were plated in a 24-well plate at 2 x 10 cells per well. 5 The cells were seeded, and the next day, 500 ng of CAR expression vector (i) and / or CAR expression vector (ii) per well was mixed with 1.5 μL of polyethyleneimine (1 mg / mL) at room temperature for 20 minutes, and then added to the HEK293 cells. Three days after gene transfection, a RIPA buffer containing a surfactant was added and the mixture was pipetted to prepare a lysate. A sample buffer containing a reducing agent was added, the mixture was boiled, and then subjected to SDS-PAGE. The mixture was transferred to a PVDF membrane and blotted with an anti-GFP antibody.

[0204] The results are shown in Figure 29. In cells transfected with only CAR expression vector (i), a band of approximately 80 kDa was detected, whereas in cells transfected with CAR expression vector (i) and CAR expression vector (ii), the intensity of the band of approximately 80 kDa was attenuated, and a band containing isopeptide-bonded trastuzumab Fab (indicated by an arrow) was detected on the higher molecular weight side. Furthermore, when HEK293 cells transfected with both vectors were observed under a fluorescence microscope, it was shown that the chimeric antigen receptor for isopeptide-bonded trastuzumab Fab was present on the cell membrane surface, as shown in Figure 30.

[0205] Measurement of antigen-binding activity An expression vector for HER2 (extracellular domain with secretory signal)-mouse Fc was transfected into HEK293 cells, and the culture supernatant was prepared two days later. The culture supernatant (containing 1 μg / mL of HER2-mouse Fc) was quantified by dot blotting using mouse IgG as a standard, and added to HEK293 cells three days after transfection with CAR expression vector (i) and / or CAR expression vector (ii), and the cells were allowed to stand at 4°C for one hour. After washing three times with PBS(-) containing 1% BSA, anti-mouse IgG-PE and Hoechst 33342 for nuclear staining were added, and the cells were similarly allowed to stand at 4°C for one hour. Next, the cells were washed three times with PBS(-) containing 1% BSA, fixed with 4% formaldehyde, and observed under a fluorescence microscope.

[0206] The results are shown in Figure 31. It was confirmed that by introducing the CAR expression vectors (i) and (ii), the HER2-mouse Fc indicated by the arrow was bound to the cell membrane of HEK293 cells.

[0207] (Example 21) The mutants in the table below were prepared in the same manner as above, and the formation of an isopeptide bond between CH1 and CL was confirmed. The heavy chain of iSoMAbv2 had SEQ ID NO: 91, and iSoMAbv2 HC mut15 had SEQ ID NO: 92.

[0208]

[0209]

[0210] As a result, as shown in Figure 32, for all mutants, bands appeared in the region of H+L molecular weight under denaturing conditions, suggesting the presence of an isopeptide bond between CH1 and CL. In these antibodies, an isopeptide bond is thought to have formed between lysine at position 183 in the heavy chain and asparagine at position 176 in the light chain.

[0211] (Example 22) The mutants in the table below were prepared in the same manner as above, and the formation of an isopeptide bond between CH1 and CL was confirmed. The heavy chain of iSoMAbv2 had SEQ ID NO: 91, iSoMAbv2 HC mut15 had SEQ ID NO: 92, and iSoMAbv2 LC mut3 had SEQ ID NO: 94.

[0212]

[0213]

[0214]

[0215]

[0216] As a result, as shown in Figure 33, all mutants (lanes 1, 2, and 3) produced bands in the region of H+L molecular weight under denaturing conditions, suggesting the presence of an isopeptide bond between CH1 and CL. The heavy chain F170E and S183K mutations and the light chain S176N mutation were sufficient to form an isopeptide bond between CH1 and CL under physiological conditions, and further mutations could improve the efficiency of isopeptide formation. In these antibodies, an isopeptide bond is thought to have formed between the lysine at position 183 in the heavy chain and the asparagine at position 176 in the light chain.

[0217] As can be seen from the above examples, the formation of a covalent bond via an isopeptide bond between CH1 and CL does not negatively affect the binding affinity of an antibody to its antigen; rather, it can stabilize the complex formed by the heavy and light chains. As a result of stabilizing the complex structure, it is clear that the introduction of an isopeptide bond between CH1 and CL into an antibody even improves the binding affinity of the antibody to its antigen. Furthermore, the reaction of forming an isopeptide bond between CH1 and CL is a bioorthogonal reaction that can proceed in vivo, which may be useful in antibody genetic engineering and antibody production processes. Furthermore, asparagine and glutamine, and aspartic acid and glutamic acid, are amino acids with very similar biochemical properties. It will be understood that isopeptide bonds can be formed not only between asparagine and lysine and between aspartic acid and lysine, but also between glutamine and lysine and between glutamic acid and lysine.

[0218] (Note) While the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure should be construed solely by the scope of the claims. It is understood that the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein.

[0219] The polypeptides comprising isopeptide bonds of the present disclosure provide one or more of the following benefits: improved stability, production of a desired substance with high purity, etc. The present invention is applicable to the production of a wide range of polypeptides, including the production of recombinant antibodies with complex structures, such as trispecific antibodies.

[0220] SEQ ID NO: 1: CH1 of h4D5 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 2: Modified CH1 of h4D5 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSS SEQ ID NO: 3: CL of h4D5 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 4: Modified CL of h4D5 RTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES SEQ ID NO: 5: CH1 of CD63 antibody ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 6: Modified CH1v1 of CD63 antibody ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 7: Modified CH1v2 of CD63 antibody ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAILQSSGLYLLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 8: CD63 antibody CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 9: Modified CLv1 of CD63 antibody RTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 10: Modified CLv2 of CD63 antibody RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLNSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11: Human_IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 12: Human IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 13: Human IgG3ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: 14: Human IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 15: Mouse_IgG1AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSGPK ・Sequence number 16: Mouse_IgG2A AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLGGPSVFIFPKIKDVLMISLSPIVTCVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK ・Sequence number 17: Mouse_IgG2BAKTTPPSVYPLAPGCGDTTGSSVTSGCLVKGYFPEPVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYTLPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK ・Sequence number 18: Mouse_IgG2C AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEVLHNHLTTKTISRSLGK ・Sequence number 19: Mouse_IgG3ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPGK SEQ ID NO: 20: Rat_IgG1 AETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRNCGGDCKPCICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEVHTAQTRPPEEQFNSTFRSVSELPILHQDWLNGRTFRCKVTSAAFPSPIEKTISKPEGRTQVPHVYTMSPTKEEMTQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPPTMDTDGSYFLYSKLNVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 21: Rat_IgG2AAETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWSSQAVTCNVAHPASSTKVDKKIVPRECNPCGCTGSEVSSVFIFPPKTKDVLTITLTPKVTCVVVDISQNDPEVRFSWFIDDVEVHTAQTHAPEKQSNSTLRSVSELPIVHRDWLNGKTFKCKVNSGAFPAPIEKSISKPEGTPRGPQVYTMAPPKEEMTQSQVSITCMVKGFYPPDIYTEWKMNGQPQENYKNTPPTMDTDGSYFLYSKLNVKKETWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 22: Rat_IgG2B AQTTAPSVYPLAPGCGDTTSSTVTLGCLVKGYFPEPVTVTWNSGALSSDVHTFPAVLQSGLYTLTSSVTSSTWPSQTVTCNVAHPASSTKVDKKVERRNGGIGHKCPTCPTCHKCPVPELLGGPSVFIFPPKPKDILLISQNAKVTCVVVDVSEEEPDVQFSWFVNNVEVHTAQTQPREEQYNSTFRVVSALPIQHQDWMSGKEFKCKVNNKALPSPIEKTISKPKGLVRKPQVYVMGPPTEQLTEQTVSLTCLTSGFLPNDIGVEWTSNGHIEKNYKNTEPVMDSDGSFFMYSKLNVERSRWDSRAPFVCSVVHEGLHNHHVEKSISRPPGK SEQ ID NO: 23: Rat_IgG2CARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGVHTFPAVLQSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPRRPKPRPPTDICSCDDNLGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDNVRVFTAQTQPHEEQLNGTFRVVSTLHIQHQDWMSGKEFKCKVNNKDLPSPIEKTISKPRGKARTPQVYTIPPPREQMSKNKVSLTCMVTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLYSKLSVDTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK - SEQ ID NO: 24: Rabbit_IgG GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSEDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHEDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK - SEQ ID NO: 25: Canine_IgG-AASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDPPCVPEPLGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELLSSDTTVSITCLIKDFYPPDILVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSGPK ・Sequence number 26: Canine_IgG-B ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKPRENGRVPRPDPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDILVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSGPK ・Sequence number 27: Canine_IgG-CASTTAPSVFPLAPSCGSQSGSTVALACLVSGYIPEPVTVSWNSVSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPATNTKVDKPVAKECECKCNCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK ・Accession No. 28: Canine_IgG-D ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKVDKPVPKESTCKCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK ・Accession No. 29: Feline_IgG1aASTTAPSVFPLAPSCGTTSGATVALACLVLGYFPEPVTVSWNSGALTSGVHTFPAVLQASGLYSLSSMVTVPSSRWLSDTFTCNVAHPPSNTKVDKTVRKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK SEQ ID NO: 30: Feline IgG2 ASTTASSVFPLAPSCGTTSGATVALACLVLGYFPEPVTVSWNSGALTSGVHTFPSVLQASGLYSLSSMVPSSRWLSDTFTCNVAHRPSSTKVDKTVPKTASTIESKTGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDN TEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK・SEQ ID NO: 31: Human_CL-kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32: Mouse_CL-kappa RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 33: Rat_CL-kappaRADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDSTYSMSSTLSLTKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC SEQ ID NO: 34: Rabbit_CL-kappa GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SEQ ID NO: 35: Canine_CL-kappa RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD SEQ ID NO: 36: Feline_CL-kappa RSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE SEQ ID NO: 37: Human_CL-lambda GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 38: Mouse_CL-lambda GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS SEQ ID NO: 39: Rat_CL-lambda GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTANPTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV ・Sequence number 40: Rabbit_CL-lambdaSEQ ID NO: 41: Canine_CL-lambda GQPVVAPLVTLFPPSSEELKANKATLVCLISDFYPGTLTVAWKADGVTITQGVDTTQPSKQSNNKYMASSFLSLSRGQWTSHSRFTCQVTHEGKTTEKSVAPARCP SEQ ID NO: 42: Feline_CL-lambda GQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKASGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS SEQ ID NO: 43: hCD63 WT HC MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP SEQ ID NO: 44: hCD63 WT LCMVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 45: hCD63 iSoMAbv1 HC MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYYCVR GGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 46: hCD63 iSoMAbv1 LC MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC・Accession No. 47: hCD63 WT VH-CH1 of Fab MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC ・Accession No. 48: hCD63 WT LC of Fab MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ・Accession No. 49: hCD63 iSoMAbv1 VH-CH1 of Fab MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC ・Accession No. 50: hCD63 iSoMAbv1 LC of FabMVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ・ Accession No. 51: h4D5 WT HC MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ・ Accession No. 52: h4D5 WT LC METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ・ Accession No. 53: h4D5iSoMAb HC MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ・Sequence number 54: h4D5 iSoMAb LC METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ・Sequence number 55: hCD63 WT VH-CH1 of FabMKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC - SEQ ID NO: 56: hCD63 WT LC of Fab MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC - SEQ ID NO: 57: hCD63 iSoMAb VH-CH1 of Fab MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC - SEQ ID NO: 58: hCD63 iSoMAb LC of FabMVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 59: h4D5 iSoMAb HC mut1 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYYCSR WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 60: h4D5 iSoMAb HC mut2MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYYCSR WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYSLNSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 61: h4D5 iSoMAb HC mut3 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYYCSR WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLNSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 62: h4D5 iSoMAb HC mut4MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYYCSR WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLDSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 63: h4D5 iSoMAb LC mut1 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 64: h4D5 iSoMAb LC mut2 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 65: h4D5 iSoMAb LC mut3 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSTLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 66: h4D5 iSoMAb LC mut4 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 67: h4D5 iSoMAb LC mut5 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 68: h4D5 iSoMAb LC mut6METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSTLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 69: h4D5 iSoMAb LC mut7 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 70: h4D5 iSoMAb LC mut8 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 71: h4D5 iSoMAb LC mut9METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 72: h4D5 iSoMAb LC mut10 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 73: h4D5 iSoMAb LC mut11 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 74: h4D5 iSoMAb LC mut12METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 75: h4D5 iSoMAb LC mut13 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 76: h4D5 iSoMAb LC mut14 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 77: h4D5 iSoMAb LC mut15METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC - SEQ ID NO: 78: h4D5 iSoMAb LC mut17 METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC - SEQ ID NO: 79: h4D5 WT VH-CH1-His MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSSHHHHHH - SEQ ID NO: 80: h4D5 iSoMAb VH-CH1-HisMEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSGSSHHHHHH ・SEQ ID NO: 81: Nivolumab iSoMAb HC MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLNSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK ・SEQ ID NO: 82: Nivolumab iSoMAb LCMEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ・Sequence number 83: Panitumumab iSoMAb HC MEFGLSWVFLVALLRGVQCQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLNSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPCPAPPVAGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ・Sequence number 84: Panitumumab iSoMAb LC MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQGSGNSQESVTEQDSKDSTYSLKSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 85: Anti-BCMA iSoMAb HC MEFGLSWLFLVAILKGVQCEVQLVESGGGLVKPGGSLRLSCAASGFTFSNSGMIWVRQAPGKGLEWVGHIRSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGGSGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 86: Anti-BCMA iSoMAb LC MAWSPLLLTLLAHCTGSWAQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKAVLECLISDFYPGAVTVAWKADGSPVKAGVETMKPSKQSNNKYAAKSLLQLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 87: h4D5VL-isoCLMSISVRFKSLIALLMTVVFLLVPSSAFADIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYT TPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES・Sequence number 88: His-h4D5VH-isoCH1 MKKRRVVNSVLLLLLLASALALTVAPMAFAADHHHHHHDDDDKEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTH SEQ ID NO: 89: h4D5VL-isoCL-CD28-CD3Zeta-EGFPMETPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECCFNGGSGGAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLV TTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 90: h4D5VH-isoCH1 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 91: hCD63 iSoMAbv2 HCKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRG GEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAILQSSGLYLLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 92: hCD63 iSoMAbv2 HC mut15 MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYYCVR GGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 93: hCD63 iSoMAbv2 LCMVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLNSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 94: hCD63 iSoMAbv2 LC mut3 MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESP PTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLNSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A polypeptide chain comprising between a first and a second polypeptide chain the following formula: (wherein the dashed lines represent a first or second polypeptide chain, respectively), the method comprising the steps of: constructing a three-dimensional structure of intermediate A comprising the first and second polypeptide chains; identifying an amino acid sequence present in a boundary region where the first and second polypeptide chains are adjacent to each other in the three-dimensional structure; mutating intermediate A by introducing at least one residue of a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid into intermediate A to prepare intermediate B, wherein intermediate B comprises a), b) and c), and the first and second polypeptide chains in intermediate B comprise a) and b) in the boundary region, respectively; and obtaining the modified polypeptide by forming the interpeptide linkage in intermediate B.

2. The method of claim 1, wherein a) and / or b) are introduced based on the distance between a first amino acid residue of the first polypeptide chain and a second amino acid residue of the second polypeptide chain present within the boundary region and the types of the first and second amino acid residues.

3. The method according to claim 1, wherein b) is asparagine or aspartic acid, and in the three-dimensional structure of intermediate A, two amino acid positions having an α-carbon distance of about 7 to 9 Å are identified as the amino acid positions of a) and b).

4. The method according to claim 1, wherein b) is glutamine or glutamic acid, and in the three-dimensional structure of intermediate A, two amino acid positions having an α-carbon distance of about 8.5 to 10 Å are identified as the amino acid positions of a) and b).

5. The method according to claim 1, wherein, in the three-dimensional structure of intermediate A, the positions of two amino acids whose side chains are located inside the three-dimensional structure are identified as amino acid positions a) and b).

6. The method of claim 1, further comprising the step of constructing a stereostructure of said intermediate B.

7. The method according to claim 6, wherein in the three-dimensional structure of intermediate B, the distance between the nitrogen atom at the ζ-position of the residue a) and the oxygen atom of the carboxy group in the side chain of the residue c) is about 2 to 5 Å.

8. The method according to claim 6, wherein in the three-dimensional structure of intermediate B, the distance between the oxygen atom of the carboxy group or amide group in the side chain of the residue of b) and the oxygen atom of the carboxy group in the side chain of the residue of c) is about 2 to 5 Å.

9. The method according to claim 6, wherein in the three-dimensional structure of intermediate B, the distance between the nitrogen atom at the ζ-position of the residue a) and the oxygen atom of the carboxyl group or amide group in the side chain of the residue b) is about 2 to 5 Å.

10. The method of claim 1, wherein the first polypeptide chain comprises c).

11. The method of claim 1, further comprising the step of determining the hydrophobicity of the boundary region.

12. The method of claim 1, further comprising the step of: deleting a hydrophilic amino acid residue in the amino acid sequence present in the boundary region, substituting an amino acid residue with a hydrophobic amino acid residue, or inserting a hydrophobic amino acid residue.

13. The method according to claim 6, wherein in the three-dimensional structure of intermediate B, a hydrophobic amino acid having an α-carbon atom located within 12 Å from the nitrogen atom at the ζ-position of the residue a) is introduced.

14. The method of claim 1, wherein hydrophobic amino acids having side chains of different volumes are introduced to fill the space in the boundary regions.

15. The method of claim 1, wherein the first and second polypeptide chains are each contained in a different molecule.

16. The method according to claim 15, further comprising the step of introducing a mutation in said intermediate A such that at least one of the two cysteine ​​residues that form a disulfide bond between said first polypeptide chain and said second polypeptide chain is deleted or substituted with another amino acid.

17. The modified polypeptide and a third polypeptide chain contained in a molecule different from the modified polypeptide, the third polypeptide chain having the following formula:

16. The method of claim 15, further comprising producing a secondary modified polypeptide comprising an interpeptide linkage represented by the formula: (wherein the dashed line represents a bond to the polypeptide backbone), the method comprising: constructing a three-dimensional structure of secondary intermediate A comprising the modified polypeptide and the third polypeptide chain; identifying an amino acid sequence present in a boundary region where the modified polypeptide and the third polypeptide chain in the three-dimensional structure are adjacent to each other; mutating the secondary intermediate A by introducing at least one residue of a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid into the secondary intermediate A to prepare a secondary intermediate B, wherein the secondary intermediate B comprises a), b) and c), and the modified polypeptide and the third polypeptide chain in the secondary intermediate B comprise a) and b), respectively, in the boundary region; and obtaining the secondary modified polypeptide by forming the interpeptide linkage in the secondary intermediate B.

18. A polypeptide chain having the following formula between the first and second polypeptide chains: wherein the dashed lines represent bonds to the polypeptide backbone, and wherein the first and second polypeptide chains each comprise a) and b) at a boundary region where the first and second polypeptide chains are adjacent to one another in a three-dimensional structure of the polypeptide.

19. The polypeptide of claim 18 which is an antibody or a T cell receptor (TCR).

20. The polypeptide of claim 19, wherein the interpeptide linkage is in a constant region.

21. The polypeptide of claim 18, comprising an interpeptide linkage between the heavy and light chain constant regions of an antibody.

22. The polypeptide of claim 18, wherein the first and second polypeptide chains are present on different molecules.

23. A CH1 region in which the amino acid at position 183 of the heavy chain is substituted with asparagine, or a nucleic acid encoding said region; a heavy chain constant region comprising said CH1 region, or a nucleic acid encoding said region; or a heavy chain comprising said CH1 region, or a nucleic acid encoding said heavy chain.

24. A CL region in which the amino acid at position 133 of the light chain is replaced with glutamic acid and the amino acid at position 176 of the light chain is replaced with lysine, or a nucleic acid encoding said region; a light chain constant region comprising said CL region, or a nucleic acid encoding said region; or a light chain comprising said CL region, or a nucleic acid encoding said light chain.

25. An antibody comprising a heavy chain according to claim 23 and a light chain according to claim 24.

26. A Fab fragment, a Fab' fragment, or an F(ab')2 fragment comprising the CH1 domain of claim 23 and the CL domain of claim 24.

27. A chimeric antigen receptor comprising a Fab fragment comprising the CH1 region of claim 23 and the CL region of claim 24, a hinge region, a transmembrane domain, and an intracellular domain.

28. A cell expressing the chimeric antigen receptor of claim 27.

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