Method for making novel polypeptides by forming isopeptide bonds

Abstract

The present invention provides novel polypeptides containing isopeptide bonds. In one aspect, a polypeptide is provided which is a polypeptide containing an isopeptide bond between a first polypeptide chain and a second polypeptide chain, said polypeptide containing a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid, wherein, in a boundary region of the above-mentioned polypeptide in which the above-mentioned first polypeptide chain and second polypeptide chain are in close proximity to each other, the above-mentioned first polypeptide chain and second polypeptide chain contain a) and b), respectively.

Description

Technical Field

[0001] This disclosure provides polypeptides containing isopeptide bonds and methods for their preparation. Background Technology

[0002] Proteins contain amide bonds as covalent bonds within their polypeptide chains, and sometimes disulfide bonds between cysteine ​​side chains. Interactions between polypeptide chains are crucial for the formation of higher-order structures, particularly covalent interactions that firmly link the chains together. Therefore, further introducing covalent bonds can be useful for protein manipulation.

[0003] As a covalent bond in proteins, a bond also known as the isopeptide bond is known, but it has only been found in a limited number of proteins, such as the Spy0128 protein of Streptococcus pyogenes (Non-Patent Literature 1).

[0004] Existing technical documents

[0005] Non-patent literature

[0006] Non-patent literature 1: J Am Chem Soc. 2011 Jan 26;133(3):478-85. Summary of the Invention

[0007] The inventors of this invention have studied the design of peptides that form isopeptide bonds between peptide chains and have produced novel peptides containing isopeptide bonds. Based on this, this disclosure provides peptides containing isopeptide bonds and methods for their production.

[0008] Therefore, the present invention provides the following solution.

[0009] (Project 1)

[0010] A method for producing a modified polypeptide, wherein the modified polypeptide contains an interpeptide linker represented by the following formula between a first polypeptide chain and a second polypeptide chain.

[0011] (In the formula, the wavy lines represent the first polypeptide chain or the second polypeptide chain, respectively)

[0012] The method includes: The steps for constructing the three-dimensional structure of intermediate A containing the first polypeptide chain and the second polypeptide chain described above; The step of determining the amino acid sequence present in the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other in the above three-dimensional structure; The step of mutating intermediate A includes preparing intermediate B by introducing at least one residue from a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid into intermediate A, wherein intermediate B contains a), b) and c), and the first polypeptide chain and the second polypeptide chain in intermediate B contain a) and b) respectively in the boundary region; and The step of obtaining the modified polypeptide by forming the above-mentioned interpeptide linkage in the above-mentioned intermediate B.

[0013] (Project 2)

[0014] According to the method of any of the above projects, a) and / or b) are introduced based on the distance between the first amino acid residue of the first polypeptide chain and the second amino acid residue of the second polypeptide chain present in the boundary region and the types of the first amino acid residue and the second amino acid residue.

[0015] (Project 3)

[0016] According to the method of any of the above projects, wherein b) is asparagine or aspartic acid, in the stereostructure of the above intermediate A, the positions of two amino acids with a distance of about 7 to 9 Å between the α carbons are determined as the amino acid positions of a) and b).

[0017] (Project 4)

[0018] According to the method of any of the above projects, wherein b) is glutamine or glutamic acid, in the stereostructure of the above intermediate A, the positions of two amino acids with a distance of about 8.5 to 10 Å between the α carbons are determined as the amino acid positions of a) and b).

[0019] (Project 5)

[0020] According to the method of any of the above projects, wherein in the stereostructure of the above intermediate A, the positions of the two amino acids in which the side chain is disposed inside the stereostructure are determined as amino acid positions a) and b).

[0021] (Project 6)

[0022] According to any of the methods in the above projects, the method further includes the step of constructing the three-dimensional structure of the intermediate B.

[0023] (Project 7)

[0024] According to the method of any of the above projects, in the stereostructure of the above intermediate B, the distance between the nitrogen atom at the ζ position of the residue of a) and the oxygen atom of the carboxyl group in the side chain of the residue of c) is about 2 to 5 Å.

[0025] (Project 8)

[0026] According to the method of any of the above projects, in the stereostructure of the above intermediate B, the distance between the oxygen atom of the carboxyl or amide group in the side chain of the residue of b) and the oxygen atom of the carboxyl group in the side chain of the residue of c) is about 2 to 5 Å.

[0027] (Project 9)

[0028] According to the method of any of the above projects, in the stereostructure of the above intermediate B, the distance between the nitrogen atom at the ζ position of the residue of a) and the oxygen atom of the carboxyl or amide group in the side chain of the residue of b) is about 2 to 5 Å.

[0029] (Project 10)

[0030] According to the method of any of the above projects, wherein the first polypeptide chain contains c).

[0031] (Project 11)

[0032] The method according to any of the above projects further includes a step of determining the hydrophobicity of the aforementioned boundary region.

[0033] (Project 12)

[0034] The method according to any of the above projects further includes the following steps: performing a mutation to delete hydrophilic amino acid residues, a mutation to replace amino acid residues with hydrophobic amino acid residues, or an insertion of hydrophobic amino acid residues in the amino acid sequence present in the above boundary region.

[0035] (Project 13)

[0036] According to the method of any of the above projects, wherein, in the stereostructure of the above intermediate B, a hydrophobic amino acid having an α carbon at a position within 12 Å from the nitrogen atom at the ζ position of the residue of a) is introduced.

[0037] (Project 14)

[0038] According to the method of any of the above projects, hydrophobic amino acids with side chains of different volumes are introduced to fill the space of the above boundary region.

[0039] (Project 15)

[0040] According to the method of any of the above projects, wherein the first polypeptide chain and the second polypeptide chain are respectively contained in different molecules.

[0041] (Project 16)

[0042] The method according to any of the above projects further includes the step of introducing a mutation that causes at least one of the two cysteine ​​residues that form a disulfide bond between the first polypeptide chain and the second polypeptide chain in the intermediate A to be deleted or replaced with another amino acid.

[0043] (Project 17)

[0044] According to the method described in Project 15, wherein, Further, a secondary modified polypeptide is prepared, comprising an interpeptide linker as shown in the following formula between the above-mentioned modified polypeptide and a third polypeptide chain contained in a molecule different from the above-mentioned modified polypeptide.

[0045] (In the formula, the wavy line represents the binding to the polypeptide backbone)

[0046] The method includes: The steps for constructing the three-dimensional structure of secondary intermediate A containing the above-described modified polypeptide and the above-described third polypeptide chain; The step of determining the amino acid sequence present in the boundary region where the modified polypeptide and the third polypeptide chain are close to each other in the above-mentioned three-dimensional structure; The step of mutating the aforementioned secondary intermediate A includes preparing secondary intermediate B by introducing at least one residue from a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid into secondary intermediate A. Here, secondary intermediate B contains a), b), and c), and the modified polypeptide and the third polypeptide chain in secondary intermediate B each contain a) and b) in the boundary region, respectively. The step of obtaining the above-mentioned modified polypeptide by forming the above-mentioned interpeptide linkage in the above-mentioned secondary intermediate B.

[0047] (Project 18)

[0048] A polypeptide having an interpeptide linker represented by the following formula between a first polypeptide chain and a second polypeptide chain.

[0049] (In the formula, the wavy line represents the binding to the polypeptide backbone)

[0050] The polypeptide contains a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid. Here, in the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other in the three-dimensional structure of the above-mentioned polypeptide, the first polypeptide chain and the second polypeptide chain respectively contain a) and b).

[0051] (Project 19)

[0052] The polypeptide of any of the above items is an antibody or a T-cell receptor (TCR).

[0053] (Project 20)

[0054] According to any of the above items, the peptides, wherein the peptide linkages exist in a constant region.

[0055] (Project 21)

[0056] According to any of the above projects, a polypeptide containing an antibody-containing heavy chain constant region and a light chain constant region, wherein there is an interpeptide linkage.

[0057] (Project 22)

[0058] According to any of the above items, the polypeptide, wherein the first polypeptide chain and the second polypeptide chain exist on different molecules.

[0059] (Project 23)

[0060] The amino acid at position 183 of the heavy chain in the EU encoding is replaced by the CH1 region of asparagine or the nucleic acid encoding that region. Heavy chain constant regions containing the CH1 region or nucleic acids encoding that region, or Heavy chains containing the CH1 region or nucleic acids encoding the heavy chain.

[0061] (Project 24)

[0062] In the EU encoding, 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 in the CL region or the nucleic acid encoding this region. Light chain constant regions containing the aforementioned CL region or nucleic acids encoding that region, or Light chains containing the aforementioned CL region or nucleic acids encoding such light chains.

[0063] (Project 25)

[0064] An antibody comprising the heavy chain described in item 23 and the light chain described in item 24.

[0065] (Project 26)

[0066] A Fab fragment, Fab' fragment, or F(ab')2 fragment containing the CH1 region described in item 23 and the CL region described in item 24.

[0067] (Project 27)

[0068] A chimeric antigen receptor having a Fab fragment containing the CH1 region described in item 23 and the CL region described in item 24, a hinge region, a transmembrane domain, and an intracellular domain.

[0069] (Project 28)

[0070] A cell that expresses the chimeric antigen receptor described in item 27.

[0071] This invention further provides a technique for forming isopeptide bonds between the CH1 and CL regions of an antibody. Surprisingly, these isopeptide bonds can spontaneously form in antibody-producing cells through amino acid substitution mutations between the CH1 and CL regions. The antibody can also maintain its functionality by substantially inhibiting its antigen-binding properties through the isopeptide bonds. Therefore, all uses of existing antibodies can be replaced by the isopeptide-bonded antibodies of this invention. The isopeptide bonds stabilize the Fab fragments and Fab' fragments, thus enabling the replacement of various uses previously only achievable with scFv using the Fab fragments and Fab' fragments stabilized by the isopeptide bonds.

[0072] It is desirable that one or more of the features described above in this invention be provided in combinations other than those explicitly stated. Those skilled in the art will recognize further embodiments and advantages of this invention upon reading and understanding the following detailed description.

[0073] This disclosure provides novel polypeptides containing isopeptide bonds. The polypeptides containing isopeptide bonds disclosed herein possess one or more advantages, such as improved stability and the ability to generate target substances with high purity. This invention enables the production of a wide range of polypeptides, including antibodies, recombinant antibodies with complex structures such as multispecific antibodies (especially bispecific or trispecific antibodies). Attached Figure Description

[0074] Figure 1 The structure of the asparagine, lysine, and glutamic acid side chain portions in the stereostructure of the associate formed by isoCH1 and isoCL before the formation of the isopeptide bond is shown.

[0075] Figure 2 The structure of the isopeptide bond portion in the three-dimensional structure of the association formed by isoCH1 and isoCL after isopeptide bond formation (left) or the association formed by isoCH1v2 and isoCLv2 (right).

[0076] Figure 3This is a Western blot showing the protein blotting results of the target conjugates. The left image shows the Coomassie Brilliant Blue staining results, the middle image shows the staining results using antibodies binding to the Fc region, and the right image shows the staining results using antibodies binding to the light chain κ region. The left lane is for molecular weight markers, the middle lane (1) is for h4D5 IgG, and the right lane (2) is for h4D5iSoMAb.

[0077] Figure 4 These are flow cytometry results demonstrating that h4D5 iSoMAb retains the binding ability of the original antibody. Fluorescently labeled h4D5 IgG and fluorescently labeled h4D5 iSoMAb showed equivalent binding ability against HER2-expressing SKBR-3 cells.

[0078] Figure 5 These are the results of SDS-polyacrylamide gel electrophoresis showing the formation of target associative compounds. The left image shows the results for molecular weight markers, the middle image shows the results for h4D5 Fab, and the right image 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 washing sample, and lane 4 is the elution sample.

[0079] Figure 6 These are the results of differential scanning fluorometry measurements of h4D5 iSoMAb Fab and h4D5 Fab with and without the addition of a reducing agent (TCEP). Under either condition, instability was observed around 75–85 °C.

[0080] Figure 7 The results are based on anti-CD63 antibody and show the formation of the target iSoMAb by SDS-polyacrylamide gel electrophoresis. Lane M shows the molecular weight marker, lane 1 shows BSA (control), lanes 2-4 show the original antibody and iSoMAb (two types) expressed in CHO cells, and lanes 5-6 show iSoMAb (two types) expressed in Expi293F cells.

[0081] Figure 8 These are flow cytometry results showing the binding of iSoMAb to CD63-expressing cells based on anti-CD63 antibodies. The top image shows the results of the original antibody, the middle image shows the results of iSoMAb (v1), and the bottom image shows the results of iSoMAb (v2).

[0082] Figure 9 This is a structural summary of the bispecific antibody HER2 / HER3 iSoMAb constructed in Example 4. It is a bispecific antibody containing a variable region of h4D5 (anti-HER2) and U1-59 (anti-HER3), where h4D5 contains an isopeptide bond.

[0083] Figure 10 This is the result of a sandwich ELISA assay confirming the binding of h4D5xU1-59 iSoMAb bispecific IgG. The left figure shows an overview of the assay. The right figure shows the results, illustrating the results for mono-binding h4D5 IgG and U1-59 IgG, as well as h4D5xU1-59 iSoMAb bispecific IgG. The vertical axis represents color intensity, and the horizontal axis represents antibody concentration.

[0084] Figure 11-1 The alignment results for various antibody heavy chain sequences (positions 118–167) are shown. Residue numbering follows Eu coding.

[0085] Figure 11-2 The alignment results for various antibody heavy chain sequences (positions 168–217) are shown. Residue numbering follows Eu coding.

[0086] Figure 11-3 The alignment results for various antibody heavy chain sequences (positions 218–220) are shown. Residue numbering follows Eu coding.

[0087] Figure 11-4 The alignment results for various antibody heavy chain sequences (positions 221–270) are shown. Residue numbering follows Eu coding.

[0088] Figure 11-5 The alignment results for various antibody heavy chain sequences (positions 271–320) are shown. Residue numbering follows Eu coding.

[0089] Figure 11-6 The alignment results for various antibody heavy chain sequences (positions 321–369) are shown. Residue numbering follows Eu coding.

[0090] Figure 11-7 The alignment results for various antibody heavy chain sequences (positions 370–417) are shown. Residue numbering follows Eu coding.

[0091] Figure 11-8 The alignment results for various antibody heavy chain sequences (positions 418–447) are shown. Residue numbering follows Eu coding.

[0092] Figure 12-1 The alignment results of the light chain (κ chain) sequences (positions 108–156) of various antibodies are shown. The residue numbering of the κ chain follows the Eu coding, and the residue numbering of the λ chain follows the Kabat coding.

[0093] Figure 12-2 The alignment results of the light chain λ chain sequences (positions 107A to 156) of various antibodies are shown. The residue numbering of the κ chain follows Eu coding, and the residue numbering of the λ chain follows Kabat coding.

[0094] Figure 13 The cancer cell killing activity of antibody-drug complexes based on anti-CD63 antibody, WT MAb, and iSoMAbv1 is shown. The vertical axis represents cell killing activity, and the horizontal axis represents the concentration of the antibody-drug complex.

[0095] Figure 14 The cancer cell killing activity of antibody-drug complexes based on anti-CD63 antibody Fab, WT Fab, and iSoMAb Fab is shown. The vertical axis represents cell killing activity, and the horizontal axis represents the concentration of the antibody-drug complex.

[0096] Figure 15 The changes in blood concentrations of h4D5 WT MAb and h4D5 iSoMAb after intraperitoneal administration to mice are shown. The vertical axis represents blood concentration, and the horizontal axis represents time after intraperitoneal administration.

[0097] Figure 16 The binding of antigen-positive cells to WT Fab and iSoMAb Fab in their respective anti-CD63 antibody Fab forms is shown. The vertical axis represents cell number, and the horizontal axis represents fluorescence intensity.

[0098] Figure 17 The results of SDS-PAGE analysis of the purified h4D5 iSoMAb mutant are shown.

[0099] Figure 18 The results of SDS-PAGE analysis of the purified h4D5 iSoMAb mutant are shown.

[0100] Figure 19 The results of SDS-PAGE and Western blot analysis of the h4D5 iSoMAb mutant are shown.

[0101] Figure 20 The binding behavior of h4D5 WT Fab and h4D5 iSoMAb Fab to the extracellular domain of HER2 is shown based on surface plasmon resonance. The vertical axis represents the binding amount, and the horizontal axis represents the time elapsed since the addition of the Fab.

[0102] Figure 21 In the middle, the left figure shows the electron density of the isopeptide bond introduction site in hCD63 iSoMAb Fab, the right figure shows the superposition of the iSoMAb Fab crystal structure and the Fab crystal structure, and the isopeptide bond site is shown by the space filling model.

[0103] Figure 22 The results of SDS-PAGE analysis and Western blot analysis of nivolumab iSoMAb are shown.

[0104] Figure 23The results of SDS-PAGE analysis and Western blot analysis of panitumumab iSoMAb are shown.

[0105] Figure 24 The results of SDS-PAGE analysis of purified 372 WT MAb and 372 iSoMAb are shown.

[0106] Figure 25 The h4D5 iSoMAb Fab short bacillus expression plasmid is shown.

[0107] Figure 26 The results of SDS-PAGE and Western blot analysis of h4D5 iSoMAb Fab produced using short bacilli are shown.

[0108] Figure 27 The results of SDS-PAGE analysis of h4D5 iSoMAb Fab expressed using short bacilli and purified by His-tagged column are shown.

[0109] Figure 28 The CAR expression plasmids (i) and (ii) produced are shown.

[0110] Figure 29 This image shows a Western blot of lysates from HEK293 cells inoculated with genes from CAR expression vectors (i) and / or (ii) using anti-GFP antibody. Cells inoculated with genes from CAR expression vectors (i) and (ii) showed a band containing trastuzumab Fab with an isopeptide bond formed on the high molecular weight side (indicated by the arrow).

[0111] Figure 30 The image shows HEK293 cells with genes introduced into CAR expression vectors (i) and (ii) using a fluorescence microscope.

[0112] Figure 31 The image shows HEK293 cells 3 days after gene delivery of CAR expression vector (i) and / or CAR expression vector (ii) were added to culture supernatant containing HER2-mouse Fc, and stained with anti-mouse IgG-PE and Hoechst 33342. Ovals represent nuclei. Arrows on the cell membrane surface indicate the presence of HER2-mouse Fc.

[0113] Figure 32 The results of SDS-PAGE analysis and Western blot analysis of the prepared iSoMAb are shown.

[0114] Figure 33 The results of SDS-PAGE analysis and Western blot analysis of the prepared iSoMAb are shown. Detailed Implementation

[0115] The following illustrates the best mode and describes this disclosure. Throughout this specification, unless otherwise stated, singular expressions should be understood to include the concept of their plural forms as well. Therefore, unless otherwise stated, articles in the singular form (e.g., "a," "an," "the," etc. in English) should be understood to include the concept of their plural forms as well. Furthermore, unless otherwise stated, the terms used in this specification should be understood to have the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0116] The following provides appropriate definitions of terms and / or basic technical content used in this specification.

[0117] In this specification, the terms "containing" and "comprise" include "consisting of" and "comprise of". Without departing from the spirit of the invention, "containing" means that it may include undescribed third components, and "comprise of" means that it substantially does not contain undescribed third components. "Substantially does not contain" is used to mean that there may be an unavoidable incorporation of third components during the manufacturing process, or the incorporation of third components below the detection limit (e.g., impurities).

[0118] In this specification, "amino acid" is used in its usual sense within the art, referring to a compound having both a carboxyl group and an amino group within a molecule. Typically, amino acids are α-amino acids, but β-amino acids and γ-amino acids, where the carboxyl group is further from the amino group, may also exist. In this specification, unless specifically indicated, amino acids represent the optical isomer of the L-body, and in the case of the D-body, are preceded by a D (or d) (e.g., D-Ala (or d-Ala)), or are indicated by a lowercase letter (e.g., a). Representative examples of amino acids are shown below, but this specification is also intended to cover other amino acids.

[0119] [Table 1]

[0120] The amino-bonded carbon in the amino acid H2N-C(R)-COOH is identified as the α-position. The following shows the positional representations of representative amino acids related to the isopeptide bond formation of this disclosure.

[0121]

[0122] In this specification, "polypeptide" refers to a molecule composed of multiple amino acids linked together. To modify 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)) and overlap extension PCR can be used. Alternatively, methods can be employed to modify the amino acid sequence by replacing it with an amino acid other than the natural one (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100(11), 6353-6357). For example, cell-free translation systems (Clover Direct (protein expression)) containing tRNAs with non-natural amino acids bound to complementary succinic repressive tRNAs that serve as one of the stop codons (succinic codons) are preferred.

[0123] In this specification, the amino acid or nucleotide referred to as "corresponding region" or "corresponding position" means an amino acid or nucleotide in a polypeptide or polynucleotide molecule that has the same function or is presumed to have the same function as an amino acid or nucleotide at a specified position in a polypeptide or polynucleotide used as a benchmark. For example, those skilled in the art can determine the corresponding position by comparing the original molecule with its variants and setting appropriate thresholds. For example, the insertion of an amino acid can increment the corresponding position of subsequent amino acid residues from the insertion position by 1. For example, when the alignment result shows that at least two consecutive amino acid residues in two regions are identical and that more than 50% of the amino acid residues in the additive sequence of the region between them are identical, a portion of the additive sequence can be identified as the corresponding region. For example, when the alignment result shows that the following sequences 1 and 2 in two amino acid sequences are identified as corresponding regions, the "A" of sequence 2 in the corresponding region can be set to position 4 and the "-" of sequence 2 can be set to position 5 to determine the corresponding position.

[0124] Sequence 1: XXXGGXX

[0125] Sequence 2: XXXA-XX

[0126] In this specification, the term "mutation" or "modification" of an amino acid refers to the insertion, substitution, or deletion of an amino acid. A mutation or modification of an amino acid refers to the insertion, substitution, or deletion of one amino acid. The addition of an amino acid to the end of an amino acid sequence is also included in the insertion of an amino acid.

[0127] In this specification, as a description of amino acid insertion, it is appropriate to use a description that simultaneously records the position of the inserted amino acid in the sequence after insertion and its single-letter code. For example, a mutation from amino acid sequence XXXXXXXX to XXXXXGGXXX can be represented as an insertion of 6G and 7G. In this specification, as a description of amino acid substitution, it is appropriate to use a description that simultaneously records the single-letter codes of the amino acid before and after the substitution, before and after the number indicating a specific position. For example, the substitution G6A used with reference to the amino acid sequence XXXXXGXXX represents a substitution from glycine at position 6 to alanine. In this specification, as a description of amino acid deletion, it is appropriate to use a description that shows the position of the deleted amino acid in the sequence before deletion (with a single-letter code recorded as needed). For example, a mutation from amino acid sequence XXXXXGXXX to XXXXXXXX can be represented as a deletion at position 6 or a deletion of 6G. Therefore, mutations of the amino acid at position 6 include the insertion of 6G, the substitution of G6A, and the deletion of 6G as described in the examples above.

[0128] The location of a mutation is not necessarily described based on the original sequence. For example, a mutation from amino acid sequence XXXXXXXX to XXXXXGXXX can be represented as the insertion of 6G into the original amino acid sequence XXXXXXXX, or the mutated amino acid sequence XXXXXGXXX can be represented as a sequence containing the 6-position insertion. The location of a mutation is not necessarily described based on the full-length polypeptide; it can also be determined based on the aforementioned "corresponding region" and "corresponding position."

[0129] In this specification, "conservative amino acid substitution" refers to replacing an amino acid in a peptide with another amino acid with similar properties, with the property of the peptide presumed to be similar before and after the conservative substitution. Specific examples of conservative amino acid substitutions will be provided elsewhere in this specification.

[0130] In this specification, unless otherwise stated, reference to a molecule (e.g., protein, nucleic acid) is understood to also refer to variants of that molecule that perform the same biological function (to varying degrees) as that molecule (e.g., variants with modifications to the amino acid sequence). For example, heavy chain proteins containing the Fc region of an antibody include not only proteins with a specific amino acid sequence, but also conjugates of the protein with a labeling molecule (fluorescent label, etc.), isotope substitutes, variants capable of associating with the light chain of an antibody and / or with other Fc regions of an antibody. 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 to the amino acid or nucleic acid sequence of the original molecule of the same size, or when compared with the sequence of the original molecule using a computer homology program known in the art. Variants may include molecules with modified amino acids (e.g., modifications based on disulfide bond formation, glycosylation, lipidation, acetylation, or phosphorylation).

[0131] In this specification, the term "antibody" is used in the broadest sense to include various antibody structures, including monoclonal antibodies, polyclonal antibodies, single-domain antibodies, and multispecific antibodies (such as bispecific or trispecific antibodies).

[0132] In this specification, "humanized antibody" refers to an antibody having a structure in which the CDR region of an antibody produced in a non-human species is integrated into a human antibody. Additionally, "chimeric antibody" refers to an antibody having a structure in which the variable region of an antibody produced in a non-human species is integrated into a human antibody.

[0133] In this specification, "antigen-binding fragment" refers to an antibody moiety capable of binding to a target antigen, typically containing at least a heavy chain variable region and a light chain variable region of the antibody. Examples of antigen-binding fragments include, for example, Fv, Fab, Fab', Fab'-SH, F(ab')2, biantibodies, and single-chain variable region fragments (scFv). In a preferred embodiment, the antigen-binding fragment contains regions corresponding to the VH and CH1 regions of the antibody, as well as regions corresponding to the VL and CL regions.

[0134] In this specification, "variable region" or "variable domain" refers to the domain of the heavy or light chain of the antibody. The variable domains (VH and VL, respectively) of the heavy and light chains of the antibody typically contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs) (e.g., see Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)).

[0135] In this specification, the "complementarity-determining region" or "CDR" refers to a region within the variable domains of an antibody that is particularly helpful for antigen binding. Typically, antibodies contain 6 CDRs: 3 for VH (H1, H2, H3) and 3 for VL (L1, L2, L3). There are several methods for determining CDR sequences, including the Kabat coding system, the IMGT coding system, the Paratome coding system, and the Chothia coding system (for example, see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat coding system), Kunik, Ashkenazi and Ofran, 2012, "Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure" Nucl.Acids Res., 40: W521-W524 (Paratome coding system), Lefranc et al., 2003, Dev Comparat Immunol 27: 55-77 (IMGT coding system), Al-Lazikani et al., 1997, J.Mol.Biol 273: 927-948 (Chothia coding system)).

[0136] "Frame" or "FR" refers to the region in a variable domain other than the complementarity-determining region (CDR). The FR of a variable domain is usually composed of four FR domains: FR1, FR2, FR3, and FR4. The sequence of CDR and FR usually appears in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0137] In this specification, "constant region" or "constant domain" refers to the portion of an antibody excluding the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains that have formed disulfide bonds. Each heavy chain has a variable region (VH) from the N-terminus to the C-terminus, as well as a heavy chain constant region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, each light chain has a variable region (VL) and a constant light chain (CL) domain from the N-terminus to the C-terminus. It is known that the light chains of naturally occurring antibodies possess two types of constant domains called κ and λ.

[0138] In this specification, the "Fc region" refers to a region containing at least a portion of the CH3, CH2, and hinge regions of the antibody's constant structural domain. In some cases, the Fc region may contain the CH4 domain present in some antibody classes. The Fc region may contain the entire hinge region of the antibody's constant structural domain. The Fc region can be 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 Pro230 to the C-terminus of the heavy chain. The C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present (the amino acid residues in the Fc region are numbered according to the EU coding 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).

[0139] Antibodies are classified into "classes" based on the types of constant structural domains contained in their heavy chains. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Furthermore, some of these can be further subdivided into subclasses (isotypes). Examples include IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0140] In this specification, "antigen" refers to the target molecule or a portion thereof to which the antibody binds. An antigenic determinant present in an antigen is called an epitope, and the antibody binds to a portion of the epitope of the antigen. Epitopes include linear epitopes and stereoepitopes. A linear epitope in a protein antigen is an epitope whose primary amino acid sequence is recognizable by the antibody. A linear epitope in a protein antigen typically consists of at least 3, at least 5, for example 8–10, or 6–20 amino acids. A stereoepitope in a protein antigen is an epitope that, in contrast to a linear epitope, cannot be defined solely by the primary amino acid sequence. Regarding the recognition of stereoepitopes, the antibody recognizes the three-dimensional structure of the protein. Methods for determining the stereostructure of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, site-specific spin labeling, and electron paramagnetic resonance spectroscopy, but are not limited to these. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0141] In this specification, "chimeric antigen receptor" (CAR) refers to a chimeric molecule containing an antigen-binding fragment of an antibody (especially scFv) and an activation domain of an immune cell. Typically, a CAR is a molecule formed by linking scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signal transduction domain (e.g., CD3ζ). CARs can be introduced into cells and expressed on the cell surface. Cells expressing CARs can target specific antigens. CARs can be introduced into immune cells such as T cells or NK cells, enabling these cells to target cancer cells. First-generation CARs link scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signal transduction domain (e.g., CD3ζ). Second-generation CARs also contain a co-stimulatory molecule signal transduction domain to activate the CAR-introduced immune cells. Co-stimulatory factors such as CD28, 4-1BB, OX40, CD27, and ICOS are used as co-stimulatory molecule signal transduction domains. Third-generation CARs integrate multiple co-stimulatory factors. Fourth-generation CARs modify cells to enable them to produce cytokines (such as inflammatory cytokines like IL-12). By releasing cytokines, they primarily improve the microenvironment and promote immune system activation. Fifth-generation CARs are characterized by structures utilizing cytokine receptor-related signal transduction pathways (such as the JAK-STAT pathway) in addition to co-stimulatory molecule signal transduction domains. Examples of such structures include the intracellular domains of the IL-2 receptor β chain, IL-7 receptor, IL-15 receptor, and IL-21 receptor. Thus, CARs incorporate modifications to facilitate the continuous proliferation of CAR-infused immune cells in vivo. The scFv, except for the CDR region, is preferably humanized, and any domain outside the scFv portion is preferably derived from human proteins. The scFv itself is sometimes derived from human antibodies. Furthermore, various modified versions of CARs have been developed. In this disclosure, the scFv region of all the aforementioned CARs can be replaced by an antigen-binding fragment of an antibody having a heavy chain VH-CH1 domain and a light chain VL-CL domain. For example, in this invention, antigen-binding fragments of antibodies such as Fab fragments and Fab' fragments can be used to replace scFv, and CARs with stabilized antigen-binding fragments of antibodies such as Fab fragments and Fab' fragments that form isopeptide bonds between CH1 and CL can be expressed on the cell surface in CAR-producing cells.

[0142] In this specification, the comparison of amino acid or base sequence similarity is performed using BLAST, a sequence analysis tool, with default parameters. Similarity searches can be performed, for example, using NCBI's BLAST 2.7.1 (released October 19, 2017). The value of "similarity" in this specification generally refers to the value when performing alignment using the aforementioned BLAST under default conditions. "Similarity" is calculated using known methods as described above to determine the proportion of identical amino acids or bases in two or more amino acid or base sequences. Specifically, before calculating the proportion, the amino acid or base sequences of the groups to be compared are arranged, and gaps are introduced into a portion of the amino acid or base sequences if necessary to maximize the proportion of identical amino acids or bases. The methods for arrangement, the methods for calculating proportions, the comparison methods, and their associated computer programs are well-known in the field (e.g., BLAST as described above). As the algorithm for comparing amino acid or base sequences using BLAST, Blastp can be used with default settings.

[0143] In this specification, "drug ingredient" refers to any component that can constitute a drug, including, for example, an active ingredient (a component that exhibits medicinal effect on its own) and additives (components that are not expected to have medicinal effect on their own, but are expected to play a certain role when included in a drug (e.g., excipients, lubricants, surfactants, etc.). A drug ingredient can be a single substance or a combination of multiple substances or agents. It can contain any combination, such as a combination of an active ingredient and an additive, or a combination of an adjuvant and an active ingredient.

[0144] In this instruction manual, "active ingredient" refers to the ingredient that exerts the desired medicinal effect, which may be a single ingredient or multiple ingredients.

[0145] In this instruction manual, "added ingredients" refers to any ingredient that is not expected to have a medicinal effect, but is expected to play a certain role when contained in a drug. Examples include pharmaceutically acceptable carriers, stabilizers, (excipients), solubility improvers, solubilizers, diluents, excipients, buffers, binders, diluents, flavorings, and lubricants.

[0146] In this specification, the terms "pharmaceutical," "agent," or "factor" (all in English, "agent") are used interchangeably in a broad sense, and can refer to any substance or other element (e.g., energy such as light, radiation, heat, electricity, etc.) as long as the desired purpose is achieved. 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, RNA such as mRNA), glycans, oligosaccharides, lipids, low-molecular-weight organic molecules (e.g., hormones, ligands, signal transducers, low-molecular-weight organic molecules, molecules synthesized through combinatorial chemistry, low-molecular-weight molecules that can be used as pharmaceuticals (e.g., low-molecular-weight ligands), complex molecules of these, and mixtures of these.

[0147] In this specification, "labeling" refers to the use of a label to identify the presence of a target molecule or substance (e.g., matter, energy, electromagnetic waves, etc.) relative to other substances. Examples of such labeling methods include RI (radioisotope) labeling, fluorescence labeling, biotin labeling, and chemiluminescence labeling. When labeling multiple target proteins or their capturing factors or means using fluorescence labeling, fluorescent substances with different fluorescence maximum wavelengths are used. The difference in fluorescence maximum wavelengths is preferably 10 nm or more. Any label that does not affect function can be used as the fluorescent substance; Alexa™ Fluor is an example. Alexa™ Fluor is a water-soluble fluorescent pigment obtained by modifying coumarin, rhodamine, fluorescein, anthocyanins, etc. It is a series corresponding to a wide range of fluorescence wavelengths, and compared with other fluorescent pigments of corresponding wavelengths, it is very stable, has high brightness, and low pH sensitivity. Examples of combinations of fluorescent pigments with fluorescence maximum wavelengths of 10 nm or more include combinations of Alexa™ 555 and Alexa™ 633, and combinations of Alexa™ 488 and Alexa™ 555. Other fluorescent labels include anthocyanins (e.g., Cy3, Cy5, etc. from the CyDye™ series), rhodamine 6G reagent, N-acetoxy-N2-acetaminofluorene (AAF), and AAIF (iodine derivatives of AAF). In this disclosure, such labels can be used to modify the target object to make it detectable by the detection method used. Such modifications are well known in the art, and those skilled in the art can appropriately implement such methods based on the label and the target object.

[0148] In this specification, "kit" refers to a unit that is typically divided into two or more sections and provides the required components (e.g., antibodies, instructions, etc.). This kit format is preferred when the purpose is to provide a composition that should not be mixed due to stability issues, etc., and is preferably mixed just before use. It is advantageous for such a kit to include instructions or manuals describing how to use the provided components or how to handle the reagents. In this specification, when the kit is used as a reagent kit, it typically contains instructions or manuals describing the use of antibodies, etc.

[0149] In this specification, "instruction manual" refers to a document that explains to doctors or other users how to use this disclosure. The instruction manual contains descriptions of the medications disclosed herein. Additionally, the instruction manual may contain descriptions of the administration method. The instruction manual is prepared in accordance with the format prescribed by the regulatory authorities of the countries where this disclosure is implemented (e.g., the Ministry of Health, Labour and Welfare of Japan, the Food and Drug Administration (FDA) of the United States), and clearly states the purpose of obtaining permission from that regulatory authority. The instruction manual is a so-called package insert, usually provided in paper form, but not limited to this; for example, it may also be provided electronically (e.g., via a webpage provided on the Internet, email).

[0150] The term "about" refers to a range of ±10% of the indicated value. When used for temperature, "about" refers to a range of ±5°C of the indicated temperature, and when used for pH, it refers to a range of ±0.5% of the indicated pH.

[0151] In this specification, "the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other" refers to the region between the backbone chains of the two polypeptides in the first polypeptide and the second polypeptide. In this specification, "the first polypeptide chain and the second polypeptide chain are close to each other" means that 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 less than 10 Å. "The boundary region where the first polypeptide chain and the second polypeptide chain are close to each other" refers to a region of less than 15 Å (a spherical region with a radius of 15 Å) starting from the midpoint of the line segment connecting the α-carbon atoms of the first amino acid and the second amino acid.

[0152] (Preferred Implementation)

[0153] Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided to better understand the present disclosure, and it should be understood that the scope of the present disclosure should not be limited by the following description. Therefore, it is understood that those skilled in the art can make appropriate modifications within the scope of the present disclosure by referring to the description in this specification. Furthermore, it is understood that the following embodiments can be used alone or in combination.

[0154] One aspect of this disclosure provides isopeptide bonds in polypeptides. It is intended that any means used to achieve this purpose be included within the scope of this disclosure. For example, even if not explicitly described, it is intended that the description of a method for producing a polypeptide that forms an isopeptide bond also encompasses embodiments reflecting other means, such as the produced polypeptide, compositions containing the polypeptide, and their applications.

[0155] (Methods for preparing polypeptides that form isopeptide bonds)

[0156] This invention provides a method for preparing polypeptides that form isopeptide bonds. In this specification, an isopeptide bond refers to an interpeptide link having the following structure, where the wavy line represents the first or second polypeptide chain, and α represents the α-carbon linking the two polypeptides.

[0157]

[0158] The peptide link is formed by bonding a lysine side chain of a first or second polypeptide chain to a side chain of asparagine, aspartic acid, glutamine, or glutamic acid. In a representative embodiment, this disclosure provides a method for preparing a modified polypeptide containing an isopeptide bond between a first and a second polypeptide chain, comprising: The steps for constructing the three-dimensional structure of intermediate A containing a first polypeptide chain and a second polypeptide chain; and The step of determining the amino acid sequence present in the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other in the three-dimensional structure; The step of mutating intermediate A, wherein intermediate B is prepared by introducing at least one residue from a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid into intermediate A, wherein intermediate B contains a), b) and c), and the first polypeptide chain and the second polypeptide chain in intermediate B contain a) and b) respectively in the boundary region; and The step of obtaining a modified polypeptide by forming peptide links in intermediate B. Here, intermediate A corresponds to the starting structure, intermediate B corresponds to the structure after mutation introduction and before isopeptide bond formation, and the modified polypeptide corresponds to the structure after mutation introduction and after isopeptide bond formation.

[0159] It is believed that if a peptide is designed properly, isopeptide bond formation can occur even in the absence of additional elements such as enzymes. However, only a limited number of proteins with isopeptide bonds are known, such as the Spy0128 protein of Streptococcus pyogenes (J AmChem Soc. 2011 Jan 26;133(3):478-85.), therefore, it is important to design peptides that can promote isopeptide bond formation. Lysine in a) can form isopeptide bonds with glutamine, glutamic acid, asparagine, or aspartic acid in b), but glutamic acid or aspartic acid in c) plays a particularly important role in promoting this reaction. c) can be present in the same molecule as the first or second polypeptide chain, or in other polypeptide chains. In one embodiment, c) is present in the same molecule as the first polypeptide chain. In one embodiment, c) is located on the first polypeptide chain further to the N-terminus than the lysine residue in a). In one embodiment, c) is located on the first polypeptide chain at a position more than 5 residues away from the lysine residue in a).

[0160] The polypeptide chain containing a) or introducing a) is referred to herein as a first polypeptide chain, and the polypeptide chain containing b) or introducing b) is referred to herein as a second polypeptide chain. The first polypeptide chain and the second polypeptide chain may exist in the same molecule or in different molecules. In one embodiment, the first polypeptide chain and the second polypeptide chain exist in different molecules. In particular, through this disclosure, heteropeptide bonds can be formed between different molecules, thereby providing advantages such as the ability to form the heteroassociates described in this specification.

[0161] In this specification, "the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other" refers to the region between the backbone chains of the two polypeptides in the first polypeptide and the second polypeptide. In this specification, "the first polypeptide chain and the second polypeptide chain are close to each other" means that there exists at least one combination of the first amino acid and the second amino acid (also called a "close amino acid pair") where 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 boundary region where the first polypeptide chain and the second polypeptide chain are close to each other" refers to a region of 15 Å or less from the midpoint of the line segment connecting the α-carbon atoms of the first amino acid and the second amino acid. When there are multiple close amino acid pairs between the first polypeptide chain and the second polypeptide chain, "the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other" means that for all close amino acid pairs, the region formed by the superposition of regions of 15 Å or less from the midpoint of the line segment connecting the α-carbon atoms of the amino acids of each close amino acid pair. In this disclosure, isopeptide bonds are not necessarily formed between the amino acids of the close amino acid pair, but their side chains are formed between the amino acids contained in the boundary region. In a representative embodiment, the boundary region can be defined as a region within approximately 25 Å, approximately 20 Å, approximately 15 Å, approximately 10 Å, approximately 7 Å, or approximately 5 Å from the midpoint of the line segment connecting the α-carbon atoms of amino acids a) and b). The orientation of the amino acid side chains extending from the polypeptide chain is generally not fixed; therefore, it is particularly preferred that the amino acid side chains of a) and b) forming the isopeptide bonds be designed to face the other polypeptide and extend into the boundary region. In one embodiment, the positions of the two amino acids whose side chains are disposed within the interior of the three-dimensional structure are defined as the positions of amino acids a) and b).

[0162] The construction of the stereostructure can be based on a known stereostructure. For example, a known stereostructure can be obtained from a protein database, etc. The stereostructure of the starting peptide (intermediate A) can be constructed, for example, by downloading the coordinate data of the crystal structure of the starting peptide registered in a protein database (https: / / www.rcsb.org / ) and using PyMOL (https: / / pymol.org / 2 / ). Based on the constructed stereostructure, a homology model can be constructed as needed using, for example, SWISS-MODEL (https: / / swissmodel.expasy.org / ), and the stereostructure of the modified peptide can be constructed using Coot (https: / / www2.mrc-lmb.cam.ac.uk / personal / pemsley / coot / ). Default values ​​are used for parameters if necessary. The stereostructure described in this specification can be constructed as such. The stereostructure of the peptide intermediate can be constructed after introducing any mutation. In one embodiment, the stereostructure of intermediate B is constructed.

[0163] In one embodiment, a) and / or b) are introduced based on the distance between the first amino acid residue (side chain) of the first polypeptide chain and the second amino acid residue (side chain) of the 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 in the stereostructure of intermediate A, the positions of two amino acids with a distance between α carbons of 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 the above values, are determined as the amino acid positions of a) and b). In one embodiment, b) is glutamine or glutamic acid, and in the stereostructure of intermediate A, the positions of two amino acids with a distance between α carbons of 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 any two of the above values ​​are determined as the amino acid positions of a) and b).

[0164] When the atoms of amino acid residues forming the interaction interface between polypeptide chains are located at relatively close distances to each other, and the distances between atoms in other polypeptide chains are less than the following: CC: 4.1 Å, CN: 3.8 Å, CO: 3.7 Å, OO: 3.3 Å, ON: 3.4 Å, NN: 3.4 Å, these atoms are referred to as "interacting atom pairs" in this specification (see S. Sheriff, W.A. Hendrickson, and J.L. Smith, J. Mol. Biol. 197, 273-296 (1987)). On the other hand, if an atom in one polypeptide chain interacts with the nearest atom in another polypeptide chain, the two polypeptide chains can interact. In one embodiment, in the stereostructure of intermediate B (after mutation introduction and before isopeptide bond formation), the side chains of amino acids surrounding a) and b) of the first and second polypeptide chains (e.g., up to 7, 6, 5, 4, 3, 2, or 1 amino acids starting from a) or b) have interacting atom pairs. In one embodiment, among the amino acid pairs surrounding a) and b), 1, 2, 3, 4, 5, or more amino acid pairs have interacting atom pairs. In one embodiment, the side chains of the amino acid pairs surrounding a) and b) have 1, 2, 3, 4, 5, or more interacting atom pairs. Here, an element (amino acid or atom) can form multiple pairs with multiple different elements. In one embodiment, the amino acid pairs between 7, 6, 5, 4, 3, 2 or 1 amino acids on the N-terminal side of a) of the first polypeptide chain and the surrounding amino acids of b) of the second polypeptide chain have interacting atomic pairs.

[0165] In one embodiment, in the stereostructure of intermediate B (after mutation introduction and before isopeptide bond formation), the distance between the nitrogen atom at the ζ-position of residue a) and the oxygen atom of the carboxyl 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 the above values. In another embodiment, in the stereostructure of intermediate B, the distance between the oxygen atom of the carboxyl or amide group in the side chain of residue b) and the oxygen atom of the carboxyl 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 the above values. In one embodiment, in the stereostructure of intermediate B, the distance between the nitrogen atom at the ζ-position of the residue a) and the oxygen atom of the carboxyl or amide group in the side chain of the residue b) 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 the above values.

[0166] In one embodiment, in the stereostructure of intermediate A, the positions of two amino acids whose distance between the α carbons of a) and c) 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 the above values, are defined as the amino acid positions of a) and c). In another embodiment, in the stereostructure of intermediate A, the positions of two amino acids whose distance between the α carbons of b) and c) 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 the above values, are defined as the amino acid positions of a) and c). In one embodiment, in the three-dimensional structure of intermediate A, the positions of three amino acids that satisfy the distance between the α carbons of a) and c) and the distance between the α carbons of b) and c) described above are determined as the amino acid positions of a), b) and c).

[0167] In one embodiment, in the three-dimensional structure of intermediate B, the angle between the orientation vector of the α carbon to the β carbon of residue a) and the orientation vector of the α carbon to the β carbon of residue b) is not particularly limited, for example, it is 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 any two of the above values. In one embodiment, in the three-dimensional structure of intermediate B, the angle between the orientation vector of the α carbon of residue a) toward the α carbon of residue b) and the orientation vector of the α carbon of residue c) toward the β carbon is not particularly limited, for example, it is 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 any two of the above values.

[0168] In one embodiment, in the stereostructure of intermediate B (after mutation introduction and before isopeptide bond formation), one, two, or three of residues a), b), and c) are absent from the domain. Spy proteins form isopeptide bonds through reactions between residues present in the domain; however, in this disclosure, isopeptide bonds can be formed independently of domain formation. For example, the presence of a residue in a domain can be determined based on its solvent contact area. The solvent contact 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 contained in a protein structure analysis package called CCP4 (https: / / www.ccp4.ac.uk / ). The coordinate data of each atom constituting the molecule can be read and calculated using default parameters. For example, in the embodiments of this application, the calculated value of isopeptide bond-forming Asn in isoCH1 (Sequence No. 1) is 0.24, the calculated value of isopeptide bond-forming Lys in isoCL (Sequence No. 2) is 0.24, and the calculated value of catalytic Glu is 0.25. In one embodiment, when the solvent contact 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 exist in the structural domain.

[0169] The isopeptide bond formation reaction between a) and b), catalyzed by c), can be promoted in a hydrophobic environment; therefore, the boundary region is preferably a hydrophobic environment. One embodiment includes a step of determining the hydrophobicity of the boundary region. In one embodiment, a mutation is introduced that deletes a hydrophilic amino acid residue from the amino acid sequence present in the boundary region, replaces an amino acid residue with a hydrophobic amino acid residue, or inserts a hydrophobic amino acid residue.

[0170] In one embodiment, the hydrophilic amino acids may include glutamic acid, aspartic acid, lysine, arginine, serine, and threonine. In another embodiment, the hydrophilic amino acids may include glutamic acid, aspartic acid, lysine, arginine, serine, threonine, tyrosine, cysteine, and histidine. In one embodiment, the hydrophobic amino acids may include phenylalanine, tryptophan, valine, leucine, and isoleucine. In yet another embodiment, the hydrophobic amino acids may include phenylalanine, tryptophan, valine, leucine, isoleucine, methionine, and proline.

[0171] In one embodiment, a hydrophobic amino acid having an α-carbon at a position within approximately 20 Å, 18 Å, 16 Å, 14 Å, 12 Å, 10 Å, 8 Å, 6 Å, or 4 Å from the nitrogen atom at the ζ-position of the residue in a) is introduced into the stereostructure of intermediate B. Depending on the requirement, in the stereostructure of intermediate B, the α-carbon of the introduced hydrophobic amino acid is located at a distance of approximately 3 Å or more from the nitrogen atom at the ζ-position of the residue in a)—approximately 3.5 Å or more, approximately 4 Å or more, approximately 4.5 Å or more, or approximately 5 Å or more.

[0172] In one embodiment, hydrophobic amino acids with side chains of different volumes are introduced (inserted or replaced) to fill the space in the boundary region. This can result in a mutation that causes the proportion of the side chain volume in the boundary region of the (recalculated) steric structure of the introduced peptide to exceed the proportion of the side chain volume in the boundary region of the peptide before introduction. Even with the introduction of amino acids with large side chains, such as tryptophan, the proportion of the side chain volume in the boundary region may not necessarily increase; sometimes, the distance between the peptide backbone chains widens, and the proportion decreases instead. The amino acids with side chains arranged from smallest to largest volume are glycine, proline, alanine, valine, leucine=isoleucine, methionine, phenylalanine, and tryptophan. In one embodiment, in the stereostructure 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 in the boundary region where the first and second polypeptide chains are close to each other is occupied by residues of the first and second polypeptide chains. Here, the following atomic radii are assigned to each atom for calculating the volume of the side chains: hydrogen: 0.53 Å, carbon: 0.67 Å, ​​nitrogen: 0.56 Å, oxygen: 0.48 Å, sulfur: 0.88 Å. When the spherical volumes of the atoms overlap, the overlapping portions are subtracted. In one embodiment, in the stereostructure 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 with a radius of 15 Å starting from the ε nitrogen atom of lysine in a), are occupied by residues of the first polypeptide chain and the second polypeptide chain.

[0173] In the steps described in this specification for obtaining a modified polypeptide by forming interpeptide links in intermediate B (the polypeptide after mutation introduction but before isopeptide bond formation), additional elements such as enzymes are not required. This step can be performed either in vitro or in vivo (intracellularly). For example, a first polypeptide chain and a second polypeptide chain can be produced in different cells, and then these polypeptide chains can be formed into isopeptide bonds in vitro. Isopeptide bonds can be formed in vivo in cells where the first and second polypeptide chains are expressed together. The formation of isopeptide bonds can be inhibited or promoted by manipulating the intracellular localization of the first and second polypeptide chains (e.g., by fusing localization signaling molecules).

[0174] Compared to the starting polypeptide (intermediate A), the final polypeptide (modified 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 to the sequence of the original molecule when compared using a computer homology program known in the art. The final polypeptide (modified polypeptide) may contain any mutations, and 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 conserved amino acid substitutions. In one embodiment, the conserved amino acid substitution is the replacement of an amino acid in the following group with another amino acid within the same group.

[0175] Group 1: Glycine, alanine, valine, leucine, and isoleucine

[0176] Group 2: Serine and Threonine

[0177] Group 3: Phenylalanine, Tyrosine, and Tryptophan

[0178] Group 4: Lysine, hydroxylysine, arginine, and histidine

[0179] Group 5: Cysteine, Methionine, Methionine sulfoxide, and Homocysteine

[0180] Group 6: Proline and hydroxyproline

[0181] Group 7: Glutamic acid and aspartic acid

[0182] Group 8: Glutamine and Asparagine

[0183] Group 9: Serine and Cysteine

[0184] (A polypeptide containing isopeptide bonds)

[0185] One aspect of this disclosure provides a polypeptide having an interpeptide linker between a first polypeptide chain and a second polypeptide chain, as shown in the following formula.

[0186] (In the formula, the wavy line represents the binding to the polypeptide backbone)

[0187] The polypeptide contains a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid. Here, in the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other in the three-dimensional structure of the polypeptide, the first polypeptide chain and the second polypeptide chain contain a) and b), respectively.

[0188] The isopeptide-containing polypeptides of this disclosure can be prepared using the method for preparing isopeptide-forming polypeptides of this disclosure. In one embodiment, the isopeptide-containing polypeptide of this disclosure has a structural difference compared to known polypeptides or natural polypeptides, which incorporates a mutation from the method for preparing isopeptide-forming polypeptides of this disclosure. In one embodiment, the isopeptide-containing polypeptide of this disclosure may have the configuration of a), b), c) and / or hydrophobic residues described above. The configuration of these residues is substantially maintained before and after isopeptide formation. In one embodiment, the isopeptide-containing polypeptide of this 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 the sequence of the original molecule being compared using a computer homology program known in the art.

[0189] In one embodiment, the polypeptide containing isopeptide bonds disclosed herein is a polypeptide molecule formed by linking different polypeptide molecules using isopeptide bonds, which can be determined by the fact that the two polypeptide chains linked by isopeptide bonds are not linked by peptide bonds.

[0190] In one embodiment, the polypeptide containing isopeptide bonds of this disclosure is an antibody or a T-cell receptor (TCR). In one embodiment, the isopeptide bond (peptide linker) is located in a constant region. For example, if an isopeptide bond is introduced into the constant region of an antibody (such as a constant region involved in the binding of heavy and light chains), antibodies that maintain the ability to form isopeptide bonds and have various target specificities can be provided by combining with various variable regions. In one embodiment, the polypeptide containing isopeptide bonds of this disclosure is a therapeutic protein, cytokine, or chemokine for enzyme replacement therapy. In one embodiment, examples of the polypeptide containing isopeptide bonds of this disclosure include Fc receptors such as FcRn and cytokine receptors such as TNFα receptors, but it is not limited to these.

[0191] To further illustrate the implementation of antibodies containing isopeptide bonds, Figures 11-12 show the alignment results of representative antibody sequences. In the case of antibodies from other biological species, sequence information can also be obtained, and the insertion site of the iSoMAb mutation can be determined similarly. These were prepared using CLUSTALW (JD Thompson, DG Higgins, and TJ Gibson, Nucleic Acids Res. 22, 4673-4680 (1994)) and numbered. The residues in the heavy chain constant region and κ chain constant region are numbered according to Eu coding, and the residues in the λ chain constant region are numbered according to Kabat coding. In this specification, antibody residues may be mentioned together with the numbering shown in Figures 11-12. Based on the successful examples of antibodies containing isopeptide bonds shown in the embodiments of this application, antibodies containing the same modifications as those in the embodiments can also form isopeptide bonds. In one embodiment, the isopeptide-containing antibody of this disclosure contains asparagine or aspartic acid at position 183 of the heavy chain, glutamic acid at position 133 of the light chain, and lysine at position 176 of the light chain, and optionally contains one or more of the following: isoleucine at position 173 of the heavy chain, leucine at position 181 of the heavy chain, valine at position 131 of the light chain, methionine at position 162 of the light chain, leucine at position 178 of the light chain, and glutamine at position 180 of the light chain. In another embodiment, the isopeptide-containing antibody of this disclosure contains lysine at position 183 of the heavy chain, glutamic acid at position 170 of the heavy chain, and asparagine or aspartic acid at position 176 of the light chain, and optionally contains one or more of the following: isoleucine at position 173 of the heavy chain, leucine at position 181 of the heavy chain, methionine at position 162 of the light chain, leucine at position 178 of the light chain, and glutamine at position 180 of the light chain. It is understood that by introducing the above mutations into antibodies, isopeptide bonds can be formed between the heavy and light chains in antibodies from different subclasses of various animal species (see Figures 11 and 12). In a preferred embodiment, the antibody may be, for example, 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.

[0192] In this invention, an antibody is further provided in which the amino acid at position 183 of the heavy chain is replaced with the CH1 region of asparagine, 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, and an isopeptide bond is formed between the asparagine and glutamic acid. In this embodiment, the replacement of the asparagine at position 183 of the heavy chain can also be a replacement with glutamine, glutamic acid, or aspartic acid.

[0193] In this invention, a heavy chain CH1 region having the amino acid at position 183 replaced by asparagine and a nucleic acid encoding that region are further provided. In this invention, a heavy chain constant region having the amino acid at position 183 replaced by asparagine and a nucleic acid encoding that region are further provided. In this invention, a heavy chain having the amino acid at position 183 replaced by asparagine and a nucleic acid encoding that heavy chain are further provided. In this embodiment, the substitution of the heavy chain at position 183 for asparagine can be a substitution for glutamine, glutamic acid, or aspartic acid.

[0194] In this invention, a CL region containing a light chain with the amino acid at position 133 replaced by glutamic acid and the amino acid at position 176 replaced by lysine, and a nucleic acid encoding this region are also provided.

[0195] In this invention, a composition is further provided in which the amino acid at position 183 of the heavy chain is replaced with the CH1 region of asparagine or a nucleic acid encoding that region, and 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 in the CL region or a nucleic acid encoding that region. In this invention, a composition is further provided in which the amino acid at position 183 of the heavy chain is replaced with the heavy chain constant region of asparagine or a nucleic acid encoding that region, and 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 in the CL region or a nucleic acid encoding that region. In this invention, a composition is further provided in which a heavy chain containing the amino acid at position 183 of the heavy chain replaced with the CH1 region of asparagine or a nucleic acid encoding that heavy chain, and a light chain containing the amino acid at position 133 of the light chain replaced with glutamic acid and the amino acid at position 176 of the light chain replaced with lysine in the CL region or a nucleic acid encoding that light chain, are provided. In this method, the substitution of asparagine at position 183 of the heavy chain can be a substitution of glutamine, glutamic acid, or aspartic acid.

[0196] The CH1 region may also have a substitution of the amino acid at position 173 to a hydrophobic amino acid residue (e.g., isoleucine). The CH1 region may also have a substitution of the amino acid at position 181 in the EU encoding to a hydrophobic amino acid residue (e.g., leucine). In a preferred embodiment, the CH1 region may also have a substitution of the amino acid at position 173 to isoleucine and a substitution of the amino acid at position 181 to, for example, leucine.

[0197] The CL region may also have a substitution at position 131 for a hydrophobic amino acid residue (e.g., valine). The CL region may also have a substitution at position 162 for methionine. The CL region may also have a substitution at position 178 for a hydrophobic amino acid residue (e.g., leucine). The CL region may also have a substitution at position 180 of the Kabat encoding for glutamine. In a preferred embodiment, the CL region may have substitutions at position 131 for valine, position 162 for methionine, position 178 for leucine, and position 180 for glutamine. 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.

[0198] In one embodiment, the antibody of this disclosure having isopeptide bonds may have stability equal to or greater than that of an antibody containing a heavy chain with the amino acid sequence described in sequence number 61 and a light chain with the amino acid sequence described in sequence number 77. In another embodiment, the antigen-binding fragment (e.g., in the form of Fab, Fab', and F(ab')2 fragments, etc.) of the antibody of this disclosure having isopeptide bonds may have an antigen-binding fragment of the corresponding form of an antibody containing a heavy chain with the amino acid sequence described in sequence number 61 and a light chain with the amino acid sequence described in sequence number 77, or greater than that of the corresponding form. In yet another embodiment, the antibody of this disclosure having isopeptide bonds and its antigen-binding fragment may have a CL region in which the amino acid at position 183 of the heavy chain is replaced with an asparagine CH1 region and 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. In one embodiment, the antibody of this disclosure having an isopeptide bond and its antigen-binding fragment may have a CL region in which at least the amino acid at position 183 of the heavy chain is replaced with the CH1 region of asparagine and at least 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. In this embodiment, the replacement of the asparagine at position 183 of the heavy chain may be a replacement of glutamine, glutamic acid, or aspartic acid.

[0199] In all the above methods, the amino acid at position 183 of the heavy chain can be glutamine or glutamic acid, the amino acid at position 133 of the light chain can be asparagine, and the amino acid at position 176 of the light chain can be lysine. Alternatively, in all the above methods, the amino acid at position 170 of the heavy chain can be glutamine or glutamic acid, the amino acid at position 183 of the heavy chain can be lysine, and the amino acid at position 176 of the light chain can be asparagine. In this method, the substitution of asparagine at position 176 of the light chain can be a substitution of glutamine, glutamic acid, or aspartic acid.

[0200] In one embodiment, the antibody of this disclosure having an isopeptide bond may have stability or yield equal to or exceeding that of an antibody containing a heavy chain with the amino acid sequence described in sequence number 92 and a light chain with the amino acid sequence described in sequence number 94. In one embodiment, the antigen-binding fragment (e.g., in the form of Fab, Fab', and F(ab')2 fragments, etc.) of the antibody of this disclosure having an isopeptide bond may have antigen-binding fragments of the corresponding form of an antibody containing a heavy chain with the amino acid sequence described in sequence number 92 and a light chain with the amino acid sequence described in sequence number 94, equal to or exceeding their stability or yield. In one embodiment, the antibody of this disclosure having an isopeptide bond and its antigen-binding fragment may have a CH1 region where position 170 of the heavy chain is replaced with glutamic acid, position 183 of the heavy chain is replaced with lysine, and position 176 of the light chain is replaced with asparagine. In one embodiment, the antibody of the present disclosure having an isopeptide bond and its antigen-binding fragment may have a CH1 region in which at least position 170 of the heavy chain is replaced with glutamic acid and position 183 of the heavy chain is replaced with lysine, and a CL region in which at least position 176 of the light chain is replaced with asparagine.

[0201] In one embodiment, a heavy chain CH1 region, a heavy chain variable region, and a heavy chain are provided, having an amino acid sequence of the CH1 region (particularly corresponding to positions 170 to 183 in the EU encoding) of the group consisting of sequence numbers 45, 47, 49, 53, 55, 57, 59, 60, 61, 62, 81, 83, 85, 88, 90, 91, and 92, and an antibody containing any of these or a nucleic acid encoding any of these.

[0202] In one manner, a CL region having an amino acid sequence selected from sequence numbers 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 the region corresponding to positions 176–180 (particularly the region corresponding to position 176 of mut3), for example the region corresponding to positions 162–180, for example the region corresponding to positions 131–180), a light chain variable region, and a light chain are provided, as well as an antibody containing any of these or a nucleic acid encoding any of these.

[0203] One approach may provide: The heavy chain CH1 region, heavy chain variable region, and heavy chain containing an amino acid sequence of the group consisting of sequence numbers 45, 47, 49, 53, 55, 57, 59, 60, 61, 62, 81, 83, 85, 88, 90, 91, and 92 (particularly the region corresponding to positions 170–183 in the EU encoding), and antibodies or nucleic acids encoding any of these, and with The CL region (especially the region corresponding to positions 176–180) of the amino acid sequence selected from the group consisting of sequence numbers 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, the light chain variable region and the light chain, and antibodies containing any of these or nucleic acids encoding any of these. The composition.

[0204] In one approach, the heavy chain CH1 region having an amino acid sequence selected from the group described above (particularly the region corresponding to positions 170-183 in the EU encoding) can be linked to the VH region of various antibodies.

[0205] In one approach, a CL region having an amino acid sequence selected from the group described above (particularly the region corresponding to positions 176–180) can be linked to various VL regions.

[0206] In one embodiment, the heavy chain CH1 region having an amino acid sequence selected from the group described above (particularly the region corresponding to positions 170-183 in the EU encoding) can be connected to the hinge and CH2-CH3. In another embodiment, instead of the CH1 region, the CL region having an amino acid sequence selected from the group described above (particularly the region corresponding to positions 176-180) can be connected to the hinge and CH2-CH3.

[0207] In this invention, the heavy chain of the antibody may be derived from any one of the groups consisting of human IgG1, human IgG2, human IgG3, and human IgG4, or their allotypes. Human IgG1, human IgG2, human IgG3, and human IgG4 may each have, for example, sequence numbers 11 to 14. In this invention, the light chain of the antibody may be derived from any one of the groups consisting of human κ and human λ chains, or their allotypes. The human κ and human λ chains may each have, for example, sequence numbers 31 and 37. Therefore, the antibody or the antigen-binding fragment of the antibody of this invention may be derived from the aforementioned human heavy chain and human light chain.

[0208] The heavy and light chain constant regions may also contain other useful mutations, such as silencing Fc mutations, CH / CL interface controls, and deletions at positions 446 and / or 447 (EU-encoded) to improve C-terminal heterogeneity (e.g., selected from the group consisting of insertions, additions, substitutions, and deletions). Mutations in the heavy chain constant region are acceptable as long as they do not significantly inhibit antibody function (especially the desired antibody function), and can be less than 15%, 10%, or 5% of the number of amino acids in the heavy chain constant region contained in any of sequences 11–14. Mutations in the light chain constant region are acceptable as long as they do not significantly inhibit antibody function (especially the desired antibody function), and can be less than 15%, 10%, or 5% of the number of amino acids in the light chain constant region contained in any of sequences 31 and 37.

[0209] According to the present invention, cells having the above-described nucleic acids (particularly antibody-producing cells, preferably Chinese hamster egg cells) and cells having combinations of the above-described nucleic acids (particularly antibody-producing cells, preferably Chinese hamster egg cells) can be provided. According to the present invention, an antigen-binding fragment of an antibody containing the above-described CH1 and CL regions is provided. Examples of antigen-binding fragments include, for example, Fab fragments, Fab' fragments, or F(ab')2 fragments, or fragments containing these. The antibody or its antigen-binding fragment can be fused with other proteins, etc. The antibody or its antigen-binding fragment can be multispecific (e.g., bispecific).

[0210] According to the present invention, a chimeric antigen receptor (CAR) comprising a Fab fragment containing the aforementioned CH1 and CL regions is provided. CARs typically have an scFv as the antigen-binding domain; however, in the present invention, the CAR may comprise a Fab fragment containing the aforementioned CH1 and CL regions instead of an scFv. In these fragments and the CAR, an isopeptide bond is preferably formed between the CH1 and CL regions. The isopeptide bond is formed intracellularly during CAR production, thus allowing the expression of stable CARs containing Fab or Fab' fragments on the cell surface. Examples of cells expressing the aforementioned CARs in the present invention include (e.g., immune cells, such as NK cells, T cells, macrophages, neutrophils, etc.). By using the Fab fragment and Fab' fragment, CARs with strong antigen-binding affinity can be readily obtained.

[0211] According to the present invention, an antibody comprising a heavy chain containing the CH1 region and a light chain containing the CL region, or an antigen-binding fragment thereof (e.g., Fab fragment and Fab' fragment, etc.) and a cytotoxic agent can be provided as an ADC. Preferably, the ADC forms an isopeptide bond between the heavy chain containing the CH1 region and the light chain containing the CL region, thereby ensuring stable binding between the heavy and light chains even when intermolecular disulfide bonds are used for linking to the drug. The drug-to-antibody ratio of the ADC can be 1 to 8, for example 4 to 8, preferably 6 to 8, more preferably 8. Preferably, the ADC with the isopeptide bond exhibits higher stability and / or higher cytotoxicity compared to the ADC without the isopeptide bond.

[0212] According to the present invention, an antigen-binding fragment (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, etc.) comprising an antibody containing a heavy chain with a CH1 region and a light chain containing a CL region, and a conjugate (ADC) with a cytotoxic agent are provided. Similarly, in the conjugate (ADC) of the antigen-binding fragment (e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, etc.) and the cytotoxic agent, the CH1-CL chains are linked by a covalent bond based on isopeptide bonds, thereby ensuring stable binding between the heavy and light chains even when intermolecular disulfide bonds are used in the connection with the drug. Therefore, the introduction of isopeptide bonds may be particularly advantageous for ADCs.

[0213] According to the present invention, a peptide comprising an amino acid sequence containing an amino acid at position 181 of the CH1 region is provided, preferably a peptide comprising an amino acid sequence containing an amino acid at positions 173 and 181 (first peptide). According to the present invention, a peptide comprising an amino acid sequence containing an amino acid at positions 133 and 176 of the CL region is also provided (second peptide). These peptides can spontaneously form heteropeptide bonds between themselves by coexisting under physiological conditions. Therefore, a stable bond can be formed between the first peptide and the second peptide. By fusing the first peptide and the second peptide, respectively, with a target protein as a tag, multiple target proteins can interact or be linked by heteropeptide bonds. The first peptide and the second peptide can be 5 or more amino acids long, 10 or more amino acids long, 15 or more amino acids long, 20 or more amino acids long, 30 or more amino acids long, 40 or more amino acids long, 50 or more amino acids long, 60 or more amino acids long, 70 or more amino acids long, 80 or more amino acids long, 90 or more amino acids long, or 100 or more amino acids long, or can be the full length of the CH1 or CL1 region. According to the present invention, the peptide may further contain a heavy chain variable region (VH) or a light chain variable region (VL). In a preferred embodiment, the peptide contains VH, CH1, VL, and CL regions, having all or part of the ability to bind to the antigen.

[0214] The modification of various antibodies is well known, and any antibody modification can be introduced into antibodies containing heteropeptide bonds. For example, according to the Eu encoding of the heavy chain as shown in Figure 11, as mutations promoting Fc heterodimerization, the following combinations can be listed: chain 1: 354C and 366W, chain 2: 349C, 366S, 368A and 407V; chain 1: 356K, 357K and 399K, chain 2: 370E, 409D and 439E; chain 1: 364H and 405A, chain 2... The mutations include: 349T and 394F; 405L and 409R; 350V, 351Y, 400E, 405A and 407V, 350V, 366L, 390R, 392M and 394W; and 351D and 368E, and 351K and 366K.Mutations that enhance the effect include combinations of 298A, 333A, and 334A; combinations of 239D, 330L, and 332E; combinations of 236A, 239D, and 332E; combinations of 236A, 330L, and 332E; combinations of 236A, 239D, 330L, and 332E; combinations of 243L, 292P, 300L, 305I, and 396L; combinations of 235V, 243L, 292P, 300L, and 396L; and, strand 1: 234Y, 235Q, 236W, 239M, 268D, 270E, and 298A; strand 2: 270E, 3... Mutation combinations of 26D, 330M, and 334E, which enhance CDC activity, include the 326W and 333S mutation combination; the 345R, 430G, and 440Y mutation combination; and the 236A, 267E, 268F, 324T, and 332E mutation combination, which enhances binding activity to FcγRIIB, includes the 267E and 328F mutation combination; and the 233D, 237D, 238D, 268D, 271G, and 330R mutation combination, which inhibits effector function, includes single mutations of 297A, 297Q, 297G, and 265A. Mutations include: 234A and 235A (LALA mutation); 235A, 237A, and 318A mutations; 234A and 235A mutations; 228P and 235E mutations; 236R and 328R mutations; 298G and 299A mutations; 234F, 235E, and 331S mutations; 268Q, 309L, 330S, and 331S mutations; 233P, 234V, 235A, 236 deletions, and 267K mutations; 234A, 235A, and 329G mutations; and 234A, 237A, 238S, 268A, 309L, 330S, and 33... Mutation combinations of 1S; and mutation combinations of 234F, 235E, and 265A; and combinations of these single mutations and / or combinations of mutations, as mutations that prolong blood half-life, include, but are not limited to, the mutation combinations of 252Y, 254T, and 256E; the mutation combinations of 250Q and 428L; the mutation combinations of 433K and 434F; the mutation combinations of 307A, 380A, and 434A; the mutation combinations of 428L and 434S; the single mutation of 308P; the mutation combinations of 252Y, 308P, and 434Y; the mutation combinations of 285D, 307Q, and 378V; and the mutation combinations of 309D, 311H, and 434S.

[0215] (Properties and further development of peptides containing isopeptide bonds)

[0216] Isopeptide bonds are more stable and robust than other forms of interpeptide bonds, such as disulfide bonds, thus imparting beneficial effects to peptides, such as stability under high temperatures, pH, high concentrations, and / or high osmotic pressures (e.g., reduced aggregate formation) and resistance to degradation by enzymes (e.g., prolonged plasma half-life). The stability of peptides is advantageous not only in their use but also in their production (e.g., allowing for the use of more stringent conditions for purification). The high concentration stability of peptides enables the administration of equivalent amounts of therapeutic peptides in relatively small volumes, thereby reducing the burden on patients.

[0217] Isopeptide bonds are formed at least between different amino acids in a) and b), thus polypeptides with isopeptide bonds have asymmetric heterozygous chains. Therefore, when different molecules associate to form heterodimers, the formation of associates other than the target associate, which is often a problem, can be reduced. In particular, for isopeptide bonds to form, paired polypeptide chains need to have structures specific to each other, so the target associate can be produced efficiently when multiple polypeptide chains, including a) or b), coexist. Taking advantage of this, associates consisting of three or more polypeptide chains can be produced efficiently (e.g., trispecific antibodies). The DuetMab technique is known to introduce new disulfide bonds at sites different from the original CH1-CL disulfide bonds in order to suppress mismatches between heavy and light chains. However, it has been reported that byproducts are generated due to the formation of disulfide bonds with the newly introduced Cys residues (Journal of Pharmaceutical Sciences 110(2021) 2904-2915). In addition, as a technique for forming specific pairings of associates, there is the mortar and pestle technique, which uses the isopeptide bonds of this disclosure to form associates with stronger binding forces between pairs and can form associates with smaller structural domains. For example, isopeptide bonds can be formed between the constant regions of the two heavy chains of a bispecific antibody.

[0218] In one embodiment, the polypeptide containing isopeptide bonds of this disclosure is a polypeptide molecule containing at least two (e.g., two, three, four, or five) isopeptide bonds. In another embodiment, the polypeptide containing isopeptide bonds of this disclosure is a polypeptide molecule composed of at least three (e.g., three, four, five, or six) different polypeptide molecules linked by isopeptide bonds. In yet another embodiment, the polypeptide containing isopeptide bonds of this disclosure is a polypeptide molecule composed of different polypeptide molecules linked by isopeptide bonds, wherein one of the different polypeptide molecules is bound to other different polypeptide molecules by at least two (e.g., two, three, four, or five) isopeptide bonds.

[0219] In one embodiment, a protein that is naturally expressed as a single molecule can be expressed as a polypeptide molecule that is different from each other by removing the linker region, etc., and the three-dimensional structure of the natural protein is maintained by isopeptide bonds.

[0220] Disulfide bonds in polypeptides are known to stabilize the polypeptide structure. It is believed that removing disulfide bonds might cause the polypeptide chains at the disulfide bond formation sites to become less close to each other. However, the inventors have unexpectedly discovered that isopeptide bonds can be introduced to replace disulfide bonds. In one embodiment, the polypeptide containing isopeptide bonds of this disclosure is introduced to replace disulfide bonds. For example, in the stereostructure of a known polypeptide, two cysteine ​​residues that form disulfide bonds are identified, and at least one of these cysteine ​​residues is deleted. Here, by removing the disulfide-bonding cysteine ​​residues, the formation of untargeted associations via disulfide bonds (which can be symmetrical reactions) can be prevented. It is generally believed that the disulfide bond-forming parts of a known polypeptide tend to be close together before disulfide bond formation, regardless of the presence of folding aids such as molecular chaperones. Therefore, by introducing a) and / or b) near the disulfide bond formation site, the closeness of a) and b) is promoted through the same mechanism as the original polypeptide. Furthermore, by introducing isopeptide bonds to replace disulfide bonds to maintain the higher-order structure of the protein, its function can be maintained. In one embodiment, a) and / or b) are introduced into the interface region between two domains that are in contact with each other via disulfide bonds. In another embodiment, a) and / or b) are introduced into the vicinity of the disulfide bond formation site (e.g., within 5 residues, 4 residues, 3 residues, 2 residues, or 1 residue from the disulfide bond formation-related cysteine).

[0221] In one embodiment, the isopeptide-containing polypeptide of this disclosure is expressed intracellularly, wherein at least one of the different polypeptide molecules interconnected by isopeptide bonds is expressed by an introduced gene. For example, CAR-T cells expressing a chimeric antigen receptor containing a Fab-type antibody as the isopeptide-containing polypeptide of this disclosure are provided.

[0222] In one embodiment, the method for preparing a polypeptide containing isopeptide bonds disclosed herein further prepares a secondary modified polypeptide having interpeptide linkages as shown in the following formula between the modified polypeptide and a third polypeptide chain contained in a molecule different from the modified polypeptide.

[0223] (In the formula, the wavy line represents the binding to the polypeptide backbone)

[0224] The method includes: Steps for constructing the stereostructure of a secondary intermediate A containing a modified polypeptide and a third polypeptide chain; The steps for determining the amino acid sequence present in the boundary region where the modified polypeptide and the third polypeptide chain are close to each other in the three-dimensional structure; The step of mutating secondary intermediate A, wherein secondary intermediate B is prepared by introducing at least one residue from a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid into secondary intermediate A, wherein secondary intermediate B contains a), b) and c), and the modified polypeptide and the third polypeptide chain in secondary intermediate B contain a) and b) respectively in the boundary region; and The secondary modified polypeptide is obtained by forming peptide links in the secondary intermediate B. Similarly, by designing peptide links between the secondary polypeptide chain and the fourth polypeptide chain, fifth polypeptide chain, etc., polypeptides with multiple polypeptide chains linked by heteropeptide bonds can be further produced.

[0225] Those skilled in the art can use any system to produce polypeptide chains that form polypeptides containing isopeptide bonds as described in this specification.

[0226] The produced peptides can be purified or separated using any purification method. Examples include size fractionation, salting-out fractionation using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration, and immunoglobulin purification methods such as protein A / G affinity chromatography. In particular, when the monoclonal antibody is mouse IgG, affinity chromatography using a protein A binding carrier or an anti-mouse immunoglobulin binding carrier can achieve highly efficient purification.

[0227] (Uses and Applications)

[0228] The polypeptides containing isopeptide bonds disclosed herein can be used for known uses of polypeptides, such as for antigen detection and / or treatment or prevention of antigen-related diseases when the polypeptide is an antibody. One aspect of this disclosure provides applications of the polypeptides containing isopeptide bonds of this disclosure. Another aspect of this disclosure provides compositions containing the polypeptides containing isopeptide bonds of this disclosure. The compositions can be used for known uses of the polypeptides.

[0229] (Dosage form, etc.)

[0230] The compositions described in this specification can be provided in various forms. These forms may include, for example, injections, capsules, tablets, granules, etc. Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers. Additionally, aqueous solutions for injection may be formulated with, for example, physiological saline, sugars (e.g., trehalose), NaCl, or NaOH.

[0231] In one embodiment, the compositions of this disclosure contain a pharmaceutically acceptable carrier or excipient. Such a carrier may be a sterile liquid, such as water and oil, including substances of petroleum, animal, plant, or synthetic origin, without limitation, and may include peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the drug is administered orally. Physiological saline and aqueous glucose are preferred carriers when the pharmaceutical composition is administered intravenously. Physiological saline solutions and aqueous glucose and glycerol solutions are preferred as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silica, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl cellulose, corn starch, and inorganic salts. Ideally, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release complexes, etc. Traditional binders and carriers, such as triglycerides, can also be used to formulate the compositions into suppositories. Oral formulations may also contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable carriers are described in EW Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, USA). In addition to these, they may contain, for example, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow promoters, and flavoring agents. In one embodiment, the pH of any liquid composition of this disclosure may 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 the above values.

[0232] Any component of the compositions disclosed herein may be provided in the form of a pharmaceutically acceptable salt, for example, a salt formed with a free carboxyl group from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; a salt formed with a free amino group from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.; and a salt formed with sodium, potassium, ammonium, calcium and ferric hydroxide.

[0233] In a preferred embodiment, the composition can be formulated in a pharmaceutical composition form suitable for human administration according to known methods. Such a composition can be administered by injection. Typically, compositions for injection are solutions in sterile isotonic buffers. If necessary, the composition may additionally contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the components can be supplied individually or in unit doses, for example, in a sealed solvent such as an ampoule or sachet showing the amount of active agent, as a lyophilized powder or anhydrous concentrate. When administering the composition by injection, it can also be dispensed using a syringe containing sterile pharmaceutical-grade water or saline. When administering the composition by injection, ampoules of sterile water or saline for injection can also be provided to allow mixing of the components before administration.

[0234] When the compositions disclosed herein are applied to test subjects, there are no particular limitations on the test subjects, which may be mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, pigs, monkeys, humans, etc.), birds, reptiles, amphibians, arthropods, fish, etc.

[0235] The amount of the compositions and / or active ingredients disclosed herein may vary depending on the nature of the disorder or condition being treated or prevented, and can be determined by those skilled in the art using standardized clinical techniques based on the description herein. In vitro studies may be used, depending on the circumstances, to help determine the optimal dosage range. The accurate dosage of the complex also varies depending on the route of administration and the severity of the disease or disorder, and should therefore be determined based on the judgment of the attending physician and the individual patient's condition. The dosage of the compositions and / or active ingredients disclosed herein 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 dose, within any two of the above values. The dosing interval is not particularly limited, and may be, for example, once or twice every 1, 7, 14, 21, or 28 days, within any two of the above values. The dosage, dosing interval, and method of administration may be appropriately selected based on the patient's age, weight, symptoms, target organs, etc.

[0236] The compositions described in this specification can be administered via routes such as intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, epidural, or oral administration. In one embodiment, the compositions of this disclosure can be used in conjunction with various delivery systems. Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules; receptor-mediated endocytosis; and the construction of therapeutic nucleic acids as part of retroviral vectors or other vectors. Drug administration can also be performed via any suitable route, such as injection, bolus, or transdermal absorption via epithelial or mucosal membranes (e.g., oral, rectal, and intestinal mucosa). Aerosols and inhalers or nebulizers may also be used as needed; additionally, they can be administered in combination with other agents. Administration can be systemic or local.

[0237] The compositions disclosed herein can be provided in the form of a kit. In one embodiment, the present disclosure provides a pharmaceutical package or kit containing one or more containers filled with one or more ingredients that can be added to the compositions of the present disclosure. Depending on the circumstances, such containers may also display information indicating government authorization for manufacture, use, or sale for human administration, as prescribed by a government authority regulating the manufacture, use, or sale of the drug or biological product.

[0238] The steps for formulating the compositions disclosed herein into drugs, etc., are well known in the art, as described in, for example, the Japanese Pharmacopoeia, the United States Pharmacopeia, and pharmacopoeias of other countries. Therefore, with the description in this specification, those skilled in the art can determine the appropriate dosage and other implementation methods without conducting excessive experiments.

[0239] The antibodies disclosed herein have intrachain mutations for introducing isopeptide bonds (e.g., amino acid substitution mutations as described above), and may also have various other mutations, provided they do not significantly inhibit or impair antibody function (e.g., the desired antibody function). Suitable mutations can be selected for introduction depending on the purpose, but since mutations may increase the antigenicity of the antibody, a minimum is desirable. Examples of mutations that can be further introduced into the antibody include, for instance, mutations that reduce the binding affinity to Fcγ receptors (binding affinity to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and FcγIIIB) (i.e., silencing Fc mutations), mutations at amino acids 297 or 299 that inhibit or eliminate glycosylation, and mutations that improve C-terminal heterogeneity (e.g., deletion of G446 and / or K447), etc.

[0240] When the antibody is a bispecific antibody, there are no particular limitations. For example, mutations for heterozygous Fc association (such as mortar mutations), mutations for controlling the CH / CL interface, and mutations for reducing the binding of protein A can be introduced.

[0241] In bispecific antibodies with two Fab arms, isopeptide bonds can be formed between the CH1-CL bonds of one of the Fab arms. This promotes the proper formation of complexes between the heavy and light chains.

[0242] In this specification, "or" is used when "at least one or more" of the items listed herein are applicable. The same applies to "or". In this specification, when described as a "range" of "two values", the range also includes the two values ​​themselves.

[0243] The scientific literature, patents, patent applications, and other references cited in this specification are referenced to the same extent as when they are described in their entirety.

[0244] For ease of understanding, preferred embodiments have been shown to illustrate this disclosure. The following description is based on examples; however, the above description and the following examples are provided for illustrative purposes only and not for limiting the scope of this disclosure. Therefore, the scope of this disclosure is not limited to the specific embodiments and examples described herein, but only to the claims.

[0245] Example

[0246] The reagents used were the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nakara, R&D Systems, USCN Life Science INC, etc.) can also be used instead.

[0247] (Example 1: Design of trastuzumab IgG to form heteropeptide bonds)

[0248] An attempt was made to form heteropeptide bonds between the heavy and light chains of the antibody. The anti-HER2 antibody h4D5 (trastuzumab) was used as the starting protein.

[0249] Download the coordinate data of the crystal structure (PDB ID: 1N8Z) of the trastuzumab Fab-HER2 complex registered in the protein database (https: / / www.rcsb.org / ) and construct the three-dimensional structure using PyMOL (https: / / pymol.org / 2 / ).

[0250] Based on the constructed stereostructure described above, a region close to the constant regions of the heavy and light chains was identified, and asparagine and lysine were introduced into this region. Asparagine was introduced into isoCH1, and lysine into isoCL. Then, hydrophobic amino acids were introduced to create a hydrophobic environment around the side chains of the introduced asparagine and lysine. Additionally, cysteine, which is associated with disulfide bond formation, was replaced with serine. Here, a homology model was suitably constructed using SWISS-MODEL (https: / / swissmodel.expasy.org / ) based on sequence data, and a structural model with introduced isopeptide bonds was created using Coot (https: / / www2.mrc-lmb.cam.ac.uk / personal / pemsley / coot / ).

[0251] The result is the design of the sequence shown below. It should be noted that in this embodiment, the polypeptide modified to form isopeptide bonds is sometimes referred to as "iso-" or "iSo-" (isoCH1, iSoMAb, etc.). It should also be noted that MAb represents a monoclonal antibody.

[0252]

[0253] The structure of the antibody with the above mutation was reconstructed by recalculation, and the results showed that the side chains of asparagine, lysine, and glutamic acid related to isopeptide bond formation were as follows: Figure 1 The configuration is as shown. Additionally, the recalculated results of the stereostructure after isopeptide bond formation are presented below. Figure 2 .

[0254] (Example 2: Preparation of trastuzumab IgG with isopeptide bond formation and evaluation of binding activity)

[0255] Prepare and evaluate peptides designed as described above.

[0256] (The creation of iSoMAb for isoCH1 and isoCL)

[0257] Preparation of h4D5 iSoMAb IgG expression vector

[0258] 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, followed by digestion with restriction enzymes Nhe I and Not I. This was then inserted into pCAGEN containing the h4D5VH gene, which had been pre-digested with the same enzymes, to create the expression vector pCA h4D5 isoHC. Conversely, the h4D5VL gene and isoCL gene were ligated by overlap extension PCR, followed by digestion with restriction enzymes Eco RI and Not I. This was then inserted into pCAGEN pre-digested with the same enzymes, to create the expression vector pCA h4D5 isoLC.

[0259] Expression of h4D5 iSoMAb IgG

[0260] 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 × 10⁻⁶ cells were recovered by centrifugation at 1500 rpm for 5 minutes. 7 Cells were resuspended in 27 mL of HE400 medium (Gmep) containing 5 mM L-alanyl-L-glutamine. 30 μg of plasmid (15 μg pCA h4D5 isoHC and 15 μg pCA h4D5 isoLC) was added to 1.5 mL of Opti-MEM medium (Gibco) and mixed. Separately, 120 μL of PEI MAX (1 mg / mL; Polysciences) was added to 1.5 mL of Opti-MEM medium and mixed, then incubated at room temperature for 5 minutes. The plasmid dilution and PEI dilution were mixed and incubated at room temperature for 20 minutes to obtain the transfection solution. The transfection solution was added to Expi293F cells resuspended in HE400 medium and the cells were cultured. Twenty hours later, 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 mixture was cultured for another 6 days.

[0261] Purification of h4D5 iSoMAb IgG

[0262] Cells were removed from the culture medium by centrifugation at 4°C, 6000 rpm, and 10 minutes. The culture supernatant was filtered using a 0.45 μm filter (Membrane Solutions) and added to a pre-equilibrated protein A column (250 μL column volume; Bipo resin protein A; protein expression). After washing with 10 mL of PBS, the target antibody was eluted with 100 mM glycine-HCl (pH 2.5). 200 μL of each elution was recovered and neutralized with 10 μL of 100 mM Tris-HCl (pH 9.0) pre-added to the sample tubes. SDS-PAGE analysis was performed on each fraction, with the fraction containing the target antibody dialyzed against PBS. The absorbance at 280 nm was measured using a NanoPhotometer NP80 (Implen), and the absorbance at 320 nm (background) was subtracted to quantify the target antibody. The absorbance was measured at an absorptivity of 195400 M. -1 ·cm -1 The results were calculated in tetrameric form. The antibody was then purified by Western blotting analysis. HRP-labeled anti-human Fc antibody (Sigma) and HRP-labeled anti-human κ chain antibody (Sigma) were used as detection antibodies.

[0263] The results are shown in Figure 3 The disulfide bonds have been reduced and cleaved, therefore wild-type h4D5 IgG did not form an association between the heavy and light chains. On the other hand, h4D5 iSoMAb showed bands at the target molecular weight portion of the heavy and light chain association, suggesting that heteropeptide bonds were formed between the heavy and light chains.

[0264] Evaluation of binding activity on HER2-positive cells

[0265] The binding activity of SKBR-3, a HER2-positive human breast cancer cell line, was evaluated by flow cytometry. SKBR-3 cells cultured in 10% FBS / D-MEM medium (Wako) at 37°C and 5% CO2 were detached from the culture dish using trypsin / EDTA (0.25% (w / v) trypsin-1mM EDTA·4Na; Gibco). Cells were recovered by centrifugation at 1000 rpm for 5 minutes at room temperature, and 5 × 10⁶ cells were aliquoted into sample tubes. 5Cells were washed with 0.1% NaN3 / PBS and reacted on ice for 30 minutes with h4D5 iSoMAb IgG at a final concentration of 500 nM as primary antibody. After washing again, 1 μL of anti-human IgG (Fc specific)-FITC antibody (2 mg / mL; Sigma) and 499 μL of 0.1% NaN3 / PBS were added as secondary antibody, and the mixture was reacted on ice for 30 minutes. After washing again, the cells were resuspended in 500 μL of 0.1% NaN3 / PBS. After filtering through a nylon filter, the cells were aliquoted into plastic tubes and analyzed using a flow cytometer (Accuri C6; BD Biosciences).

[0266] The results are shown in Figure 4 iSoMAb was found to have the same HER2 binding ability as the original antibody.

[0267] (Example 3: Preparation of antibodies formed by isopeptide bonds in Fab form)

[0268] Antibody fragments in Fab form were fabricated based on the iSoMAb prepared above and evaluated.

[0269] Preparation of h4D5 iSoMAb Fab heavy chain expression vector

[0270] Using the synthesized isoCH1 gene as a template, a histidine tag sequence was added to the C-terminus of the CH1 region via PCR. The PCR product was digested with restriction enzymes Nhe I and Not I. This product was then inserted into pCAGEN containing the h4D5VH gene, which had been pre-digested with the same enzymes, thereby creating the expression vector pCA h4D5VH-isoCH1.

[0271] Expression of h4D5 iSoMAb Fab

[0272] h4D5 iSoMAb Fab was expressed using the same method as described above. pCA h4D5VH-isoCH1 and pCA h4D5 isoLC were used as plasmids for gene introduction.

[0273] Purification of h4D5 iSoMAb Fab

[0274] Cells were removed from the culture supernatant by centrifugation at 4°C, 6000 rpm, and 10 minutes. The culture supernatant was filtered using a 0.45 μm filter (Membrane Solutions) and added to a Ni Sepharose excel column (500 μL column volume; 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. 200 μL of each fraction was recovered and analyzed by SDS-PAGE. The fraction containing the target antibody was dialyzed against PBS. The absorbance at 280 nm was measured using a NanoPhotometer NP80, and the absorbance at 320 nm (for background) was subtracted to quantify the target antibody. With an absorption coefficient of 64900M -1 ·cm -1 The calculation is performed in dimer form.

[0275] The results are shown in Figure 5 The disulfide bonds have been reduced and cleaved, therefore wild-type h4D5 Fab did not form an association between the heavy and light chains. On the other hand, bands were identified in the target molecular weight region of the heavy and light chain association in h4D5 iSoMAb Fab, suggesting that an isopeptide bond was formed between the heavy and light chains.

[0276] • Evaluation of thermal stability using differential scanning fluorescence

[0277] Add 17.5 μL of a 50-fold diluted fluorescent dye (SYPRO (registered trademark) dye, used for protein gel staining; Merck) to 70 μL of a 0.1 mg / mL antibody solution and mix. Dispense 25 μL of this antibody-dye mixture into each of three wells in a 48-well PCR plate. Using a real-time PCR apparatus, the temperature is increased from 25 °C to 99 °C over approximately 40 minutes, and the fluorescence intensity is measured. Additionally, to investigate thermal stability under reducing conditions, 1 mM TCEP (tris(2-carboxyethyl)phosphine) is added to the antibody solution, and after incubation on ice for 3 hours, the same measurements are performed.

[0278] The results are shown in Figure 6And the following table. Compared with h4D5 Fab, a slight decrease in thermal stability was observed in h4D5 iSoMAb Fab, but this decrease in thermal stability is not significant enough to hinder purification or use in vivo. Furthermore, unlike h4D5 Fab, no decrease in stability was observed in h4D5iSoMAb Fab under reducing conditions (in the presence of TCEP), confirming that the isopeptide bonds are resistant to cleavage under reducing conditions.

[0279] [Table 2]

[0280] (Example 4: Design of anti-CD63 antibody formed by isopeptide bond)

[0281] Similar to the example of anti-HER2 antibody h4D5 mentioned above, an attempt was made to create a modified antibody based on anti-CD63 antibody (clone No. 2 described in Monoclon. Antib. Immunodiagn. Immunother. 39, 74-76 (2020)) with heavy chain-light chain binding via isopeptide bonds.

[0282] A stereochemical model was constructed in the same manner as in Example 1, and the following sequence was 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. Glutamic acid was then introduced in such a manner that its side chains were close to the side chains of the introduced asparagine and lysine.

[0283]

[0284] The positional relationships between atoms of a), b), c) and hydrophobic mutant residues in the three-dimensional structure are as follows.

[0285] [Table 3]

[0286] Preparation of hCD63 iSoMAb IgG expression vector

[0287] Eurofins Genomics was commissioned to synthesize IgG and CL (isoCL) genes containing mutant CH1 (isoCH1). The synthesized genes were then seamlessly cloned and inserted into pCEC4.3W to create pCEC4.3W-isoCH1vC (Afl II) or pCEC4.3W-isoCLvC (BsrGI). The vectors were then digested with restriction enzymes Afl II or BsrGI, and the humanized hCD63 antibody gene was seamlessly cloned and inserted into the heavy and light chains, respectively, to create expression vectors pCEC4.3W-isoCH1vChCD63 H1 and pCEC4.3W-isoCLvC hCD63 K1.

[0288] hCD63 iSoMAb IgG expression

[0289] 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 procedures for the ExpiCHO expression system (Gibco, Thermo Fisher Scientific). Cells with a density of 18.0 × 10⁻⁶ were recovered by centrifugation at 300 × g for 5 minutes. 7 Cells were suspended in 30 mL of ExpiCHO expression medium. 24 μg of plasmid (12 μg of pCEC4.3W-isoCH1vC hCD63 H1 and 12 μg of pCEC4.3W-isoCLvC hCD63 K1) was added to 1.2 mL of OptiPRO SFM composite 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 composite medium and mixed. The plasmid dilution and ExpiFectamine (trademark) CHO Reagent dilution were then incubated at room temperature for 5 minutes to obtain the transfection solution. The transfection solution was added to the CHO-S cells suspended in ExpiCHO expression medium and the cells were cultured. Eighteen hours later, 180 μL of ExpiFectamine (registered trademark) CHO enhancer and 7.2 mL of ExpiCHO (registered trademark) feed were added, and the mixture was cultured for another 12 days.

[0290] Purification of hCD63 iSoMAb IgG

[0291] Cells were removed from the culture supernatant by centrifugation at 4°C, 12000×g, for 10 minutes. 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. The reaction was carried out overnight at 4°C with stirring, and then the mixture was added to the column along with the gel. After washing with 30 mL of PBS, the target antibody was eluted with 0.1 M citrate elution buffer (pH 3.0). Recovery was performed in 1.0 mL increments, and each fraction was analyzed by SDS-PAGE. For fractions containing the target antibody, the solvent was replaced with PBS using Pierce Protein Concentrators PES, 10K MWCO, and 2–6 mL. The absorbance at 280 nm was measured using an Eppendorf BioPhotometer plus spectrophotometer, and the absorbance at 340 nm (for background) was subtracted to quantify the target antibody. Then, the Pierce (registered trademark) BCA protein assay kit was used to determine a more accurate concentration.

[0292] • Evaluation of binding activity on hCD63-positive cells

[0293] The binding activity of Expi293F cells, derived from hCD63-positive human fetal kidneys, was evaluated by flow cytometry. Expi293F cells cultured in Expi293 expression medium (Gibco, Thermo Fisher Scientific) at 37°C, 5% CO2, and 125 rpm were recovered by centrifugation at room temperature, 300 × g, for 5 minutes, and 5 × 10⁶ cells were aliquoted into sample tubes. 5 Cells were washed with 0.2% BSA / PBS and incubated with hCD63iSoMAb IgG at a final concentration of 2 μg / mL as primary antibody at 4°C for 60 min. After similar washing, cells were incubated with goat F(ab')2 anti-human IgG-Fc(PE) and pre-adsorbed (ab98596, Abcam) at a final concentration of 0.5 μg / mL as secondary antibody at 4°C for 30 min. Cells were washed again and resuspended in 200 μL of 0.2% BSA / PBS. Cells were aliquoted into plastic tubes and analyzed using flow cytometry (FACS Verse; BDBiosciences).

[0294] Results of gel electrophoresis ( Figure 7Yes, under reducing conditions, bands were identified at the target molecular weight region of the iSoMAb heavy and light chain conjugate, suggesting the formation of isopeptide bonds between the heavy and light chains (lanes 3 and 4). Furthermore, iSoMAb expression in Expi293F cells was also confirmed (lanes 5 and 6). Further, based on the results of flow cytometry (… Figure 8 The study confirmed that both iSoMAb strains maintained binding to CD63-expressing cells (293 strains). Specifically, when either the amino acid at position 186 of the heavy chain or the amino acid at position 176 of the light chain was lysine and the other was asparagine, these amino acids formed heteropeptide bonds.

[0295] (Example 5: Preparation of bispecific antibodies containing isopeptide bonds)

[0296] An attempt was made to create a bispecific antibody containing one isopeptide bond. A summary of the structure of the constructed bispecific antibody is shown below. Figure 9 It is a bispecific antibody containing variable regions of h4D5 (anti-HER2) and U1-59 (anti-HER3), and h4D5 was modified in accordance with Examples 1-2 to form isopeptide bonds.

[0297] Preparation of h4D5xU1-59 iSoMAb bispecific IgG expression vector

[0298] The isoCH1 gene and the hinge-Fc(knob) gene were ligated by overlap extension PCR and digested with restriction enzymes Nhe I and Not I. The ligation was then inserted into pCAGEN containing the h4D5VH gene, which had been pre-digested with the same enzymes, to create the expression vector pCA h4D5isoHC(knob). Next, the U1-59VH and VL genes were synthesized by Eurofins Genomics. The plasmid containing the U1-59VH gene was digested with restriction enzymes Afl II and Nhe I and then inserted into pCAGEN containing the CH1-Fc(hole) gene, which had been pre-digested with the same enzymes, to create the expression vector pCA U1-59 HC(hole). Additionally, the U1-59VL gene and the CL gene were ligated by overlap extension PCR and digested with restriction enzymes Eco RI and Not I. The ligation was then inserted into pCAGEN pre-digested with the same enzymes, to create the expression vector pCA U1-59 LC.

[0299] Expression of h4D5xU1-59 iSoMAb bispecific IgG expression vector

[0300] 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 × 10⁻⁶ cells were recovered by centrifugation at 1500 rpm for 5 minutes. 7 One cell was suspended in 27 mL of HE400 medium (Gmep) containing 5 mM L-alanyl-L-glutamine. Add 30 μg of plasmid (7.5 μg pCA h4D5 isoHC (knob), 7.5 μg pCA U1-59 HC (hole), 7.5 μg pCA h4D5 isoLC, and 7.5 μg pCA U1-59 LC) to 1.5 mL of Opti-MEM medium (Gibco) and mix. Separately, add 120 μL of PEI MAX (1 mg / mL; Polysciences) to 1.5 mL of Opti-MEM medium and mix, then incubate at room temperature for 5 minutes. Mix the plasmid dilution and PEI dilution, and incubate at room temperature for 20 minutes to obtain the transfection solution. Add the transfection solution to Expi293F cells suspended in HE400 medium and culture. After 20 hours, add 75 μL of 0.5 M sodium valproate, 120 μL of 1 M sodium propionate, and 750 μL of 20% (w / v) tryptone, and culture for another 6 days.

[0301] Purification of h4D5xU1-59 iSoMAb bispecific IgG

[0302] Cells were removed from the culture supernatant by centrifugation at 4°C, 6000 rpm, and 10 minutes. The culture supernatant was filtered using a 0.45 μm filter (Membrane Solutions) and added to a pre-equilibrated protein A column (250 μL column volume; Bipo resin protein A; protein expression) 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 of each elution was recovered and neutralized with 10 μL of 100 mM Tris-HCl (pH 9.0) pre-added to the sample tubes. SDS-PAGE analysis was performed on each fraction, with the fraction containing the target antibody dialyzed against PBS. The absorbance at 280 nm was measured using a NanoPhotometer NP80 (Implen), and the absorbance at 320 nm (for background) was subtracted to quantify the target antibody. The absorbance was measured at an absorptivity of 220500 M. -1 cm -1 / Calculation in tetramer form.

[0303] • Evaluation of bispecificity using sandwich ELISA

[0304] Add 50 μL of 10 μg / mL HER2-ECD-human Fc to each well of a 96-well EIA / RIA plate (Corning), and incubate at 37°C for 30 minutes. After removing the solution, add 200 μL of PBS (PBST) containing 0.05% (v / v) Tween 20 and wash three times. Add 100 μL of PBST containing 1% (w / v) BSA as blocking buffer. After incubating at 37°C for 1 hour, remove the blocking buffer, add 200 μL of PBST, and wash three times. Add 50 μL of each concentration of bispecific antibody solution diluted with PBST containing 1% (w / v) BSA, and incubate at 37°C for 30 minutes. After removing the antibody solution, add 200 μL of PBST and wash three times. Add 50 μL of 10 μg / mL HER3-ECD-mouse Fc. After standing at 37°C for 30 minutes, the solution was removed, and 200 μL of PBST was added, followed by three washes. Then, 50 μL of 0.25 μg / mL anti-IgG (H+L), Mouse, Goat-Poly, or HRP (SeraCare Life Sciences, Inc.) as detection antibodies were added, and the solution was incubated at 37°C for 30 minutes. After removing the antibody solution, 200 μL of PBST was added, followed by three washes. Then, 50 μL of chromogenic substrate solution [50 μL of 10 mg / mL TMB, 4 μL of 3% H2O2, or 5 mL of substrate solution A (50 mM Na2HPO4·12H2O, 25 mM citric acid, pH 5.5)] was added, and the solution was incubated for 15 minutes. Finally, 50 μL of reaction stop solution (1N H2SO4) was added. The absorbance at 450 nm was measured using an Infinite 200 PRO (TECAN) microplate reader.

[0305] The results are shown in Figure 10 No color development was observed in the case of single-binding h4D5 IgG and U1-59 IgG, while significant color development was observed in the case of h4D5xU1-59 iSoMAb bispecific IgG. This indicates that antibodies containing isopeptide bonds are suitable for use as bispecific antibodies. It is also thought that bispecific antibodies containing isopeptide bonds at each of the two antigen-binding sites could be constructed similarly. It should be noted that, in order to efficiently obtain the target bispecific antibody, a mortar and pestle structure was used to promote pairing between the heavy chains of the specific antibody; however, this mortar and pestle structure can be replaced with the isopeptide bonds of this disclosure.

[0306] (Example 6: Creation of a modified organism without hydrophobic mutation)

[0307] Peptides with mutants K, N, and E introduced into isoCH1v1, isoCLv2, isoCLv1, and isoCH1v2 were prepared. The predicted results confirmed the formation of isopeptide bonds.

[0308] (Example 7: Preparation of various forms of antibodies containing isopeptide bonds)

[0309] Based on the sequences with introduced isopeptide bond formation mutations, antibodies in Fab, Fab', or F(ab')2 forms were prepared. It is predicted that isopeptide bonds can still be formed in these different forms.

[0310] (Example 8: Determination of the cytotoxic activity of anti-CD63 antibody formed by isopeptide bond)

[0311] • Preparation of antibody-drug complexes (conjugates)

[0312] Anti-hCD63 iSoMAbv1 (heavy chain with SEQ ID NO: 45, light chain with SEQ ID NO: 46) and non-mutant anti-hCD63 WT (wild-type) MAb (heavy chain with SEQ ID NO: 43, light chain with 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) to a final concentration of 100 μM, and the mixture was incubated at 37°C for 2 hours. After incubating the reaction solution at room temperature for 30 minutes, VcMMAE (MedChemExpress) was added to a final concentration of 100 μM, and the mixture was incubated at room temperature for 30 minutes. Then, NAC (N-acetylcysteine) was added to a final concentration of 1 mM, and the reaction was stopped by incubating on ice for 30 minutes. The mixture was then dialyzed against PBS(-) to prepare the antibody-drug complex. The drug-antibody ratio of the obtained antibody-drug complexes was determined by UV absorption method. The results showed that the drug-antibody ratio of both anti-hCD63 WT Mab-VcMMAE and anti-hCD63 iSoMAbv1-MMAE was approximately 4.

[0313] • Assay of cell-killing activity

[0314] Human pancreatic cancer cell line SUIT-2 was used at a density of 4 × 10⁻⁶ cells / well. 3Cells were seeded in 96-well plates, and the antibody-drug complex was added to a final concentration of 0.1–3 μg / mL the following day. The plates were then incubated at 37°C for 96 hours in a CO2 incubator. An equal volume of culture medium was added to the negative control, and the plates were also incubated for 96 hours. After incubation, as a positive control, 0.1% Tween 20 was added to each well, and the plates were incubated in a CO2 incubator for 30 minutes. Then, Cell Counting Kit-8 (Dojindo) was added to each well, and after incubation in a CO2 incubator for 1 hour, the absorbance at 450 nm was measured using a microplate reader. Cell killing activity (%) was calculated using the following formula.

[0315] Cell-killing activity (%) = ((Absorbance of negative control - Absorbance of antibody-drug complex) / (Absorbance of negative control - Absorbance of positive control)) × 100

[0316] The results are shown in Figure 13 At antibody-drug complex concentrations of 1 μg / mL and 3 μg / mL, anti-hCD63 iSoMAbv1-MMAE showed significantly higher cytotoxic activity compared to anti-hCD63 WT MAb-VcMMAE.

[0317] (Example 9: Determination of the cytotoxic activity of the anti-CD63 antibody Fab form formed by isopeptide bonds)

[0318] • Preparation of antibody-drug complexes

[0319] An antibody-drug complex was prepared based on the anti-hCD63 iSoMAbv1 antibody fragment (heavy chain fragment with sequence number 49 and light chain fragment with sequence number 50) in Fab form, prepared in Example 8. The control was an antibody-drug complex of anti-hCD63 WT Fab (heavy chain with sequence number 47 and light chain with sequence number 48). The drug-antibody ratio of the obtained antibody-drug complexes was determined by UV absorptiometry. The results showed that the drug-antibody ratio of both anti-hCD63 WT Fab-VcMMAE and anti-hCD63 iSoMAbv1 Fab-MMAE was approximately 1.

[0320] • Assay of cell-killing activity

[0321] The cytotoxic activity of the antibody-drug complex containing the Fab-form antibody fragment was determined according to the method described in Example 8.

[0322] The results are shown in Figure 14 At antibody-drug complex concentrations of 1 μg / mL and 3 μg / mL, anti-hCD63 iSoMAbv1Fab-MMAE showed significantly higher cytotoxic activity compared to anti-hCD63 WT Fab-VcMMAE.

[0323] (Example 10: Determination of the concentration of trastuzumab IgG in mouse blood that forms isopeptide bonds)

[0324] The trastuzumab IgG (h4D5 iSoMAb) with formed isopeptide bonds (heavy chain serial number 53, light chain serial number 54) and the WT (wild-type) trastuzumab IgG (h4D5 WT MAb) without formed isopeptide bonds (heavy chain serial number 51, light chain serial number 52) prepared in Example 2 were administered to mice. Blood samples were collected after a period of time to determine the highest blood concentration and blood half-life.

[0325] • Drug administration and plasma preparation

[0326] Five 5-week-old male ICR mice were intraperitoneally administered 10 mg / kg of IgG in PBS solution (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 3500 rpm, 4°C, for 15 minutes to prepare plasma, which was immediately stored at -30°C.

[0327] • Determination of human IgG concentration in mouse plasma

[0328] The concentration of human IgG in mouse plasma was determined using a human IgG ELISA kit (Assaypro).

[0329] The results are shown in Figure 15 The highest serum concentrations of h4D5 WT MAb were 107.8 ± 10.6 μg / mL (after 1 day) and h4D5 iSoMAb were 102.0 ± 10.8 μg / mL (after 2 days), which were approximately the same. On the other hand, the serum half-life of h4D5 WT MAb was 6.3 ± 4.1 days, while the serum half-life of h4D5 iSoMAb was 8.2 ± 3.7 days, indicating that h4D5 iSoMAb has a longer serum half-life compared to h4D5 WT MAb.

[0330] (Example 11: Determination of the binding of anti-CD63 antibody in Fab form formed by isopeptide bonds to antigen-positive cells)

[0331] Expression of hCD63 iSoMAb Fab

[0332] hCD63 iSoMAb Fab (heavy chain fragment with SEQ ID NO: 57, light chain with 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 introduction.

[0333] Purification of hCD63 iSoMAb Fab

[0334] Cells were removed from the culture supernatant by centrifugation at 4°C, 12000×g, and 10 minutes. The culture supernatant was filtered through a 0.45 μm pore size filter (GVS) and added to a KanCap G column (1.0 mL column volume; 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 wash fraction (5 mL) and eluted fraction (1 mL each) were subjected to CBB staining and analysis after SDS-PAGE. For fractions containing the target antibody, 2–6 mL of Pierce staining solution was used. TM Protein Concentrators: PES, 10K MWCO, with the solvent replaced by PBS. Absorbance at 280 nm was measured using an Eppendorf BioPhotometer plus spectrophotometer; the absorbance at 340 nm (for background) was subtracted to quantify the target antibody. Then, Pierce... TM The BCA protein assay kit can determine a more accurate concentration.

[0335] The same method was used to prepare purified antibodies for hCD63 WT (wild-type) Fab (heavy chain fragment with sequence number 55, light chain with sequence number 56).

[0336] • Evaluation of binding activity on hCD63-positive cells

[0337] The binding activity of the hCD63-positive human fetal kidney-derived cell line 293 was evaluated by flow cytometry. Expi293F cells cultured in Expi293 expression medium (Gibco, Thermo Fisher Scientific) at 37°C, 5% CO2, and 125 rpm were recovered by centrifugation at room temperature, 300 × g, for 5 minutes, washed with 0.2% BSA / PBS, and aliquoted into sample tubes at 5 × 10⁻⁶ cells / mL. 5 Cells were incubated with hCD63 iSoMAbFab at a final primary antibody concentration of 2 μg / mL for 60 min at 4°C. After washing, cells were incubated with mouse secondary antibody human IgKappa (PE) at a final secondary antibody concentration of 12.5 μg / mL (TB28-2, BD Biosciences) for 30 min at 4°C. After washing again, cells were resuspended in 200 μL of 0.2% BSA / PBS. Cells were aliquoted into plastic tubes and analyzed using flow cytometry (FACS Verse; BD Biosciences).

[0338] The results are shown in Figure 16 hCD63 iSoMAb Fab and hCD63 WT (wild-type) Fab bind equally to antigen-positive cells.

[0339] (Example 12: Analysis of heteropeptide bond formation in h4D5 iSoMAbv1 mutant)

[0340] Preparation of h4D5 iSoMAb mutant expression plasmid

[0341] To investigate the effects of amino acid mutations (especially those shown in Tables 4-6) at positions 173, 181, 183 of the heavy chain and positions 131, 133, 162, 176, 178, 180 of the light chain) on isopeptide bond formation, Eurofins Genomics was commissioned to create mutant expression plasmids based on the h4D5 iSoMAb heavy chain (SEQ ID NO. 53) and light chain (SEQ ID NO. 54) expression plasmids according to Tables 4-6, with any amino acid substitution at positions 173, 181, 183 of the heavy chain and positions 131, 133, 162, 176, 178, 180 of the light chain. The h4D5 iSoMAb heavy chain mut1–mut4 have SEQ ID NO. 59–62, and the h4D5 iSoMAb light chain mut1–mut15 have SEQ ID NO. 63–77.

[0342] [Table 4]

[0343] [Table 5]

[0344] [Table 6]

[0345] Expression of h4D5 iSoMAb mutant

[0346] HEK293T cells were used as host cells. Culture was performed entirely at 37°C and 5% CO2. First, HEK293T cells were cultured in 96-well plates in DMEM containing 10% FBS. Transfection was performed when the cells reached 80-90% confluence. For transfection, 2 μL of 10 mg / ml polyethyleneimine solution was mixed into 1100 μL of FBS-free DMEM, and 13 μL was dispensed into each of the 96 wells, along with 0.12 μg of plasmid (heavy chain: 0.065 μg, light chain: 0.065 μg, combination as shown in Table 7).

[0347] [Table 7]

[0348] After standing for 20 minutes, 200 μL of DMEM containing 2% FBS was added as the transfection solution. After removing the culture medium from the 96-well plate seeded with cells, 200 μL of the transfection solution was injected into each well, and the plate was incubated for 8 days. The culture supernatant was recovered, and protein expression was investigated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with anti-human Fc antibodies (A0170, SIGMA-ALDRICH). Additionally, band intensity was calculated using ImageJ to predict the percentage of isopeptide bonds formed. The percentage (%) was calculated using the following formula.

[0349] Formation ratio (%) = ((intensity of bands with cross-linked heavy and light chains) / (band intensity of peptides with cross-linked heavy and light chains + band intensity of heavy chain)) × 100

[0350] The results are shown in Table 8. Mutants (mutants 3-15) in which the heavy chain 183, light chain 133, and light chain 176 were replaced with asparagine, glutamic acid, and lysine, respectively, showed peptides in which the heavy and light chains were cross-linked by isopeptide bonds.

[0351] [Table 8]

[0352] (Example 13: Preparation of h4D5 iSoMAbv1 mutant)

[0353] Expression and purification of h4D5 iSoMAb mutant

[0354] The mutant expression plasmid prepared in Example 12 was expressed using the same method 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. Additionally, ImageJ was used to calculate band intensity and predict the formation rate of isopeptide bonds. The formation rate (%) was calculated using the following formula.

[0355] Formation ratio (%) = ((intensity of bands with cross-linked heavy and light chains) / (band intensity of cross-linked peptides with cross-linked heavy and light chains + band intensity of heavy chain + band intensity of light chain)) × 100

[0356] The results are shown in Figure 17 and 18 Mutants with positions 183 of the heavy chain, 133 of the light chain, and 176 of the light chain replaced by asparagine, glutamic acid, and lysine, respectively (mutants 3-15) Figure 17 In lane 1, under physiological conditions, the formation of isopeptide bonds between CH1-CL occurs sufficiently. Additionally, with h4D5 iSoMAbv1 ( Figure 17 Compared to lane 4), these antibodies showed higher yields, but the proportion of isopeptide bonds formed was slightly reduced. It is believed that these antibodies formed isopeptide bonds between asparagine at position 183 of the heavy chain and lysine at position 176 of the light chain.

[0357] [Table 9]

[0358] On the other hand, in addition to the heavy chain positions 183, 133, and 176 being replaced with asparagine, glutamic acid, and lysine respectively, mutants also had leucine replaced at position 181 of the heavy chain (mutants 1-15). Figure 18 Lane 2) showed a similar ratio of isopeptide bonds to h4D5 iSoMAbv1. It is believed that these antibodies formed isopeptide bonds between asparagine at position 183 of the heavy chain and lysine at position 176 of the light chain.

[0359] [Table 10]

[0360] (Example 14: Analysis of isopeptide bond formation in h4D5 iSoMAbv1 H-S183D mutant)

[0361] Preparation of expression plasmid for h4D5 iSoMAbv1 H-S183D mutant

[0362] Eurofins Genomics was commissioned to produce expression plasmids for heavy chain mutants, with positions 173, 181, and 183 of the heavy chain replaced by isoleucine, leucine, and aspartic acid, respectively.

[0363] Expression of the h4D5 iSoMAbv1 H-S183D mutant

[0364] 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 replaced with glutamic acid and lysine, respectively) (the heavy chain has sequence number 45, and the light chain has sequence number 78), and expressed using the same method as in Example 12. Analysis was performed by SDS-PAGE and Western blotting. The proportion of isopeptide bonds formed was predicted in the same manner as in Example 12.

[0365] The results are shown in Figure 19 When the heavy chain at position 183 was replaced with aspartic acid, a polypeptide was detected in which the heavy and light chains were cross-linked via isopeptide bonds. The isopeptide bond formation rate was approximately 10%. It is believed that these antibodies formed isopeptide bonds between asparagine or aspartic acid at position 183 of the heavy chain and lysine at position 176 of the light chain.

[0366] [Table 11]

[0367] (Example 15: Analysis of the binding of h4D5 iSoMAb Fab to the extracellular domain of HER2)

[0368] • Surface plasmon resonance analysis of interactions between extracellular domains of h4D5 Fab-HER2

[0369] For the h4D5 iSoMAb Fab (heavy chain with serial number 80, light chain with serial number 46) prepared in Example 3, the binding to the HER2 extracellular domain was analyzed by surface plasmon resonance. As a comparison, h4D5 WT (wild-type) Fab (heavy chain with serial number 79, light chain with serial number 46) was used. Measurements were performed using a BIACORE3000 (Cytiva) intermolecular interaction analysis device. Using a mouse antibody capture kit (Cytiva), the HER2 extracellular domain fused with the mouse-derived Fc region was captured onto a sensor chip (CM5, Cytiva), and Fab solutions prepared at various concentrations were added to investigate binding behavior. The obtained sensor maps were fitted using a 1:1 Langmuir binding model with BIAevaluation software (Cytiva) to calculate interaction-related kinetic parameters.

[0370] The results are shown in Figure 20 The binding activity of h4D5 iSoMAb Fab to the extracellular domain of HER2 is equal to or higher than that of h4D5 WT (wild-type) Fab.

[0371] [Table 12]

[0372] (Example 16: Crystal structure analysis of hCD63 iSoMAb Fab)

[0373] Crystallization of hCD63 iSoMAb Fab

[0374] The hCD63 iSoMAb Fab sample prepared in Example 11 at a concentration of 16.0 mg / mL was crystallized. The crystallization buffer consisted of 0.1 M sodium dimethylarsinate (pH 6.5), 0.2 M calcium acetate, and 43% PEG300. Crystallization was carried out at 20°C by hanging drop vapor diffusion.

[0375] Crystallization of hCD63 WT Fab

[0376] Crystallization was performed using the 15.8 mg / mL hCD63 WT Fab sample prepared in Example 11. The crystallization buffer consisted of 0.1 M sodium dimethylarsinate pH 6.5, 0.2 M calcium acetate, and 42% PEG300. Crystallization was carried out at 20°C by hanging drop vapor diffusion.

[0377] Structural analysis of hCD63 iSoMAb Fab and hCD63 WT Fab

[0378] For the obtained crystals, diffraction data were acquired using the Tsukuba KEK Photon Factory BL5A beamline. Molecular substitution was performed using similar molecular structures, and refinement was achieved using Phenix Refine. Molecular modeling was performed using Coot.

[0379] The results are shown in Figure 21 Electron density indicating isopeptide bond formation was observed between the asparagine residue at position 183 of the heavy chain and the lysine residue at position 176 of the light chain. Additionally, a glutamic acid residue at position 133 of the light chain was present nearby. Superimposing the structures of iSoMAb Fab and wild-type Fab yielded an RMSD (root mean square deviation) of 0.34 Å, with no significant changes observed in the overall structure.

[0380] (Example 17: Formation of isopeptide bonds in human IgG4κ)

[0381] • Creation of expression plasmids

[0382] Based on the amino acid sequence information of nivolumab (human IgG4κ), mutations were introduced into the isopeptide bond formation-related sites (replacing heavy chain position 183, light chain position 133, and light chain position 176 with asparagine, glutamic acid, and lysine, respectively), to create the amino acid sequence of nivolumab iSoMAb. Eurofins Genomics was commissioned to create artificial genes for the heavy chain (sequence number 81) and light chain (sequence number 82) based on the amino acid sequence and insert them into the expression plasmid.

[0383] Expression of nivolumab iSoMAb

[0384] 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 recovered, and expression was investigated by SDS-PAGE and Western blotting. Anti-human Fc antibody (SIGMA: A0170) and anti-human κ chain antibody (SIGMA: A7164) were used as detection antibodies.

[0385] The results are shown in Figure 22When using anti-human Fc antibody and anti-human κ chain antibody as detection antibodies, peptides in which the heavy chain and light chain are cross-linked through isopeptide bonds were detected.

[0386] (Example 18: Formation of heteropeptide bonds in human IgG2κ)

[0387] • Creation of expression plasmids

[0388] Based on the amino acid sequence information of panitumumab (human IgG2κ), mutations were introduced into the isopeptide bond formation-related sites (replacing heavy chain position 183, light chain position 133, and light chain position 176 with asparagine, glutamic acid, and lysine, respectively), thereby generating the amino acid sequence of panitumumab iSoMAb. Eurofins Genomics was commissioned to create artificial genes for the heavy chain (serial number 83) and light chain (serial number 84) based on the amino acid sequence and insert them into the expression plasmid.

[0389] Expression of panitumumab iSoMAb

[0390] 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 recovered, and expression was investigated by SDS-PAGE and Western blotting. Anti-human Fc antibody (SIGMA: A9544) and anti-human κ chain antibody (SIGMA: A7164) were used as detection antibodies.

[0391] The results are shown in Figure 23 When anti-human Fc antibody and anti-human κ chain antibody were used as detection antibodies, peptides in which the heavy chain and light chain were cross-linked through isopeptide bonds were detected.

[0392] (Example 19: Formation of isopeptide bonds in human IgG1λ)

[0393] • Creation of expression plasmids

[0394] The heavy chain (serial number 85) and light chain variable region (serial number 86) of the anti-BCMA antibody (clone name 372, human IgG1λ) were synthesized artificially by Eurofins Genomics. The heavy chain expression plasmid of h4D5 iSoMAbv1 prepared in Example 12 was digested with restriction enzymes EcoRI and NotI, and inserted into the 372 heavy chain variable region digested with the same enzymes, thereby preparing the 372 heavy chain expression plasmid. Additionally, the light chain constant region (with positions 131, 133, 162, 176, 178, and 180 replaced with valine, glutamic acid, methionine, lysine, leucine, and glutamine, respectively) and the 372 light chain variable region, also synthesized artificially, were ligated by overlap extension PCR, digested with restriction enzymes EcoRI and NotI, and inserted into the expression plasmid pre-digested with the same enzymes, thereby preparing the 372 light chain expression plasmid.

[0395] • Expression and purification of 372 iSoMAb

[0396] Using the expression plasmid described above, recombinant antibodies were expressed using the same method as in Example 2, and purified from the culture supernatant using protein A affinity chromatography. The purified recombinant antibodies were analyzed by SDS-PAGE under reducing conditions.

[0397] The results are shown in Figure 24 In the purified 372 iSoMAb, a polypeptide in which the heavy and light chains were cross-linked via isopeptide bonds was detected.

[0398] (Example 20: Production of h4D5 iSoMAb Fab using short bacteria)

[0399] • Creation of expression plasmids

[0400] Eurofins Genomics was commissioned to prepare plasmids containing the light chain sequence of h4D5 iSoMAb (amino acid substitution sequences at positions 133 and 176 in Table 6) (Sequence No. 87) and the heavy chain sequence (amino acid substitution sequence at position 183 in Table 4) (Sequence No. 88). Using this plasmid as a template, PCR was performed with primers (5'-AGTTCCGCATTCGCTGACATC-3' and 5'-CATCCTGTTAAGCTTAGTGGGTCTTGTCGCTGC-3') to amplify the target sequence containing the His tag sequence, which was then inserted into the pBIC3 DNA vector (vector sequence shown in [reference needed]). Figure 25 ).

[0401] Purification of h4D5 iSoMAb Fab

[0402] The expression vector obtained above was introduced into short bacilli to prepare transformants, which were then cultured in MT medium at 30°C for 3 days. h4D5 iSoMAb Fab was purified by adding Ni-NTA resin (Ni Sepharose Excel, Cytiva) to the culture supernatant obtained after centrifugation. The culture supernatant was analyzed by SDS-PAGE, CBB staining, and Western blotting. Anti-His-tagged antibody (MBL: Code No. D291-3) and anti-human κ chain antibody (SIGMA: A7164) were used as detection antibodies. CBB staining was performed on the purified antibodies. Similarly, purified antibodies were prepared and CBB stained for h4D5 WT (wild-type) Fab.

[0403] The results are shown in Figure 26 and 27 When using anti-His-tagged antibody and anti-human κ chain antibody as detection antibodies, peptides with heavy and light chains cross-linked via isopeptide bonds were detected in both cases. Additionally, peptides with heavy and light chains cross-linked were also detected in purified h4D5 iSoMAb Fab.

[0404] (Example 20): Preparation of chimeric antigen receptor for trastuzumab Fab with isopeptide bond formation and determination of its binding activity with antigen)

[0405] Typically, chimeric antigen receptors (CARs) are artificial chimeric proteins formed by fusing a single-chain antibody that recognizes cell surface antigens of cancer cells with a signal transduction region that induces T cell activation. However, the fabrication of single-chain antibodies that recognize cell surface antigens is known to be challenging. Therefore, CARs containing antibodies that form isopeptide bonds were fabricated, and their binding activity with the antigen was measured.

[0406] • Fabrication and expression validation of CAR expression vector for trastuzumab Fab that forms isopeptide bonds

[0407] CAR expression vectors containing human CD28, CD3zeta, and high-sensitivity green fluorescent protein (EGFP) downstream of the h4D5 iSoMAb Fab light chain prepared in Example 3 were prepared (i). Additionally, CAR expression vectors containing the h4D5 iSoMAb Fab heavy chain were prepared (ii) (as shown in Example 3). Figure 28 (As shown). The obtained CAR has serial number 89.

[0408] HEK293 cells were spaced at 2 x 10⁻⁶ cells per well. 510 cells were seeded in 24-well plates. The following day, for each well, a total of 500 ng of CAR expression vector (i) and / or CAR expression vector (ii) was mixed with 1.5 μL of polyethyleneimine (1 mg / mL) and added to HEK293 cells at room temperature for 20 minutes. Three days after gene introduction, RIPA buffer containing surfactant was added, and lysates were prepared by pipetting. Sample buffer containing reducing agent was added, and the mixture was boiled before SDS-PAGE. The lysate was transferred to a PVDF membrane and blotted with anti-GFP antibody.

[0409] The results are shown in Figure 29 Cells in which only the gene from CAR expression vector (i) was introduced showed a band of approximately 80 kDa. In cells in which both CAR expression vectors (i) and (ii) were introduced, the intensity of the approximately 80 kDa band decreased, but a band containing trastuzumab Fab, which forms isopeptide bonds, was detected on the high molecular weight side (indicated by the arrow). Furthermore, HEK293 cells in which both vectors were introduced were observed under a fluorescence microscope, and the results are as follows: Figure 30 As shown, the chimeric antigen receptor of trastuzumab Fab, which forms an isopeptide bond, exists on the cell membrane surface.

[0410] • Determination of binding activity with antigen

[0411] The gene of the HER2 (extracellular domain with secretion signaling)-mouse Fc expression vector was introduced into HEK293 cells, and a culture supernatant was prepared after 2 days. This culture supernatant (containing 1 μg / mL HER2-mouse Fc), quantified by dot blot analysis using mouse IgG as a standard, was added to HEK293 cells 3 days after gene introduction using CAR expression vector (i) and / or CAR expression vector (ii), and incubated at 4°C for 1 hour. After washing three times with PBS (-) containing 1% BSA, Hoechst 33342 (for staining against mouse IgG-PE and nuclei) was added, and the cells were incubated at 4°C for 1 hour. Then, after washing three times with PBS (-) containing 1% BSA, the cells were fixed with 4% formaldehyde and observed under a fluorescence microscope.

[0412] The results are shown in Figure 31 It was confirmed that HER2-mouse Fc, indicated by the arrows, bound to the cell membrane of HEK293 cells after the introduction of CAR expression vectors (i) and (ii).

[0413] (Example 21)

[0414] The mutants listed in the table below were prepared in the same manner as described above to confirm the formation of isopeptide bonds between CH1 and CL. The heavy chain of iSoMAbv2 has sequence number 91, and iSoMAbv2 HC mut15 has sequence number 92.

[0415] [Table 13]

[0416] [Table 14]

[0417] [Table 15]

[0418] [Table 16]

[0419] The results are as follows Figure 32 As shown, under denaturing conditions, any mutant produced a band in the H+L molecular weight region, indicating the presence of isopeptide bonds between CH1 and CH1. It is believed that these antibodies formed isopeptide bonds between lysine residue at position 183 of the heavy chain and asparagine residue at position 176 of the light chain.

[0420] (Example 22)

[0421] The mutants listed in the table below were prepared in the same manner as described above to confirm the formation of isopeptide bonds between CH1 and CL. The heavy chain of iSoMAbv2 has sequence number 91, iSoMAbv2 HC mut15 has sequence number 92, and iSoMAbv2 LC mut3 has sequence number 94.

[0422] [Table 17]

[0423] [Table 18]

[0424] [Table 19]

[0425] [Table 20]

[0426] The results are as follows Figure 33 As shown, under denaturing conditions, all mutants (lanes 1, 2, and 3) produced bands in the H+L molecular weight region, indicating the existence of isopeptide bonds between CH1 and CH1C. If mutations in the heavy chain F170E and S183K and the light chain S176N are present, isopeptide bond formation between CH1 and CH1C is sufficient under physiological conditions. Further mutation introduction can improve isopeptide formation efficiency. It is believed that these antibodies form isopeptide bonds between lysine at position 183 of the heavy chain and asparagine at position 176 of the light chain.

[0427] As can be understood from the above examples, the formation of covalent bonds based on isopeptide bonds between CH1-CLs does not negatively affect the binding affinity of the antibody to the antigen; on the contrary, it stabilizes the complex formed by the heavy and light chains. The result of this complex stabilization is that introducing isopeptide bonds between the CH1-CLs of the antibody even increases the antibody's binding affinity to the antigen. Furthermore, the formation reaction of isopeptide bonds between CH1-CLs is a bioorthogonal reaction and can occur in vivo, which is beneficial in antibody genetic engineering and antibody manufacturing processes. Moreover, asparagine and glutamine, as well as aspartic acid and glutamic acid, are amino acids with very similar biochemical properties. It can 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.

[0428] (Postscript)

[0429] The present disclosure has been illustrated above using preferred embodiments, but the scope of the disclosure should be interpreted solely by the claims. The contents of the patents, patent applications, and documents referenced in this specification are incorporated herein by reference as they are specifically described herein.

[0430] Industrial availability

[0431] The polypeptides containing isopeptide bonds disclosed herein offer one or more advantages, such as improved stability and the ability to generate target substances with high purity. This invention can be used for a wide range of polypeptide production applications, including the generation of recombinant antibodies with complex structures, such as trispecific antibodies.

[0432] Sequence List Free Text

[0433] • Serial number 1: CH1 of h4D5

[0434] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0435] • Serial Number 2: Modification of h4D5 CH1

[0436] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSS

[0437] Serial number 3: h4D5's CL

[0438] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0439] ·Sequence No. 4: Engineered CL of h4D5

[0440] RTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0441] ·Sequence No. 5: CH1 of CD63 antibody

[0442] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0443] ·Sequence No. 6: Engineered CH1v1 of CD63 antibody

[0444] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0445] ·Sequence No. 7: Engineered CH1v2 of CD63 antibody

[0446] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAILQSSGLYLLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0447] ·Sequence No. 8: CL of CD63 antibody

[0448] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0449] • Serial number 9: Modified CD63 antibody CLv1

[0450] RTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0451] • Serial number 10: Modified CD63 antibody CLv2

[0452] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLNSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0453] • Serial number 11: Human IgG1

[0454] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0455] • Serial number 12: Human IgG2

[0456] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0457] ·Sequence No. 13: Human_IgG3

[0458] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0459] ·Sequence No. 14: Human_IgG4

[0460] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0461] · serial number 15: mouse_IgG1

[0462] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPKPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRKPAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0463] · serial number 16: mouse_IgG2A

[0464] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPCIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFCKKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0465] · serial number 17: mouse_IgG2B

[0466] AKTTPPSVYPLAPGCGDTTGSSVTSGCLVKGYFPPVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTVPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPNIKDVLMISLTKPVTVVVVSEDDPDVQISWFV NNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFCKVNNKDLPSPIERTISKIKGLVRAPQVYTLPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

[0467] · serial number 18: mouse_IgG2C

[0468] AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEVLHNHLTTKTISRSLGK

[0469] ·Sequence No. 19: Mouse_IgG3

[0470] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPGK

[0471] ·Sequence No. 20: Rat_IgG1

[0472] AETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRNCGGDCKPCICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEVHTAQTRPPEEQFNSTFRSVSELPILHQDWLNGRTFRCKVTSAAFPSPIEKTISKPEGRTQVPHVYTMSPTKEEMTQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPPTMDTDGSYFLYSKLNVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0473] • Sequence No. 21: Rat_IgG2A

[0474] AETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWSSQAVTCNVAHPASSTKVDKKIVPRECNPCGCTGSEVSSVFIFPPKTKDVLTITLTPKVTCVVVDISQNDPEVRFSWFIDDVEVHTAQTHAPEKQSNSTLRSVSELPIVHRDWLNGKTFKCKVNSGAFPAPIEKSISKPEGTPRGPQVYTMAPPKEEMTQSQVSITCMVKGFYPPDIYTEWKMNGQPQENYKNTPPTMDTDGSYFLYSKLNVKKETWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0475] • Sequence No. 22: Rat_IgG2B

[0476] AQTTAPSVYPLAPGCGDTTSSTVTLGCLVKGYFPEPVTVTWNSGALSSDVHTFPAVLQSGLYTLTSSVTSSTWPSQTVTCNVAHPASSTKVDKKVERRNGGIGHKCPTCPTCHKCPVPELLGGPSVFIFPPKPKDILLISQNAKVTCVVVDVSEEEPDVQFSWFVNNVEVHTAQTQPREEQYNSTFRVVSALPIQHQDWMSGKEFKCKVNNKALPSPIEKTISKPKGLVRKPQVYVMGPPTEQLTEQTVSLTCLTSGFLPNDIGVEWTSNGHIEKNYKNTEPVMDSDGSFFMYSKLNVERSRWDSRAPFVCSVVHEGLHNHHVEKSISRPPGK

[0477] • Sequence No. 23: Rat_IgG2C

[0478] ARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGVHTFPAVLQSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPRRPKPRPPTDICSCDDNLGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDNVRVFTAQTQPHEEQLNGTFRVVSTLHIQHQDWMSGKEFKCKVNNKDLPSPIEKTISKPRGKARTPQVYTIPPPREQMSKNKVSLTCMVTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLYSKLSVDTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK

[0479] • Sequence No. 24: Rabbit_IgG

[0480] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSEDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHEDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0481] • Sequence No. 25: Canine_IgG-A

[0482] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK

[0483] • Sequence No. 26: Canine_IgG-B

[0484] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGK

[0485] ·Sequence No. 27: Canine_IgG-C

[0486] ASTTAPSVFPLAPSCGSQSGSTVALACLVSGYIPEPVTVSWNSVSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPATNTKVDKPVAKECECKCNCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPGK

[0487] ·Sequence No. 28: Canine_IgG-D

[0488] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKVDKPVPKESTCKCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGK

[0489] ·Sequence No. 29: Feline_IgG1a

[0490] ASTTAPSVFPLAPSCGTTSGATVALACLVLGYFPEPVTVSWNSGALTSGVHTFPAVLQASGLYSLSSMVTVPSSRWLSDTFTCNVAHPPSNTKVDKTVRKTDHPPGPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK

[0491] ·Sequence No. 30: Feline_IgG2

[0492] ASTTASSVFPLAPSCGTTSGATVALACLVLGYFPEPVTVSWNSGALTSGVHTFPSVLQASGLYSLSSMVTVPSSRWLSDTFTCNVAHRPSSTKVDKTVPKTASTIESKTGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK

[0493] • Sequence No. 31: human_CL-kappa

[0494] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0495] • Sequence No. 32: mouse_CL-kappa

[0496] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0497] • Sequence No. 33: rat_CL-kappa

[0498] RADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDSTYSMSSTLSLTKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC

[0499] • Sequence No. 34: rabbit_CL-kappa

[0500] GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0501] • Sequence No. 35: Canine_CL-kappa

[0502] RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0503] • Sequence No. 36: Feline_CL-kappa

[0504] RSDAQPSVFLFQPSLDELHTGSASIVCILNDFYPKEVNVKWKVDGVVQNKGIQESTTEQNSKDSTYSLSSTLTMSSTEYQSHEKFSCEVTHKSLASTLVKSFNRSECQRE

[0505] • Sequence No. 37: Human_CL-lambda

[0506] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0507] • Sequence No. 38: Mouse_CL-lambda

[0508] GQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS

[0509] • Sequence No. 39: Rat_CL-lambda

[0510] GQPKSTPTLTVFPPSTEELQGNKATLVCLISDFYPSDVEVAWKANGAPISQGVDTANPTKQGNKYIASSFLRLTAEQWRSRNSFTCQVTHEGNTVEKSLSPAECV

[0511] Serial Number 40: Rabbit_CL-lambda

[0512] GQPVVAPLVTLFPPSSEELKANKATLVCLISDFYPGTLTVAWKADGVTITQGVDTTQPSKQSNNKYMASSFLSLSRGQWTSHSRFTCQVTHEGKTTEKSVAPARCP

[0513] Serial Number 41: Dog_CL-lambda

[0514] GQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKASGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS

[0515] Serial Number 42: Cat_CL-lambda

[0516] GQPKSAPSVTLFPPSNEELSANKATLVCLISDFYPSGLTVAWKADGTPITQGVETTKPSKQSNNKYAASSYLSLSPNEWKSRSRFTCQVTHEGSTVEKNVVPAECS

[0517] Serial Number 43: hCD63 WT HC

[0518] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

[0519] • Sequence No. 44: hCD63 WT LC

[0520] MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0521] • Sequence No. 45: hCD63 iSoMAbv1 HC

[0522] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0523] ·Sequence No. 46: hCD63 iSoMAbv1 LC

[0524] MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0525] ·Sequence No. 47: hCD63 WT VH-CH1 of Fab

[0526] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0527] • Sequence No. 48: hCD63 WT LC of Fab

[0528] MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0529] • Sequence No. 49: hCD63 iSoMAbv1 VH-CH1 of Fab

[0530] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0531] • Sequence No. 50: hCD63 iSoMAbv1 LC of Fab

[0532] MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0533] • Sequence No. 51: h4D5 WT HC

[0534] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0535] • Sequence No. 52: h4D5 WT LC

[0536] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0537] • Sequence No. 53: h4D5 iSoMAb HC

[0538] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0539] • Sequence No. 54: h4D5 iSoMAb LC

[0540] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0541] • Sequence No. 55: hCD63 WT VH-CH1 of Fab

[0542] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0543] • Sequence No. 56: hCD63 WT LC of Fab

[0544] MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0545] • Sequence No. 57: hCD63 iSoMAb VH-CH1 of Fab

[0546] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0547] ·Sequence No. 58: hCD63 iSoMAb LC of Fab

[0548] MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0549] ·Sequence No. 59: h4D5 iSoMAb HC mut1

[0550] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0551] SEQ ID NO: 60: h4D5 iSoMAb HC mut2

[0552] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYSLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0553] Sequence No. 61: h4D5 iSoMAb HC mut3

[0554] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0555] SEQ ID NO: 62: h4D5 iSoMAb HC mut4

[0556] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLDSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0557] • Sequence No. 63: h4D5 iSoMAb LC mut1

[0558] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0559] • Sequence No. 64: h4D5 iSoMAb LC mut2

[0560] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0561] • Sequence No. 65: h4D5 iSoMAb LC mut3

[0562] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSTLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0563] • Sequence No. 66: h4D5 iSoMAb LC mut4

[0564] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0565] • Sequence No. 67: h4D5 iSoMAb LC mut5

[0566] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0567] • Sequence No. 68: h4D5 iSoMAb LC mut6

[0568] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSTLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0569] • Sequence No. 69: h4D5 iSoMAb LC mut7

[0570] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0571] • Sequence No. 70: h4D5 iSoMAb LC mut8

[0572] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0573] • Sequence No. 71: h4D5 iSoMAb LC mut9

[0574] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0575] • Sequence No. 72: h4D5 iSoMAb LC mut10

[0576] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0577] • Sequence No. 73: h4D5 iSoMAb LC mut11

[0578] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0579] • Sequence No. 74: h4D5 iSoMAb LC mut12

[0580] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSLLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0581] • Sequence No. 75: h4D5 iSoMAb LC mut13

[0582] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0583] • Sequence No. 76: h4D5 iSoMAb LC mut14

[0584] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAVVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0585] ·Sequence No. 77: h4D5 iSoMAb LC mut15

[0586] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0587] ·Sequence No. 78: h4D5 iSoMAb LC mut17

[0588] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0589] ·Sequence No. 79: h4D5 WT VH-CH1-His

[0590] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSSHHHHHH

[0591] • Sequence No. 80: h4D5 iSoMAb VH-CH1-His

[0592] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSGSSHHHHHH

[0593] • Sequence No. 81: Nivolumab iSoMAb HC

[0594] MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLNSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0595] ·Sequence No. 82: Nivolumab iSoMAb LC

[0596] MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0597] ·Sequence No. 83: Panitumumab iSoMAb HC

[0598] MEFGLSWVFLVALLRGVQCQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLNSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0599] • Sequence No. 84: Panitumumab iSoMAb LC

[0600] MEAPAQLLFLLLLWLPDTTGDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0601] • Sequence No. 85: Anti-BCMA iSoMAb HC

[0602] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVKPGGSLRLSCAASGFTFSNSGMIWVRQAPGKGLEWVGHIRSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGGSGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAILQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0603] • Sequence No. 86: Anti-BCMA iSoMAb LC

[0604] MAWSPLLLTLLAHCTGSWAQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKAVLECLISDFYPGAVTVAWKADGSPVKAGVETMKPSKQSNNKYAAKSLLQLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0605] • Sequence No. 87: h4D5VL-isoCL

[0606] MSISVRFKSLIALLMTVVFLLVPSSAFADIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0607] • Sequence No. 88: His-h4D5VH-isoCH1

[0608] MKKRRVVNSVLLLLLLASALALTVAPMAFAADHHHHHHDDDDKEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTH

[0609] • Sequence No. 89: h4D5VL-isoCL-CD28-CD3Zeta-EGFP

[0610] METPAQLLFLLLLWLPESTGDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLKSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECCFNGGSGGAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0611] ·Sequence Number 90: h4D5VH-isoCH1

[0612] MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYLLNSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0613] • Sequence No. 91: hCD63 iSoMAbv2 HC

[0614] KHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAILQSSGLYLLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0615] • Sequence No. 92: hCD63 iSoMAbv2 HC mut15

[0616] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYYVQWIRQPPGKGLEWMGFIRSGGNTDYNSEFKSRLTISRDTSKNQVSLKLSSVTAADTAVYYCVRGGEYNWDYFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAVLQSSGLYSLKSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0617] • Sequence No. 93: hCD63 iSoMAbv2 LC

[0618] MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQEMVTEQDSKDSTYSLNSLLQLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0619] • Sequence No. 94: hCD63 iSoMAbv2 LC mut3

[0620] MVLQTQVFISLLLWISGAYGDIVLTQSPASLAVSPGERATISCRASQSVTIFSINLMQWFQQKPGQPPKLLIYRASNLASGVPDRFSGSGSGTDFTLTISRVEAEDVAVYYCQQTRESPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLNSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A method for preparing a modified polypeptide, wherein the modified polypeptide contains an interpeptide linker of the following formula between a first polypeptide chain and a second polypeptide chain. In the formula, the wavy lines represent the first polypeptide chain or the second polypeptide chain, respectively. The method includes: The steps of constructing a three-dimensional structure of intermediate A containing the first polypeptide chain and the second polypeptide chain; The step of determining the amino acid sequence present in the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other in the stereostructure; The step of mutating intermediate A, wherein intermediate B is prepared 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, wherein intermediate B contains a), b) and c), and the first polypeptide chain and the second polypeptide chain in intermediate B contain a) and b) respectively in the boundary region; as well as The step of obtaining the modified polypeptide by forming the interpeptide linkage in the intermediate B.

2. The method according to claim 1, wherein, Based on the distance between the first amino acid residue of the first polypeptide chain and the 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, a) and / or b) are introduced.

3. The method according to claim 1, wherein, b) is asparagine or aspartic acid, and in the stereostructure of intermediate A, the positions of two amino acids with a distance of about 7 to 9 Å between the α carbons are determined 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 stereostructure of the intermediate A, the positions of the two amino acids with a distance of about 8.5 to 10 Å between the α carbons are determined as the amino acid positions of a) and b).

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

6. The method according to claim 1, wherein, It also includes the step of constructing the three-dimensional structure of the intermediate B.

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

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

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

10. The method according to claim 1, wherein, The first polypeptide chain contains c).

11. The method according to claim 1, wherein, It also includes a step of determining the hydrophobicity of the boundary region.

12. The method according to claim 1, wherein, It also includes the following steps: in the amino acid sequence present in the boundary region, performing a mutation to delete hydrophilic amino acid residues, a mutation to replace amino acid residues with hydrophobic amino acid residues, or inserting hydrophobic amino acid residues.

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

14. The method according to claim 1, wherein, Hydrophobic amino acids with side chains of different volumes are introduced to fill the space in the boundary region.

15. The method according to claim 1, wherein, The first polypeptide chain and the second polypeptide chain are contained in different molecules.

16. The method according to claim 15, wherein, It also includes the step of introducing a mutation that causes at least one of the two cysteine ​​residues that form a disulfide bond between the first polypeptide chain and the second polypeptide chain in the intermediate A to be deleted or replaced with another amino acid.

17. The method according to claim 15, wherein, Further, a secondary modified polypeptide is prepared, comprising an interpeptide linker as shown in the following formula, between the modified polypeptide and a third polypeptide chain contained in a molecule different from the modified polypeptide. In the formula, the wavy line represents the binding to the polypeptide backbone. The method includes: The step of constructing a three-dimensional structure of a secondary intermediate A containing the modified polypeptide and the third polypeptide chain; The step of determining the amino acid sequence present in the boundary region where the modified polypeptide and the third polypeptide chain are close to each other in the stereostructure; The step of mutating the secondary intermediate A includes preparing secondary intermediate B by introducing at least one residue from a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid into the secondary intermediate A, wherein the secondary intermediate B contains a), b) and c), and the modified polypeptide and the third polypeptide chain in the secondary intermediate B each contain a) and b) in the boundary region; and The step of obtaining the secondary modified polypeptide by forming the interpeptide linkage in the secondary intermediate B.

18. A polypeptide having an interpeptide linker represented by the following formula between a first polypeptide chain and a second polypeptide chain. In the formula, the wavy line represents the binding to the polypeptide backbone. The polypeptide contains a) lysine, b) glutamine, glutamic acid, asparagine or aspartic acid, and c) glutamic acid or aspartic acid. Here, in the three-dimensional structure of the polypeptide, at the boundary region where the first polypeptide chain and the second polypeptide chain are close to each other, the first polypeptide chain and the second polypeptide chain respectively contain a) and b).

19. The polypeptide according to claim 18, wherein it is an antibody or a T-cell receptor (TCR).

20. The polypeptide according to claim 19, wherein, The peptide linkages exist in the constant region.

21. The polypeptide according to claim 18, wherein, Peptide linkages between the heavy chain constant region and the light chain constant region containing antibodies.

22. The polypeptide according to claim 18, wherein, The first polypeptide chain and the second polypeptide chain exist on different molecules.

23. The amino acid at position 183 of the heavy chain is replaced by the CH1 region of asparagine or the nucleic acid encoding that region. Heavy chain constant regions containing the CH1 region or nucleic acids encoding that region, or Heavy chains containing the CH1 region or nucleic acids encoding the heavy chain.

24. A CL region or nucleic acid encoding this region in which the amino acid at position 133 of the light chain is replaced by glutamic acid and the amino acid at position 176 of the light chain is replaced by lysine. The light chain constant region containing the CL region or the nucleic acid encoding the region, or A light chain containing the CL region or a nucleic acid encoding the light chain.

25. An antibody comprising the heavy chain of claim 23 and the light chain of claim 24.

26. A Fab fragment, Fab' fragment, or F(ab')2 fragment containing the CH1 region of claim 23 and the CL region of claim 24.

27. A chimeric antigen receptor having a Fab fragment containing 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 that expresses the chimeric antigen receptor of claim 27.

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